Mutant Human Nax Proteins and Screening Methods

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Nov. 14, 2023, is named “01164-0016-00US.xml” and is 178,992 bytes in size.

FIELD

The present disclosure relates to mutant human Na_Xion channel (“Na_X”) proteins and screening methods using a mutant human Na_Xthat may be used to identify molecules that modulate the activity of human Na_X. For example, in some embodiments, screening methods involve performing an ion channel assay on a mutant human Na_Xin the presence of a potential Na_Xmodulator. The disclosure also relates to molecules that act as modulators of human Na_Xand associated kits for detecting such molecules.

BACKGROUND

Prototypical voltage-gated sodium (Na_V) channels perform key roles in electrical signaling by initiating and propagating action potentials. Nine closely related mammalian channel subtypes, Na_V1.1-Na_V1.9, are expressed in a range of tissues where they contribute to essential physiological processes including nervous system function, muscle contraction, and hormone secretion. A tenth Na_Vchannel-like gene, SCN7A, was cloned nearly three decades ago, but atypical sequence features and the inability to record voltage-activated sodium (Na⁺)-currents from this Na_V2.1 protein raised speculation that it might have distinct physiological roles, resulting in the designation Na_X.

In contrast to canonical Na_Vchannels, Na_Xhas been proposed to function as a Na⁺-channel activated by the extracellular Na⁺ concentration. Multiple lines of evidence suggest that Na_Xmay contribute to Na⁺ homeostasis^1,2. Na_Xshows restricted expression in a brain area that specializes in monitoring blood composition⁵. Na_X-knockout mice ingest salt despite dehydration and are resistant to hypertension caused by elevated blood Na⁺ levels^5,6. In humans, autoimmunity against Na_Xcauses chronic hypernatremia with impaired thirst perception and salt appetite⁷. In the periphery, Na_Xregulates Na⁺ homeostasis upstream of the epithelial Na⁺ channel (ENaC) and has been suggested as a target to treat atopic dermatitis and hypertrophic scarring⁸.

Na_Xshares highest sequence identity (˜55%) with the Na_V1.7 channel¹⁶but it is the most divergent member of the Na_Vchannel family. In conventional Na_Vchannels, the voltage sensor domains (VSDs) contain 4-8 gating charge residues conserved along the S4 segment that undergo outward movement upon membrane depolarization to open the channel gate (VSD1-VSD3) or initiate fast inactivation (VSD4). In contrast to this paradigm, Na_Xhas been reported to be voltage-insensitive and contain a reduced number of S4 gating charge residues. Thus, the function and conformational state of the voltage sensor-like domains (VSLDs) in Na_Xis unclear, and how the VSLDs contribute to channel gating is unknown. In addition, the DIII-DIV linker segment, which is divergent in Na_Xin comparison to Na_V, is known to be required for fast inactivation in Na_Vchannels.

SUMMARY

The present disclosure relates to mutant human Na_Xion channel (“Na_X”) proteins and screening methods using a mutant human Na_Xthat may be used to identify molecules that modulate the activity of human Na_X, and to molecules identified by such methods and kits for performing the methods. The mutant human Na_Xproteins and associated methods may be used, for example, to identify molecules that not only modulate Na_Xactivity, but also to test molecules known to bind to or modulate Na_Vproteins to test their selectivity to their target proteins. Modulators identified in assays described herein using a mutant Na_X, may be useful as modulators/candidate modulators of human Na_X. The present inventors have surprisingly found mutant Na_Xproteins that allow ion channel measurements relevant to human Na_Xby mimicking an open, conductive state of the ion channel. Without being bound by theory, mutant Na_Xmay mimic an open, conductive state of the Na_Xion channel by mimicking, e.g., phosphorylation of the ion channel protein(s) and/or other relevant physiological or structural changes that normally allow the Na_Xion channel to open. The present disclosure includes, for example, any one or a combination of the following embodiments.

In some embodiments, the disclosure herein relates to methods of identifying a human Na_Xion channel protein (Na_X) modulator, comprising: (a) providing a mutant human Na_Xin which at least a portion of the DIII domain of human Na_Xis replaced by a corresponding portion of the DIII domain of a human or mammalian Na_Vprotein (Na_V); (b) performing an ion channel assay on the mutant human Na_Xin the presence of a potential Na_Xmodulator; and (c) identifying the potential modulator as a human Na_Xmodulator if the activity of the mutant human Na_Xin the assay in the presence of the potential modulator is higher or lower than the activity in the absence of the potential modulator. In some cases, the mutant human Na_Xcomprises all or a portion of the DIII S1-S6 region from a human or mammalian Na_V, optionally wherein the at least a portion of the DIII domain of human Na_Xthat is replaced comprises or consists of: (a) DIII (residues 920-1200), (b) DIII voltage-sensor domain III (VSD3) and S4-S5 linker (residues 920-1058), (c) DIII VSD3, S4-S5 linker and S5 (residues 920-1078), (d) DIII and DII-DIII linker (residues 733-1200), (e) DIII and DIII-DIV linker (920-1237), (f) DIII and DII-DIII linker and DIII-DIV linker (733-1237). In some cases, the at least a portion of the DIII domain of human Na_Xthat is replaced by a corresponding portion of the DIII domain of the human or mammalian Na_Vdoes not comprise S5 and/or does not comprise S6. In some cases, the at least a portion of the DIII domain of human Na_Xthat is replaced by a corresponding portion of the DIII domain of the human or mammalian Na_Vdoes not comprise any of S1, S2, S3, S4, and/or S4-S5 linker. In some cases, the at least a portion of the DIII domain of human Na_Xis replaced by a corresponding portion of mammalian Na_V1.1, mammalian Na_V1.2, mammalian Na_V1.3, mammalian Na_V1.4, mammalian Na_V1.5, mammalian Na_V1.6, mammalian Na_V1.7, mammalian Na_V1.8, mammalian Na_V1.9, human Na_V1.1, human Na_V1.2, human Na_V1.3, human Na_V1.4, human Na_V1.5, human Na_V1.6, human Na_V1.7, human Na_V1.8, or human Na_V1.9. For example, in some cases, the at least a portion of the DIII domain of human Na_Xis replaced by a corresponding portion of human Na_V1.7. In some cases, the mutant human Na_Xis chimera construct 2, chimera construct 3, chimera construct 7, chimera construct 9, chimera construct 11, chimera construct 15 or chimera construct 19, or comprises an amino acid sequence of any one of SEQ ID Nos: 3, 4, 8, 10, 12, 16, or 20.

The present disclosure also encompasses methods of identifying a human Na_Xion channel protein (Na_X) modulator, comprising: (a) providing a mutant human Na_Xcomprising a substitution of at least one residue on each of two, three, or four S6 alpha helices of human Na_Xwith a glycine, proline, or polar or charged residue; (b) performing an ion channel assay on the mutant human Na_Xin the presence of the potential modulator; and (c) identifying the potential modulator as a human Na_Xmodulator if the activity of the mutant human Na_Xin the assay in the presence of the potential modulator is higher or lower than the activity in the absence of the potential modulator. In some cases, the mutant human Na_Xcomprises a substitution of at least one residue on two of the four S6 alpha helices with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises a substitution of at least one residue on three of the four S6 alpha helices with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises a substitution of at least one residue on all four S6 alpha helices with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises one substitution of an amino acid residue on at least three of the four S6 alpha helices with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises one or two substitutions of an amino acid residue with a glycine, proline, or polar or charged residue within at least two of following segments of SEQ ID NO: 1: residues 383-397 (in S6 of domain I (D1)), residues 717-731 (in S6 of DII), residues 1182-1196 (in S6 of DIII), and/or residues 1485-1499 (in S6 of DIV). In some cases, the mutant human Na_Xcomprises one or two substitutions of an amino acid residue with a glycine, proline, or polar or charged residue within at least three of the following segments of SEQ ID NO: 1: residues 383-397 (in S6 of domain I (D1)), residues 717-731 (in S6 of DII), residues 1182-1196 (in S6 of DIII), and/or residues 1485-1499 (in S6 of DIV). In some cases, the mutant human Na_Xcomprises one or two substitutions of an amino acid residue with a glycine, proline, or polar or charged residue within each of the following segments of SEQ ID NO: 1: residues 383-397 (in S6 of domain I (D1)), residues 717-731 (in S6 of DII), residues 1182-1196 (in S6 of DIII), and/or residues 1485-1499 (in S6 of DIV). In some cases, the mutant human Na_Xcomprises substitutions of an amino acid residue at two or more of residues L390, F724, I1189, and I1492 with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises substitutions of an amino acid residue at three or more of residues L390, F724, I1189, and I1492 with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises substitutions of amino acid residues L390, F724, and I1189 or of amino acid residues F724, I1189, and I1492 with glycine, proline, or polar or charged residues. In some cases, the mutant human Na_Xcomprises substitutions F724Q, I1189T, and I1492T compared to wild-type human Na_X. In some cases, the human Na_Xcomprises substitutions L390E, I1189E, and I1492E compared to wild-type human Na_X.

In any of the above methods, the ion channel assay may also be performed in the presence of an identified modulator of the mutant human Na_X, for example, a modulator identified herein or otherwise identified in performance of the methods herein. In some such cases, the identified modulator of the mutant human Na_Xis one or more of tetrodotoxin (TTX), quinidine, flecainide, tetracaine, or lidocaine.

In any of the above methods, the potential modulator may be a peptide or macrocycle or antibody. For example, in some cases, the potential modulator is a small molecule. In some cases, a small molecule may be a derivative of tetrodotoxin (TTX), quinidine, flecainide, tetracaine, or lidocaine, for example.

Any of the above methods may also further comprise determining the binding affinity of the potential modulator identified in part (c) to either or both of the mutant human Na_Xand wild-type human Na_X. In some cases, the potential modulator binds to either or both of the mutant human Na_Xand wild-type human Na_Xan EC50 or IC50 of 10 μM or less, 10 μM to 50 nM, 10 M to 500 nM, 1 μM or less, 1 μM to 50 nM, or 100 nM or less. In some cases, an EC50 or IC50 may be determined in an ELISA assay.

In any of the above methods, the ion channel assay may be a patch clamp or an automated patch clamp assay, an ion flux assay, or an ion- or voltage-sensitive dye assay. In any of the above methods, the potential modulator identified in part (c) may reduce the activity of the mutant human Na_Xin the assay by at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In some cases, the potential modulator identified in part (c) reduces the activity of the mutant human Na_Xin the assay with a half-maximal concentration of 10 nM to 500 μM, 50 nM to 500 μM, 10 nM to 50 μM, 100 nM to 500 μM, 100 nM to 50 μM, 1-500 μM, 1-50 μM, 10-500 μM, or 50-250 μM. In other cases, the potential modulator identified in part (c) increases the activity of the mutant human Na_Xin the assay.

The present disclosure also relates to modulators of human Na_Xidentified by the methods described herein, which may be optionally peptides, macrocycles, small molecules, or antibodies.

The present disclosure also encompasses mutant human Na_Xion channel (Na_X) proteins, wherein at least a portion of the DIII domain of human Na_Xis replaced by a corresponding portion of the DIII domain of a human or mammalian Na_Vprotein (Na_V). In some cases, the mutant human Na_Xcomprises all or a portion of the DIII S1-S6 region from a human or mammalian Na_V, optionally wherein the at least a portion of the DIII domain of human Na_Xthat is replaced comprises or consists of: (a) DIII (residues 920-1200), (b) DIII voltage-sensor domain III (VSD3) and S4-S5 linker (residues 920-1058), (c) DIII VSD3, S4-S5 linker and S5 (residues 920-1078), (d) DIII and DII-DIII linker (residues 733-1200), (e) DIII and DIII-DIV linker (920-1237), (f) DIII and DII-DIII linker and DIII-DIV linker (733-1237). In some cases, the at least a portion of the DIII domain of human Na_Xthat is replaced by a corresponding portion of the DIII domain of the human or mammalian Na_Vdoes not comprise S5 and/or does not comprise S6. In some cases, the at least a portion of the DIII domain of human Na_Xthat is replaced by a corresponding portion of the DIII domain of the human or mammalian Na_Vdoes not comprise any of S1, S2, S3, S4, and/or S4-S5 linker. In some cases, the at least a portion of the DIII domain of human Na_Xis replaced by a corresponding portion of mammalian Na_V1.1, mammalian Na_V1.2, mammalian Na_V1.3, mammalian Na_V1.4, mammalian Na_V1.5, mammalian Na_V1.6, mammalian Na_V1.7, mammalian Na_V1.8, mammalian Na_V1.9, human Na_V1.1, human Na_V1.2, human Na_V1.3, human Na_V1.4, human Na_V1.5, human Na_V1.6, human Na_V1.7, human Na_V1.8, or human Na_V1.9. For example, in some cases, the at least a portion of the DIII domain of human Na_Xis replaced by a corresponding portion of human Na_V1.7. In some cases, the mutant human Na_Xis chimera construct 2, chimera construct 3, chimera construct 7, chimera construct 9, chimera construct 11, chimera construct 15 or chimera construct 19, or comprises an amino acid sequence of any one of SEQ ID Nos: 3, 4, 8, 10, 12, 16, or 20.

The present disclosure further includes mutant human Na_Xion channel (Na_X) proteins, comprising a substitution of at least one residue on each of the two, three, or four S6 alpha helices with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises a substitution of at least one residue on two of the four S6 alpha helices with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises a substitution of at least one residue on three of the four S6 alpha helices with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises a substitution of at least one residue on all four S6 alpha helices with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises one substitution of an amino acid residue on at least three of the four S6 alpha helices with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises one or two substitutions of an amino acid residue with a glycine, proline, or polar or charged residue within at least two of following segments of SEQ ID NO: 1: residues 383-397 (in S6 of domain I (D1)), residues 717-731 (in S6 of DII), residues 1182-1196 (in S6 of DIII), and/or residues 1485-1499 (in S6 of DIV). In some cases, the mutant human Na_Xcomprises one or two substitutions of an amino acid residue with a glycine, proline, or polar or charged residue within at least three of the following segments of SEQ ID NO: 1: residues 383-397 (in S6 of domain I (D1)), residues 717-731 (in S6 of DII), residues 1182-1196 (in S6 of DIII), and/or residues 1485-1499 (in S6 of DIV). In some cases, the mutant human Na_Xcomprises one or two substitutions of an amino acid residue with a glycine, proline, or polar or charged residue within each of the following segments of SEQ ID NO: 1: residues 383-397 (in S6 of domain I (D1)), residues 717-731 (in S6 of DII), residues 1182-1196 (in S6 of DIII), and/or residues 1485-1499 (in S6 of DIV). In some cases, the mutant human Na_Xcomprises substitutions of an amino acid residue at two or more of residues L390, F724, I1189, and I1492 with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises substitutions of an amino acid residue at three or more of residues L390, F724, I1189, and I1492 with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises substitutions of amino acid residues L390, F724, and I1189 or of amino acid residues F724, I1189, and I1492 with glycine, proline, or polar or charged residues. In some cases, the mutant human Na_Xcomprises substitutions F724Q, I1189T, and I1492T compared to wild-type human Na_X. In some cases, the human Na_Xcomprises substitutions L390E, I1189E, and I1492E compared to wild-type human Na_X.

The present disclosure further includes methods of determining whether a test molecule that modulates the activity of a human ion channel protein modulates the activity of human Na_Xion channel (Na_X) protein, comprising: (a) providing a mutant human Na_Xin which at least a portion of the DIII domain of human Na_Xis replaced by a corresponding portion of the DIII domain of a human or mammalian Na_Vprotein (Na_V); (b) performing an ion channel assay on the mutant human Na_Xin the presence of the test molecule; and (c) determining that the test molecule is a human Na_Xmodulator if the activity of the mutant human Na_Xin the assay in the presence of the test molecule is higher or lower than the activity in the absence of the test molecule; and optionally, (d) selecting the test molecule for additional screening if it is not a human Na_Xmodulator according to part (c).

The present disclosure additionally includes methods of determining whether a test molecule that modulates the activity of a human ion channel protein modulates the activity of human Na_Xion channel (Na_X) protein, comprising: (a) determining that the test molecule modulates the activity of the human ion channel protein; (b) providing a mutant human Na_Xin which at least a portion of the DIII domain of human Na_Xis replaced by a corresponding portion of the DIII domain of a human or mammalian Na_Vprotein (Na_V); (c) performing an ion channel assay on the mutant human Na_Xin the presence of the test molecule; and (d) determining that the test molecule is a human Na_Xmodulator if the activity of the mutant human Na_Xin the assay in the presence of the test molecule is higher or lower than the activity in the absence of the test molecule; and optionally, (e) selecting the test molecule for additional screening if it is not a human Na_Xmodulator according to part (d).

In the two additional methods above, in some cases the mutant human Na_Xcomprises all or a portion of the DIII S1-S6 region from a human or mammalian Na_V, optionally wherein the at least a portion of the DIII domain of human Na_Xthat is replaced comprises or consists of: (a) DIII (residues 920-1200), (b) DIII voltage-sensor domain III (VSD3) and S4-S5 linker (residues 920-1058), (c) DIII VSD3, S4-S5 linker and S5 (residues 920-1078), (d) DIII and DII-DIII linker (residues 733-1200), (e) DIII and DIII-DIV linker (920-1237), (f) DIII and DII-DIII linker and DIII-DIV linker (733-1237). In some cases, the at least a portion of the DIII domain of human Na_Xthat is replaced by a corresponding portion of the DIII domain of the human or mammalian Na_Vdoes not comprise S5 and/or does not comprise S6. In some cases, the at least a portion of the DIII domain of human Na_Xthat is replaced by a corresponding portion of the DIII domain of the human or mammalian Na_Vdoes not comprise any of S1, S2, S3, S4, and/or S4-S5 linker. In some cases, the at least a portion of the DIII domain of human Na_Xis replaced by a mammalian Na_V1.4, mammalian Na_V1.5, mammalian Na_V1.6, mammalian Na_V1.7, mammalian Na_V1.8, mammalian Na_V1.9, human Na_V1.1, human Na_V1.2, human Na_V1.3, human Na_V1.4, human Na_V1.5, human Na_V1.6, human Na_V1.7, human Na_V1.8, or human Na_V1.9. In some such cases, the at least a portion of the DIII domain of human Na_Xis replaced by a corresponding portion of human Na_V1.7. In some cases, the mutant human Na_Xis chimera construct 2, chimera construct 3, chimera construct 7, chimera construct 9, chimera construct 11, chimera construct 15 or chimera construct 19, or comprises an amino acid sequence of any one of SEQ ID Nos: 3, 4, 8, 10, 12, 16, or 20.

The present disclosure also includes methods of determining whether a test molecule that modulates the activity of a human ion channel protein modulates the activity of human Na_Xion channel (Na_X) protein, comprising: (a) providing a mutant human Na_Xcomprising a substitution of at least one residue on each of two, three, or four S6 alpha helices of human Na_Xwith a glycine, proline, or polar or charged residue; (b) performing an ion channel assay on the mutant human Na_Xin the presence of the test molecule; and (c) determining that the test molecule is a human Na_Xmodulator if the activity of the mutant human Na_Xin the assay in the presence of the test molecule is higher or lower than the activity in the absence of the test molecule; and optionally, (d) selecting the test molecule for additional screening if it is not a human Na_Xmodulator according to part (c).

The disclosure further encompasses methods of determining whether a test molecule that modulates the activity of a human ion channel protein also modulates the activity of human Na_Xion channel (Na_X) protein, comprising: (a) determining that the test molecule modulates the activity of the human ion channel protein; (b) providing a mutant human Na_Xcomprising a substitution of at least one residue on each of two, three, or four S6 alpha helices of human Na_Xwith a glycine, proline, or polar or charged residue; (c) performing an ion channel assay on the mutant human Na_Xin the presence of the test molecule; and (d) determining that the test molecule is a human Na_Xmodulator if the activity of the mutant human Na_Xin the assay in the presence of the test molecule is higher or lower than the activity in the absence of the test molecule; and optionally, (e) selecting the test molecule for additional screening if it is not a human Na_Xmodulator according to part (d).

In either of the two methods above, in some cases, the mutant human Na_Xcomprises a substitution of at least one residue on two of the four S6 alpha helices with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises a substitution of at least one residue on three of the four S6 alpha helices with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises a substitution of at least one residue on all four S6 alpha helices with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises one substitution of an amino acid residue on at least three of the four S6 alpha helices with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises one or two substitutions of an amino acid residue with a glycine, proline, or polar or charged residue within at least two of following segments of SEQ ID NO: 1: residues 383-397 (in S6 of domain I (D1)), residues 717-731 (in S6 of DII), residues 1182-1196 (in S6 of DIII), and/or residues 1485-1499 (in S6 of DIV). In some cases, the mutant human Na_Xcomprises one or two substitutions of an amino acid residue with a glycine, proline, or polar or charged residue within at least three of the following segments of SEQ ID NO: 1: residues 383-397 (in S6 of domain I (D1)), residues 717-731 (in S6 of DII), residues 1182-1196 (in S6 of DIII), and/or residues 1485-1499 (in S6 of DIV). In some cases, the mutant human Na_Xcomprises one or two substitutions of an amino acid residue with a glycine, proline, or polar or charged residue within each of the following segments of SEQ ID NO: 1: residues 383-397 (in S6 of domain I (D1)), residues 717-731 (in S6 of DII), residues 1182-1196 (in S6 of DIII), and/or residues 1485-1499 (in S6 of DIV). In some cases, the mutant human Na_Xcomprises substitutions of an amino acid residue at two or more of residues L390, F724, I1189, and I1492 with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises substitutions of an amino acid residue at three or more of residues L390, F724, I1189, and I1492 with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises substitutions of amino acid residues L390, F724, and I1189 or of amino acid residues F724, I1189, and I1492 with glycine, proline, or polar or charged residues. In some cases, the mutant human Na_Xcomprises substitutions F724Q, I1189T, and I1492T compared to wild-type human Na_X. In some cases, the human Na_Xcomprises substitutions L390E, I1189E, and I1492E compared to wild-type human Na_X.

The present disclosure also includes molecules identified by the methods above, which optionally may be peptides, macrocycles, small molecules, or antibodies.

The disclosure also includes molecular complexes comprising a chimeric or mutant human Na_Xprotein as described herein and a modulator of the protein or a potential modulator of the protein.

The disclosure also includes kits for assaying the activity of human Na_Xor for identifying a human Na_Xmodulator and/or a molecule that binds to human Na_X, comprising a mutant human Na_Xas described herein, and further comprising at least one of: one or more reagents for conducting an ion channel assay, a modulator of the mutant human Na_X, and instructions for use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-H show characterization of human Na_Xand overall structure of the β3-Na_Xchannel complex. FIG. 1A shows representative currents from Xenopus laevis oocytes expressing human Na_Xor Na_V1.7. FIG. 1B shows representative currents from oocytes expressing Na_Xin response to extracellular application of indicated compounds. FIG. 1C shows representative currents from oocytes expressing Na_Xand co-expression of Na_Vor Ca_Vchannel auxiliary subunits, Na⁺/K⁺-ATPase subunits and synapse-associated protein 97 (SAP97), and in the presence of the indicated extracellular Na⁺ concentration. Voltage protocols for FIGS. 1A-C are as follows: Na_X: steps between +80 to −100 mV, in 20 mV increments from a HP of 0 mV; Na_V1.7: depolarizing steps between −80 and +65 mV, in 5 mV increments, from a HP of −100 mV. FIG. 1D shows data summary of independent experiments performed as in FIGS. 1A-C. FIG. 1E shows representative currents from murine Neuro-2 cells expressing human Na_Xor Na_V1.7 in response to changes of the extracellular Na⁺ concentration (HP=−60 mV), as indicated, or voltage: depolarizing steps between −60 to +60 mV, in 10 mV increments from a HP of −100 mV. FIG. 1F shows data summary of independent experiments performed as in FIG. 1E. FIG. 1G shows Western blots of total lysate and surface fraction of proteins extracted from Neuro-2 cells probed for the indicated proteins. FIG. 1H shows side and extracellular view of the β3-Na_Xchannel complex. Approximate membrane boundaries are indicated. DI, DII, DIII and DIV are colored in green, blue, orange and pink, respectively, with the β3 subunit in grey surface representation.

FIGS. 2A-E show structure of the Na_Xpore module reveals a non-conductive state. FIG. 2A show Na_Xpore volume is shown as grey surface with DII and DIV shown in cartoon rendering (DI and DIII omitted for clarity). FIG. 2B show view of the S6-helices with side-chains lining the activation gate in stick and semi-transparent surface representation. Orthogonal view provides a wider perspective with DIII and DIV colored orange and pink, with the IFI motif (green, in dashed box) from the DIII-DIV linker shown in stick and semi-transparent surface representation. FIG. 2C show orthogonal views sliced through the pore module highlighting lateral fenestrations and bound lipids. The phosphatidylethanolamine that crosses the S6-gate is in purple stick representation. In side view, a lipid in the foreground has been removed for clarity. The view in FIG. 2D is similar to middle panel C, but with cryo-EM map shown around modeled lipids in blue mesh representation. FIG. 2E shows representative currents from Xenopus laevis oocytes expressing human Na_X, human Na_V1.7 or double- and single-domain-swapped Na_V1.7-Na_Xchimeras, including chimera constructs 1-7 shown in Table 4 herein. Steps between +80 to −100 mV, in 20 mV increments, from a HP of 0 mV.

FIGS. 3A-F show characterization of human Na_Xcarrying targeted pore-wetting S6-mutations. FIG. 3A shows location and zoomed view of targeted hydrophobic side-chains lining the S6-gate. FIG. 3B shows representative currents from Xenopus laevis oocytes expressing the Na_X-QTT construct under indicated voltage protocols. FIG. 3C shows representative currents from oocytes expressing the indicated Na_Xconstruct. FIG. 3D shows representative currents from oocytes expressing the Na_X-EEE construct. Voltage protocols for FIGS. 3C-D were as above in FIG. 2E. FIG. 3E shows representative currents from oocytes expressing the Na_X-QTT construct and co-expressing indicated Na_Vauxiliary subunit. Voltage protocols as above. FIG. 3F shows data summary of independent experiments performed in FIGS. 2B-E.

FIGS. 4A-C show structure and characterization of the Na_Xselectivity filter. FIG. 4A shows Na_XDENA-motif residue side-chains shown as sticks. FIG. 4B, top panels, shows Na_Xside-chains and analogous residues that form an interaction network around the selectivity filter in Na_Vchannels are shown in stick representation (Na_V1.7, PDB 6J8J). FIG. 4B, bottom panels show the same view of the Na_Xand Na_V1.7 selectivity filters (as in FIG. 4B, top panels) but as electrostatic surface rending. Central cavity and activation gate excluded for clarity. FIG. 4C shows superimposed and zoomed view of the DI-DIV interface comparing Na_Xand Na_V1.7 (grey, PDB 6J8J) structures with select side-chains shown as sticks. FIG. 4D shows representative currents from HEK293 cells expressing human Na_X-QTT with a C-terminal GFP-Flag tag in a physiological (FIG. 4D, left panel) or NMDG⁺-only extracellular solution (FIG. 4D, middle panel). Steps between +80 to −100 mV, in 20 mV increments, from a HP of 0 mV.

FIG. 4D, right panel, shows I-V curve data summary from independent experiments. FIG. 4E, left panel, shows representative currents from HEK293 cells expressing human Na_X-QTT with indicated monovalent cations in the extracellular solution. Voltage ramp from −80 to +80 mV was applied. FIG. 4E, right panel, shows summary of reversal potentials and permeability ratios measured from independent experiments.

FIGS. 5A-C show pharmacology of the human Na_X-QTT channel. FIG. 5A, left and middle panels, show representative I-V currents from HEK293 cells expressing human Na_X-QTT with indicated extracellular monovalent cations with or without indicated amounts of CaCl₂in the extracellular solution. Voltage ramp from −80 to +80 mV was applied. FIG. 5A, right panel, shows percentage of block of outward Na⁺ by indicated concentrations of Ca²⁺ at 80 mV. FIG. 5B shows representative currents from Xenopus laevis oocytes expressing human Na_X-QTT in standard extracellular solution with or without indicated divalent and trivalent cations (unit in mM), when stepping from 0 to +80 mV. FIG. 5C shows representative currents from oocytes expressing human Na_X-QTT in standard extracellular solution with or without indicated blockers added, when stepping from 0 to +80 mV (left panel) or from 0 to −100 mV (right panel). FIG. 5C, middle panel, shows summary of currents measured from independent experiments.

FIGS. 6A-E show structure of the atypical Na_Xvoltage sensor-like domains. FIG. 6A show VSLD1 with gating charges (blue), HCS (green), and ENC/INC (red) side-chains shown in stick representation. Inset highlights a unique proline in Na_X, with Na_V1.7 VSD1 (PDB 6J8J) superimposed for comparison. FIG. 6B show VSLD2 rendered as in FIG. 6A. Insets highlight His579-S4 interaction (FIG. 6B, top panel) and S4-S5 linker displacements (FIG. 6B, bottom panel), with Na_V1.7 DII (PDB 6J8J) superimposed for comparison. FIG. 6C shows VSLD3 rendered as in FIG. 6A. Inset highlights Phe1011 from the S3 helix. FIG. 6D shows VSLD4, rendered as in FIG. 6A. Inset shows a comparison of the S4/S4-S5 linker region between Na_Xand Na_V1.7 VSD4 (PDB 6J8J), with Ca of gating charge residues indicated as blue spheres. FIG. 6E shows representative currents from Xenopus laevis oocytes expressing Na_V1.7 and various Na_V1.7-Na_X-VSLD in response to depolarizing steps between −80 and +65 mV in 5 mV increments from a HP of −100 mV.

FIGS. 7A-F show functional evaluation of human Na_Xin different expression systems. FIG. 7A shows Western blots of total lysate and surface fraction of proteins extracted from HEK293T cells expressing the indicated constructs. FIG. 7B shows representative currents from HEK293T cells expressing human Na_Xor Na_V1.7 (with C-terminal GFP and Flag tags) with indicated voltage protocol. FIG. 7C shows Western blots of total lysate and surface fraction of proteins extracted from Xenopus laevis oocytes expressing the indicated constructs. FIG. 7D shows representative currents from oocytes expressing human Na_Xor Na_V1.7 with indicated voltage protocol. FIG. 7E shows Western blots of total lysate and surface fraction of proteins extracted from murine Neuro-2A cells expressing the indicated constructs. FIG. 7F shows representative currents from Neuro-2A cells expressing human Na_X(*, with C-terminal GFP and Flag tags) under indicated extracellular Na⁺ concentrations (HP=−60 mV). Seal resistances (Rm) of individual cells are provided.

FIGS. 8A-D show functional evaluation of human Na_Xin Xenopus laevis oocytes. FIGS. 8A, left panel, and 8B, left panel, show representative currents from Xenopus laevis oocytes expressing human Na_Xwith the indicated pharmacological modulator present in the extracellular solution. FIG. 8A, right panel, shows I-V plots with depolarizing steps between +80 and −100 mV, in 20 mV increments, from a HP of 0 mV. FIG. 8B, left panel, shows I-V plots with depolarizing steps between −100 and +100 mV, in 20 mV increments, from a HP of −100 mV. FIGS. 8C, left panel, and 8D, left panel, show representative currents from oocytes expressing human Na_Xwith the indicated proteins co-expressed. FIG. 8C, right panel, show I-V plots with steps between +80 and −100 mV, in 20 mV increments, from a HP of 0 mV. FIG. 8D shows I-V plots with depolarizing steps between −100 and +100 mV, in 20 mV increments, from a HP of −100 mV.

FIG. 9 shows multiple-sequence alignment of Na_Xand Na_Vchannels. Na_Xdomain boundaries are shown for reference. Na_Xgating charges are denoted by asterisks and selectivity filter residues are boxed. The Na_XDIII-DIV linker IFI-motif is denoted by three consecutive asterisks.

FIGS. 10A-C show β3-Na_Xchannel sample purification. FIG. 10A show β3-Na_Xexpression, purification and reconstitution scheme. FIG. 10B show size-exclusion chromatography elution profile for β3-Na_Xsample in lipid nanodiscs (MSP1E3D1). FIG. 10C show SDS-PAGE analysis of size exclusion chromatography elution fractions.

FIGS. 11A-L show cryo-EM data processing workflow. FIG. 11A shows representative cryo-EM micrograph of β3-Na_X-nanodisc sample. FIG. 11B shows representative 2D class averages. FIG. 11C shows heat map showing the overall distribution of assigned particle orientations in the final reconstruction. FIG. 11D shows global resolution estimate based off the Fourier Shell Correlation (FSC) between two half datasets. FIG. 11E shows isosurface rendering of the final 3D reconstruction colored by local resolution, as estimated by windowed FSCs. FIG. 11F shows cryo-EM map shown over transmembrane regions of DI. FIG. 11G shows representative cryo-EM micrograph of β3-Na_X-detergent (GDN) sample. FIG. 11H shows representative 2D class averages. FIG. 11I shows heat map showing the overall distribution of assigned particle orientations in the final reconstruction.

FIG. 11J shows global resolution estimate based off the Fourier Shell Correlation (FSC) between two half datasets. FIG. 11K shows isosurface rendering of the final 3D reconstruction colored by local resolution, as estimated by windowed FSCs. FIG. 11L shows cryo-EM map shown over transmembrane regions of DI.

FIGS. 12A-B show cryo-EM data processing workflows. FIG. 12A shows schematic of the cryo-EM data processing workflow for the β3-Na_X-nanodisc sample. FIG. 12B shows schematic of the cryo-EM data processing workflow for the β3-Na_X-detergent sample.

FIGS. 13A-F show the overall Na_Xstructure, β3 interactions, and comparison to Na_Vchannels. FIG. 13A shows cryo-EM reconstruction of the β3-Na_X-nanodisc complex. The Na_XNTD (N-terminal domain) is on the intracellular side and the β3-subunit ECD (extracellular domain) is on the extracellular side. Approximate membrane boundaries are indicated. FIG. 13B shows side-view of the β3-Na_Xcomplex. Insets highlight β3 interactions with Na_X. FIGS. 13C-E show close-up views of 3-Na_Xaligned with human Na_V1.2 (PDB 6J8E), Na_V1.4 (PDB 6AGF), Na_V1.5 (PDB 6UZ0) and Na_V1.7 (PDB 6J8J) structures from various perspectives. FIG. 13F shows close-up view into the central cavity highlighting the DIV W1484 side-chain of Na_Xwith Na_V1.7 superimposed (PDB 6J8J). DIV Phe of Na_V1.7 is shown in stick representation.

FIGS. 14A-F show DIII-DI linker, DIII chimeric channels, and lipid bound pore structures of Na_X. FIG. 14A shows side- and intracellular view of DIII and DIV from β3-Na_X-nanodisc structure with the IFI-motif of the DIII-DIV linker shown in spheres, with equivalent region of Na_V1.7 (PDB 6J8J) shown for comparison. FIG. 14B shows multi-sequence alignment of the DIII-DIV linker region with the IFI/IFM-motif. FIG. 14C shows representative currents from oocytes expressing wild-type or IFI>QQQ-mutant human Na_Xwith voltage steps between +80 and −100 mV, in 20 mV increments, from a HP of 0 mV, or depolarizing steps between −100 and +120 mV, in 20 mV increments, from a HP of −100 mV. I-V plots are shown in FIG. 14C, right panel. FIG. 14D shows schematic and representative currents from oocytes expressing various DIII human Na_V1.7-Na_Xchannel chimeras in response to voltage steps between +80 and −100 mV, in 20 mV increments, from a HP of 0 mV. FIG. 14D, right panel, maximal current amplitudes at +80 mV (top panel) and −100 mV (bottom panel) of various DIII Na_V1.7-Na_Xchimeras. FIG. 14E shows side-view of the β3-Na_X-nanodisc S6-gate structure with gate-lining side-chains shown in stick and semi-transparent surface representation and assigned phosphatidylethanolamine shown in sticks. FIG. 14F shows top-view of the pore comparing the β3-Na_X-nanodisc and β3-Na_X-detergent-based structures with assigned lipids in the pore shown in stick representations. Cryo-EM map is shown around modeled lipids in mesh representation for the β3-Na_X-GDN structure.

FIGS. 15A-C show structure and comparison of the Na_Xand Na_V1.7 selectivity filters. FIG. 15A shows equivalent views of the Na_Xand Na_V1.7 (PDB 6J8J) selectivity filters. Tetrodotoxin bound in Na_V1.7 is shown with select interactions. FIG. 15B shows representative currents from Xenopus laevis oocytes expressing wild-type or mutant human Na_V1.7 and Na_Xchannel constructs in the presence of 115 mM extracellular Na+, K+, Li+ or Cs+. Steps between −50 and +50 mV, in 10 mV steps, from a HP of −100 mV are shown. FIG. 15C shows representative currents from HEK293 cells expressing human Na_X-QTT (containing a C-terminal GFP-Flag tag) in response to a voltage ramp from −80 to +80 mV in the presence (left) or absence (right) of chloride (Cl−) ions (substituted with methanesulfonate, MS−).

FIGS. 16A-E show structure and comparison of the Na_Xvoltage sensor-like domains. FIG. 16A shows equivalent views of Na_V1.7 VSD4 (PDB 6J8J) and Na_XVSLD4 with gating charge side-chains shown in sticks and R3-R5 labeled for reference. FIG. 16B shows voltage dependence of activation and steady-state inactivation of wild-type Na_V1.7 and various mutant channels. Activation, currents were obtained following depolarizing steps between −80 and +65 mV, in 5 mV increments, from a HP of −100 mV. Inactivation, currents were obtained during the test pulse at 0 or −20 mV (25 ms) after a series of conditioning pulses (between −120 and −10 mV, in 5 mV increments, 500 ms) from a HP of −100 mV. Normalized activation and inactivation curves are shown to the right of the current traces. FIG. 16C shows superposition of Na_XVSDL3 with the KCNQ1 VSD (PDB 6UZZ) with Na_Xgating charges and HCS shown as sticks. Na_XS3 F1101 and the corresponding S3 serine from KCNQ1 are shown in surface representation; note, this position is also conserved as a serine in all human Na_Vchannels. FIG. 16D shows a view of Na_XVSDL4 highlighting the proline side-chains that are unique when compared with human Na_Vchannel VSD4 sequences. FIG. 16E shows voltage dependence of activation and steady-state inactivation of wild-type Na_V1.5 and triple-proline VSD4 mutant channel. Activation, currents were obtained following depolarizing steps between −80 and +40 mV, in 5 mV increments, from a HP of −100 mV. Inactivation, currents were obtained during the test pulse at 0 or −20 mV (25 ms) after a series of conditioning pulses (between −120 and −10 mV, in 5 mV increments, 500 ms) from a HP of −100 mV. Normalized activation and inactivation curves are shown to the right of the current traces.

FIGS. 17A-B shows structure and comparison of the Na_Xselectivity filter and S6-gate regions. FIG. 17A shows a close-up view of the selectivity filer with a putative ion binding site (DEE motif) in Na_Xshown with a portion of the cryo-EM map (mesh) and a Na+ ion modeled as a sphere. The Na+ ion assigned in the DEE motif binding site in Na_V1.2 (PDB 6J8E) and NaVPaS (PDB 6NT4) are shown for comparison. FIG. 17B shows an intracellular view of the Na_XS6-gate with Tyr1491 (S6) and bound phosphatidylethanolamine shown and compared to Na_V1.4 (assumed to be in a non-conductive, inactivated state; PDB 6AGF) and drug-bound Na_V1.5 (assumed to be in a non-conductive, drug blocked state; PDB 6UZ0). Assigned lipid, detergent and drug molecules (PE, GDN, or flecainide) shown as stick representations, respectively.

FIG. 18 shows current amplitudes of Xenopus laevis oocytes expressing the constructs 1-25. (*p<0.05; **p<0.01; ****p<0.0001; one-way ANOVA, Dunnett's test against Na_X.)

FIGS. 19A-B show structures of the Na_Xchannel. In FIG. 19A, the DIII+ DIII-IV linker (920-1237) is shown. In FIG. 19B, the S6 gate region is shown.

FIG. 20 shows a multiple-sequence alignment of the Na_Xchannel S6 segment (partial) and the Na_Vchannels.

DETAILED DESCRIPTION
I. Definitions

Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

In this application, the use of “or” means “and/or” unless stated otherwise. In the context of a multiple dependent claim, the use of “or” refers back to more than one preceding independent or dependent claim in the alternative only. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.

As used herein, the transition term “consisting essentially of,” when referring to steps of a claimed process signifies that the process comprises no additional steps beyond those specified that would materially affect the basic and novel characteristics of the process. As used herein, the transition term “consisting essentially of,” when referring to a composition or product, such as a kit, signifies that it comprises no additional components beyond those specified that would materially affect its basic and novel characteristics.

As used herein, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise.

As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

Units, prefixes, and symbols are denoted in their Système International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

All proteins described herein are human proteins unless expressly described otherwise, as in “mammalian Na_V” or the like phrases. A “mammalian” protein includes the human protein as well as the equivalent protein from other mammalian species.

An “Na_V” protein or “Nav” or “NaV” protein, as described herein, is a member of a voltage-gated sodium channel protein family. Examples of Na_Vfamily members in humans and other mammals include Na_V1.1, Na_V1.2, Na_V1.3, Na_V1.4, Na_V1.5, Na_V1.6, Na_V1.7, Na_V1.8, and Na_V1.9. Human Na_Vfamily member proteins comprise four domains (DI, DII, DIII, and DIV), each comprising six transmembrane alpha helices (S1, S2, S3, S4, S5, and S6), in some cases with loops or linkers in between certain alpha helices within a domain and between the four domains, for a total of 24 transmembrane alpha helices.

An “Na_X” ion channel protein or “Na_X” or “NaX” protein, as described herein, is a member of the voltage-gated sodium channel protein family and is also known as Na_V2.1 or scn7a. Unless specified otherwise, the “Na_X” protein means the “human Na_X” protein. The human Na_Xprotein has been reported to act as a sodium sensor in the central nervous system and also in the skin and other epithelia. The amino acid sequence of wild-type human Na_Xis provided in the UniProt database (Accession No. Q01118), and is provided herein as SEQ ID NO: 1. A human Na_Xprotein also includes naturally occurring variants of the protein, examples of which may also be found in the UniProt database under No. Q01118, for example. Like the human Na_Vproteins, human Na_Xcomprises four domains (DI, DII, DIII, and DIV), each comprising six transmembrane alpha helices (S1, S2, S3, S4, S5, and S6).

A “mutant human Na_X” as used herein refers to a human Na_Xprotein that comprises at least one engineered amino acid substitution, insertion or deletion compared to the wild-type Na_Xprotein of SEQ ID NO: 1. In some cases a “mutant human Na_X” comprises at least one amino acid substitution in which at least one residue of wild-type human Na_Xis substituted with the equivalent/corresponding residue of a mammalian or human Na_V. In some cases, a region or a complete domain of wild-type human Na_Xis substituted for the equivalent region or domain of a mammalian or human Na_V, e.g., some or all of DIII and/or the DIII-DIV linker. In such cases, the mutant human Na_Xmay alternatively be referred to herein as a “chimeric human Na_X” or a “chimeric Na_X” or “chimera,” as it contains amino acid sequence segments from a different protein, a human or mammalian Na_V.

The term “chimeric” herein, when referring to a protein, means that the protein is made up of amino acid sequences from more than one native protein. For instance, as described above, a “chimeric human Na_X” as used herein refers to a type of mutant human Na_Xin which at least one region of the Na_Xprotein has been exchanged with a corresponding region from a human Na_Vfamily member protein.

The term “chimeric” herein, when referring to a protein, means that the protein is made up of amino acid sequences from more than one native protein. For instance, a “chimeric human Na_X” as used herein refers to a type of mutant human Na_Xin which at least one region of the Na_Xprotein has been exchanged with a corresponding region from a Na_Vfamily member protein, such as from a mammalian or human Na_Vfamily member protein, such as human or mammalian Na_V1.1, Na_V1.2, Na_V1.3, Na_V1.4, Na_V1.5, Na_V1.6, Na_V1.7, Na_V1.8, or Na_V1.9. In some cases, the exchanged regions may be complete domains, or one of more individual transmembrane alpha helices or linker regions or loops within a domain, or a portion of an alpha helix and/or loop within a domain.

As used herein, the terms “corresponding” or “equivalent” are used interchangeably when referring to a residue or region from a human or mammalian Na_Vprotein that replaces a residue or region deleted from human Na_Xin making a mutant or chimeric human Na_X. As used herein, “corresponding” or “equivalent” residues or regions are those residues or regions that are in the same location within the two proteins when properly folded. In some cases, such corresponding or equivalent regions or amino acid residues may be identified using sequence alignments and structural information for the two proteins.

A “modulator” as used herein refers to a molecule that is capable of altering the behavior of a protein, such as an ion channel protein like human Na_X. For example, in some cases a modulator may alter the behavior of a target protein through binding to the protein. A modulator herein may act to either increase or decrease the activity of the protein, such as, for example, the degree to which the protein regulates the flow of ions across a cellular membrane. A modulator that decreases the activity of human Na_X, for example, is an “inhibitor” of human Na_Xactivity. A modulator that increases the activity of human Na_X, for example, is an “activator” of human Na_Xactivity. In some embodiments, a modulator may only modulate the activity of human Na_Xunder certain conditions, such as in the presence of certain ions or in the presence of certain secondary molecules.

As used herein, a “potential modulator” of human Na_Xis a molecule that is to be tested to determine if it acts as a modulator of human Na_X.

An “ion channel assay” herein refers to an assay that is used to measure the activity of an ion channel protein, such as a voltage-gated sodium channel. A variety of assays and assay formats are regularly used in the art, and many are provided in automated formats for high-throughput screening (HTS) analysis. Examples include ion flux assays, for example, using radioactive ions such as radioactive Na⁺ ions, an ion- or voltage-sensitive dye assay such as fluorescence assays for example using fluorescent indicator molecules whose fluorescence signal increases or decreases with changes in ion concentration, and various types of patch clamp assays. Such assays may directly or indirectly measure changes in ionic currents across a membrane comprising an ion channel protein in a variety of conditions. In some cases, such assays are used to assess the “activity” of the ion channel protein in the presence or absence of a potential or identified modulator. The term “activity” in this sense is meant in the broadest sense, given that these different assays measure the activity of the protein either directly or indirectly and through the measurement of different parameters, such as changes in fluorescence, radioactivity, or ionic current.

A “patch clamp assay” is used herein in the broadest sense to refer to an assay that is used to assesses changes in the movement of ions across a small patch of cell membrane containing an ion channel protein such as Na_Xor Na_Vunder different solution conditions, for example.

The term “peptide” as used herein refers to a chain of fifty amino acids or less linked by peptide bonds, including amino acid chains of 2 to 50, 2 to 15, 2 to 10, 2 to 8, or 6 to 14 amino acids.

The term “small molecule” as used herein refers to an organic molecule having a molecular weight of 50 Daltons to 2500 Daltons.

The term “macrocycle” or “macrocylic molecule” as used herein refers to a cyclic macromolecule or a macromolecular cyclic portion of a macromolecule. Macrocycles range in size from 500 Daltons to 7500 Daltons. In some cases herein, macrocycles are cyclic peptides or peptide derivatives.

The term “binding fragment” as used herein refers to a portion of a larger molecule, such as a small molecule, peptide, or antibody, that is expected to directly contact a target protein. Binding fragments may be used in high-throughput screens.

In this disclosure, “binds” or “binding” or “specific binding” and similar terms, when referring to a molecule that “binds” to a protein such as a Na_Xor Na_Vprotein, for example, means that the binding affinity is sufficiently strong that the interaction between the members of the binding pair cannot be due to random molecular associations (i.e. “nonspecific binding”). Thus, the binding is selective or specific.

The term “competition assay” as used herein refers to an assay in which a molecule being tested prevents or inhibits specific binding of a reference molecule to a common target. Other definitions are included in the sections below, as appropriate.

II. Mutant Na_XProteins

In some embodiments, the invention comprises a mutant human Na_Xion channel protein.

Human Na_Xis a member of the voltage-gated sodium channel protein family and is also known as Na_V2.1 or scn7a. Information on the genomic and amino acid sequences of the protein may be found, for example, in the UniProt database under Accession No. Q01118. In some embodiments, the amino acid sequence of human Na_X, including the signal sequence, is that of SEQ ID NO: 1. The human Na_Xprotein has been reported to act as a sodium sensor in the central nervous system and also in the skin and other epithelia. The human Na_Xand Na_Vproteins comprise four domains (DI, DII, DIII, and DIV), each comprising six transmembrane alpha helices (S1, S2, S3, S4, S5, and S6). Within those domains are further regions, as described in the Table 1 below, with the corresponding amino acid residue positions for each. For example, each domain also comprises a “voltage-sensor domain” or “VSD” (e.g., VSD1, VSD2, VSD3, VSD4) or “voltage-sensor-like domain” or “VSLD” (e.g. VSLD1, VSLD2, VLSD3, VSLD4), comprising the S1-S4 helices and their intervening linkers or loops; specific linkers or loops between the alpha helices, which occur on the extracellular or intracellular face of the protein, and which include, for example, an S4-S5 linker and a pore-forming selectivity filter loop between S5 and S6; and linkers between the four domains. Specific helices, linkers, loops, and other regions of human Na_Xof SEQ ID NO: 1 are as shown below:

TABLE 1

Human SCN7A, Na_xor Nav2.1 [UniProtKB - Q01118]

Topology

Sequence

Sequence

(based on

region
Description
cryoEM)

1-118
Cytoplasmic

119-137
Transmembrane, Name = S1 of DI
[118-137]

138-144
Extracellular

145-165
Transmembrane; Name = S2 of DI
[146-165]

166-179
Cytoplasmic

180-197
Transmembrane; Name = S3 of DI
[179-194]

198-203
Extracellular

204-220
Transmembrane; Name = S4 of DI
[204-218]

221-239
Cytoplasmic; Name = S4-S5 linker of DI

240-259
Transmembrane; Name = S5 of DI
[237-258]

260-338
Extracellular

339-363
Pore-forming selectivity filter loop;

Name = P1 and P2 of DI

364-370
Extracellular

371-391
Transmembrane; Name = S6 of DI
[371-402]

392-505
Cytoplasmic; Name = DI-DII linker
[404-495]

506-524
Transmembrane, Name = S1 of DII
[509-523]

525-535
Extracellular

536-555
Transmembrane; Name = S2 of DII
[531-556]

556-569
Cytoplasmic

570-589
Transmembrane; Name = S3 of DII
[569-583]

590-591
Extracellular

592-609
Transmembrane; Name = S4 of DII
[594-608]

610-625
Cytoplasmic; Name = S4-S5 linker of DII

626-644
Transmembrane; Name = S5 of DII
[626-648]

645-673
Extracellular

674-694
Pore-forming selectivity filter loop;

Name = P1 and P2 of DII

695-707
Extracellular

708-727
Transmembrane; Name = S6 of DII
[705-732]

728-935
Cytoplasmic; Name = DII-DIII linker
[733-920]

936-953
Transmembrane; Name = S1 of DIII
[934-950]

954-966
Extracellular

967-985
Transmembrane; Name = S2 of DIII
[965-987]

986-999
Cytoplasmic

1000-1018
Transmembrane; Name = S3 of DIII
[1000-1017]

1019-1021
Extracellular

1022-1040
Transmembrane; Name = S4 of DIII
[1026-1039]

1041-1057
Cytoplasmic; Name = S4-S5 linker of DIII

1058-1077
Transmembrane; Name = S5 of DIII
[1059-1078]

1078-1128
Extracellular

1129-1150
Pore-forming selectivity filter loop;

Name = P1 and P2 of DIII

1151-1167
Extracellular

1168-1189
Transmembrane; Name = S6 of DIII
[1170-1200]

1190-1252
Cytoplasmic; Name = DIII-DIV linker
[1201-1237]

1253-1270
Transmembrane; Name = S1 of DIV
[1251-1269]

1271-1281
Extracellular

1282-1300
Transmembrane; Name = S2 of DIV
[1280-1301]

1301-1312
Cytoplasmic

1313-1330
Transmembrane; Name = S3 of DIV
[1314-1327]

1331-1343
Extracellular

1344-1360
Transmembrane; Name = S4 of DIV
[1344-1357]

1361-1379
Cytoplasmic; Name = S4-S5 linker of DIV

1380-1397
Transmembrane; Name = S5 of DIV
[1378-1400]

1398-1419
Extracellular

1420-1442
Pore-forming selectivity filter loop;

Name = P1 and P2 of DIV

1443-1472
Extracellular

1473-1495
Transmembrane; Name = S6 of DIV
[1471-1502]

1496-1682
Cytoplasmic
[1503-1682]

The present disclosure includes, for example, a mutant human Na_Xin which at least one amino acid residue is substituted with at least one corresponding residue of a wild-type mammalian or human Na_V. In some cases, a region or a complete domain of wild-type human Na_Xis substituted for the equivalent region or domain of a mammalian or human Na_V, e.g., some or all of DIII or the DIII/DIV linker. In such cases, the mutant human Na_Xmay alternatively be referred to herein as a “chimeric human Na_X” or a “chimeric Na_X” or “chimera,” as it contains amino acid sequence segments from a different protein, a mammalian or human Na_V. In some cases, the substituted residue or region is from mammalian or human Na_V1.1, Na_V1.2, Na_V1.3, Na_V1.4, Na_V1.5, Na_V1.6, Na_V1.7, Na_V1.8, or Na_V1.9. In some cases, the substituted residue or region is from a human Na_V, such as from human Na_V1.1, Na_V1.2, Na_V1.3, Na_V1.4, Na_V1.5, Na_V1.6, Na_V1.7, Na_V1.8, or Na_V1.9, for example, from human Na_V1.7.

In some embodiments, at least a portion of the DIII domain of human Na_Xis replaced by a corresponding portion of the DIII domain of a human or mammalian Na_Vprotein (Na_V). In some cases, the mutant human Na_Xcomprises all or a portion of the DIII S1-S6 region from a human or mammalian Na_V, optionally wherein the at least a portion of the DIII domain of human Na_Xthat is replaced comprises: (a) DIII (residues 920-1200), (b) DIII voltage-sensor domain III (VSD3) and S4-S5 linker (residues 920-1058), (c) DIII VSD3, S4-S5 linker and S5 (residues 920-1078), (d) DIII and DII-DIII linker (residues 733-1200), (e) DIII and DIII-DIV linker (920-1237), (f) DIII and DII-DIII linker and DIII-DIV linker (733-1237). In some cases, the mutant human Na_Xcomprises all or a portion of the DIII S1-S6 region from a human or mammalian Na_V, optionally wherein the at least a portion of the DIII domain of human Na_Xthat is replaced consists of: (a) DIII (residues 920-1200), (b) DIII voltage-sensor domain III (VSD3) and S4-S5 linker (residues 920-1058), (c) DIII VSD3, S4-S5 linker and S5 (residues 920-1078), (d) DIII and DII-DIII linker (residues 733-1200), (e) DIII and DIII-DIV linker (920-1237), (f) DIII and DII-DIII linker and DIII-DIV linker (733-1237). In some cases, the at least a portion of the DIII domain of human Na_Xthat is replaced by a corresponding portion of the DIII domain of the human or mammalian Na_Vdoes not comprise S5. In some cases, the at least a portion of the DIII domain of human Na_Xthat is replaced by a corresponding portion of the DIII domain of the human or mammalian Na_Vdoes not comprise S6. In some cases, the at least a portion of the DIII domain of human Na_Xthat is replaced by a corresponding portion of the DIII domain of the human or mammalian Na_Vdoes not comprise any of S1, S2, S3, S4, and/or S4-S5 linker. In some cases, the at least a portion of the DIII domain of human Na_Xis replaced by a mammalian Na_V1.4, mammalian Na_V1.5, mammalian Na_V1.6, mammalian Na_V1.7, mammalian Na_V1.8, mammalian Na_V1.9, human Na_V1.1, human Na_V1.2, human Na_V1.3, human Na_V1.4, human Na_V1.5, human Na_V1.6, human Na_V1.7, human Na_V1.8, or human Na_V1.9. For example, in some cases, the at least a portion of the DIII domain of human Na_Xis replaced by a corresponding portion of human Na_V1.7. In some cases, the mutant human Na_Xis chimera construct 2, chimera construct 3, chimera construct 7, chimera construct 9, chimera construct 11, chimera construct 15 or chimera construct 19, or comprises an amino acid sequence of any one of SEQ ID Nos: 3, 4, 8, 10, 12, 16, or 20. (See Table 4 below and FIG. 18.)

In other cases, a mutant human Na_Xhas at least one substitution of an amino acid residue on one, two, three, or all four of its S6 segments with a polar or charged residue or a glycine or proline residue. In some cases, the substituted residue is polar or charged. Exemplary polar amino acid residues include serine, threonine, tyrosine, asparagine, glutamine, histidine. Exemplary charged amino acid residues include aspartic acid, glutamic acid, lysine, and arginine. These amino acid substitutions can span across the intracellular to the mid-transmembrane region of one, two, three, or all four of the S6 segments (i.e. up to halfway across the membrane bilayer). Predictions of Na_Xtransmembrane segments, for the design of mutations, can be made based on available experimental structures available from the Protein Data Bank (PDB) and standard structure-based multi-sequences alignments with Na_Vchannels (e.g. ClustalW). FIG. 20 depicts the potential location of the amino acid substitutions in human Na_X, at S6 of each of DI, DII, DIII, and DIV, as well as their alignment with corresponding residues in other human Na_Vproteins. As shown by the dashed box on the right side of each sequence, amino acid substitutions may be made at any one of residues L390, F724, I1189, or I1492 or at any residue up to 7 residues in either direction, e.g., at residues 383-397 (in S6 of domain I (D1)), residues 717-731 (in S6 of DII), residues 1182-1196 (in S6 of DIII), and/or residues 1485-1499 (in S6 of DIV). Thus, in some embodiments, at least one of S6 of D1, S6 of DII, S6 of DIII, and S6 of DIV contains at least one substitution of an amino acid residue within the ranges above for a glycine, proline, polar, or charged residue.

In some cases, the mutant human Na_Xcomprises a substitution of at least one residue on one of the four S6 alpha helices with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises a substitution of at least one residue on two of the four S6 alpha helices with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises a substitution of at least one residue on three of the four S6 alpha helices with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises a substitution of at least one residue on all four S6 alpha helices with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises one substitution of an amino acid residue on at least three of the four S6 alpha helices with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises one or two substitutions of an amino acid residue with a glycine, proline, or polar or charged residue within at least two of following segments of SEQ ID NO: 1: residues 383-397 (in S6 of domain I (D1)), residues 717-731 (in S6 of DII), residues 1182-1196 (in S6 of DIII), and/or residues 1485-1499 (in S6 of DIV). In some cases, the mutant human Na_Xcomprises one or two substitutions of an amino acid residue with a glycine, proline, or polar or charged residue within at least two of the following segments of SEQ ID NO: 1: residues 383-397 (in S6 of domain I (D1)), residues 717-731 (in S6 of DII), residues 1182-1196 (in S6 of DIII), and/or residues 1485-1499 (in S6 of DIV). In some cases, the mutant human Na_Xcomprises one or two substitutions of an amino acid residue with a glycine, proline, or polar or charged residue within at least three of the following segments of SEQ ID NO: 1: residues 383-397 (in S6 of domain I (D1)), residues 717-731 (in S6 of DII), residues 1182-1196 (in S6 of DIII), and/or residues 1485-1499 (in S6 of DIV). In some cases, the mutant human Na_Xcomprises one or two substitutions of an amino acid residue with a glycine, proline, or polar or charged residue within each of the following segments of SEQ ID NO: 1: residues 383-397 (in S6 of domain I (D1)), residues 717-731 (in S6 of DII), residues 1182-1196 (in S6 of DIII), and/or residues 1485-1499 (in S6 of DIV). In some cases, the mutant human Na_Xcomprises substitutions of an amino acid residue at two or more of residues L390, F724, I1189, and I1492 with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises substitutions of an amino acid residue at three of residues L390, F724, I1189, and I1492 with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises substitutions of amino acid residues L390, F724, and I1189 or of amino acid residues F724, I1189, and I1492 with glycine, proline, or polar or charged residues. In some cases, the mutant human Na_Xcomprises substitutions F724Q, I1189T, and I1492T compared to wild-type human Na_X. In some cases, the human Na_Xcomprises substitutions L390E, I1189E, and I1492E compared to wild-type human Na_X. In some cases, the mutant human Na_Xcomprises substitutions of an amino acid residue at all four of residues L390, F724, I1189, and I1492 with a glycine, proline, or polar or charged residue.

In some embodiments, the mutant human Na_Xprotein is active in an ion channel assay. For example, in some cases, an ion channel assay involves expressing the mutant protein in Xenopus laevis oocytes. In some such cases, the current amplitude of Xenopus laevis oocytes expressing the constructs elicited at +80 mV or −100 mV from a holding potential of 0 m V is significantly higher than that from oocytes expressing human Na_Xwild-type. (See FIG. 18 for illustration of several active and inactive mutant human Na_Xprotein constructs, wherein asterisks denote statistical significance).

III. Screening Methods Using Mutant Human Na_XProteins

The present disclosure also encompasses, inter alia, methods of identifying molecules that act as modulators of human Na_X, comprising screening potential modulators against a mutant human Na_X, for example, in an ion channel assay, and/or in a binding assay.

In some embodiments, at least a portion of the DIII domain of human Na_Xis replaced by a corresponding portion of the DIII domain of a human or mammalian Na_Vprotein (Na_V). In some cases, the mutant human Na_Xcomprises all or a portion of the DIII S1-S6 region from a human or mammalian Na_V, optionally wherein the at least a portion of the DIII domain of human Na_Xthat is replaced comprises: (a) DIII (residues 920-1200), (b) DIII voltage-sensor domain III (VSD3) and S4-S5 linker (residues 920-1058), (c) DIII VSD3, S4-S5 linker and S5 (residues 920-1078), (d) DIII and DII-DIII linker (residues 733-1200), (e) DIII and DIII-DIV linker (920-1237), (f) DIII and DII-DIII linker and DIII-DIV linker (733-1237). In some cases, the mutant human Na_Xcomprises all or a portion of the DIII S1-S6 region from a human or mammalian Na_V, optionally wherein the at least a portion of the DIII domain of human Na_Xthat is replaced consists of: (a) DIII (residues 920-1200), (b) DIII voltage-sensor domain III (VSD3) and S4-S5 linker (residues 920-1058), (c) DIII VSD3, S4-S5 linker and S5 (residues 920-1078), (d) DIII and DII-DIII linker (residues 733-1200), (e) DIII and DIII-DIV linker (920-1237), (f) DIII and DII-DIII linker and DIII-DIV linker (733-1237). In some cases, the at least a portion of the DIII domain of human Na_Xthat is replaced by a corresponding portion of the DIII domain of the human or mammalian Na_Vdoes not comprise S5. In some cases, the at least a portion of the DIII domain of human Na_Xthat is replaced by a corresponding portion of the DIII domain of the human or mammalian Na_Vdoes not comprise S6. In some cases, the at least a portion of the DIII domain of human Na_Xthat is replaced by a corresponding portion of the DIII domain of the human or mammalian Na_Vdoes not comprise any of S1, S2, S3, S4, and/or S4-S5 linker. In some cases, the at least a portion of the DIII domain of human Na_Xis replaced by a corresponding portion of mammalian Na_V1.1, mammalian Na_V1.2, mammalian Na_V1.3, mammalian Na_V1.4, mammalian Na_V1.5, mammalian Na_V1.6, mammalian Na_V1.7, mammalian Na_V1.8, mammalian Na_V1.9, human Na_V1.1, human Na_V1.2, human Na_V1.3, human Na_V1.4, human Na_V1.5, human Na_V1.6, human Na_V1.7, human Na_V1.8, or human Na_V1.9. For example, in some cases, the at least a portion of the DIII domain of human Na_Xis replaced by a corresponding portion of human Na_V1.7. In some cases, the mutant human Na_Xis chimera construct 2, chimera construct 3, chimera construct 7, chimera construct 9, chimera construct 11, chimera construct 15 or chimera construct 19, or comprises an amino acid sequence of any one of SEQ ID Nos: 3, 4, 8, 10, 12, 16, or 20. (See Table 4 below and FIG. 18.)

The present disclosure also encompasses methods of identifying a human Na_Xion channel protein (Na_X) modulator, comprising: (a) providing a mutant human Na_Xcomprising a substitution of at least one residue on each of two, three, or four S6 alpha helices of human Na_Xwith a glycine, proline, or polar or charged residue; (b) performing an ion channel assay on the mutant human Na_Xin the presence of the potential modulator; and (c) identifying the potential modulator as a human Na_Xmodulator if the activity of the mutant human Na_Xin the assay in the presence of the potential modulator is higher or lower than the activity in the absence of the potential modulator. In some cases, the mutant human Na_Xcomprises a substitution of at least one residue on two of the four S6 alpha helices with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises a substitution of at least one residue on one of the four S6 alpha helices with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises a substitution of at least one residue on two of the four S6 alpha helices with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises a substitution of at least one residue on three of the four S6 alpha helices with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises a substitution of at least one residue on all four S6 alpha helices with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises one substitution of an amino acid residue on at least three of the four S6 alpha helices with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises one or two substitutions of an amino acid residue with a glycine, proline, or polar or charged residue within at least two of following segments of SEQ ID NO: 1: residues 383-397 (in S6 of domain I (D1)), residues 717-731 (in S6 of DII), residues 1182-1196 (in S6 of DIII), and/or residues 1485-1499 (in S6 of DIV). In some cases, the mutant human Na_Xcomprises one or two substitutions of an amino acid residue with a glycine, proline, or polar or charged residue within at least two of the following segments of SEQ ID NO: 1: residues 383-397 (in S6 of domain I (D1)), residues 717-731 (in S6 of DII), residues 1182-1196 (in S6 of DIII), and/or residues 1485-1499 (in S6 of DIV). In some cases, the mutant human Na_Xcomprises one or two substitutions of an amino acid residue with a glycine, proline, or polar or charged residue within at least three of the following segments of SEQ ID NO: 1: residues 383-397 (in S6 of domain I (D1)), residues 717-731 (in S6 of DII), residues 1182-1196 (in S6 of DIII), and/or residues 1485-1499 (in S6 of DIV). In some cases, the mutant human Na_Xcomprises one or two substitutions of an amino acid residue with a glycine, proline, or polar or charged residue within each of the following segments of SEQ ID NO: 1: residues 383-397 (in S6 of domain I (D1)), residues 717-731 (in S6 of DII), residues 1182-1196 (in S6 of DIII), and/or residues 1485-1499 (in S6 of DIV). In some cases, the mutant human Na_Xcomprises substitutions of an amino acid residue at two or more of residues L390, F724, I1189, and I1492 with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises substitutions of an amino acid residue at three of residues L390, F724, I1189, and I1492 with a glycine, proline, or polar or charged residue. In some cases, the mutant human Na_Xcomprises substitutions of amino acid residues L390, F724, and I1189 or of amino acid residues F724, I1189, and I1492 with glycine, proline, or polar or charged residues. In some cases, the mutant human Na_Xcomprises substitutions F724Q, I1189T, and I1492T compared to wild-type human Na_X. In some cases, the human Na_Xcomprises substitutions L390E, I1189E, and I1492E compared to wild-type human Na_X. In some cases, the mutant human Na_Xcomprises substitutions of an amino acid residue at all four of residues L390, F724, I1189, and I1492 with a glycine, proline, or polar or charged residue.

The present disclosure further includes methods of determining whether a test molecule that modulates the activity of a human ion channel protein, e.g. a human Na_Vprotein, also modulates the activity of human Na_Xion channel (Na_X) protein, comprising: (a) providing a mutant human Na_Xin which at least a portion of the DIII domain of human Na_Xis replaced by a corresponding portion of the DIII domain of a human or mammalian Na_Vprotein (Na_V); (b) performing an ion channel assay on the mutant human Na_Xin the presence of the test molecule; and (c) determining that the test molecule is a human Na_Xmodulator if the activity of the mutant human Na_Xin the assay in the presence of the test molecule is higher or lower than the activity in the absence of the test molecule; and optionally, (d) selecting the test molecule for additional screening if it is not a human Na_Xmodulator according to part (c).

The present disclosure additionally includes methods of determining whether a test molecule that modulates the activity of a human ion channel protein, such as a human Na_Vprotein, also modulates the activity of human Na_Xion channel (Na_X) protein, comprising: (a) determining that the test molecule modulates the activity of the human ion channel protein; (b) providing a mutant human Na_Xin which at least a portion of the DIII domain of human Na_Xis replaced by a corresponding portion of the DIII domain of a human or mammalian Na_Vprotein (Na_V); (c) performing an ion channel assay on the mutant human Na_Xin the presence of the test molecule; and (d) determining that the test molecule is a human Na_Xmodulator if the activity of the mutant human Na_Xin the assay in the presence of the test molecule is higher or lower than the activity in the absence of the test molecule; and optionally, (e) selecting the test molecule for additional screening if it is not a human Na_Xmodulator according to part (d).

In some embodiments, at least a portion of the DIII domain of human Na_Xis replaced by a corresponding portion of the DIII domain of a human or mammalian Na_Vprotein (Na_V). In some cases, the mutant human Na_Xcomprises all or a portion of the DIII S1-S6 region from a human or mammalian Na_V, optionally wherein the at least a portion of the DIII domain of human Na_Xthat is replaced comprises: (a) DIII (residues 920-1200), (b) DIII voltage-sensor domain III (VSD3) and S4-S5 linker (residues 920-1058), (c) DIII VSD3, S4-S5 linker and S5 (residues 920-1078), (d) DIII and DII-DIII linker (residues 733-1200), (e) DIII and DIII-DIV linker (920-1237), (f) DIII and DII-DIII linker and DIII-DIV linker (733-1237). In some cases, the mutant human Na_Xcomprises all or a portion of the DIII S1-S6 region from a human or mammalian Na_V, optionally wherein the at least a portion of the DIII domain of human Na_Xthat is replaced consists of: (a) DIII (residues 920-1200), (b) DIII voltage-sensor domain III (VSD3) and S4-S5 linker (residues 920-1058), (c) DIII VSD3, S4-S5 linker and S5 (residues 920-1078), (d) DIII and DII-DIII linker (residues 733-1200), (e) DIII and DIII-DIV linker (920-1237), (f) DIII and DII-DIII linker and DIII-DIV linker (733-1237). In some cases, the at least a portion of the DIII domain of human Na_Xthat is replaced by a corresponding portion of the DIII domain of the human or mammalian Na_Vdoes not comprise S5. In some cases, the at least a portion of the DIII domain of human Na_Xthat is replaced by a corresponding portion of the DIII domain of the human or mammalian Na_Vdoes not comprise S6. In some cases, the at least a portion of the DIII domain of human Na_Xthat is replaced by a corresponding portion of the DIII domain of the human or mammalian Na_Vdoes not comprise any of S1, S2, S3, S4, and/or S4-S5 linker. In some cases, the at least a portion of the DIII domain of human Na_Xis replaced by a corresponding portion of mammalian Na_V1.1, mammalian Na_V1.2, mammalian Na_V1.3, mammalian Na_V1.4, mammalian Na_V1.5, mammalian Na_V1.6, mammalian Na_V1.7, mammalian Na_V1.8, mammalian Na_V1.9, human Na_V1.1, human Na_V1.2, human Na_V1.3, human Na_V1.4, human Na_V1.5, human Na_V1.6, human Na_V1.7, human Na_V1.8, or human Na_V1.9. For example, in some cases, the at least a portion of the DIII domain of human Na_Xis replaced by a corresponding portion of human Na_V1.7. In some cases, the mutant human Na_Xis chimera construct 2, chimera construct 3, chimera construct 7, chimera construct 9, chimera construct 11, chimera construct 15 or chimera construct 19, or comprises an amino acid sequence of any one of SEQ ID Nos: 3, 4, 8, 10, 12, 16, or 20. (See Table 4 below and FIG. 18.)

In certain methods of determining whether a test molecule that is known or discovered to modulate the activity of a human ion channel protein also modulates the activity of human Na_Xion channel (Na_X) protein, the methods may be used as counter selections. For example, if an experimenter wishes to ensure that a modulator of a particular Na_Vprotein is specific for that Na_Vprotein and/or related Na_Vproteins, the experimenter may perform the methods herein to confirm that a test molecule is not a modulator of the mutant human Na_X.

In any of the above methods, the ion channel assay may be, for example, any suitable assay used to detect the activity of the mutant Na_Xprotein as an ion channel. Examples include, but are not limited to, a patch clamp assay, including an automated patch clamp assay, an ion flux assay, and an ion- or voltage-sensitive dye assay. Exemplary assays are described, for example, in H. Yu et al, “high throughput screening technologies for ion channels,” Acta Pharm. Sinica 37:34-43 (2016), and materials are available from commercial manufacturers. In an ion flux assay, for example, radioactive isotopes, such as sodium 22 (²²Na⁺), can be used to trace the cellular influx and efflux of sodium ions or other ions. Another type of assay is a voltage-sensitive or ion-sensitive dye assay. In a voltage-sensitive dye assay, voltage changes across a membrane comprising the ion channel protein are measured using fluorescence resonance energy transfer (FRET), for example, using dyes such as oxonol derivatives such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC₄) or FMP. In some cases, a FRET dye may be localized or tethered to the membrane. Ion-sensitive dye assays may use dyes that show a difference in signal depending on ion concentration. An example is the sodium indicator dye SBFI. In other embodiments, a patch-clamp assay may be used in the screening methods. In some embodiments, an automated patch-clamp assay may be used. Exemplary platforms and instrumentation for performing patch-clamp assays are sold by several manufacturers. Examples platform assays include IonWorks™ platform assays, PatchXpress™ and IonFlux™ (Molecular Devices), Qpatch™ HT/HTX (Sophion), and Patchliner™ and SynchroPatch™ (Nanion Technologies).

In any of the above methods, the potential modulator identified in the method modulates the activity of the mutant human Na_X, e.g., has an activity that is lower or higher than that of wild-type human Na_X. In some cases, the potential modulator reduces the activity of the mutant human Na_X, such as having an activity that is lower than that of wild-type human Na_X. In some cases, the activity may be reduced by at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, or at least 90%. In some cases, the activity the mutant human Na_Xin the presence of the modulator may be, for example, 1-90%, 10-90%, 1-10%, 1-20%, 25-90%, 50-90%, 25-75%, 40-80%, or 50-75% of the activity of the mutant human Na_Xin the absence of the modulator. In some cases, the potential modulator identified in the assay reduces the activity of the mutant human Na_Xin the assay with a half-maximal concentration of 10 nM to 500 M, 50 nM to 500 μM, 10 nM to 50 μM, 100 nM to 500 μM, 100 nM to 50 μM, 1-500 μM, 1-50 μM, 10-500 μM, or 50-250 μM. In other cases, the potential modulator identified in the assay increases the activity of the mutant human Na_Xin the assay, such as having an activity that is higher than that of wild-type human Na_X. In some cases, the activity may be increased by at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, or at least 100% (i.e. two-fold), up to 100%, or up to three-fold. In some cases, the activity the mutant human Na_Xin the presence of the modulator, may be, for example, 10-100%, 10-80%, 20-80%, 25-100%, 25-75%, 50-100%, two to three fold, or 100-200% higher than the activity of the mutant human Na_Xin the absence of the modulator.

In any of the above methods, the ion channel assay may also be performed in the presence of both a potential modulator and an identified modulator of the mutant human Na_X. For example, in such a case, one might determine if the identified modulator competes with the potential modulator for altering the activity of the protein. In some such cases, the identified modulator of the mutant human Na_Xis one or more of tetrodotoxin (TTX), quinidine, flecainide, tetracaine, or lidocaine.

Any of the above methods may also further comprise determining the binding affinity of the potential modulator identified in the assay to either or both of the mutant human Na_Xand wild-type human Na_X, and in the case of a chimeric human Na_X, to the mammalian or human Na_Vprotein from which the substituted amino acids are derived. For instance, an ELISA assay may be used to determine binding affinity, for example, using mutant human Na_Xprotein in a lipid-stabilized form on a matrix, such as a solid surface, such as beads, plates or the like. Beads can have any shape, such as flakes or chips, spheres, pellets, etc. In some embodiments, such beads are streptavidin-coated beads, avidin-coated beads, or deglycosylated-avidin-coated beads. In some embodiments, such beads are magnetic beads. In some cases, the potential modulator binds to either or both of the mutant human Na_Xand wild-type human Na_Xan EC50 or IC50 of 10 μM or less, 10 μM to 50 nM, 10 μM to 500 nM, 1 μM or less, 1 μM to 50 nM, or 100 nM or less.

In some embodiments, further experiments may be performed on molecules selected in the above screens, for example, to determine other biological activities of the molecules. For instance, further assays may be used to determine if the molecules also bind to other ion channel proteins, such as a human or mammalian Na_Vprotein, thus determining the specificity of the molecules as ion channel modulators or binders. For example, in methods using chimeric human Na_X, further assays may be performed to determine if the molecules also modulate the activity of the Na_Vprotein from which the chimeric portions of the mutant Na_Xwere derived, or if the molecules bind to that Na_Vprotein.

IV. Test Molecules for Screening Methods

In any of the above methods, the potential modulator (i.e., the test molecule) may be a peptide or macrocycle or antibody. For example, in some cases, the potential modulator is a small molecule. The present disclosure also relates to modulators of human Na_Xidentified by the methods described herein, which may be optionally peptides, macrocycles, small molecules, or antibodies.

In some embodiments, the potential modulator molecule to be tested is a peptide. In some embodiments, the peptide is a 6-14-mer peptide, such as a 6-12-mer, a 6-10-mer, a 6-8-mer, an 8-12-mer, an 8-10-mer, or the like. See slide 10. In some embodiments, the peptide is a 14-mer. See slides 15-16. In some embodiments, the peptide is an 6-10 mer. In some embodiments, the peptide is an 8-10 mer. In some embodiments, the peptide is an 6-8 mer. In some embodiment, the peptide is an 8-mer. See slides 21-22. In some embodiments, the peptide is a 3-40-mer, a 3-20-mer, a 4-16-mer, a 4-14-mer, or a 6-14-mer, such as a 3-mer, 4-mer, 5-mer, 6-mer, 7-mer, 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, 25-mer, 26-mer, 27-mer, 28-mer, 29-mer, 30-mer, 31-mer, 32-mer, 33-mer, 34-mer, 35-mer, 36-mer, 37-mer, 38-mer, 39-mer, or 40-mer.

In some embodiments, the peptide is a macrocycle. In some embodiments, the macrocycle is a 6-14 mer macrocycle, such as a 6-12-mer, a 6-10-mer, a 6-8-mer, an 8-12-mer, an 8-10-mer, or the like. See slide 10. In some embodiments, the macrocycle is a 14-mer macrocycle. See slides 15-16. In some embodiments, the macrocycle is an 6-10 mer macrocycle. In some embodiments, the macrocycle is an 8-10 mer macrocycle. In some embodiments, the macrocycle is an 6-8 mer macrocycle. In some embodiments, the macrocycle is an 8-mer macrocycle. See slides 21-22. In some embodiments, the macrocycle is a 3-40-mer, a 3-20-mer, a 4-16-mer, a 4-14-mer, or a 6-14-mer, such as a 3-mer, 4-mer, 5-mer, 6-mer, 7-mer, 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, 25-mer, 26-mer, 27-mer, 28-mer, 29-mer, 30-mer, 31-mer, 32-mer, 33-mer, 34-mer, 35-mer, 36-mer, 37-mer, 38-mer, 39-mer, or 40-mer macrocycle. In some embodiments, the macrocycle has at least one lipophilic side-chain and at least one positively charged side-chain.

In some embodiments, the molecule to be tested in a screen herein is a small molecule. In some embodiments, the molecule to be tested is an antibody, which may include not only full length antibodies of any of IgG, IgM, IgA, IgD, and IgE, but also an antigen binding fragment of an antibody, such as an Fv, Fab′, (Fab′)2, scFv, or the like, a nanobody, single-chain antibody, bispecific or multispecific antibody.

In some embodiments, the molecule to be tested is a binding fragment of a peptide, a binding fragment of a small molecule, or a binding fragment of an antibody (e.g., an antigen binding fragment).

V. Molecular Complexes

In some embodiments, the disclosure comprises a molecular complex comprising a mutant human Na_Xas described herein bound to a molecule, such as a potential modulator molecule, such as a peptide, small molecule, antibody, or binding fragment of a peptide, small molecule, or antibody.

In some embodiments, the molecule is a peptide. In some embodiments, the peptide is a 6-14 mer peptide, such as a 6-12-mer, a 6-10-mer, a 6-8-mer, an 8-12-mer, an 8-10-mer, or the like. See slide 10. In some embodiments, the peptide is a 14-mer. See slides 15-16. In some embodiments, the peptide is an 6-10 mer. In some embodiments, the peptide is an 8-10 mer. In some embodiments, the peptide is an 6-8 mer. In some embodiment, the peptide is an 8-mer. See slides 21-22. In some embodiments, the peptide is a 3-40-mer, a 3-20-mer, a 4-16-mer, a 4-14-mer, or a 6-14-mer, such as a 3-mer, 4-mer, 5-mer, 6-mer, 7-mer, 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, 25-mer, 26-mer, 27-mer, 28-mer, 29-mer, 30-mer, 31-mer, 32-mer, 33-mer, 34-mer, 35-mer, 36-mer, 37-mer, 38-mer, 39-mer, or 40-mer.

In some embodiments, the molecule in the complex is a small molecule. In some embodiments, the molecule is an antibody, which may include not only full length antibodies of any of IgG, IgM, IgA, IgD, and IgE, but also an antigen binding fragment of an antibody, such as an Fv, Fab′, (Fab′)2, scFv, or the like, a nanobody, single-chain antibody, bispecific or multispecific antibody.

In some embodiments, the molecule is a binding fragment of a peptide, a binding fragment of a small molecule, or a binding fragment of an antibody (e.g., an antigen binding fragment).

VII. Kits

The present disclosure also includes kits comprising reagents associated with screening methods herein. In some cases, kits comprise one or more species of mutant human Na_Xas described herein. In some cases, kits comprise reagents used in screening methods herein, either with or without a particular mutant human Na_X. In some cases, kits comprise more than one type of mutant human Na_X. In some cases, the kits also comprise at least one mammalian or human Na_Vand/or wild type human Na_X, for example, as a control.

In some embodiments, kits herein may comprise one or more reagents for performing an ion channel assay. In some cases, kits may comprise particular modulators of the mutant human Na_X, for instance, as positive controls. Kits herein may also include negative controls that are identified as not modulating the mutant human Na_X. Where kits are used to determine whether a molecule binds to the mutant protein, the kits may include mutant human Na_Xattached to matrix particles such as beads or to another type of matrix such as a plate. Beads can have any shape, such as flakes or chips, spheres, pellets, etc. In some embodiments, such beads are streptavidin-coated beads, avidin-coated beads, or deglycosylated-avidin-coated beads. In some embodiments, such beads are magnetic beads. The mutant human Na_Xmay or may not be pre-attached to a matrix. In some embodiments, reagents are included to facilitate attachment of the protein to a matrix, such as through biotin-streptavidin or a similar system.

In some embodiments, kits may comprise reagents associated with screening methods herein, such as some or all of the reagents needed to perform an ion channel assay as described in the methods herein. In some cases, one or more control reagents, such as modulators of the protein, may also be included.

In some embodiments, kits may comprise test molecules or libraries of test molecules, such as peptides, small molecules, and/or antibodies. In some embodiments, peptides in the kit may be macrocycles. In some embodiments, the kit may comprise test molecules that are a binding fragment of a peptide, small molecule, or antibody.

In some embodiments, kits may also comprise directions for use.

EXAMPLES

The following are examples of methods and compositions of the disclosure. It is understood that these Examples are not meant to limit the disclosure, but only to exemplify it, and that various other embodiments may be practiced, given the general description provided above.

Example 1—Materials and Methods
Example 1.1—Experimental Model and Subject Details

The cell line Expi293F is a human embryonic kidney cell line (female) transformed and adopted to grow in suspension (Thermo Fisher Scientific) and was used for protein expression. Expi293F cells were cultured in SMM 293T-I medium at 37° C. and 5% CO2. Expi293F cells were authenticated and tested for Mycoplasma contamination.

Example 1.2-Protein Expression and Purification

Full-length human Na_Xcontaining tandem 2× StrepII and 2×FLAG tags at its N-terminus and untagged full-length human β3 were each cloned into pRK vectors under the control of a CMV promotor. Both constructs were transfected with PEI into Expi293 cells and cultured for 48 hours in SMM 293T-I medium under 5% CO2 at 37° C. Cells were harvested by centrifugation at 800 g for 10 minutes and resuspended and lysed in Buffer A (25 mM ADA pH 6.0, 200 mM NaCl, 1 mM PMSF, 1 μg/mL benzonase, and 1x Roche protease inhibitor cocktail) by dounce homogenization. To solubilize the cell membranes, glyco-diosgenin (GDN) and cholesterol hemisuccinate (CHS) were added to the sample to final concentrations of 2% and 0.3%, respectively, and the sample was gently stirred at 4° C. for two hours. Insoluble material was then collected by ultracentrifugation at 125,000 g for 1 hour at 4° C. Supernatants were applied to anti-FLAG M2 agarose resin that had been pre-equilibrated in Buffer B (25 mM ADA pH 6.0, 200 mM NaCl, 0.01% GDN) and bound in batch at 4° C. for 1 hour. The sample was then applied to a gravity column and the collected resin was washed with 10 CV Buffer B followed by 10 CV supplemented with 5 mM ATP and 10 mM MgCl₂. Protein was eluted with 5 CV Buffer B supplemented with 300 μg/mL FLAG peptide. Eluates were applied directly to Strep-Tactin XT Superflow high capacity resin that had been pre-equilibrated in Buffer B and bound in batch for three hours, then washed with 10 CV Buffer B prior to sample elution in 5 CV supplemented with 50 mM biotin. For nanodisc incorporation, the sample was concentrated to 15 μM using an Amicon Ultra centrifugal filter device (100 kDa MWCO). For structural analysis in detergent (GDN), the eluate was instead concentrated to 100 μL and applied to a Superose 6 3.2/300 column that had been pre-equilibrated in Buffer B.

Example 1.3—Mass Spectrometry Analysis

Following size exclusion chromatography, 10 μg of the Na_X-containing peak fraction (after co-expression with β1- or β3-subunits, respectively) was denatured with 8 M guanidine (1:1 v/v), reduced with 1M dithiothreitol (DTT, Sigma-Aldrich, St Louis, MO) to a final concentration of 100 mM DTT and incubated at 95° C. for 10 minutes, then centrifuged. 10 μL of 1 M Tris, pH 8 was added to the solution and water was used to dilute the guanidine to a 2 M final concentration. Deglycosylation was performed with 2 μL of PNGaseF (15000 U, New England Biolabs) followed by overnight incubation at 37° C. Samples were further reduced with 10 mM DTT at 60° C. (15 minutes) followed by alkylation with 20 mM iodoacetamide at room temperature. Proteins were digested with 0.2 μg trypsin (Promega) or chymotrypsin in 50 mM ammonium bicarbonate, pH 8 at 37° C. overnight. Digestions were quenched with formic acid and the supernatants were subjected to desalting on C18 PhyTips (PhyNexus), lyophilized, reconstituted in 0.1% formic acid containing 2% acetonitrile and analyzed without further processing by reversed phase nano-LC/MS/MS on a Waters NanoAcquity HPLC system (Waters Corp.) interfaced to an Elite Orbitrap mass spectrometer (ThermoFisher Scientific). Peptides were loaded onto a Symmetry C18 column (1.7 mm BEH-130, 0.1×100 mm, Waters) and separated with a 60 minute gradient from 2% to 25% solvent B (0.1% formic acid, 98% acetonitrile) at 1 μL/min flow rate. Peptides were eluted directly into the mass spectrometer with a spray voltage of 1.2 kV. Full MS data were acquired in FT for 350-1250 m/z with a 60,000-resolution. The most abundant ions from full MS scans were selected for MS/MS through a 2-Da isolation window.

Acquired tandem MS spectra were searched using the Mascot algorithm (Matrix Sciences) with trypsin or chymotrypsin enzyme specificity. Search criteria included a full MS tolerance of 50 ppm, MS/MS tolerance of 0.5 Da with oxidation of methionine (+15.9949 Da) as variable modification and carbamidomethylation (+57.0215 Da) of cysteine as static modification. Data were searched against the specific sequences of in-house constructs overlaid onto the Uniprot mammalian database, including reverse protein sequences. Peptide assignments were first filtered to a 2% false discovery rate (FDR) at the peptide level and subsequently to a 2% FDR at the protein level.

Example 1.4—Reconstitution of β3-Na_Xinto Lipid Nanodiscs

For reconstitution into nanodiscs, multiple 50 μL sample aliquots were each applied to 2 mL tubes with a 200-fold molar excess of a POPC:POPE:POPG lipid mix (3:1:1 ratio solubilized in a sonication bath at 10 mg/mL in a buffer containing 50 mM HEPES pH 7.5, 100 mM NaCl, 5 mM MgCl₂, and 1% CHAPS) and incubated for 30 minutes at 4° C. The membrane scaffold protein MSP1E3D1 (Sigma) was then applied to the samples in a 4-fold molar excess and incubated for an additional 30 minutes at 4° C. Samples were then diluted to 1.5 mL and Bio-Beads were applied to a final concentration of 0.25 mg/mL. Samples were then mutated overnight at 4° C. prior to Bio-Bead removal. The samples were then pooled and passed over 1 mL Strep-Tactin XT resin in a column that had been pre-equilibrated in Buffer C (25 mM HEPES pH 7.0, 200 mM NaCl) to remove empty nanodiscs. The column was washed with 5 CV Buffer C prior to sample elution with 5 CV containing 50 mM biotin. The sample was then concentrated to 100 μL and applied to a Superose 6 3.2/300 column that had been pre-equilibrated in Buffer C. Peak fractions were pooled and concentrated to 3 mg/mL.

Example 1.5—Cryo-EM Sample Preparation and Data Acquisition

Samples were cross-linked with a final concentration of 0.05% EM-grade glutaraldehyde at room temperature for 10 minutes. The cross-linking reactions were quenched by the addition of 1 M Tris pH 7.0. Ultrafoil R2/2 (200 mesh) cryoEM grids (Quantifoil GMBH) were plasma cleaned for 5 seconds using the Solarus plasma cleaner (Gatan, Pleasanton, CA). Three microliters of the final samples at 2.25 mg/mL (nanodiscs) or 1.5 mg/mL (GDN) were applied to the cryoEM grids and blotted for 2.5 seconds using a Vitribot Mark IV (ThermoFIsher Scientific, Waltham, MA) using a blot force setting of 8 at 100% humidity, and plunged into liquid ethane. Grids were then imaged on a Titan Krios electron microscope (ThermoFisher Scientific, Waltham, MA) operated at 300 keV with a bioquantum energy filter using a K2 (nanodiscs) or K3 (GDN) Summit direct electron detector (Gatan, Pleasanton, CA). Images of the nanodisc were recorded at a magnification of 165,000x, which corresponded to 0.824 Å/pixel using a 20 eV energy slit. Image stacks contained 50 images recorded at 0.2 second intervals over 10 seconds, giving a total exposure of ˜50 e/Å². Images of the GDN sample were recorded in super resolution mode at 105,000× magnification, corresponding to 0.419 Å/pixel, using a 20 eV energy slit. Image stacks contained 60 images recorded at 0.05 second intervals over 3 seconds, giving a total exposure of ˜64 e/Å². All data collection was done using serialEM⁶⁷.

Example 1.6—Cryo-EM Data Processing
Nanodisc Sample

Cryo-EM data was processed using a combination of the WARP, RELION, and cisTEM software packages^68-71. For the first dataset, 11,658 movies were corrected for frame movement using MotionCor2⁷²in RELION. The resulting images were filtered to retain only those with an accumulated motion total below a value of 250 and contrast-transfer function (CTF) parameters were fit using the 30-4.5 Å band of the spectrum using CTFFIND4.1⁷³. Images were filtered to include only those with a detected fit resolution better than 5 Å, giving a total of 9,988 good images for further processing. 1,669,107 particles were picked using a deep learning-based algorithm in WARP⁶⁸. Particles were subjected to two rounds of 2D classification in cisTEM, and the best 30 classes were chosen (76,392 particles) and exported to RELION for 3D classification. A 20 Å low-pass filtered (LPF) Kv1.2 reconstruction solved in nanodiscs (EMD-9024)⁷⁴for which all density outside of the nanodisc had been erased was used as the initial 3D reference. The best obtained 3D volume was then used as the reference for a second round of 3D classification using a broader selection of particles from the 2D classification in cisTEM (350,901 particles). The best 3D volume and its corresponding 91,435 particles were then imported back into cisTEM for iterative rounds of auto-refine without a mask and manual refinement with iteratively adjusted masks and 20 Å LPF outside the mask (outside weight of 0.8). The resulting 4.5 Å map was then used as the reference in a final round of unmasked auto-refinements and masked manual refinements in cisTEM using the particle stack from only a single round of 2D classification in cisTEM, giving the final 3.2 Å map.

Detergent (GDN) Sample

Cryo-EM data was processed using a combination of the RELION, Gautomatch [K. Zhang, MRC LMB (mrc-lmb.cam.ac.uk/kzhang/)], and cisTEM software packages. 10,850 movies were corrected for frame movement using MotionCor272 in RELION and binned to 1 Å/pixel. The resulting images were filtered to retain only those with an accumulated motion total below a value of 250 and contrast-transfer function (CTF) parameters were fit using the 30-4.5 Å band of the spectrum using CTFFIND4.1⁷³. Images were filtered to include only those with a detected fit resolution better than 5 Å, giving a total of 10,569 good images for further processing. A total of 2,780,663 particles were automatically picked using Gautomatch using 16 uniform projections of the reconstruction of Na_V1.4 in complex with the β1 auxiliary subunit (EMD-9617) as templates. Particles were subjected to a single round of 2D classification into 250 classes in RELION and a particle stack containing 1,420,422 particles from the best 55 classes were extracted and exported to cisTEM. The structure of β3-Na_Xsolved in nanodiscs was used as an initial reference in an initial round of auto-refine without a mask, followed by several rounds of manual refinement with iterative mask refinement and 20 Å LPF outside the mask (outside weight of 0.8). Manual refinement runs included defocus refinement and used a score threshold of 0.2. Iterative rounds of unmasked auto-refinements and masked manual refinements yielded a final reconstruction of 2.85 Å resolution.

Example 1.7—Model Building and Structure Analysis

The structure of Na_V1.4 in complex with the β1 auxiliary subunit (PDB 6AGF) 28 was used as a template to generate a Na_Xhomology model using the Phyre2 server⁵⁸. This model along with β1 was rigid body docked into the cryo-EM map for manual rebuilding in Coot⁵⁹to give an initial model of the β3-Na_Xcomplex. Multiple rounds of real space refinement in Phenix⁶⁰and manual rebuilding in Coot were followed by molecular dynamics-assisted manual refinement in UCSF ChimeraX⁶¹with ISOLDE⁶². After a final refinement in Phenix, the model was validated using the Phenix validation package. Structure figures were generated using PyMol⁶³, UCSF Chimera⁶⁴, and UCSF ChimeraX. Caver3.0 was used to analyze the channel pore⁶⁵. Sequence alignments were performed using Clustal Omega⁶⁶and rendered with ESPript 3.0⁶⁷.

Example 1.8—Molecular Biology for Biochemical and Electrophysiological Experiments

Complementary DNAs (cDNAs) of human Na_X, Na_X-eGFP-2×FLAG, Na_V1.7, Na_X-Na_V1.7 domain-swapped chimeras, ATP1A1, ATP1B1 and SAP97, codon optimized for Homo sapiens, were cloned into the pcDNA3.1/Hygro (+) vector were used for this study. The human Na_Vβ1, Na_Vβ3, Ca_Vβ1, Ca_Vγ2, Ca_Vα281, ATP1A1, ATP1B1, ATP1G1 and SAP97 constructs were cloned into the pcDNA3.1 (+) vector. Na_X, Na_V1.5 and Na_V1.7 mutants (Na_XN1142K, QTT (F724Q/I1189T/I1492T); Na_V1.5 S1610P/S1618P/A1640P; Na_V1.7 S211P, AL966, S1269F, K1406N and A1596P/S1605P/A1627P) were generated using site-directed mutagenesis with custom-designed primers (Eurofins Genomics), PfuUltra II Fusion HS DNA Polymerase (Agilent Technologies) and DNA sequences were verified by Sanger DNA sequencing (Eurofins Genomics). For expression in Xenopus laevis oocytes, cDNAs were linearized using either NotI, BamHI or XbaI restriction enzyme and then transcribed to capped RNAs with the T7 mMessage mMachine Kit (Ambion). For expression in HEK293T and Neuro-2a cells, plasmid DNAs purified with the NucleoBond Xtra Midi Plus kit (Macherey-Nagel) were used.

Example 1.9—Two-Electrode Voltage-Clamp Electrophysiology

Oocytes were prepared as previously described⁶⁸. Healthy-looking stage V-VI oocytes were injected with 2.5-40 ng of RNA (in 5-41 nL) using a Nanoliter 2010 injector (World Precision Instruments). When excluding one or more construct, an equivalent volume of nuclease-free water was added to maintain a constant RNA concentration across different construct combinations. Injected oocytes were kept at 18° C., 140 rpm, in ND96 supplemented with 50 μg/mL gentamicin and 50 μg/mL tetracycline (in mM: 96 NaCl, 2 KCl, 1 MgCl₂, 1.8 CaCl₂), 2.5 sodium pyruvate, 0.5 theophylline, 5 HEPES; pH 7.4 with NaOH) for two to five days. Two-electrode voltage-clamp measurements were performed at room temperature using a Warner OC-725C Oocyte Clamp amplifier (Warner Instrument Corp, USA) and under constant perfusion with ND96 solution (in mM: 96 NaCl, 2 KCl, 1 MgCl₂, 1.8 CaCl₂, 5 HEPES; pH 7.4 with NaOH). For ion selectivity experiments, external solutions contained 115 mM of test cations as chloride salts, 1.2 mM CaCl₂, 2 mM MgCl₂, 5 mM HEPES (pH 7.4 with the corresponding hydroxide). Data acquisition was performed using a Digidata 1550 digitizer (Molecular devices; sampled at 10 kHz) and pCLAMP 10 software (Molecular Devices). Microelectrodes from borosilicate glass capillaries (Harvard Apparatus) were prepared to have resistances around 0.2-1.0 MQ2 using a P-1000 Flaming/Brown Micropipette Puller System (Sutter Instrument) and backfilled with 3 M KCl. Aconitine and veratridine (Alomone Labs) were dissolved in DMSO to make 50 mM stocks, and were further diluted to 300 and 100 μM in ND96 for electrophysiological experiments. Other Na_Vactivators (Alomone Labs; FIGS. 8A-D) were dissolved in water to make stock solutions. The voltage dependence of ion current was determined using a protocol consisting of steps from a holding potential of 0 mV to voltages ranging from +80 to −100 mV for 1 s in 20 mV increment.

Example 1.10—Cell Culture, Cell Surface Biotinylation and Western Blots

HEK293T and Neuro-2a cells (ATCC) were grown and maintained as described previously⁶⁸. Approximately 800,000 HEK293T and 250,000 Neuro-2a cells were seeded in 35 mm cell culture dishes ˜20 hours before transient transfection with 1 μg of cDNA using LipoD293 ver. II (tebu-bio). For biochemical experiments, the cDNAs of Na_X-eGFP-2×FLAG and β3 were mixed in a mass ratio of 1:1. Equal amount of empty vector DNA was added to keep the total cDNA amount constant when β3 was excluded from transfection. Transfected HEK293T cells were used for biochemical experiments 24 hours post-transfection. To induce differentiation, Neuro-2a cells were serum starved 24 hours post-transfection (cultured in DMEM for additional 24-30 hours) before they were used for biochemical and electrophysiological experiments. Cell surface biotinylation and Western blots were performed as described previously⁶⁸, with only slight modifications: 1) The quenching step was performed under gentle agitation on ice for 30 min, 2) Xenopus laevis oocytes were washed twice with tris-buffered saline before lysis, and 3) the total lysate fraction for oocytes was diluted 1:5 in SDS sample buffer before loading onto the gel due to excess protein. Antibodies were used as stated in⁶⁸, except the mouse anti-Na⁺/K⁺-ATPase antibody was from Santa Cruz Biotechnology (sc-21712). Blots are representative of minimum three individual experiments.

Example 1.11—Patch Clamp Electrophysiology

Differentiated Neuro-2a cells expressing Na_X-eGFP-2×FLAG were seeded on glass cover slips an hour before patch clamping experiments. Cells were voltage-clamped at −60 m V in whole-cell configuration (FIGS. 1E-F and 7F) using an Axopatch 200B amplifier (Molecular Devices). Digidata 1550A digitizer (Molecular devices; sampled at 10 kHz) and pCLAMP 10 software (Molecular Devices). Glass pipettes for patch-clamp experiments were pulled from borosilicate glass capillaries (World Precision Instruments; Kwik-Fil 1.5/1.12; OD/ID), and fire polished to final resistances between 2.0-5.5 MΩ. Extracellular solutions contained (in mM): 140 or 190 NaCl, 5 KCl, 2.5 CaCl₂), 1 MgCl₂, 5 HEPES, 20 glucose (pH 7.4 with NaOH). The osmolarity values of these solutions were ˜302 mOsm/L (for 140 NaCl) and ˜395 mOsm/L (for 190 NaCl). The intracellular solution contained (in mM): 120 K-gluconate, 20 TEA-C1, 2 MgCl₂, 2Na₂ATP, 1 EGTA and 10 HEPES, pH 7.3 with KOH (˜281 mOsm/L).

HEK293T cells expressing Na_X- or Na_X-QTT-eGFP-2×FLAG were seeded on glass cover slips an hour before whole-cell patch-clamping experiments. For a more physiological condition (FIGS. 4D and 7B), the extracellular solution contained 150 mM NaCl, 5 mM KCl, 0.5 mM CaCl₂, 1.2 mM MgCl₂, 10 mM HEPES, and 13 mM D-(+)-glucose (pH 7.4) with NaOH, ˜320 mOsm/L, and the intracellular solution contained 140 mM CsCl, 10 mM CsF, 5 mM EGTA, 10 mM HEPES, and 2 mM Na₂ATP (pH 7.2) with CsOH, ˜304 mOsm/L. To determine if Na_X-QTT was cation-permeable, we first substituted all extracellular cations with NMDG⁺ (FIG. 4E). Extracellular solution contained 150 mM NMDG and 10 mM HEPES (D-(+)-glucose was added accordingly to achieve osmolarity ˜325 mOsm/L and pH was adjusted to 7.4 with hydrochloric acid). To determine if Na_X-QTT was anion-permeable, we substituted all Cl⁻ ions in both intra- and extracellular solutions with the large anion methanesulfonate (MS⁻) (FIG. 15C). The extracellular solution contained 150 mM NaMS and 10 mM HEPES (D-(+)-glucose was added accordingly to achieve osmolarity ˜325 mOsm/L, and pH was adjusted to 7.4 with NaOH). The intracellular solution contained 136 mM NaMS, 10 mM NaF, 5 mM EGTA, 10 mM HEPES, and 2 mM Na₂ATP (pH 7.2 with NaOH, ˜309 mOsm/L). The extracellular NMDG solution contained 150 NMDG and 10 mM HEPES (D-(+)-glucose was added accordingly to achieve osmolarity ˜325 mOsm/L, and pH was adjusted to 7.4 with methanesulfonic acid).

For ion-selectivity experiments (FIGS. 4E and 5A) (i) involving monovalent cations, the extracellular solution contained 150 mM XCl, 10 mM HEPES, and D-(+)-glucose was added accordingly to achieve osmolarity ˜325 mOsm/L, and the pH was adjusted to 7.4 with XOH, where X indicates the cation of interest (Na⁺, K⁺, Li+ or Cs+); (ii) involving Ca²⁺, the extracellular solution contained 110 mM CaCl₂, 10 mM HEPES, and D-(+)-glucose was added accordingly to achieve osmolarity ˜325 mOsm/L, and the pH was adjusted to 7.4 with Ca(OH)₂; (iii) the intracellular solution contained 136 mM NaCl, 10 mM NaF, 5 mM EGTA, 10 mM HEPES, and 2 mM Na₂ATP (pH 7.2) with NaOH, ˜309 mOsm/L.

Example 1.12—Data Analysis

For data analysis, raw current traces were typically filtered using 8-pole Bessel low-pass filter at 500-800 Hz. Current traces were subjected to factor 5 data reduction for display in figures. Data are presented as mean±standard deviation (SD) from at least 3 cells from at least two batches of cells. For ion-selectivity experiments performed with Na⁺ intracellular solution (FIGS. 4E and 5A), liquid junction potential (LJP) was measured with reference to the Na⁺ intracellular solution and corrected after recording. Relative ion permeabilities (PNa/PX) were calculated using the equation P_X/P_Na=[Na⁺]_ICexp(E_revF/RT)/[X⁺]_EC(X=Na, K, Li or Cs), where F is Faraday's constant, R is the gas constant, T=274 K, and E_revs were measured from the −80- to +80-m V ramp (corrected for LJP).

Example 2—Evaluation of Human Na_XFunction

Reliable ionic currents were not detected following expression of human Na_Xin HEK293T cells or Xenopus laevis oocytes in response to changes in membrane voltage (FIGS. 1A-D, 7A-D, and 8A-D). The application of classical Na_Vchannel activators like aconitine, veratridine, pompilidotoxins, blood-depressing substance-I, and neurotoxin-II during different voltage-step protocols in oocytes did not invoke any clear human Na_X-mediated currents (FIGS. 1B, 1D, and 8A-B). Despite robust surface expression of human Na_X, co-expression with Na_Vor voltage-gated calcium (Ca_V) channel auxiliary-subunits (i.e. β1, β3, α2δ1, γ2) also failed to produce dependable currents (FIGS. 1C, 1D, 7A, 7C and 8C-D). These results considerably extend prior findings that Na_Xdoes not operate as a conventional voltage-gated channel^14,15.

Increases in the extracellular sodium concentration, [Na⁺]_e, have been reported to activate a non-inactivating Na⁺ current in glial and neuroblastoma (Neuro-2a) cell lines expressing murine Na_X. Murine Na_Xhas also been suggested to interact with the Na⁺/K⁺-ATPase and synapse-associated protein 97 (SAP97) at the plasma membrane. Despite surface expression and our ability to also co-immunoprecipitate the ATP1A1 a-subunit under some conditions (Table 2), no reliable inward Na⁺ currents were detected in oocytes co-expressing human Na_Xwith the Na⁺/K⁺-ATPase subunits and SAP97 upon exposure to 200 mM [Na⁺]_e(FIGS. 1C and 1D).

TABLE 2

Na_xChannel Sample Analyzed

Protein ID:
#PSMs
% Coverage

SCN7A + SCN1B

SCN7A
254
36

SCN1B
62
40

HSP71_HUMAN
32
43

DNJC7_HUMAN
27
46

F10A1_HUMAN
22
31

SCN7A + SCN3B

SCN7A
234
35

SCN3B
19
44

HSP71_HUMAN
24
30

DNJC7_HUMAN
19
31

AT1A1_HUMAN
24
21

When human Na_Xwas expressed in Neuro-2a cells, we observed small and flickery inward currents upon changes in [Na⁺]_efrom 140 to 190 mM in the whole-cell patch-clamp configuration (FIGS. 1E-G and 7E-F). Neuro-2a cells expressing the Na_V1.7 channel showed similar flicker behavior following osmolarity changes, indicating that this phenomenon was not specific to cells expressing human Na_X(FIGS. 1E-G). A non-inactivating inward current was only observed when seal resistances were low (<1 G (2), and this phenotype was found in both mock- and human Na_X-transfected Neuro-2a cells (FIG. 7F). Overall, it is unclear if our findings reflect a functional divergence between human and murine Na_Xchannels (FIG. 9), or slight differences in experimental protocols, but we failed to measure any reliable currents through human Na_X. We next considered if structure determination of human Na_Xmight provide insight into its function or regulation.

Example 3—Structure Determination of the β3-Na_XComplex in Lipid Nanodiscs

Human Na_Xwas recombinantly expressed in HEK293 cells and purified in the mild detergent glyco-diosgenin (FIGS. 10A-C). Inspired by reports that the Na_V1.7 channel forms stable complexes with auxiliary β-subunits^23-25, β1- or β3-subunits were co-expressed and found to co-purify with Na_X(Table 2). To provide a more native environment, the β3-Na_Xcomplex was reconstituted into nanodiscs using a common phospholipid mixture (FIGS. 10A-C). Prior to sample vitrification, the Na⁺ concentration was raised above the threshold reported to activate Na_X, to 200 mM. The resulting cryo-EM reconstruction of the human β3-Na_Xcomplex extended to 3.2 Å resolution (FIGS. 11A-F, 12A, and Table 3) and represents the first structure of a eukaryotic Na_Vchannel family member determined in a reconstituted lipid membrane environment. In the first, title row of Table 3 below, the EMDB reference for β3-Na_X-nanodisc is EMDB-25919, and the PDB reference is PDB 7TJ8. The EMDB reference for β3-Na_X-GDN is EDMB-25920 and the PDB reference is PDB 7TJ9.

TABLE 3

Cryo-EM Data Collection, Refinement and Validation Statistics

β3-Na_x-nanodisc
β3-Na_x-GDN

(EMDB-25919)
(EMDB-25920)

(PDB 7TJ8)
(PDB 7TJ9)

Data collection and processing

Magnification
165,000
105,000

Voltage (kV)
300
300

Electron exposure (e-/Å²)
48.579
64.009

Defocus range (μm)
0.5-1.5
0.5-1.5

Pixel size (Å)
0.849
1.1648

Symmetry imposed
C1
C1

Initial particle images (no.)
1,968,741
2,780,663

Final particle images (no.)
1,238,338
1,420,422

Map resolution (Å)
3.2
2.9

FSC threshold
0.143
0.143

Map resolution range (Å)
3.2-46.6
2.9-33.13

Refinement

Initial model used
6AGF
β3-Na_x-nanodisc

(PDB code)

Model resolution (Å)
3.4
3.1

FSC threshold
0.5
0.5

Model resolution range (Å)
3.2-46.6
2.9-33.13

Map sharpening B factor (Å²)
−90
−90

Model composition

Non-hydrogen atoms
21502
22342

Protein residues
1242
1273

Ligands
18
23

B factors (Å²)

Protein
67.58
77.83

Ligand
64.16
70.41

R.m.s. deviations

Bond lengths (Å)
0.004
0.006

Bond angles (°)
0.906
0.611

Validation

MolProbity score
1.78
1.66

Clashscore
5.79
5.15

Poor rotamers (%)
0.80
0.09

Ramachandran plot

Favored (%)
93.72
94.85

Allowed (%)
5.79
5.15

Disallowed (%)
0.49
0.00

Example 4—Overall Structure of the β3-Na_XChannel Complex

The β3-Na_Xchannel complex resembles a four-leaf clover with the VSLDs arranged in a domain-swapped organization around the central pore module (FIG. 1H), which conforms to the architecture shared among Na_V, Ca_Vand NALCN channels. In Na_X, a soluble intracellular amino-terminal domain is docked beneath VSLD1, the intracellular DIII-DIV linker is bound alongside the pore, and the intracellular carboxyl-terminal domain (CTD) is not well resolved (FIG. 13A), features reminiscent of available human Na_Vchannel structures. On the extracellular side, two and four N-linked glycans are found to extend from the Na_Xand β3 subunits, respectively.

The β3-subunit forms extensive interactions by wedging its immunoglobulin-domain between elaborated extracellular loops of the Na_Xpore module and positioning its single transmembrane helix against VSLD3 (FIGS. 1H and 13B). Although contemporary β1- or β3-auxiliary subunits appear to form stable complexes with Na_X(Table 2), its pore module does not possess the cysteine residue required to form covalent complexes with β2- or β4-subunits^23,27.

The overall architecture of human Na_Xreconstituted in lipid nanodiscs is highly similar to Na_V1.1, Na_V1.2, Na_V1.4, Na_V1.5 and Na_V1.7 channel structures determined in detergent^23,28-31(FIG. 13C). Conversely, Na_Xdoes display substantial deviations in regions that are frequently implicated in Na_Vchannel gating, including a ˜5 Å positional shift of VSLD4, and >6 Å displacements along the DII S4-S5 linker (FIGS. 13D and 13E). It seems plausible that the observed structural changes in Na_Xare a consequence of its high sequence divergence and contribute to its distinct function (FIG. 9).

Example 5—the Na_XPore Module is in a Non-Conductive State

The Na_Xpore module contains an outer vestibule, an ion selectivity filter, a central cavity, and an S6-activation gate (FIG. 2A). The S6-gate is narrow and lined by a double-ring of hydrophobic side-chains that would form a barrier to the passage of hydrated ions, which unequivocally defines a non-conductive state (FIGS. 2A, 2B and 13C)^23,28-31. However, because our Na_Xstructure was determined under high Na⁺ concentrations, these findings appear inconsistent with reports that murine Na_Xforms a Na⁺-activated, non-inactivating channel. Instead, the non-conductive state observed for the pore module aligns perfectly with predictions from our physiological studies on human Na_X(FIGS. 1A-F, 7A-F and 8A-D).

The Na_Xcentral cavity is similar in volume to those in Na_Vchannels, although the DIV-S6 phenylalanine commonly targeted by pore blocking drugs^32,33is replaced by DIV-Trp1484 (FIG. 13F). Na_Xreveals four lateral fenestrations that penetrate the pore module, identifying membrane access pathways for hydrophobic drugs or lipids to directly enter into the central cavity (FIGS. 2C and 2D), as first suggested in Na_Vchannels^28-30,34. It is therefore conceivable that classic Na_Vor Ca_Vchannel antagonists might target Na_Xwithin the central cavity.

Example 6—The Na_XDIII-DIV Linker Restricts S6-gate Dilation

The intracellular DIII-DIV linker of Na_Xinteracts with a pore module receptor site in a manner which closely resembles the fast-inactivated state structure of human Na_Vchannels (FIGS. 2B and 14A). The Na_XIFI-motif is bound in-between the DIII-S4-S5 linker and the DIV-S5 and S6, analogous to complexes that restrict S6-gate dilation in Na_Vchannels (FIGS. 2B and 14A). Although the DIII-DIV linker of Na_Xhas long been recognized as a site of substantial sequence divergence (FIG. 14B), it clearly shares high structural homology with this key functional region in Na_Vchannels (FIG. 14A). Nevertheless, mutating the IFI-motif of Na_Xinto a classical non-inactivating Na_Vchannel QQQ-motif was insufficient to generate functional channels in injected oocytes (FIG. 14C), which further highlights the existence of distinct requirements to gate the Na_Xchannel.

We exploited Na_Xand Na_V1.7 structural models to construct a panel of precisely-targeted chimeric channels to evaluate the determinants responsible for the non-conductive state of human Na_X. Double-domain DII-DIII and DIII-DIV Na_V1.7-Na_Xchimeras and the single-domain DIII Na_V1.7-Na_Xchimera produced large inward and outward currents when expressed alone in Xenopus oocytes (FIG. 2E). Ultimately, swapping both Na_V1.7-VSD3 and the associated DIII-S4-S5 linker were the minimal regions sufficient to transfer the leak channel phenotype (FIG. 14D). Because these regions are required to form the receptor site for the IFM-motif which restricts S6-dilation in Na_Vchannels (FIG. 14A), and their substitution into Na_Xproduces robust leak currents (FIGS. 2E and 14D), we propose that the DIII-DIV linker of Na_Xserves to restrict S6-dialation and stabilize the non-conductive state of the pore module.

Example 7—The Na_XPore is Infiltrated by Membrane Lipids

By definition, a closed or inactivated state of an ion channel is non-conductive. In the β3-Na_Xstructure, four lipids penetrate through the lateral pore fenestrations to enter the central cavity above the narrow, hydrophobic S6-gate. These striking densities can be unambiguously assigned as three phospholipids and one cholesterol (FIGS. 2C and 2D). One phospholipid even straddles and seals the intracellular S6-activation gate (FIGS. 2C and 14E).

To evaluate if the lipid-nanodisc environment was responsible for the robust visualization of membrane lipids in β3-Na_X, we prepared a new sample in the detergent glyco-diosgenin without exogenous phospholipid supplementation and determined its structure to 2.85 Å resolution (FIGS. 11G-L, 12B and Table 3). We observed an identical Na_Xpore structure penetrated by four well-defined co-purifying lipids (FIG. 14F). The ion conduction pathway in β3-Na_Xis therefore unequivocally non-conductive in this pore module conformation because it is infiltrated and occluded by lipids (FIGS. 2A-D and 14E-F). The presence of well-resolved, co-purifying lipids bound within Na_Xmay reflect the intrinsic stability of its non-conductive state and hydrophobicity of the S6 gate and central cavity.

Example 8—Targeted Pore-Wetting Mutations can Activate Human Na_X

Based on our collective observations that the S6-gate is narrow, hydrophobic, and lipid-bound, and that disruption of the IFI-motif receptor site may promote S6-dilation, we reasoned that the targeted introduction of polar residues around the S6-gate might promote pore hydration, destabilization of bound lipids, and transition of Na_Xto an open, conductive state (FIGS. 3A-F). Accordingly, a triple-mutant Na_Xchannel construct (F724Q-I1189T-I1492T) containing polar substitutions at three S6-gate lining positions, Na_X-QTT, produced robust ionic currents when expressed alone in Xenopus oocytes in response to voltage-step protocols (FIGS. 3A and 3B). The corresponding single-point mutant Na_Xchannel constructs failed to produce robust currents, signifying a pore-wetting threshold exists to achieve S6-gate dilation through destabilization of the non-conductive pore module state (FIG. 3C). The L390E-I189E-I1492E triple-mutant Na_Xchannel construct (Na_X-EEE) also displayed function but with much lower current amplitudes and higher current variability relative to Na_X-QTT (FIGS. 3D and 3F).

In injected oocytes, Na_X-QTT currents are outward-rectifying with no signs of inactivation even during long (5 sec) depolarization test pulses (FIG. 3B). Because Na_Xcan interact stably with the β1- or β3-subunits (FIG. 1H and Table 2), the functional effects of these auxiliary subunits were evaluated. No difference in current amplitudes or kinetics were observed when Na_X-QTT was expressed in the presence of the β1- or β3-subunits, respectively (FIGS. 3E and 3F). In HEK293T cells, Na_X-QTT also behaves as a non-inactivating channel in whole cell patch-clamp experiments when expressed with a C-terminal GFP-2×FLAG tag (see below). Critically, the Na_X-QTT channel construct contains only three mutations targeted around the intracellular S6-gate (FIG. 3A), far removed from the selectivity filter and other important channel regions, so below we characterize Na_X-QTT as a proxy to evaluate ion selectivity and pharmacology of the human Na_Xchannel.

Example 9—The Na_XSelectivity Filter

The unique ion selectivity filter of Na_X(DENA-motif) is supported by a local architecture that differs radically from the scaffold which supports the classic DEKA-motif in Na_Vchannels (FIGS. 4A-C). Four landmark tryptophan residues in Na_Vchannels form interdomain interactions involving three conserved threonine side-chains (FIG. 4B)^{23,28-30,35,36}. In Na_X, Pro355 replaces the DI tryptophan, while Ala351 and Ile1432 substitute for the DI and DIV threonine counterparts, respectively (FIG. 4B). Substitutions at positions equivalent to Na_XPro355 and Ile1432 are in Na_V1.5 are pathogenic, highlighting the potential functional relevance of these alterations. Consequently, the Na_Xselectivity filter is wider and more electronegative than the selectivity filter in Na_Vchannels (FIG. 4B).

At the DENA-motif of Na_X, Asp353 (DI) hydrogen bonds to the backbone of DII-Trp689, Glu688 (DII) points into the ion permeation pathway, and Asn1142 (DIII) bonds to the DIV-Phe1433 carbonyl to impose a ˜1.5 Å radial shift onto Ala1434 (DIV) compared to the DIV alanine in Na_Vchannels (FIGS. 4A and 4C). The relative displacement of Ala1434 is accommodated by DI-Pro355 in Na_X, which also permits DI-Tyr359 to reorient ˜90° to fill the volume vacated by the absent conventional DI tryptophan (FIG. 4C). This structural remodeling displaces DI-Glu356 and DII-Glu691 in the outer vestibule by 2.5-3.5 Å, which modifies the canonical tetrodotoxin (TTX)-binding site and may highlight the potential to discover Na_X-selective inhibitors (FIG. 15A).

Example 10—Na_Xdoes not Discriminate Monovalent Cations

The signature DIII-Asn1142 side-chain dramatically alters the structure and chemical profile of the Na_Xselectivity filter relative to the conventional DIII-lysine that plays a key role in establishing Na⁺-selectivity in Na_Vchannels (FIGS. 4A-C). Accordingly, we introduced a DENA-motif into the human Na_V1.7 channel through a single mutation and observed a pronounced loss in Na⁺ and Lit selectivity in injected Xenopus oocytes, whereas measurable K+ currents with unusual kinetics were recorded (FIG. 15B). In reciprocal experiments, mutational transfer of a canonical DEKA-motif into Na_Xproduced no measurable currents (FIG. 15B), confirming that molecular features beyond the Na_XDENA-motif contribute to its activation.

We examined currents from the Na_X-QTT construct expressed in HEK293T cells and found that when all cations within the extracellular solution were replaced with the large cation N-methyl-D-glucamine (NMDG), the reversal potential shifted from +5 mV to below −100 mV (FIG. 4D), establishing that this construct behaves as a cation channel, and that large cations do not permeate. Replacement of all extracellular Cl-ions with the large anion methansulfonate did not change the current profile (FIG. 15C), indicating that Na_Xbehaves as a cation-selective channel, which is consistent with the size and electronegative profile of the selectivity filter (FIG. 4B). As expected, using a ramp protocol (−80 to +80 mV) with a simple Na⁺ intracellular solution (150 mM), the reversal potential was close to 0 mV under symmetrical NaCl/NaCl conditions (FIG. 4E). Notably, only slight changes in the reversal potentials were measured when cells were exposed to different extracellular monovalent cations (150 mM), establishing that Na_X-QTT does not effectively discriminate between Na⁺, K⁺, Cs⁺ or Li⁺ ions (FIG. 4E)

Example 11—Na_Xis Inhibited by Extracellular Ca²⁺

We measured currents from Na_X-QTT and observed that Ca²⁺ does not permeate (FIG. 5A). However, extracellular Ca²⁺ showed an inhibitory effect, where a physiologically-relevant concentration of Ca²⁺ (1 mM) inhibited ˜70% of the outward Na⁺ current (FIG. 5A). Notably, extracellular Ca²⁺ block is ˜10-fold more potent on Na_Xrelative to traditional Na_Vchannels, confirming physiochemical distinctions within the selectivity filter (FIGS. 4A and 4B). The divalent cations Zn²⁺ and Co²⁺ inhibited Na_X-QTT currents at 1 mM, whereas inhibition by the trivalent cation Gd³⁺ was more potent (FIG. 5B), demonstrating that pore block by multivalent cations is not substantially impacted by the wider and more electronegative selectivity filter structure of Na_X(FIG. 4B).

Example 12—Na_Xis Sensitive to Classic Na_VChannel Blockers

We tested a range of classical Na_Vchannel blockers to establish the pharmacological properties of Na_X-QTT. Remarkably, and strongly contrasting prior reports that Na_Xis a TTX-resistant channel, tetrodotoxin showed inhibitory effects on Na_X-QTT at concentrations as low as 10 nM (FIG. 5C). The potency of TTX block suggests that it occurs at the selectivity filter, consistent with substantial conservation of side-chains implicated in TTX-block of sensitive Na_Vchannel subtypes (FIGS. 9 and 15A).

The general anaesthetic lidocaine was effective at blocking Na_X-QTT currents at low mM concentrations (FIG. 5C), presumably by binding within the central cavity of the channel. Other classical pore-blocking drugs like quinidine and loperamide were equally potent inhibitors of Na_X-QTT currents at high μM concentrations (FIG. 5C). By contrast, strong inhibitory effects were not observed for flecainide, ranolazine or phenytoin on Na_X-QTT currents at concentrations up to 300 μM (FIG. 5C).

Example 13—Na_XReveals Atypical Voltage Sensor-Like Domains

Conditions under which murine or human Na_Xchannels produce voltage-activated currents have not yet been identified, and this apparent lack of voltage sensitivity remains a puzzle. Paradoxically, the number of S4-gating charges is only modestly reduced in human Na_X^14,15,22(FIG. 9), and its VSLDs shares a high overall structural similarity with the VSDs of Na_Vand Ca_Vchannels (FIGS. 13D and 13E). Intriguingly, while VSLD1, VSLD2 and VSLD3 in Na_Xare all found in activated conformations, VSLD4 is deactivated (FIG. 6A-D). Closer evaluation of the Na_XVSLDs should provide insight into its apparent defective voltage-sensitivity.

Relative to human Na_Vchannels, VSLD1 in Na_Xcontains only three of four expected S4-gating charges (₂₀₃PTLQTARTLRILKIIP₂₁₈; SEQ ID NO: 87; FIGS. 6A and 9). K4 is coordinated by the intracellular negative charge cluster (INC) beneath the hydrophobic construction site (HCS), akin to an activated conformation (FIG. 6A)^37,38. Notably, glutamine neutralization at R1 is known to reduce voltage-sensitivity in other channels⁴⁰, and Pro203 imparts a radial bend within the extracellular S3-S4 loop that may impact S4 movement in Na_X, at a position conserved as serine in Na_Vchannels (FIGS. 6A and 9).

Na_XVSLD2 contains all five anticipated S4-gating charges (₅₉₃ALLRLFRMLRIFKLGK₆₀₈; SEQ ID NO: 88), and its activated state appears stabilized by a unique S3 His579 side-chain that bonds to the S4 carbonyl backbone (FIGS. 6B and 9). Furthermore, a radical shift of the DII S4-S5 linker towards the cytosol (3-7 Å) relative to human Na_Vchannel structures likely alters VSLD2 coupling (FIGS. 6B and 13D). This is propagated by a single residue deletion in the Na_XDII-S6 helix, which repositions Phe731 to undergo a ˜180° reorientation to enforce S4-S5 linker displacement (FIGS. 6B and 9).

Na_XVSLD3 displays only four of six canonical S4-gating charges (₁₀₂₄KPLISMKFLRPLRVLSQ₁₀₄₀; SEQ ID NO: 89), an alteration predicted to stabilize its activated conformation and decrease intrinsic voltage-sensitivity (FIGS. 6C and 9). Additionally, Phe1011 (S3) substantially and uniquely extends the HCS in VSLD3 (FIGS. 6C and 9), which would be expected to further impact S4-gating charge movement.

Na_XVSLD4 contains only four S4 gating charges (₁₃₄₆QLILLSRIIHMLRLGKGPKVFH₁₃₆₇; SEQ ID NO: 90), since H4 and H8 are unlikely to be charged at physiological pH^37,39,44,45(FIGS. 6D and 9). By comparison, VSD4 and the associated DIV S4-S5 linker in canonical Na_Vchannels conserve eight S4-gating charges that are inextricably linked to the fast inactivation mechanism. In stark contrast to human Na_Vchannel structures^{23,28,29,31,37}and expectations at 0 mV, VSLD4 of Na_Xexists in a deactivated-like state, as indicated by a relative ˜7 Å displacement of the S4 towards the cytosol (FIGS. 6D and 16A). This observation is surprising, but consistent with the deficient voltage-sensitivity so far described for Na_X.

Example 14—Probing Function of the Divergent Na_XVSLDs

Structural features identified in the VSLDs of Na_Xsuggest non-canonical contributions to channel function. Moreover, a number of prominent sequence peculiarities occur at positions in Na_Xthat have been reported as disease-causing alterations in related human channels. Pro203 in Na_XVSLD1 is equivalent to the pathogenic Ser211Pro mutation in Na_V1.7 reported to hyperpolarize channel activation by −12 mV (FIGS. 6A and 16B). Structure-based sequence alignments reveal that an analogous in-frame DII-S6 deletion is pathogenic in Na_V1.7 (ΔLeu966) and produces a −16 mV hyperpolarization of activation (FIGS. 6B and 16B). Phe1011 in Na_XVSLD3 corresponds to the pathogenic Ser209Phe mutation in KCNQ1 reported to impart a strong hyperpolarization (−45 mV) on channel activation (FIG. 16C) 43, while the equivalent Ser1269Phe substitution in Na_V1.7 produced a ˜8 mV depolarizing shift (FIGS. 6C and 16B). In addition, three prolines are uniquely present in VSLD4 of Na_X(FIGS. 9 and 16D), and mutation of the corresponding VSD4 residues in Na_V1.5 to proline imparted a strong depolarizing shift (15 mV) onto steady-state inactivation, consistent with the deactivated conformation observed in VSLD4 (FIGS. 6D, 16A and 16E).

To further probe Na_XVSLD function, we engineered chimeric constructs through targeted transfer of individual VSLDs into Na_V1.7. Remarkably, robust currents were recorded from injected Xenopus laevis oocytes expressing the Na_V1.7-Na_X-VSLD2 channel chimera (FIG. 6E), indicating that VSLD2 is either constitutively activated or intrinsically capable of electromechanical-coupling. By contrast, wholesale transfer of Na_X-VSLD1, VSLD3 or VSLD4 rendered Na_V1.7 chimeric channels non-functional (FIG. 6E). Thus, despite Na_Xsharing global structural similarities with Na_V, Ca_Vand Kv channels, our findings suggest that cumulative sequence adaptations may result in highly divergent functional outcomes across the channel superfamily.

Example 15—Exemplary Na_X/Na_V1.7 Chimeric Constructs

A series of chimeric human Na_Xproteins were constructed in which one or more regions from human Na_V1.7 were inserted in place of the corresponding Na_Xsequences, as noted in Table 4 below. A construct is considered as functional (see Table 4) if the current amplitude of Xenopus laevis oocytes expressing the construct elicited at +80 mV or −100 mV from a holding potential of 0 mV is significantly higher than that from oocytes expressing human Na_Xwild-type (see FIG. 18). Data for the first 7 chimeras of Table 4, and for a hNa_X-hNa_V1.7 DIV chimera, are also described in Example 6 and are shown in FIG. 2E.

TABLE 4

Constructs

SEQ
Construct

ID
No.

NO
(FIG. 18)
Construct
Function*

2
1
hNa_x_—hNa_v1.7_DI-DII chimera
No

3
2
hNa_x_—hNa_v1.7_DIII-DIV chimera
Yes

4
3
hNa_x_—hNa_v1.7_DII-DIII chimera
Yes

5
4
hNa_x_—hNa_v1.7_DI-DIV chimera
No

6
5
hNa_x-hNa_v1.7_DI chimera
No

7
6
hNa_x-hNa_v1.7_DII chimera
No

8
7
hNa_x-hNa_v1.7_DIII chimera
Yes

9
8
hNa_x-hNa_v1.7_DIV-DIII-DIVlinker chimera
No

10
9
hNa_x_—hNa_v1.7_DIII_Chimera_SF chimera
Yes

11
10
hNa_x_—hNa_v1.7_D3-D4link chimera
No

12
11
hNa_x_—hNa_v1.7_D3-D4link-PM chimera
Yes

13
12
hNa_x_—hNa_v1.7_D3-D4link-PM-S4S5link chimera
No

14
13
hNa_x_—hNa_v1.7_VSD3 chimera
No

15
14
hNa_x_—hNa_v1.7_VSD3-S4S5link chimera
No

16
15
hNa_x_—hNa_v1.7_VSD3-S4S5link-PM chimera
Yes

17
16
hNa_x_—hNa_v1.7_DIII_VSD3-S3-S4 chimera
No

18
17
hNa_x_—hNa_v1.7_DIII_VSD3-S5-S6 chimera
No

19
18
hNa_x_—hNa_v1.7_DIII_VSD3-S3-S4loop_S4-S5link_S5 chimera
No

20
19
hNa_x_—hNa_v1.7_DIII_DIVS6-CTD chimera
Yes

21
20
Na_x_—Na_v1.7_D1-D2_linker chimera
No

22
21
Na_x_—Na_v1.7_D2-D3_linker chimera
No

23
22
Na_x_—Na_v1.7_D3-D4_linker chimera
No

24
23
Na_x_—Na_v1.7_ultimateloop chimera
No

25
24
Na_xF724Q/I1189T/I1492T (QTT)
Yes

26
25
Na_xL390E/I1189E/I1492E (EEE)
Yes

Discussion

The above Examples pursue an integrated analysis of structure, function and pharmacology to provide insight into the Na_Xchannel. Through comprehensive assessment of different expression systems, cofactors, and pharmacological activators, we demonstrated that human Na_Xdoes not function as a voltage-activated channel. The molecular features responsible for the altered voltage sensitivity of Na_Xmight include single residue changes within the selectivity filter, voltage sensors, and pore module, as analogous changes can drastically impact the gating properties of Na_V1.5 and Na_V1.7 channels (FIGS. 6A-E, 9, 16B and 16E). In fact, many positional changes which impart structural alterations in Na_Xappear to underlie disease hotspot positions in Na_Vchannels, but the significance of these observations remains unknown.

Using structure-guided protein engineering, we found that the transfer of entire homologous domains or subdomains (e.g. VSLDs) between Na_Xand Na_V1.7 produces either non-functional constructs or channels with highly unusual gating characteristics (FIGS. 2E and 6E). These results suggest that the coupling or energetics of Na_Xgating has diverged substantially from Na_Vchannels, which likely points to structural differences that burden the coupling interfaces between these closely related channel scaffolds. Notably, our studies do not exclude the possibility that Na_Xmay function as a voltage-modulated channel in specific cellular contexts. It is conceivable that binding of a putative auxiliary subunit or cofactor may impart some degree of voltage sensitivity onto Na_X, as recently described for the NALCN or TMEM16A channels.

Reconciling Differences Over Na⁺-Sensing

Murine Na_Xhas been reported to produce non-inactivating inward currents upon increasing the extracellular Na⁺ concentration >150 mM. We only observed qualitatively similar recordings with low seal resistance and failed to reconstitute Na⁺-activated currents upon heterologous expression of human Na_X, even when co-expressed with factors expected to stabilize it on the plasma membrane (i.e. SAP97) (FIGS. 1D and 7F). It is plausible that sequence divergence or cellular context differences between murine and human Na_Xproteins account for these seemingly conflicting functional outcomes (FIG. 9).

Our cryo-EM samples of β3-Na_Xwere prepared above the reported threshold required for Na⁺-dependent gating, in 200 mM NaCl^9-11. No clear Na⁺-sensing locus or activation-based mechanism was observed upon structural analyses, and the only map feature that we might assign as an extracellular Na⁺ ion is bound near the Na_Xselectivity filter to a site also found in Na_Vchannels (FIG. 17A)^29,49. We infer that the Na⁺-sensing mechanism described for Na_Xis most likely contributed by an exogenous protein factor or pathway, such as the Na⁺/K⁺ ATPase, the endothelin receptor, or a yet to be identified system. It is conceivable that post-translational modification of the predicted phosphorylation sites along the Na_XDIII-DIV linker (FIG. 14B) might help dislodge the IFI motif from its pore module receptor site to facilitate S6-dilation and channel gating. Alternatively, the presence of unusual but highly conserved histidine residues found within DIV and VSLD4 of Na_Xcould also raise speculation of a potential intracellular divalent cation- or pH-sensitive activation or modulation mechanism (FIGS. 6D and 9).

The Na_XHypothesis: A Non-Selective Na⁺ Leak Channel

Guided by structural analyses and reiterative protein-engineering experiments, we found that targeted mutation of three residues around the hydrophobic S6-gate region is sufficient to produce robust ionic currents through human Na_Xexpressed in Xenopus laevis oocytes and HEK293T cells (FIGS. 3, 4D, 4E and 5). Presumably, the introduction of three polar side-chains into the Na_X-QTT channel construct will hydrate the S6-gate, dislodge bound membrane lipids, and allow for pore dilation and ion conduction (FIGS. 2B-2D). This conceptual and technical breakthrough allowed us to characterize the basic properties of human Na_Xfor the first time.

Distinct from Na_Vchannels, Na_X-QTT effectively conducts monovalent cations as an Ohmic leak channel in the absence of extracellular Ca²⁺ ions (FIGS. 3, 4D, 4E). Ca²⁺ does not permeate but blocks Na_X-QTT, so in the presence of physiological levels of extracellular Ca²⁺, Na_X-QTT currents are outward-rectifying with only a small inward leak component at negative membrane potentials (FIG. 5A). This unique current phenotype has pharmacological sensitivities expected for a Na_V-like channel, including apparent selectivity filter block by TTX, Zn²⁺, Co²⁺ and Gd³⁺, and presumed pore block by lidocaine, quinidine and loperamide (FIGS. 5B and 5C). Flecainide, ranolazine or phenytoin did not produce strong inhibition of Na_X-QTT currents (FIG. 5C), indicating a distinctive pharmacological profile of human Na_Xrelative to canonical Na_Vchannels, and possibly reflecting unique central cavity-lining residues like DIV-Trp1484 (FIG. 13F), or the potential absence of an inactivated state in Na_X(FIGS. 3B and 4D). Although Na_X-QTT currents were non-inactivating and not modulated by voltage, future studies will be required to define these important characteristics in wild-type human Na_Xchannels because the pore-wetting QTT-mutations are targeted to the S6-gate region. Nevertheless, it is noteworthy that an earlier study had suggested Na_Xdoes function as a leak channel in vivo.

The physical, chemical and electrostatic profile of the Na_Xselectivity filter structure meets expectations of a cation-selective channel (FIGS. 4A and 4B), but it lacks the DIII-lysine side-chain that imparts Na⁺-selectivity in Na_Vchannels (FIG. 9). Indeed, our electrophysiological characterization of Na_X-QTT revealed no selectivity among monovalent cations examined (Na⁺, K⁺, Cs⁺ and Li⁺) (FIG. 4E). Therefore, we hypothesize that human Na_Xfunctions as Na⁺-leak channel which is sensitive to modulation by extracellular Ca²⁺ under physiological conditions (FIG. 5A). Notably, Ca²⁺ block of Na_X-QTT is ˜10-fold more potent than Ca²⁺ block on Na_Vchannels (FIG. 5A), but similar to the Ca²⁺ block sensitivity of the distantly related non-selective NALCN Na⁺-leak channel, perhaps reflecting a shared control mechanism to avoid excessive depolarizing Na⁺ influx into cells.

Visualizing Pore-Bound Membrane Lipids

A striking feature of our Na_Xchannel structures determined in detergent or lipid nanodiscs was the visualization of a non-conductive state where lipids completely occlude the ion conduction pathway and seal the S6-gate (FIGS. 2C, 2D and 14F). The phospholipid which plugs the S6-gate is reminiscent of cholesterol-like molecules bound at the S6-gate in Na_Vchannel structures^28,29,31(FIG. 17B). Occupancy of these hydrophobic ligands at the S6-gate appears to correlate with the “up” conformation of a conserved DIV-S6 tyrosine side-chain that has been classically implicated in drug block in Na_Vchannels (FIGS. 9 and 17B)^23,31-33. Notably, drug binding within the central cavity can enforce a “down” conformation of this conserved S6 tyrosine^30,31, concurrent with displacement of the lipid-like density at the S6-gate (FIG. 17B)³¹. Our Na_Xstructures therefore support an emerging paradigm whereby membrane lipids can seal the S6-gate, and that lipid occupancy may modulate ion conductance, drug binding and pore structure. This developing model strongly contrasts with the long-held view that the S6-gate must close tightly and completely shut in Na_Vchannels³⁰.

Summary and Outlook

The basic physiological and pharmacological characteristics of the Na_Xchannel have remained mysterious since its cloning nearly three decades ago. We find that human Na_Xdoes not function independently as a Na⁺-activated channel, nor as a voltage-activated channel, under the conditions that we have examined. Through structure-guided protein engineering efforts, we uncovered an approach to characterize a human Na_Xchannel variant using traditional heterologous expression systems. The Na_X-QTT channel construct displayed a distinctive but Na_Vchannel-like pharmacological profile, including potent inhibition by Ca²⁺ and TTX, and revealed a non-selective cationic conductance suggesting that Na_Xchannel may operate as a Ca²⁺-modulated, Na⁺ leak channel in vivo. Overall, this study provides essential tools to further interrogate the physiology and pharmacology of Na_X, and greatly advances our understanding of this atypical ion channel.

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SEQUENCE TABLE

The following table further describes certain sequences referenced herein.

TABLE 5

Sequences

SEQ

ID NO.
Identity
Sequence

1
Wild-type human
MLASPEPKGLVPFTKESFELIKQHIAKTHNEDHEEEDLKPTPDLEVGKKLPF

Nax (hNax)
IYGNLSQGMVSEPLEDVDPYYYKKKNTFIVLNKNRTIFRFNAASILCTLSPF

(Uniprot Q01118-
NCIRRTTIKVLVHPFFQLFILISVLIDCVEMSLTNLPKWRPVLENTLLGIYT

1)
FEILVKLFARGVWAGSFSFLGDPWNWLDESVTVFEVIIRYSPLDFIPTLQTA

RTLRILKIIPLNQGLKSLVGVLIHCLKQLIGVIILTLFFLSIFSLIGMGLFM

GNLKHKCFRWPQENENETLHNRTGNPYYIRETENFYYLEGERYALLCGNRTD

AGQCPEGYVCVKAGINPDQGFTNEDSFGWALFALFRLMAQDYPEVLYHQILY

ASGKVYMIFFVVVSFLFSFYMASLFLGILAMAYEEEKQRVGEISKKIEPKFQ

QTGKELQEGNETDEAKTIQIEMKKRSPISTDTSLDVLEDATLRHKEELEKSK

KICPLYWYKFAKTFLIWNCSPCWLKLKEFVHRIIMAPFTDLFLIICIILNVC

FLTLEHYPMSKQTNTLLNIGNLVFIGIFTAEMIFKIIAMHPYGYFQVGWNIF

DSMIVFHGLIELCLANVAGMALLRLFRMLRIFKLGKYWPTFQILMWSLSNSW

VALKDLVLLLFTFIFFSAAFGMKLFGKNYEEFVCHIDKDCQLPRWHMHDFFH

SFLNVERILCGEWVETLWDCMEVAGQSWCIPFYLMVILIGNLLVLYLFLALV

SSFSSCKDVTAEENNEAKNLQLAVARIKKGINYVLLKILCKTQNVPKDTMDH

VNEVYVKEDISDHTLSELSNTQDFLKDKEKSSGTEKNATENESQSLIPSPSV

SETVPIASGESDIENLDNKEIQSKSGDGGSKEKIKQSSSSECSTVDIAISEE

EEMFYGGERSKHLKNGCRRGSSLGQISGASKKGKIWQNIRKTCCKIVENNWF

KCFIGLVTLLSTGTLAFEDIYMDQRKTIKILLEYADMIFTYIFILEMLLKWM

AYGFKAYFSNGWYRLDFVVVIVFCLSLIGKTREELKPLISMKFLRPLRVLSQ

FERMKVVVRALIKTTLPTLNVFLVCLMIWLIFSIMGVDLFAGRFYECIDPTS

GERFPSSEVMNKSRCESLLFNESMLWENAKMNFDNVGNGELSLLQVATENGW

ITIMNSAIDSVAVNIQPHFEVNIYMYCYFINFIIFGVFLPLSMLITVIIDNF

NKHKIKLGGSNIFITVKQRKQYRRLKKLMYEDSQRPVPRPLNKLQGFIFDVV

TSQAFNVIVMVLICFQAIAMMIDTDVQSLQMSIALYWINSIFVMLYTMECIL

KLIAFRCFYFTIAWNIFDFMVVIFSITGLCLPMTVGSYLVPPSLVQLILLSR

IIHMLRLGKGPKVFHNLMLPLMLSLPALLNIILLIFLVMFIYAVFGMYNFAY

VKKEAGINDVSNFETFGNSMLCLFQVAIFAGWDGMLDAIFNSKWSDCDPDKI

NPGTQVRGDCGNPSVGIFYFVSYILISWLIIVNMYIVVVMEFLNIASKKKNK

TLSEDDFRKFFQVWKRFDPDRTQYIDSSKLSDFAAALDPPLFMAKPNKGQLI

ALDLPMAVGDRIHCLDILLAFTKRVMGQDVRMEKVVSEIESGELLANPFKIT

CEPITTTLKRKQEAVSATIIQRAYKNYRLRRNDKNTSDIHMIDGDRDVHATK

EGAYFDKAKEKSPIQSQI

2
hNax_hNav1.7_DI
MAMLPPPGPQSFVHFTKQSLALIEQRIAERKSKEPKEEKKDDDEEAPKPSSD

-DII_chimera
LEAGKQLPFIYGDIPPGMVSEPLEDLDPYYADKKTFIVLNKGKTIFRFNATP

ALYMLSPFSPLRRISIKILVHSLESMLIMCTILTNCIFMTMNNPPDWTKNVE

YTFTGIYTFESLVKILARGFCVGEFTFLRDPWNWLDFVVIVFAYLTEFVNLG

NVSALRTFRVLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVFCLSVFA

LIGLQLFMGNLKHKCFRNSLENNETLESIMNTLESEEDFRKYFYYLEGSKDA

LLCGFSTDSGQCPEGYTCVKIGRNPDYGYTSFDTFSWAFLALFRLMTQDYWE

NLYQQTLRAAGKTYMIFFVVVIFLGSFYLINLILAVVAMAYEEQNQANIEEA

KQKELEFQQMLDRLKKEQEEAEAIAAAAAEYTSIRRSRIMGLSESSSETSKL

SSKSAKERRNRRKKKNQKKLSSGEEKGDAEKLSKSESEDSIRRKSFHLGVEG

HRRAHEKRLSTPNQSPLSIRGSLFSARRSSRTSLFSFKGRGRDIGSETEFAD

DEHSIFGDNESRRGSLFVPHRPQERRSSNISQASRSPPMLPVNGKMHSAVDC

NGVVSLVDGRSALMLPNGQLLPEVIIDKATSDDSGTTNQIHKKRRCSSYLLS

EDMLNDPNLRQRAMSRASILTNTVEELEESRQKCPPWWYRFAHKFLIWNCSP

YWIKFKKCIYFIVMDPFVDLAITICIVLNTLFMAMEHHPMTEEFKNVLAIGN

LVFTGIFAAEMVLKLIAMDPYEYFQVGWNIFDSLIVTLSLVELFLADVEGLS

VLRSFRLLRVFKLAKSWPTLNMLIKIIGNSVGALGNLTLVLAIIVFIFAVVG

MQLFGKSYKECVCKINDDCTLPRWHMNDFFHSFLIVFRVLCGEWIETMWDCM

EVAGQAMCLIVYMMVMVIGNLVVLNLFLALLLSSFSSDNLTAIEEDPDANNL

QIAVTRIKKGINYVKQTLREFILKAFSKKPKISREIRQAEDLNTKKENYISN

HTLAEMSKGHNFLKEKDKISGFGSSVDKHLMEDSDGQSFIHNPSLTVTVPIA

PGESDLENMNAEELSSDSDSEYSKVRLNRSSSSECSTVDNPLPGEGEEAEAE

PMNSDEPEACFTDGCVWRFSCCQVNIESGKGKIWQNIRKTCCKIVENNWFKC

FIGLVTLLSTGTLAFEDIYMDQRKTIKILLEYADMIFTYIFILEMLLKWMAY

GFKAYFSNGWYRLDFVVVIVFCLSLIGKTREELKPLISMKFLRPLRVLSQFE

RMKVVVRALIKTTLPTLNVFLVCLMIWLIFSIMGVDLFAGRFYECIDPTSGE

RFPSSEVMNKSRCESLLFNESMLWENAKMNFDNVGNGFLSLLQVATENGWIT

IMNSAIDSVAVNIQPHFEVNIYMYCYFINFIIFGVFLPLSMLITVIIDNENK

HKIKLGGSNIFITVKQRKQYRRLKKLMYEDSQRPVPRPLNKLQGFIFDVVTS

QAFNVIVMVLICFQAIAMMIDTDVQSLQMSIALYWINSIFVMLYTMECILKL

IAFRCFYFTIAWNIFDFMVVIFSITGLCLPMTVGSYLVPPSLVQLILLSRII

HMLRLGKGPKVFHNLMLPLMLSLPALLNIILLIFLVMFIYAVFGMYNFAYVK

KEAGINDVSNFETFGNSMLCLFQVAIFAGWDGMLDAIFNSKWSDCDPDKINP

GTQVRGDCGNPSVGIFYFVSYILISWLIIVNMYIVVVMEFLNIASKKKNKTL

SEDDFRKFFQVWKRFDPDRTQYIDSSKLSDFAAALDPPLFMAKPNKGQLIAL

DLPMAVGDRIHCLDILLAFTKRVMGQDVRMEKVVSEIESGELLANPFKITCE

PITTTLKRKQEAVSATIIQRAYKNYRLRRNDKNTSDIHMIDGDRDVHATKEG

AYFDKAKEKSPIQSQI

3
hNax_hNav1.7_DI
MLASPEPKGLVPFTKESFELIKQHIAKTHNEDHEEEDLKPTPDLEVGKKLPF

II-DIV_chimera
NCIRRTTIKVLVHPFFQLFILISVLIDCVEMSLTNLPKWRPVLENTLLGIYT

IYGNLSQGMVSEPLEDVDPYYYKKKNTFIVLNKNRTIFRENAASILCTLSPF

FEILVKLFARGVWAGSFSFLGDPWNWLDFSVTVFEVIIRYSPLDFIPTLQTA

RTLRILKIIPLNQGLKSLVGVLIHCLKQLIGVIILTLFFLSIFSLIGMGLFM

GNLKHKCFRWPQENENETLHNRTGNPYYIRETENFYYLEGERYALLCGNRTD

AGQCPEGYVCVKAGINPDQGFTNFDSFGWALFALFRLMAQDYPEVLYHQILY

ASGKVYMIFFVVVSFLESFYMASLFLGILAMAYEEEKQRVGEISKKIEPKFQ

QTGKELQEGNETDEAKTIQIEMKKRSPISTDTSLDVLEDATLRHKEELEKSK

KICPLYWYKFAKTFLIWNCSPCWLKLKEFVHRIIMAPFTDLFLIICIILNVC

FLTLEHYPMSKQTNTLLNIGNLVFIGIFTAEMIFKIIAMHPYGYFQVGWNIF

DSMIVFHGLIELCLANVAGMALLRLFRMLRIFKLGKYWPTFQILMWSLSNSW

VALKDLVLLLFTFIFFSAAFGMKLFGKNYEEFVCHIDKDCQLPRWHMHDFFH

SFLNVFRILCGEWVETLWDCMEVAGQSWCIPFYLMVILIGNLLVLYLFLALV

SSFSSCKDVTAEENNEAKNLQLAVARIKKGINYVLLKILCKTQNVPKDTMDH

VNEVYVKEDISDHTLSELSNTQDELKDKEKSSGTEKNATENESQSLIPSPSV

SETVPIASGESDIENLDNKEIQSKSGDGGSKEKIKQSSSSECSTVDIAISEE

EEMFYGGERSKHLKNGCRRGSSLGQISGASKKGKIWWNIRKTCYKIVEHSWF

ESFIVLMILLSSGALAFEDIYIERKKTIKIILEYADKIFTYIFILEMLLKWI

AYGYKTYFTNAWCWLDFLIVDVSLVTLVANTLGYSDLGPIKSLRTLRALRPL

RALSRFEGMRVVVNALIGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGKFYEC

INTTDGSRFPASQVPNRSECFALMNVSQNVRWKNLKVNFDNVGLGYLSLLQV

ATFKGWTIIMYAAVDSVNVDKQPKYEYSLYMYIYFVVFIIFGSFFTLNLFIG

VIIDNFNQQKKKLGGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPGNKIQG

CIFDLVTNQAFDISIMVLICLNMVTMMVEKEGQSQHMTEVLYWINVVFIILF

TGECVLKLISLRHYYFTVGWNIFDFVVVIISIVGMFLADLIETYFVSPTLER

VIRLARIGRILRLVKGAKGIRTLLFALMMSLPALFNIGLLLFLVMFIYAIFG

MSNFAYVKKEDGINDMFNFETFGNSMICLFQITTSAGWDGLLAPILNSKPPD

CDPKKVHPGSSVEGDCGNPSVGIFYFVSYIIISFLVVVNMYIAVILENFSVA

TEESTEPLSEDDFEMFYEVWEKFDPDATQFIEFSKLSDFAAALDPPLLIAKP

NKVQLIAMDLPMVSGDRIHCLDILFAFTKRVLGESGEMDSLRSQMEERFMSA

NPSKVSYEPITTTLKRKQEDVSATVIQRAYRRYRLRQNVKNISSIYIKDGDR

DDDLLNKKDMAFDNVNENSSPEKTDATSSTTSPPSYDSVTKPDKEKYEQDRT

EKEDKGKDSKESKK

4
hNax_hNav1.7_DI
MLASPEPKGLVPFTKESFELIKQHIAKTHNEDHEEEDLKPTPDLEVGKKLPF

I-DIII_chimera
IYGNLSQGMVSEPLEDVDPYYYKKKNTFIVLNKNRTIFRFNAASILCTLSPF

NCIRRTTIKVLVHPFFQLFILISVLIDCVEMSLTNLPKWRPVLENTLLGIYT

FEILVKLFARGVWAGSFSFLGDPWNWLDFSVTVFEVIIRYSPLDFIPTLQTA

RTLRILKIIPLNQGLKSLVGVLIHCLKQLIGVIILTLFFLSIFSLIGMGLFM

GNLKHKCFRWPQENENETLHNRTGNPYYIRETENFYYLEGERYALLCGNRTD

AGQCPEGYVCVKAGINPDQGFTNEDSFGWALFALFRLMAQDYPEVLYHQILY

ASGKVYMIFFVVVSFLFSFYMASLFLGILAMAYEEEKQRVGEISKKIEPKFQ

QTGKELQEGNETDEAKTIQIEMKKRSPISTDTSLDVLEDATLRHKEELEKSK

KICPLYWYKFAKTFLIWNCSPYWIKFKKCIYFIVMDPFVDLAITICIVLNTL

FMAMEHHPMTEEFKNVLAIGNLVFTGIFAAEMVLKLIAMDPYEYFQVGWNIE

DSLIVTLSLVELFLADVEGLSVLRSFRLLRVFKLAKSWPTLNMLIKIIGNSV

GALGNLTLVLAIIVFIFAVVGMQLFGKSYKECVCKINDDCTLPRWHMNDFFH

SFLIVFRVLCGEWIETMWDCMEVAGQAMCLIVYMMVMVIGNLVVLNLFLALL

LSSFSSDNLTAIEEDPDANNLQIAVTRIKKGINYVKQTLREFILKAFSKKPK

ISREIRQAEDLNTKKENYISNHTLAEMSKGHNFLKEKDKISGFGSSVDKHLM

EDSDGQSFIHNPSLTVTVPIAPGESDLENMNAEELSSDSDSEYSKVRLNRSS

SSECSTVDNPLPGEGEEAEAEPMNSDEPEACFTDGCVWRFSCCQVNIESGKG

KIWWNIRKTCYKIVEHSWFESFIVLMILLSSGALAFEDIYIERKKTIKIILE

YADKIFTYIFILEMLLKWIAYGYKTYFTNAWCWLDFLIVDVSLVTLVANTLG

YSDLGPIKSLRTLRALRPLRALSRFEGMRVVVNALIGAIPSIMNVLLVCLIF

WLIFSIMGVNLFAGKFYECINTTDGSRFPASQVPNRSECFALMNVSQNVRWK

NLKVNFDNVGLGYLSLLQVATFKGWTIIMYAAVDSVNVDKQPKYEYSLYMYI

YFVVFIIFGSFFTLNLFIGVIIDNFNQQKKKLGGQDIFMTEEQKKYYNAMKK

LGSKKPQKPIPRPGNKLQGFIFDVVTSQAFNVIVMVLICFQAIAMMIDTDVQ

SLQMSIALYWINSIFVMLYTMECILKLIAFRCFYFTIAWNIFDFMVVIFSIT

GLCLPMTVGSYLVPPSLVQLILLSRIIHMLRLGKGPKVFHNLMLPLMLSLPA

LLNIILLIFLVMFIYAVFGMYNFAYVKKEAGINDVSNFETFGNSMLCLFQVA

IFAGWDGMLDAIFNSKWSDCDPDKINPGTQVRGDCGNPSVGIFYFVSYILIS

WLIIVNMYIVVVMEFLNIASKKKNKTLSEDDFRKFFQVWKRFDPDRTQYIDS

SKLSDFAAALDPPLFMAKPNKGQLIALDLPMAVGDRIHCLDILLAFTKRVMG

QDVRMEKVVSEIESGFLLANPFKITCEPITTTLKRKQEAVSATIIQRAYKNY

RLRRNDKNTSDIHMIDGDRDVHATKEGAYFDKAKEKSPIQSQI

5
hNax_hNav1.7_DI
MAMLPPPGPQSFVHFTKQSLALIEQRIAERKSKEPKEEKKDDDEEAPKPSSD

-DIV_chimera
LEAGKQLPFIYGDIPPGMVSEPLEDLDPYYADKKTFIVLNKGKTIFRENATP

ALYMLSPFSPLRRISIKILVHSLFSMLIMCTILTNCIFMTMNNPPDWTKNVE

YTFTGIYTFESLVKILARGFCVGEFTFLRDPWNWLDFVVIVFAYLTEFVNLG

NVSALRTFRVLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVFCLSVFA

LIGLQLFMGNLKHKCFRNSLENNETLESIMNTLESEEDFRKYFYYLEGSKDA

LLCGFSTDSGQCPEGYTCVKIGRNPDYGYTSFDTFSWAFLALFRLMTQDYWE

NLYQQTLRAAGKTYMIFFVVVIFLGSFYLINLILAVVAMAYEEQNQANIEEA

KQKELEFQQMLDRLKKEQEEAEAIAAAAAEYTSIRRSRIMGLSESSSETSKL

SSKSAKERRNRRKKKNQKKLSSGEEKGDAEKLSKSESEDSIRRKSFHLGVEG

HRRAHEKRLSTPNQSPLSIRGSLFSARRSSRTSLFSFKGRGRDIGSETEFAD

DEHSIFGDNESRRGSLFVPHRPQERRSSNISQASRSPPMLPVNGKMHSAVDC

NGVVSLVDGRSALMLPNGQLLPEVIIDKATSDDSGTTNQIHKKRRCSSYLLS

EDMLNDPNLRQRAMSRASILTNTVEELEESRQKCPPWWYRFAHKFLIWNCSP

CWLKLKEFVHRIIMAPFTDLFLIICIILNVCFLTLEHYPMSKQTNTLLNIGN

LVFIGIFTAEMIFKIIAMHPYGYFQVGWNIFDSMIVFHGLIELCLANVAGMA

LLRLFRMLRIFKLGKYWPTFQILMWSLSNSWVALKDLVLLLFTFIFFSAAFG

MKLFGKNYEEFVCHIDKDCQLPRWHMHDFFHSFLNVFRILCGEWVETLWDCM

EVAGQSWCIPFYLMVILIGNLLVLYLFLALVSSFSSCKDVTAEENNEAKNLQ

LAVARIKKGINYVLLKILCKTQNVPKDTMDHVNEVYVKEDISDHTLSELSNT

QDFLKDKEKSSGTEKNATENESQSLIPSPSVSETVPIASGESDIENLDNKEI

QSKSGDGGSKEKIKQSSSSECSTVDIAISEEEEMFYGGERSKHLKNGCRRGS

SLGQISGASKKGKIWQNIRKTCCKIVENNWFKCFIGLVTLLSTGTLAFEDIY

MDQRKTIKILLEYADMIFTYIFILEMLLKWMAYGFKAYFSNGWYRLDFVVVI

VFCLSLIGKTREELKPLISMKFLRPLRVLSQFERMKVVVRALIKTTLPTLNV

FLVCLMIWLIFSIMGVDLFAGRFYECIDPTSGERFPSSEVMNKSRCESLLEN

ESMLWENAKMNFDNVGNGFLSLLQVATENGWITIMNSAIDSVAVNIQPHFEV

NIYMYCYFINFIIFGVFLPLSMLITVIIDNENKHKIKLGGSNIFITVKQRKQ

YRRLKKLMYEDSQRPVPRPLNKIQGCIFDLVTNQAFDISIMVLICLNMVTMM

VEKEGQSQHMTEVLYWINVVFIILFTGECVLKLISLRHYYFTVGWNIFDFVV

VIISIVGMFLADLIETYFVSPTLERVIRLARIGRILRLVKGAKGIRTLLFAL

MMSLPALFNIGLLLFLVMFIYAIFGMSNFAYVKKEDGINDMENFETFGNSMI

CLFQITTSAGWDGLLAPILNSKPPDCDPKKVHPGSSVEGDCGNPSVGIFYFV

SYIIISFLVVVNMYIAVILENFSVATEESTEPLSEDDFEMFYEVWEKFDPDA

TQFIEFSKLSDFAAALDPPLLIAKPNKVQLIAMDLPMVSGDRIHCLDILFAF

TKRVLGESGEMDSLRSQMEERFMSANPSKVSYEPITTTLKRKQEDVSATVIQ

RAYRRYRLRQNVKNISSIYIKDGDRDDDLLNKKDMAFDNVNENSSPEKTDAT

SSTTSPPSYDSVTKPDKEKYEQDRTEKEDKGKDSKESKK

6
hNax-
MAMLPPPGPQSFVHFTKQSLALIEQRIAERKSKEPKEEKKDDDEEAPKPSSD

hNav1.7_DI_
LEAGKQLPFIYGDIPPGMVSEPLEDLDPYYADKKTFIVLNKGKTIFRENATP

chimera
ALYMLSPFSPLRRISIKILVHSLFSMLIMCTILTNCIFMTMNNPPDWTKNVE

YTFTGIYTFESLVKILARGFCVGEFTFLRDPWNWLDFVVIVFAYLTEFVNLG

NVSALRTFRVLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVFCLSVFA

LIGLQLFMGNLKHKCFRNSLENNETLESIMNTLESEEDFRKYFYYLEGSKDA

LLCGFSTDSGQCPEGYTCVKIGRNPDYGYTSFDTFSWAFLALFRLMTQDYWE

NLYQQTLRAAGKTYMIFFVVVIFLGSFYLINLILAVVAMAYEEQNQANIEEA

KQKELEFQQMLDRLKKEQEEAEAIAAAAAEYTSIRRSRIMGLSESSSETSKL

SSKSAKERRNRRKKKNQKKLSSGEEKGDAEKLSKSESEDSIRRKSFHLGVEG

HRRAHEKRLSTPNQSPLSIRGSLFSARRSSRTSLFSFKGRGRDIGSETEFAD

DEHSIFGDNESRRGSLFVPHRPQERRSSNISQASRSPPMLPVNGKMHSAVDC

NGVVSLVDGRSALMLPNGQLLPEVIIDKATSDDSGTTNQIHKKRRCSSYLLS

EDMLNDPNLRQRAMSRASILTNTVEELEESRQKCPPWWYRFAHKFLIWNCSP

CWLKLKEFVHRIIMAPFTDLFLIICIILNVCFLTLEHYPMSKQTNTLLNIGN

LVFIGIFTAEMIFKIIAMHPYGYFQVGWNIFDSMIVFHGLIELCLANVAGMA

LLRLFRMLRIFKLGKYWPTFQILMWSLSNSWVALKDLVLLLFTFIFFSAAFG

MKLFGKNYEEFVCHIDKDCQLPRWHMHDFFHSELNVFRILCGEWVETLWDCM

EVAGQSWCIPFYLMVILIGNLLVLYLFLALVSSFSSCKDVTAEENNEAKNLQ

LAVARIKKGINYVLLKILCKTQNVPKDTMDHVNEVYVKEDISDHTLSELSNT

QDFLKDKEKSSGTEKNATENESQSLIPSPSVSETVPIASGESDIENLDNKEI

QSKSGDGGSKEKIKQSSSSECSTVDIAISEEEEMFYGGERSKHLKNGCRRGS

SLGQISGASKKGKIWQNIRKTCCKIVENNWFKCFIGLVTLLSTGTLAFEDIY

MDQRKTIKILLEYADMIFTYIFILEMLLKWMAYGFKAYFSNGWYRLDFVVVI

VFCLSLIGKTREELKPLISMKFLRPLRVLSQFERMKVVVRALIKTTLPTLNV

FLVCLMIWLIFSIMGVDLFAGRFYECIDPTSGERFPSSEVMNKSRCESLLEN

ESMLWENAKMNFDNVGNGFLSLLQVATENGWITIMNSAIDSVAVNIQPHFEV

NIYMYCYFINFIIFGVFLPLSMLITVIIDNFNKHKIKLGGSNIFITVKORKQ

YRRLKKLMYEDSQRPVPRPLNKLQGFIFDVVTSQAFNVIVMVLICFQAIAMM

IDTDVQSLQMSIALYWINSIFVMLYTMECILKLIAFRCFYFTIAWNIFDFMV

VIFSITGLCLPMTVGSYLVPPSLVQLILLSRIIHMLRLGKGPKVFHNLMLPL

MLSLPALLNIILLIFLVMFIYAVFGMYNFAYVKKEAGINDVSNFETFGNSML

CLFQVAIFAGWDGMLDAIFNSKWSDCDPDKINPGTQVRGDCGNPSVGIFYFV

SYILISWLIIVNMYIVVVMEFLNIASKKKNKTLSEDDFRKFFQVWKREDPDR

TQYIDSSKLSDFAAALDPPLFMAKPNKGQLIALDLPMAVGDRIHCLDILLAF

TKRVMGQDVRMEKVVSEIESGELLANPFKITCEPITTTLKRKQEAVSATIIQ

RAYKNYRLRRNDKNTSDIHMIDGDRDVHATKEGAYFDKAKEKSPIQSQI

7
hNax-
MLASPEPKGLVPFTKESFELIKQHIAKTHNEDHEEEDLKPTPDLEVGKKLPF

hNav1.7_DII_
IYGNLSQGMVSEPLEDVDPYYYKKKNTFIVLNKNRTIFRFNAASILCTLSPF

chimera
NCIRRTTIKVLVHPFFQLFILISVLIDCVFMSLTNLPKWRPVLENTLLGIYT

FEILVKLFARGVWAGSFSFLGDPWNWLDFSVTVFEVIIRYSPLDFIPTLQTA

RTLRILKIIPLNQGLKSLVGVLIHCLKOLIGVIILTLFFLSIFSLIGMGLFM

GNLKHKCFRWPQENENETLHNRTGNPYYIRETENFYYLEGERYALLCGNRTD

AGQCPEGYVCVKAGINPDQGFTNEDSFGWALFALFRLMAQDYPEVLYHQILY

ASGKVYMIFFVVVSFLFSFYMASLFLGILAMAYEEEKORVGEISKKIEPKFQ

QTGKELQEGNETDEAKTIQIEMKKRSPISTDTSLDVLEDATLRHKEELEKSK

KICPLYWYKFAKTFLIWNCSPYWIKFKKCIYFIVMDPFVDLAITICIVLNTL

FMAMEHHPMTEEFKNVLAIGNLVFTGIFAAEMVLKLIAMDPYEYFQVGWNIF

DSLIVTLSLVELFLADVEGLSVLRSFRLLRVFKLAKSWPTLNMLIKIIGNSV

GALGNLTLVLAIIVFIFAVVGMQLFGKSYKECVCKINDDCTLPRWHMNDFFH

SFLIVFRVLCGEWIETMWDCMEVAGQAMCLIVYMMVMVIGNLVVLNLFLALL

LSSFSSDNLTAIEEDPDANNLQIAVTRIKKGINYVKOTLREFILKAFSKKPK

ISREIRQAEDLNTKKENYISNHTLAEMSKGHNFLKEKDKISGFGSSVDKHLM

EDSDGQSFIHNPSLTVTVPIAPGESDLENMNAEELSSDSDSEYSKVRLNRSS

SSECSTVDNPLPGEGEEAEAEPMNSDEPEACFTDGCVWRFSCCQVNIESGKG

KIWQNIRKTCCKIVENNWFKCFIGLVTLLSTGTLAFEDIYMDORKTIKILLE

YADMIFTYIFILEMLLKWMAYGFKAYFSNGWYRLDFVVVIVFCLSLIGKTRE

ELKPLISMKFLRPLRVLSQFERMKVVVRALIKTTLPTLNVFLVCLMIWLIES

IMGVDLFAGRFYECIDPTSGERFPSSEVMNKSRCESLLFNESMLWENAKMNF

DNVGNGFLSLLQVATFNGWITIMNSAIDSVAVNIQPHFEVNIYMYCYFINFI

IFGVFLPLSMLITVIIDNFNKHKIKLGGSNIFITVKORKQYRRLKKLMYEDS

QRPVPRPLNKLQGFIFDVVTSQAFNVIVMVLICFQAIAMMIDTDVOSLOMSI

ALYWINSIFVMLYTMECILKLIAFRCFYFTIAWNIFDFMVVIFSITGLCLPM

TVGSYLVPPSLVQLILLSRIIHMLRLGKGPKVFHNLMLPLMLSLPALLNIIL

LIFLVMFIYAVFGMYNFAYVKKEAGINDVSNFETFGNSMLCLFQVAIFAGWD

GMLDAIFNSKWSDCDPDKINPGTQVRGDCGNPSVGIFYFVSYILISWLIIVN

MYIVVVMEFLNIASKKKNKTLSEDDFRKFFQVWKRFDPDRTQYIDSSKLSDF

AAALDPPLFMAKPNKGQLIALDLPMAVGDRIHCLDILLAFTKRVMGQDVRME

KVVSEIESGFLLANPFKITCEPITTTLKRKQEAVSATIIQRAYKNYRLRRND

KNTSDIHMIDGDRDVHATKEGAYFDKAKEKSPIQSQI

8
hNax-
MLASPEPKGLVPFTKESFELIKQHIAKTHNEDHEEEDLKPTPDLEVGKKLPF

hNav1.7_DIII_
IYGNLSQGMVSEPLEDVDPYYYKKKNTFIVLNKNRTIFRFNAASILCTLSPF

chimera
NCIRRTTIKVLVHPFFQLFILISVLIDCVFMSLTNLPKWRPVLENTLLGIYT

FEILVKLFARGVWAGSFSFLGDPWNWLDFSVTVFEVIIRYSPLDFIPTLQTA

RTLRILKIIPLNQGLKSLVGVLIHCLKQLIGVIILTLFFLSIFSLIGMGLFM

GNLKHKCFRWPQENENETLHNRTGNPYYIRETENFYYLEGERYALLCGNRTD

AGQCPEGYVCVKAGINPDQGFTNFDSFGWALFALFRLMAQDYPEVLYHQILY

ASGKVYMIFFVVVSFLESFYMASLFLGILAMAYEEEKQRVGEISKKIEPKFQ

QTGKELQEGNETDEAKTIQIEMKKRSPISTDTSLDVLEDATLRHKEELEKSK

KICPLYWYKFAKTFLIWNCSPCWLKLKEFVHRIIMAPFTDLFLIICIILNVC

FLTLEHYPMSKQTNTLLNIGNLVFIGIFTAEMIFKIIAMHPYGYFQVGWNIF

DSMIVFHGLIELCLANVAGMALLRLFRMLRIFKLGKYWPTFQILMWSLSNSW

VALKDLVLLLFTFIFFSAAFGMKLFGKNYEEFVCHIDKDCQLPRWHMHDFFH

SFLNVFRILCGEWVETLWDCMEVAGQSWCIPFYLMVILIGNLLVLYLFLALV

SSFSSCKDVTAEENNEAKNLQLAVARIKKGINYVLLKILCKTQNVPKDTMDH

VNEVYVKEDISDHTLSELSNTQDFLKDKEKSSGTEKNATENESQSLIPSPSV

SETVPIASGESDIENLDNKEIQSKSGDGGSKEKIKQSSSSECSTVDIAISEE

EEMFYGGERSKHLKNGCRRGSSLGQISGASKKGKIWWNIRKTCYKIVEHSWF

ESFIVLMILLSSGALAFEDIYIERKKTIKIILEYADKIFTYIFILEMLLKWI

AYGYKTYFTNAWCWLDFLIVDVSLVTLVANTLGYSDLGPIKSLRTLRALRPL

RALSRFEGMRVVVNALIGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGKFYEC

INTTDGSRFPASQVPNRSECFALMNVSQNVRWKNLKVNFDNVGLGYLSLLQV

ATFKGWTIIMYAAVDSVNVDKQPKYEYSLYMYIYFVVFIIFGSFFTLNLFIG

VIIDNFNQQKKKLGGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPGNKLQG

FIFDVVTSQAFNVIVMVLICFQAIAMMIDTDVQSLQMSIALYWINSIFVMLY

TMECILKLIAFRCFYFTIAWNIFDFMVVIFSITGLCLPMTVGSYLVPPSLVQ

LILLSRIIHMLRLGKGPKVFHNLMLPLMLSLPALLNIILLIFLVMFIYAVFG

MYNFAYVKKEAGINDVSNFETFGNSMLCLFQVAIFAGWDGMLDAIENSKWSD

CDPDKINPGTQVRGDCGNPSVGIFYFVSYILISWLIIVNMYIVVVMEFLNIA

SKKKNKTLSEDDFRKFFQVWKRFDPDRTQYIDSSKLSDFAAALDPPLFMAKP

NKGQLIALDLPMAVGDRIHCLDILLAFTKRVMGQDVRMEKVVSEIESGELLA

NPFKITCEPITTTLKRKQEAVSATIIQRAYKNYRLRRNDKNTSDIHMIDGDR

DVHATKEGAYFDKAKEKSPIQSQI

9
hNax-
MLASPEPKGLVPFTKESFELIKQHIAKTHNEDHEEEDLKPTPDLEVGKKLPF

hNav1.7_DIV-
IYGNLSQGMVSEPLEDVDPYYYKKKNTFIVLNKNRTIFRFNAASILCTLSPF

DIII-
NCIRRTTIKVLVHPFFQLFILISVLIDCVFMSLTNLPKWRPVLENTLLGIYT

DIVlinker_
FEILVKLFARGVWAGSFSFLGDPWNWLDFSVTVFEVIIRYSPLDFIPTLQTA

chimera
RTLRILKIIPLNQGLKSLVGVLIHCLKQLIGVIILTLFFLSIFSLIGMGLFM

GNLKHKCFRWPQENENETLHNRTGNPYYIRETENFYYLEGERYALLCGNRTD

AGQCPEGYVCVKAGINPDQGFTNEDSFGWALFALFRLMAQDYPEVLYHQILY

ASGKVYMIFFVVVSFLFSFYMASLFLGILAMAYEEEKQRVGEISKKIEPKFQ

QTGKELQEGNETDEAKTIQIEMKKRSPISTDTSLDVLEDATLRHKEELEKSK

KICPLYWYKFAKTFLIWNCSPCWLKLKEFVHRIIMAPFTDLFLIICIILNVC

FLTLEHYPMSKQTNTLLNIGNLVFIGIFTAEMIFKIIAMHPYGYFQVGWNIF

DSMIVFHGLIELCLANVAGMALLRLFRMLRIFKLGKYWPTFQILMWSLSNSW

VALKDLVLLLFTFIFFSAAFGMKLFGKNYEEFVCHIDKDCQLPRWHMHDFFH

SFLNVFRILCGEWVETLWDCMEVAGQSWCIPFYLMVILIGNLLVLYLFLALV

SSFSSCKDVTAEENNEAKNLQLAVARIKKGINYVLLKILCKTQNVPKDTMDH

VNEVYVKEDISDHTLSELSNTQDFLKDKEKSSGTEKNATENESQSLIPSPSV

SETVPIASGESDIENLDNKEIQSKSGDGGSKEKIKQSSSSECSTVDIAISEE

EEMFYGGERSKHLKNGCRRGSSLGQISGASKKGKIWQNIRKTCCKIVENNWE

KCFIGLVTLLSTGTLAFEDIYMDQRKTIKILLEYADMIFTYIFILEMLLKWM

AYGFKAYFSNGWYRLDFVVVIVFCLSLIGKTREELKPLISMKFLRPLRVLSQ

FERMKVVVRALIKTTLPTLNVFLVCLMIWLIFSIMGVDLFAGRFYECIDPTS

GERFPSSEVMNKSRCESLLENESMLWENAKMNFDNVGNGFLSLLQVATENGW

ITIMNSAIDSVAVNIQPHFEVNIYMYCYFINFIIFGVFLPLSMLITVIIDNF

NQQKKKLGGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPGNKIQGCIFDLV

TNQAFDISIMVLICLNMVTMMVEKEGQSQHMTEVLYWINVVFIILFTGECVL

KLISLRHYYFTVGWNIFDFVVVIISIVGMFLADLIETYFVSPTLFRVIRLAR

IGRILRLVKGAKGIRTLLFALMMSLPALFNIGLLLFLVMFIYAIFGMSNFAY

VKKEDGINDMFNFETFGNSMICLFQITTSAGWDGLLAPILNSKPPDCDPKKV

HPGSSVEGDCGNPSVGIFYFVSYIIISFLVVVNMYIAVILENFSVATEESTE

PLSEDDFEMFYEVWEKFDPDATQFIEFSKLSDFAAALDPPLLIAKPNKVQLI

AMDLPMVSGDRIHCLDILFAFTKRVLGESGEMDSLRSQMEERFMSANPSKVS

YEPITTTLKRKQEDVSATVIQRAYRRYRLRQNVKNISSIYIKDGDRDDDLLN

KKDMAFDNVNENSSPEKTDATSSTTSPPSYDSVTKPDKEKYEQDRTEKEDKG

KDSKESKK

10
hNax_hNav1.7_
MLASPEPKGLVPFTKESFELIKQHIAKTHNEDHEEEDLKPTPDLEVGKKLPF

DIII_Chimera_SF_
IYGNLSQGMVSEPLEDVDPYYYKKKNTFIVLNKNRTIFRFNAASILCTLSPF

chimera
NCIRRTTIKVLVHPFFQLFILISVLIDCVFMSLTNLPKWRPVLENTLLGIYT

FEILVKLFARGVWAGSFSFLGDPWNWLDFSVTVFEVIIRYSPLDFIPTLQTA

RTLRILKIIPLNQGLKSLVGVLIHCLKQLIGVIILTLFFLSIFSLIGMGLFM

GNLKHKCFRWPQENENETLHNRTGNPYYIRETENFYYLEGERYALLCGNRTD

AGQCPEGYVCVKAGINPDQGFTNFDSFSWAFLALFRLMTQDYWENLYQQTLR

ASGKVYMIFFVVVSFLESFYMASLFLGILAMAYEEEKQRVGEISKKIEPKFQ

QTGKELQEGNETDEAKTIQIEMKKRSPISTDTSLDVLEDATLRHKEELEKSK

KICPLYWYKFAKTFLIWNCSPCWLKLKEFVHRIIMAPFTDLFLIICIILNVC

FLTLEHYPMSKQTNTLLNIGNLVFIGIFTAEMIFKIIAMHPYGYFQVGWNIF

DSMIVFHGLIELCLANVAGMALLRLERMLRIFKLGKYWPTFQILMWSLSNSW

VALKDLVLLLFTFIFFSAAFGMKLFGKNYEEFVCHIDKDCQLPRWHMHDFFH

SFLIVFRVLCGEWIETMWDCMEVAGQSWCIPFYLMVILIGNLLVLYLFLALV

SSFSSCKDVTAEENNEAKNLQLAVARIKKGINYVLLKILCKTQNVPKDTMDH

VNEVYVKEDISDHTLSELSNTQDFLKDKEKSSGTEKNATENESQSLIPSPSV

SETVPIASGESDIENLDNKEIQSKSGDGGSKEKIKQSSSSECSTVDIAISEE

EEMFYGGERSKHLKNGCRRGSSLGQISGASKKGKIWWNIRKTCYKIVEHSWF

ESFIVLMILLSSGALAFEDIYIERKKTIKIILEYADKIFTYIFILEMLLKWI

AYGYKTYFTNAWCWLDFLIVDVSLVTLVANTLGYSDLGPIKSLRTLRALRPL

RALSRFEGMRVVVNALIGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGKFYEC

INTTDGSRFPASQVPNRSECFALMNVSQNVRWKNLKVNFDNVGLGYLSLLQV

ATFKGWTIIMYAAVDSVNVDKQPKYEYSLYMYIYFVVFIIFGSFFTLNLFIG

VIIDNFNQQKKKLGGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPGNKLQG

FIFDVVTSQAFNVIVMVLICFQAIAMMIDTDVQSLQMSIALYWINSIFVMLY

TMECILKLIAFRCFYFTIAWNIFDFMVVIFSITGLCLPMTVGSYLVPPSLVQ

LILLSRIIHMLRLGKGPKVFHNLMLPLMLSLPALLNIILLIFLVMFIYAVFG

MYNFAYVKKEAGINDVSNFETFGNSMICLFQITTSAGWDGLLAPILNSKWSD

CDPDKINPGTQVRGDCGNPSVGIFYFVSYILISWLIIVNMYIVVVMEFLNIA

SKKKNKTLSEDDFRKFFQVWKRFDPDRTQYIDSSKLSDFAAALDPPLFMAKP

NKGQLIALDLPMAVGDRIHCLDILLAFTKRVMGQDVRMEKVVSEIESGELLA

NPFKITCEPITTTLKRKQEAVSATIIQRAYKNYRLRRNDKNTSDIHMIDGDR

DVHATKEGAYFDKAKEKSPIQSQI

11
hNax_hNav1.7_D3
MLASPEPKGLVPFTKESFELIKQHIAKTHNEDHEEEDLKPTPDLEVGKKLPF

-D4link_chimera
IYGNLSQGMVSEPLEDVDPYYYKKKNTFIVLNKNRTIFRFNAASILCTLSPF

NCIRRTTIKVLVHPFFQLFILISVLIDCVEMSLTNLPKWRPVLENTLLGIYT

FEILVKLFARGVWAGSFSFLGDPWNWLDESVTVFEVIIRYSPLDFIPTLQTA

RTLRILKIIPLNQGLKSLVGVLIHCLKQLIGVIILTLFFLSIFSLIGMGLFM

GNLKHKCFRWPQENENETLHNRTGNPYYIRETENFYYLEGERYALLCGNRTD

AGQCPEGYVCVKAGINPDQGFTNEDSFGWALFALFRLMAQDYPEVLYHQILY

ASGKVYMIFFVVVSFLFSFYMASLFLGILAMAYEEEKQRVGEISKKIEPKFQ

QTGKELQEGNETDEAKTIQIEMKKRSPISTDTSLDVLEDATLRHKEELEKSK

KICPLYWYKFAKTFLIWNCSPCWLKLKEFVHRIIMAPFTDLFLIICIILNVC

FLTLEHYPMSKQTNTLLNIGNLVFIGIFTAEMIFKIIAMHPYGYFQVGWNIF

DSMIVFHGLIELCLANVAGMALLRLERMLRIFKLGKYWPTFQILMWSLSNSW

VALKDLVLLLFTFIFFSAAFGMKLFGKNYEEFVCHIDKDCQLPRWHMHDFFH

SFLNVFRILCGEWVETLWDCMEVAGQSWCIPFYLMVILIGNLLVLYLFLALV

SSFSSCKDVTAEENNEAKNLQLAVARIKKGINYVLLKILCKTQNVPKDTMDH

VNEVYVKEDISDHTLSELSNTQDFLKDKEKSSGTEKNATENESQSLIPSPSV

SETVPIASGESDIENLDNKEIQSKSGDGGSKEKIKQSSSSECSTVDIAISEE

EEMFYGGERSKHLKNGCRRGSSLGQISGASKKGKIWWNIRKTCYKIVEHSWF

ESFIVLMILLSSGALAFEDIYIERKKTIKIILEYADKIFTYIFILEMLLKWI

AYGYKTYFTNAWCWLDFLIVDVSLVTLVANTLGYSDLGPIKSLRTLRALRPL

RALSRFEGMRVVVNALIGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGKFYEC

INTTDGSRFPASQVPNRSECFALMNVSQNVRWKNLKVNFDNVGLGYLSLLQV

ATFKGWTIIMYAAVDSVNVDKQPKYEYSLYMYIYFVVFIIFGSFFTLNLFIG

VIIDNFNQQKKKLGGSNIFITVKQRKQYRRLKKLMYEDSQRPVPRPLNKLQG

FIFDVVTSQAFNVIVMVLICFQAIAMMIDTDVQSLQMSIALYWINSIFVMLY

TMECILKLIAFRCFYFTIAWNIFDFMVVIFSITGLCLPMTVGSYLVPPSLVQ

LILLSRIIHMLRLGKGPKVFHNLMLPLMLSLPALLNIILLIFLVMFIYAVFG

MYNFAYVKKEAGINDVSNFETFGNSMLCLFQVAIFAGWDGMLDAIFNSKWSD

CDPDKINPGTQVRGDCGNPSVGIFYFVSYILISWLIIVNMYIVVVMEFLNIA

SKKKNKTLSEDDFRKFFQVWKRFDPDRTQYIDSSKLSDFAAALDPPLFMAKP

NKGQLIALDLPMAVGDRIHCLDILLAFTKRVMGQDVRMEKVVSEIESGELLA

NPFKITCEPITTTLKRKQEAVSATIIQRAYKNYRLRRNDKNTSDIHMIDGDR

DVHATKEGAYFDKAKEKSPIQSQI

12
hNax_hNav1.7_D3
MLASPEPKGLVPFTKESFELIKQHIAKTHNEDHEEEDLKPTPDLEVGKKLPF

-D4link-
IYGNLSQGMVSEPLEDVDPYYYKKKNTFIVLNKNRTIFRFNAASILCTLSPF

PM_chimera
NCIRRTTIKVLVHPFFQLFILISVLIDCVFMSLTNLPKWRPVLENTLLGIYT

FEILVKLFARGVWAGSFSFLGDPWNWLDFSVTVFEVIIRYSPLDFIPTLQTA

RTLRILKIIPLNQGLKSLVGVLIHCLKQLIGVIILTLFFLSIFSLIGMGLEM

GNLKHKCFRWPQENENETLHNRTGNPYYIRETENFYYLEGERYALLCGNRTD

AGQCPEGYVCVKAGINPDQGFTNEDSFGWALFALFRLMAQDYPEVLYHQILY

ASGKVYMIFFVVVSFLESFYMASLFLGILAMAYEEEKQRVGEISKKIEPKFQ

QTGKELQEGNETDEAKTIQIEMKKRSPISTDTSLDVLEDATLRHKEELEKSK

KICPLYWYKFAKTFLIWNCSPCWLKLKEFVHRIIMAPFTDLFLIICIILNVC

FLTLEHYPMSKQTNTLLNIGNLVFIGIFTAEMIFKIIAMHPYGYFQVGWNIF

DSMIVFHGLIELCLANVAGMALLRLERMLRIFKLGKYWPTFQILMWSLSNSW

VALKDLVLLLFTFIFFSAAFGMKLFGKNYEEFVCHIDKDCQLPRWHMHDFFH

SFLNVERILCGEWVETLWDCMEVAGQSWCIPFYLMVILIGNLLVLYLFLALV

SSFSSCKDVTAEENNEAKNLQLAVARIKKGINYVLLKILCKTQNVPKDTMDH

VNEVYVKEDISDHTLSELSNTQDFLKDKEKSSGTEKNATENESQSLIPSPSV

SETVPIASGESDIENLDNKEIQSKSGDGGSKEKIKQSSSSECSTVDIAISEE

EEMFYGGERSKHLKNGCRRGSSLGQISGASKKGKIWWNIRKTCYKIVEHSWF

ESFIVLMILLSSGALAFEDIYIERKKTIKIILEYADKIFTYIFILEMLLKWI

AYGYKTYFTNAWCWLDFLIVDVSLVTLVANTLGYSDLGPIKSLRTLRALRPL

RALSRFEGMRVVVNALIGAILPTLNVFLVCLMIWLIFSIMGVDLFAGRFYEC

IDPTSGERFPSSEVMNKSRCESLLFNESMLWENAKMNFDNVGNGELSLLQVA

TFNGWITIMNSAIDSVAVNIQPHFEVNIYMYCYFINFIIFGVFLPLSMLITV

IIDNFNKHKIKLGGSNIFITVKQRKQYRRLKKLMYEDSQRPVPRPLNKLQGF

IFDVVTSQAFNVIVMVLICFQAIAMMIDTDVQSLQMSIALYWINSIFVMLYT

MECILKLIAFRCFYFTIAWNIFDFMVVIFSITGLCLPMTVGSYLVPPSLVQL

ILLSRIIHMLRLGKGPKVFHNLMLPLMLSLPALLNIILLIFLVMFIYAVFGM

YNFAYVKKEAGINDVSNFETFGNSMLCLFQVAIFAGWDGMLDAIFNSKWSDC

DPDKINPGTQVRGDCGNPSVGIFYFVSYILISWLIIVNMYIVVVMEFLNIAS

KKKNKTLSEDDFRKFFQVWKRFDPDRTQYIDSSKLSDFAAALDPPLFMAKPN

KGQLIALDLPMAVGDRIHCLDILLAFTKRVMGQDVRMEKVVSEIESGELLAN

PFKITCEPITTTLKRKQEAVSATIIQRAYKNYRLRRNDKNTSDIHMIDGDRD

VHATKEGAYFDKAKEKSPIQSQI

13
hNax_hNav1.7_D3
MLASPEPKGLVPFTKESFELIKQHIAKTHNEDHEEEDLKPTPDLEVGKKLPF

-D4link-PM-
IYGNLSQGMVSEPLEDVDPYYYKKKNTFIVLNKNRTIFRFNAASILCTLSPF

S4S5link_chimera
NCIRRTTIKVLVHPFFQLFILISVLIDCVFMSLTNLPKWRPVLENTLLGIYT

FEILVKLFARGVWAGSFSFLGDPWNWLDFSVTVFEVIIRYSPLDFIPTLQTA

RTLRILKIIPLNQGLKSLVGVLIHCLKQLIGVIILTLFFLSIFSLIGMGLFM

GNLKHKCFRWPQENENETLHNRTGNPYYIRETENFYYLEGERYALLCGNRTD

AGQCPEGYVCVKAGINPDQGFTNEDSFGWALFALFRLMAQDYPEVLYHQILY

ASGKVYMIFFVVVSFLFSFYMASLFLGILAMAYEEEKQRVGEISKKIEPKFQ

QTGKELQEGNETDEAKTIQIEMKKRSPISTDTSLDVLEDATLRHKEELEKSK

KICPLYWYKFAKTFLIWNCSPCWLKLKEFVHRIIMAPFTDLFLIICIILNVC

FLTLEHYPMSKQTNTLLNIGNLVFIGIFTAEMIFKIIAMHPYGYFQVGWNIF

DSMIVFHGLIELCLANVAGMALLRLERMLRIFKLGKYWPTFQILMWSLSNSW

VALKDLVLLLFTFIFFSAAFGMKLFGKNYEEFVCHIDKDCQLPRWHMHDFFH

SFLNVFRILCGEWVETLWDCMEVAGQSWCIPFYLMVILIGNLLVLYLFLALV

SSFSSCKDVTAEENNEAKNLQLAVARIKKGINYVLLKILCKTQNVPKDTMDH

VNEVYVKEDISDHTLSELSNTQDFLKDKEKSSGTEKNATENESQSLIPSPSV

SETVPIASGESDIENLDNKEIQSKSGDGGSKEKIKQSSSSECSTVDIAISEE

EEMFYGGERSKHLKNGCRRGSSLGQISGASKKGKIWWNIRKTCYKIVEHSWF

ESFIVLMILLSSGALAFEDIYIERKKTIKIILEYADKIFTYIFILEMLLKWI

AYGYKTYFTNAWCWLDFLIVDVSLVTLVANTLGYSDLGPIKSLRTLRALRPL

RALSRFERMKVVVRALIKTTLPTLNVFLVCLMIWLIFSIMGVDLFAGRFYEC

IDPTSGERFPSSEVMNKSRCESLLFNESMLWENAKMNFDNVGNGFLSLLQVA

TFNGWITIMNSAIDSVAVNIQPHFEVNIYMYCYFINFIIFGVFLPLSMLITV

IIDNFNKHKIKLGGSNIFITVKQRKQYRRLKKLMYEDSQRPVPRPLNKLQGF

IFDVVTSQAFNVIVMVLICFQAIAMMIDTDVQSLQMSIALYWINSIFVMLYT

MECILKLIAFRCFYFTIAWNIFDFMVVIFSITGLCLPMTVGSYLVPPSLVQL

ILLSRIIHMLRLGKGPKVFHNLMLPLMLSLPALLNIILLIFLVMFIYAVFGM

YNFAYVKKEAGINDVSNFETFGNSMLCLFQVAIFAGWDGMLDAIFNSKWSDC

DPDKINPGTQVRGDCGNPSVGIFYFVSYILISWLIIVNMYIVVVMEFLNIAS

KKKNKTLSEDDFRKFFQVWKRFDPDRTQYIDSSKLSDFAAALDPPLFMAKPN

KGQLIALDLPMAVGDRIHCLDILLAFTKRVMGQDVRMEKVVSEIESGELLAN

PFKITCEPITTTLKRKQEAVSATIIQRAYKNYRLRRNDKNTSDIHMIDGDRD

VHATKEGAYFDKAKEKSPIQSQI

14
hNax_hNav1.7_V
MLASPEPKGLVPFTKESFELIKQHIAKTHNEDHEEEDLKPTPDLEVGKKLPF

SD3_chimera
NCIRRTTIKVLVHPFFQLFILISVLIDCVEMSLTNLPKWRPVLENTLLGIYT

IYGNLSQGMVSEPLEDVDPYYYKKKNTFIVLNKNRTIFRFNAASILCTLSPF

FEILVKLFARGVWAGSFSFLGDPWNWLDESVTVFEVIIRYSPLDFIPTLQTA

RTLRILKIIPLNQGLKSLVGVLIHCLKQLIGVIILTLFFLSIFSLIGMGLFM

GNLKHKCFRWPQENENETLHNRTGNPYYIRETENFYYLEGERYALLCGNRTD

AGQCPEGYVCVKAGINPDQGFTNEDSFGWALFALFRLMAQDYPEVLYHQILY

ASGKVYMIFFVVVSFLFSFYMASLFLGILAMAYEEEKQRVGEISKKIEPKFQ

QTGKELQEGNETDEAKTIQIEMKKRSPISTDTSLDVLEDATLRHKEELEKSK

KICPLYWYKFAKTFLIWNCSPCWLKLKEFVHRIIMAPFTDLFLIICIILNVC

FLTLEHYPMSKQTNTLLNIGNLVFIGIFTAEMIFKIIAMHPYGYFQVGWNIF

DSMIVFHGLIELCLANVAGMALLRLFRMLRIFKLGKYWPTFQILMWSLSNSW

VALKDLVLLLFTFIFFSAAFGMKLFGKNYEEFVCHIDKDCQLPRWHMHDFFH

SFLNVFRILCGEWVETLWDCMEVAGQSWCIPFYLMVILIGNLLVLYLFLALV

SSFSSCKDVTAEENNEAKNLQLAVARIKKGINYVLLKILCKTQNVPKDTMDH

VNEVYVKEDISDHTLSELSNTQDELKDKEKSSGTEKNATENESQSLIPSPSV

SETVPIASGESDIENLDNKEIQSKSGDGGSKEKIKQSSSSECSTVDIAISEE

EEMFYGGERSKHLKNGCRRGSSLGQISGASKKGKIWWNIRKTCYKIVEHSWF

ESFIVLMILLSSGALAFEDIYIERKKTIKIILEYADKIFTYIFILEMLLKWI

AYGYKTYFTNAWCWLDFLIVDVSLVTLVANTLGYSDLGPIKSLRTLRALRPL

RALSRFEGMKVVVRALIKTTLPTLNVFLVCLMIWLIFSIMGVDLFAGRFYEC

IDPTSGERFPSSEVMNKSRCESLLFNESMLWENAKMNFDNVGNGFLSLLQVA

TFNGWITIMNSAIDSVAVNIQPHFEVNIYMYCYFINFIIFGVFLPLSMLITV

IIDNFNKHKIKLGGSNIFITVKQRKQYRRLKKLMYEDSQRPVPRPLNKLQGF

IFDVVTSQAFNVIVMVLICFQAIAMMIDTDVQSLQMSIALYWINSIFVMLYT

MECILKLIAFRCFYFTIAWNIFDFMVVIFSITGLCLPMTVGSYLVPPSLVQL

ILLSRIIHMLRLGKGPKVFHNLMLPLMLSLPALLNIILLIFLVMFIYAVFGM

YNFAYVKKEAGINDVSNFETFGNSMLCLFQVAIFAGWDGMLDAIFNSKWSDC

DPDKINPGTQVRGDCGNPSVGIFYFVSYILISWLIIVNMYIVVVMEFLNIAS

KKKNKTLSEDDFRKFFQVWKRFDPDRTQYIDSSKLSDFAAALDPPLFMAKPN

KGQLIALDLPMAVGDRIHCLDILLAFTKRVMGQDVRMEKVVSEIESGELLAN

PFKITCEPITTTLKRKQEAVSATIIQRAYKNYRLRRNDKNTSDIHMIDGDRD

VHATKEGAYFDKAKEKSPIQSQI

15
hNax_hNav1.7_
MLASPEPKGLVPFTKESFELIKQHIAKTHNEDHEEEDLKPTPDLEVGKKLPF

VSD3-
IYGNLSQGMVSEPLEDVDPYYYKKKNTFIVLNKNRTIFRFNAASILCTLSPF

S4S5link_chimera
NCIRRTTIKVLVHPFFQLFILISVLIDCVEMSLTNLPKWRPVLENTLLGIYT

FEILVKLFARGVWAGSFSFLGDPWNWLDFSVTVFEVIIRYSPLDFIPTLQTA

RTLRILKIIPLNQGLKSLVGVLIHCLKQLIGVIILTLFFLSIFSLIGMGLFM

GNLKHKCFRWPQENENETLHNRTGNPYYIRETENFYYLEGERYALLCGNRTD

AGQCPEGYVCVKAGINPDQGFTNFDSFGWALFALFRLMAQDYPEVLYHQILY

ASGKVYMIFFVVVSFLESFYMASLFLGILAMAYEEEKQRVGEISKKIEPKFQ

QTGKELQEGNETDEAKTIQIEMKKRSPISTDTSLDVLEDATLRHKEELEKSK

KICPLYWYKFAKTFLIWNCSPCWLKLKEFVHRIIMAPFTDLFLIICIILNVC

FLTLEHYPMSKQTNTLLNIGNLVFIGIFTAEMIFKIIAMHPYGYFQVGWNIF

DSMIVFHGLIELCLANVAGMALLRLFRMLRIFKLGKYWPTFQILMWSLSNSW

VALKDLVLLLFTFIFFSAAFGMKLFGKNYEEFVCHIDKDCQLPRWHMHDFFH

SFLNVFRILCGEWVETLWDCMEVAGQSWCIPFYLMVILIGNLLVLYLFLALV

SSFSSCKDVTAEENNEAKNLQLAVARIKKGINYVLLKILCKTQNVPKDTMDH

VNEVYVKEDISDHTLSELSNTQDFLKDKEKSSGTEKNATENESQSLIPSPSV

SETVPIASGESDIENLDNKEIQSKSGDGGSKEKIKQSSSSECSTVDIAISEE

EEMFYGGERSKHLKNGCRRGSSLGQISGASKKGKIWWNIRKTCYKIVEHSWF

ESFIVLMILLSSGALAFEDIYIERKKTIKIILEYADKIFTYIFILEMLLKWI

AYGYKTYFTNAWCWLDFLIVDVSLVTLVANTLGYSDLGPIKSLRTLRALRPL

RALSRFEGMRVVVNALIGAIPSTLNVFLVCLMIWLIFSIMGVDLFAGRFYEC

IDPTSGERFPSSEVMNKSRCESLLFNESMLWENAKMNFDNVGNGFLSLLQVA

TFNGWITIMNSAIDSVAVNIQPHFEVNIYMYCYFINFIIFGVFLPLSMLITV

IIDNFNKHKIKLGGSNIFITVKQRKQYRRLKKLMYEDSQRPVPRPLNKLQGF

IFDVVTSQAFNVIVMVLICFQAIAMMIDTDVQSLQMSIALYWINSIFVMLYT

MECILKLIAFRCFYFTIAWNIFDFMVVIFSITGLCLPMTVGSYLVPPSLVQL

ILLSRIIHMLRLGKGPKVFHNLMLPLMLSLPALLNIILLIFLVMFIYAVFGM

YNFAYVKKEAGINDVSNFETFGNSMLCLFQVAIFAGWDGMLDAIFNSKWSDC

DPDKINPGTQVRGDCGNPSVGIFYFVSYILISWLIIVNMYIVVVMEFLNIAS

KKKNKTLSEDDFRKFFQVWKRFDPDRTQYIDSSKLSDFAAALDPPLEMAKPN

KGQLIALDLPMAVGDRIHCLDILLAFTKRVMGQDVRMEKVVSEIESGELLAN

PFKITCEPITTTLKRKQEAVSATIIQRAYKNYRLRRNDKNTSDIHMIDGDRD

VHATKEGAYFDKAKEKSPIQSQI

16
hNax_hNav1.7_V
MLASPEPKGLVPFTKESFELIKQHIAKTHNEDHEEEDLKPTPDLEVGKKLPF

SD3-S4S5link-
IYGNLSQGMVSEPLEDVDPYYYKKKNTFIVLNKNRTIFRFNAASILCTLSPF

PM_chimera
NCIRRTTIKVLVHPFFQLFILISVLIDCVFMSLTNLPKWRPVLENTLLGIYT

FEILVKLFARGVWAGSFSFLGDPWNWLDFSVTVFEVIIRYSPLDFIPTLQTA

RTLRILKIIPLNQGLKSLVGVLIHCLKQLIGVIILTLFFLSIFSLIGMGLFM

GNLKHKCFRWPQENENETLHNRTGNPYYIRETENFYYLEGERYALLCGNRTD

AGQCPEGYVCVKAGINPDQGFTNEDSFGWALFALFRLMAQDYPEVLYHQILY

ASGKVYMIFFVVVSFLFSFYMASLFLGILAMAYEEEKQRVGEISKKIEPKFQ

QTGKELQEGNETDEAKTIQIEMKKRSPISTDTSLDVLEDATLRHKEELEKSK

KICPLYWYKFAKTFLIWNCSPCWLKLKEFVHRIIMAPFTDLFLIICIILNVC

FLTLEHYPMSKQTNTLLNIGNLVFIGIFTAEMIFKIIAMHPYGYFQVGWNIF

DSMIVFHGLIELCLANVAGMALLRLFRMLRIFKLGKYWPTFQILMWSLSNSW

VALKDLVLLLFTFIFFSAAFGMKLFGKNYEEFVCHIDKDCQLPRWHMHDFFH

SFLNVFRILCGEWVETLWDCMEVAGQSWCIPFYLMVILIGNLLVLYLFLALV

SSFSSCKDVTAEENNEAKNLQLAVARIKKGINYVLLKILCKTQNVPKDTMDH

VNEVYVKEDISDHTLSELSNTQDFLKDKEKSSGTEKNATENESQSLIPSPSV

SETVPIASGESDIENLDNKEIQSKSGDGGSKEKIKQSSSSECSTVDIAISEE

EEMFYGGERSKHLKNGCRRGSSLGQISGASKKGKIWWNIRKTCYKIVEHSWF

ESFIVLMILLSSGALAFEDIYIERKKTIKIILEYADKIFTYIFILEMLLKWI

AYGYKTYFTNAWCWLDFLIVDVSLVTLVANTLGYSDLGPIKSLRTLRALRPL

RALSRFEGMRVVVNALIGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGKFYEC

INTTDGSRFPASQVPNRSECFALMNVSQNVRWKNLKVNFDNVGLGYLSLLQV

ATFKGWTIIMYAAVDSVNVDKQPKYEYSLYMYIYFVVFIIFGSFFTLNLFIG

VIIDNFNQQKKKLGGQDIFMTVKQRKQYRRLKKLMYEDSQRPVPRPLNKLQG

FIFDVVTSQAFNVIVMVLICFQAIAMMIDTDVQSLQMSIALYWINSIFVMLY

TMECILKLIAFRCFYFTIAWNIFDFMVVIFSITGLCLPMTVGSYLVPPSLVQ

LILLSRIIHMLRLGKGPKVFHNLMLPLMLSLPALLNIILLIFLVMFIYAVFG

MYNFAYVKKEAGINDVSNFETFGNSMLCLFQVAIFAGWDGMLDAIFNSKWSD

CDPDKINPGTQVRGDCGNPSVGIFYFVSYILISWLIIVNMYIVVVMEFLNIA

SKKKNKTLSEDDFRKFFQVWKRFDPDRTQYIDSSKLSDFAAALDPPLFMAKP

NKGQLIALDLPMAVGDRIHCLDILLAFTKRVMGQDVRMEKVVSEIESGELLA

NPFKITCEPITTTLKRKQEAVSATIIQRAYKNYRLRRNDKNTSDIHMIDGDR

DVHATKEGAYFDKAKEKSPIQSQI

17
hNax_hNav1.7_DI

II_VSD3-S3-
MLASPEPKGLVPFTKESFELIKQHIAKTHNEDHEEEDLKPTPDLEVGKKLPF

S4_chimera
IYGNLSQGMVSEPLEDVDPYYYKKKNTFIVLNKNRTIFRFNAASILCTLSPF

NCIRRTTIKVLVHPFFQLFILISVLIDCVFMSLTNLPKWRPVLENTLLGIYT

FEILVKLFARGVWAGSFSFLGDPWNWLDFSVTVFEVIIRYSPLDFIPTLQTA

RTLRILKIIPLNQGLKSLVGVLIHCLKQLIGVIILTLFFLSIFSLIGMGLFM

GNLKHKCFRWPQENENETLHNRTGNPYYIRETENFYYLEGERYALLCGNRTD

AGQCPEGYVCVKAGINPDQGFTNEDSFGWALFALFRLMAQDYPEVLYHQILY

ASGKVYMIFFVVVSFLFSFYMASLFLGILAMAYEEEKQRVGEISKKIEPKFQ

QTGKELQEGNETDEAKTIQIEMKKRSPISTDTSLDVLEDATLRHKEELEKSK

KICPLYWYKFAKTFLIWNCSPCWLKLKEFVHRIIMAPFTDLFLIICIILNVC

FLTLEHYPMSKQTNTLLNIGNLVFIGIFTAEMIFKIIAMHPYGYFQVGWNIF

DSMIVFHGLIELCLANVAGMALLRLERMLRIFKLGKYWPTFQILMWSLSNSW

VALKDLVLLLFTFIFFSAAFGMKLFGKNYEEFVCHIDKDCQLPRWHMHDFFH

SFLNVFRILCGEWVETLWDCMEVAGQSWCIPFYLMVILIGNLLVLYLFLALV

SSFSSCKDVTAEENNEAKNLQLAVARIKKGINYVLLKILCKTQNVPKDTMDH

VNEVYVKEDISDHTLSELSNTQDFLKDKEKSSGTEKNATENESQSLIPSPSV

SETVPIASGESDIENLDNKEIQSKSGDGGSKEKIKQSSSSECSTVDIAISEE

EEMFYGGERSKHLKNGCRRGSSLGQISGASKKGKIWQNIRKTCCKIVENNWF

KCFIGLVTLLSTGTLAFEDIYMDQRKTIKILLEYADMIFTYIFILEMLLKWM

AYGFKAYFTNAWCWLDFLIVDVSLVTLVANTLGYSDLGPIKSLRTLRALRPL

RALSRFERMKVVVRALIKTTLPTLNVFLVCLMIWLIFSIMGVDLFAGRFYEC

IDPTSGERFPSSEVMNKSRCESLLFNESMLWENAKMNFDNVGNGFLSLLQVA

TFNGWITIMNSAIDSVAVNIQPHFEVNIYMYCYFINFIIFGVFLPLSMLITV

IIDNFNKHKIKLGGSNIFITVKQRKQYRRLKKLMYEDSQRPVPRPLNKLQGF

IFDVVTSQAFNVIVMVLICFQAIAMMIDTDVQSLQMSIALYWINSIFVMLYT

MECILKLIAFRCFYFTIAWNIFDFMVVIFSITGLCLPMTVGSYLVPPSLVQL

ILLSRIIHMLRLGKGPKVFHNLMLPLMLSLPALLNIILLIFLVMFIYAVFGM

YNFAYVKKEAGINDVSNFETFGNSMLCLFQVAIFAGWDGMLDAIFNSKWSDC

DPDKINPGTQVRGDCGNPSVGIFYFVSYILISWLIIVNMYIVVVMEFLNIAS

KKKNKTLSEDDFRKFFQVWKRFDPDRTQYIDSSKLSDFAAALDPPLFMAKPN

KGQLIALDLPMAVGDRIHCLDILLAFTKRVMGQDVRMEKVVSEIESGELLAN

PFKITCEPITTTLKRKQEAVSATIIQRAYKNYRLRRNDKNTSDIHMIDGDRD

VHATKEGAYFDKAKEKSPIQSQI

18
hNax_hNav1.7_DI
MLASPEPKGLVPFTKESFELIKQHIAKTHNEDHEEEDLKPTPDLEVGKKLPF

II_VSD3-S5-
IYGNLSQGMVSEPLEDVDPYYYKKKNTFIVLNKNRTIFRFNAASILCTLSPF

S6_chimera
NCIRRTTIKVLVHPFFQLFILISVLIDCVFMSLTNLPKWRPVLENTLLGIYT

FEILVKLFARGVWAGSFSFLGDPWNWLDFSVTVFEVIIRYSPLDFIPTLQTA

RTLRILKIIPLNQGLKSLVGVLIHCLKQLIGVIILTLFFLSIFSLIGMGLFM

GNLKHKCFRWPQENENETLHNRTGNPYYIRETENFYYLEGERYALLCGNRTD

AGQCPEGYVCVKAGINPDQGFTNFDSFGWALFALFRLMAQDYPEVLYHQILY

ASGKVYMIFFVVVSFLFSFYMASLFLGILAMAYEEEKQRVGEISKKIEPKFQ

QTGKELQEGNETDEAKTIQIEMKKRSPISTDTSLDVLEDATLRHKEELEKSK

KICPLYWYKFAKTFLIWNCSPCWLKLKEFVHRIIMAPFTDLFLIICIILNVC

FLTLEHYPMSKQTNTLLNIGNLVFIGIFTAEMIFKIIAMHPYGYFQVGWNIF

DSMIVFHGLIELCLANVAGMALLRLFRMLRIFKLGKYWPTFQILMWSLSNSW

VALKDLVLLLFTFIFFSAAFGMKLFGKNYEEFVCHIDKDCQLPRWHMHDFFH

SFLNVFRILCGEWVETLWDCMEVAGQSWCIPFYLMVILIGNLLVLYLFLALV

SSFSSCKDVTAEENNEAKNLQLAVARIKKGINYVLLKILCKTQNVPKDTMDH

VNEVYVKEDISDHTLSELSNTQDFLKDKEKSSGTEKNATENESQSLIPSPSV

SETVPIASGESDIENLDNKEIQSKSGDGGSKEKIKQSSSSECSTVDIAISEE

EEMFYGGERSKHLKNGCRRGSSLGQISGASKKGKIWQNIRKTCCKIVENNWF

KCFIGLVTLLSTGTLAFEDIYMDQRKTIKILLEYADMIFTYIFILEMLLKWM

AYGFKAYFSNGWYRLDFVVVIVFCLSLIGKTREELKPLISMKFLRPLRVLSQ

FEGMRVVVNALIGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGKFYECINTTD

GSRFPASQVPNRSECFALMNVSQNVRWKNLKVNFDNVGLGYLSLLQVATFKG

WTIIMYAAVDSVNVDKQPKYEYSLYMYIYFVVFIIFGSFFTLNLFIGVIIDN

FNQQKKKLGGSNIFITVKQRKQYRRLKKLMYEDSQRPVPRPLNKLQGFIFDV

VTSQAFNVIVMVLICFQAIAMMIDTDVQSLQMSIALYWINSIFVMLYTMECI

LKLIAFRCFYFTIAWNIFDFMVVIFSITGLCLPMTVGSYLVPPSLVQLILLS

RIIHMLRLGKGPKVFHNLMLPLMLSLPALLNIILLIFLVMFIYAVFGMYNFA

YVKKEAGINDVSNFETFGNSMLCLFQVAIFAGWDGMLDAIFNSKWSDCDPDK

INPGTQVRGDCGNPSVGIFYFVSYILISWLIIVNMYIVVVMEFLNIASKKKN

KTLSEDDFRKFFQVWKRFDPDRTQYIDSSKLSDFAAALDPPLFMAKPNKGQL

IALDLPMAVGDRIHCLDILLAFTKRVMGQDVRMEKVVSEIESGFLLANPEKI

TCEPITTTLKRKQEAVSATIIQRAYKNYRLRRNDKNTSDIHMIDGDRDVHAT

KEGAYFDKAKEKSPIQSQI

19
hNax_hNav1.7_DI
MLASPEPKGLVPFTKESFELIKQHIAKTHNEDHEEEDLKPTPDLEVGKKLPF

II_VSD3-S3-
IYGNLSQGMVSEPLEDVDPYYYKKKNTFIVLNKNRTIFRFNAASILCTLSPF

S4loop_S4-
NCIRRTTIKVLVHPFFQLFILISVLIDCVFMSLTNLPKWRPVLENTLLGIYT

S5link_S5_
FEILVKLFARGVWAGSFSFLGDPWNWLDFSVTVFEVIIRYSPLDFIPTLQTA

chimera
RTLRILKIIPLNQGLKSLVGVLIHCLKQLIGVIILTLFFLSIFSLIGMGLFM

GNLKHKCFRWPQENENETLHNRTGNPYYIRETENFYYLEGERYALLCGNRTD

AGQCPEGYVCVKAGINPDQGFTNEDSFGWALFALFRLMAQDYPEVLYHQILY

ASGKVYMIFFVVVSFLESFYMASLFLGILAMAYEEEKQRVGEISKKIEPKFQ

QTGKELQEGNETDEAKTIQIEMKKRSPISTDTSLDVLEDATLRHKEELEKSK

KICPLYWYKFAKTFLIWNCSPCWLKLKEFVHRIIMAPFTDLFLIICIILNVC

FLTLEHYPMSKQTNTLLNIGNLVFIGIFTAEMIFKIIAMHPYGYFQVGWNIF

DSMIVFHGLIELCLANVAGMALLRLFRMLRIFKLGKYWPTFQILMWSLSNSW

VALKDLVLLLFTFIFFSAAFGMKLFGKNYEEFVCHIDKDCQLPRWHMHDFFH

SFLNVFRILCGEWVETLWDCMEVAGQSWCIPFYLMVILIGNLLVLYLFLALV

SSFSSCKDVTAEENNEAKNLQLAVARIKKGINYVLLKILCKTQNVPKDTMDH

VNEVYVKEDISDHTLSELSNTQDFLKDKEKSSGTEKNATENESQSLIPSPSV

SETVPIASGESDIENLDNKEIQSKSGDGGSKEKIKQSSSSECSTVDIAISEE

EEMFYGGERSKHLKNGCRRGSSLGQISGASKKGKIWQNIRKTCCKIVENNWF

KCFIGLVTLLSTGTLAFEDIYMDQRKTIKILLEYADMIFTYIFILEMLLKWM

AYGFKAYFSNGWYRLDFVVVIVFCLSLIGGYSDLGLKPLISMKFLRPLRVLS

QFEGMRVVVNALIGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGRFYECIDPT

SGERFPSSEVMNKSRCESLLENESMLWENAKMNFDNVGNGFLSLLQVATENG

WITIMNSAIDSVAVNIQPHFEVNIYMYCYFINFIIFGVFLPLSMLITVIIDN

FNKHKIKLGGSNIFITVKQRKQYRRLKKLMYEDSQRPVPRPLNKLQGFIFDV

VTSQAFNVIVMVLICFQAIAMMIDTDVQSLQMSIALYWINSIFVMLYTMECI

LKLIAFRCFYFTIAWNIFDFMVVIFSITGLCLPMTVGSYLVPPSLVQLILLS

RIIHMLRLGKGPKVFHNLMLPLMLSLPALLNIILLIFLVMFIYAVFGMYNFA

YVKKEAGINDVSNFETFGNSMLCLFQVAIFAGWDGMLDAIFNSKWSDCDPDK

INPGTQVRGDCGNPSVGIFYFVSYILISWLIIVNMYIVVVMEFLNIASKKKN

KTLSEDDFRKFFQVWKRFDPDRTQYIDSSKLSDFAAALDPPLFMAKPNKGQL

IALDLPMAVGDRIHCLDILLAFTKRVMGQDVRMEKVVSEIESGELLANPFKI

TCEPITTTLKRKQEAVSATIIQRAYKNYRLRRNDKNTSDIHMIDGDRDVHAT

KEGAYFDKAKEKSPIQSQI

20
hNax_
MLASPEPKGLVPFTKESFELIKQHIAKTHNEDHEEEDLKPTPDLEVGKKLPF

hNav1.7_DI
IYGNLSQGMVSEPLEDVDPYYYKKKNTFIVLNKNRTIFRFNAASILCTLSPF

II_DIVS6-
NCIRRTTIKVLVHPFFQLFILISVLIDCVFMSLTNLPKWRPVLENTLLGIYT

CTD_chimera
FEILVKLFARGVWAGSFSFLGDPWNWLDFSVTVFEVIIRYSPLDFIPTLQTA

RTLRILKIIPLNQGLKSLVGVLIHCLKQLIGVIILTLFFLSIFSLIGMGLFM

GNLKHKCFRWPQENENETLHNRTGNPYYIRETENFYYLEGERYALLCGNRTD

AGQCPEGYVCVKAGINPDQGFTNEDSFGWALFALFRLMAQDYPEVLYHQILY

ASGKVYMIFFVVVSFLFSFYMASLFLGILAMAYEEEKQRVGEISKKIEPKFQ

QTGKELQEGNETDEAKTIQIEMKKRSPISTDTSLDVLEDATLRHKEELEKSK

KICPLYWYKFAKTFLIWNCSPCWLKLKEFVHRIIMAPFTDLFLIICIILNVC

FLTLEHYPMSKQTNTLLNIGNLVFIGIFTAEMIFKIIAMHPYGYFQVGWNIF

DSMIVFHGLIELCLANVAGMALLRLERMLRIFKLGKYWPTFQILMWSLSNSW

VALKDLVLLLFTFIFFSAAFGMKLFGKNYEEFVCHIDKDCQLPRWHMHDFFH

SFLNVFRILCGEWVETLWDCMEVAGQSWCIPFYLMVILIGNLLVLYLFLALV

SSFSSCKDVTAEENNEAKNLQLAVARIKKGINYVLLKILCKTQNVPKDTMDH

VNEVYVKEDISDHTLSELSNTQDFLKDKEKSSGTEKNATENESQSLIPSPSV

SETVPIASGESDIENLDNKEIQSKSGDGGSKEKIKQSSSSECSTVDIAISEE

EEMFYGGERSKHLKNGCRRGSSLGQISGASKKGKIWWNIRKTCYKIVEHSWF

ESFIVLMILLSSGALAFEDIYIERKKTIKIILEYADKIFTYIFILEMLLKWI

AYGYKTYFTNAWCWLDFLIVDVSLVTLVANTLGYSDLGPIKSLRTLRALRPL

RALSRFEGMRVVVNALIGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGKFYEC

INTTDGSRFPASQVPNRSECFALMNVSQNVRWKNLKVNFDNVGLGYLSLLQV

ATFKGWTIIMYAAVDSVNVDKQPKYEYSLYMYIYFVVFIIFGSFFTLNLFIG

VIIDNFNQQKKKLGGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPGNKLQG

FIFDVVTSQAFNVIVMVLICFQAIAMMIDTDVQSLQMSIALYWINSIFVMLY

TMECILKLIAFRCFYFTIAWNIFDFMVVIFSITGLCLPMTVGSYLVPPSLVQ

LILLSRIIHMLRLGKGPKVFHNLMLPLMLSLPALLNIILLIFLVMFIYAVFG

MYNFAYVKKEAGINDVSNFETFGNSMLCLFQVAIFAGWDGMLDAIENSKWSD

CDPDKINPGTQVRGDCGNPSVGIFYFVSYILISWLIIVNMYIAVILENFSVA

TEESTEPLSEDDFEMFYEVWEKFDPDATQFIEFSKLSDFAAALDPPLLIAKP

NKVQLIAMDLPMVSGDRIHCLDILFAFTKRVLGESGEMDSLRSQMEERFMSA

NPSKVSYEPITTTLKRKQEDVSATVIQRAYRRYRLRQNVKNISSIYIKDGDR

DDDLLNKKDMAFDNVNENSSPEKTDATSSTTSPPSYDSVTKPDKEKYEQDRT

EKEDKGKDSKESKK

21
Nax_Nav1.7_D1-
MLASPEPKGLVPFTKESFELIKQHIAKTHNEDHEEEDLKPTPDLEVGKKLPF

D2_linker_chimera
IYGNLSQGMVSEPLEDVDPYYYKKKNTFIVLNKNRTIFRFNAASILCTLSPF

NCIRRTTIKVLVHPFFQLFILISVLIDCVFMSLTNLPKWRPVLENTLLGIYT

FEILVKLFARGVWAGSFSFLGDPWNWLDESVTVFEVIIRYSPLDFIPTLQTA

RTLRILKIIPLNQGLKSLVGVLIHCLKQLIGVIILTLFFLSIFSLIGMGLFM

GNLKHKCFRWPQENENETLHNRTGNPYYIRETENFYYLEGERYALLCGNRTD

AGQCPEGYVCVKAGINPDQGFTNEDSFGWALFALFRLMAQDYPEVLYHQILY

ASGKVYMIFFVVVSFLFSFYMASLFLGQKELEFQQMLDRLKKEQEEAEAIAA

AAAEYTSIRRSRIMGLSESSSETSKLSSKSAKERRNRRKKKNQKKLSSGEEK

GDAEKLSKSESEDSIRRKSFHLGVEGHRRAHEKRLSTPNQSPLSIRGSLFSA

RRSSRTSLFSFKGRGRDIGSETEFADDEHSIFGDNESRRGSLFVPHRPQERR

SSNISQASRSPPMLPVNGKMHSAVDCNGVVSLVDGRSALMLPNGQLLPEVII

DKATSDDSGTTNQIHKKRRCSSYLLSEDMLNDPNLRQRAMSRASILTNTVEE

LEESRQKCPPWWYRFAHKFLIWNFTDLFLIICIILNVCFLTLEHYPMSKQTN

TLLNIGNLVFIGIFTAEMIFKIIAMHPYGYFQVGWNIFDSMIVFHGLIELCL

ANVAGMALLRLFRMLRIFKLGKYWPTFQILMWSLSNSWVALKDLVLLLFTFI

FFSAAFGMKLFGKNYEEFVCHIDKDCQLPRWHMHDFFHSFLNVFRILCGEWV

ETLWDCMEVAGQSWCIPFYLMVILIGNLLVLYLFLALVSSFSSCKDVTAEEN

NEAKNLQLAVARIKKGINYVLLKILCKTQNVPKDTMDHVNEVYVKEDISDHT

LSELSNTQDFLKDKEKSSGTEKNATENESQSLIPSPSVSETVPIASGESDIE

NLDNKEIQSKSGDGGSKEKIKQSSSSECSTVDIAISEEEEMFYGGERSKHLK

NGCRRGSSLGQISGASKKGKIWQNIRKTCCKIVENNWFKCFIGLVTLLSTGT

LAFEDIYMDQRKTIKILLEYADMIFTYIFILEMLLKWMAYGFKAYFSNGWYR

LDFVVVIVFCLSLIGKTREELKPLISMKFLRPLRVLSQFERMKVVVRALIKT

TLPTLNVFLVCLMIWLIFSIMGVDLFAGRFYECIDPTSGERFPSSEVMNKSR

CESLLENESMLWENAKMNFDNVGNGELSLLQVATENGWITIMNSAIDSVAVN

IQPHFEVNIYMYCYFINFIIFGVFLPLSMLITVIIDNENKHKIKLGGSNIFI

TVKQRKQYRRLKKLMYEDSQRPVPRPLNKLQGFIFDVVTSQAFNVIVMVLIC

FQAIAMMIDTDVQSLQMSIALYWINSIFVMLYTMECILKLIAFRCFYFTIAW

NIFDFMVVIFSITGLCLPMTVGSYLVPPSLVQLILLSRIIHMLRLGKGPKVE

HNLMLPLMLSLPALLNIILLIFLVMFIYAVFGMYNFAYVKKEAGINDVSNFE

TFGNSMLCLFQVAIFAGWDGMLDAIFNSKWSDCDPDKINPGTQVRGDCGNPS

VGIFYFVSYILISWLIIVNMYIVVVMEFLNIASKKKNKTLSEDDFRKFFQVW

KRFDPDRTQYIDSSKLSDFAAALDPPLEMAKPNKGQLIALDLPMAVGDRIHC

LDILLAFTKRVMGQDVRMEKVVSEIESGELLANPFKITCEPITTTLKRKQEA

VSATIIQRAYKNYRLRRNDKNTSDIHMIDGDRDVHATKEGAYFDKAKEKSPI

QSQI

22
Nax_Nav1.7_D2-
MLASPEPKGLVPFTKESFELIKQHIAKTHNEDHEEEDLKPTPDLEVGKKLPF

D3_linker_chimera
NCIRRTTIKVLVHPFFQLFILISVLIDCVFMSLTNLPKWRPVLENTLLGIYT

IYGNLSQGMVSEPLEDVDPYYYKKKNTFIVLNKNRTIFRENAASILCTLSPF

FEILVKLFARGVWAGSFSFLGDPWNWLDFSVTVFEVIIRYSPLDFIPTLQTA

RTLRILKIIPLNQGLKSLVGVLIHCLKQLIGVIILTLFFLSIFSLIGMGLFM

GNLKHKCFRWPQENENETLHNRTGNPYYIRETENFYYLEGERYALLCGNRTD

AGQCPEGYVCVKAGINPDQGFTNEDSFGWALFALFRLMAQDYPEVLYHQILY

ASGKVYMIFFVVVSFLFSFYMASLFLGILAMAYEEEKQRVGEISKKIEPKFQ

QTGKELQEGNETDEAKTIQIEMKKRSPISTDTSLDVLEDATLRHKEELEKSK

KICPLYWYKFAKTFLIWNCSPCWLKLKEFVHRIIMAPFTDLFLIICIILNVC

FLTLEHYPMSKQTNTLLNIGNLVFIGIFTAEMIFKIIAMHPYGYFQVGWNIF

DSMIVFHGLIELCLANVAGMALLRLFRMLRIFKLGKYWPTFQILMWSLSNSW

VALKDLVLLLFTFIFFSAAFGMKLFGKNYEEFVCHIDKDCQLPRWHMHDFFH

SFLNVFRILCGEWVETLWDCMEVAGQSWCIPFYLMVILIGNLLVLYLFLALS

FSSDNLTAIEEDPDANNLQIAVTRIKKGINYVKQTLREFILKAFSKKPKISR

EIRQAEDLNTKKENYISNHTLAEMSKGHNFLKEKDKISGFGSSVDKHLMEDS

DGQSFIHNPSLTVTVPIAPGESDLENMNAEELSSDSDSEYSKVRLNRSSSSE

CSTVDNPLPGEGEEAEAEPMNSDEPEACFTDGCVWRFSCCQVNIESGKGKKG

KIWQNIRKTCCKIVENNWFKCFIGLVTLLSTGTLAFEDIYMDQRKTIKILLE

YADMIFTYIFILEMLLKWMAYGFKAYFSNGWYRLDFVVVIVFCLSLIGKTRE

ELKPLISMKFLRPLRVLSQFERMKVVVRALIKTTLPTLNVFLVCLMIWLIFS

IMGVDLFAGRFYECIDPTSGERFPSSEVMNKSRCESLLFNESMLWENAKMNF

DNVGNGFLSLLQVATFNGWITIMNSAIDSVAVNIQPHFEVNIYMYCYFINFI

IFGVFLPLSMLITVIIDNFNKHKIKLGGSNIFITVKQRKQYRRLKKLMYEDS

QRPVPRPLNKLQGFIFDVVTSQAFNVIVMVLICFQAIAMMIDTDVQSLQMSI

ALYWINSIFVMLYTMECILKLIAFRCFYFTIAWNIFDFMVVIFSITGLCLPM

TVGSYLVPPSLVQLILLSRIIHMLRLGKGPKVFHNLMLPLMLSLPALLNIIL

LIFLVMFIYAVFGMYNFAYVKKEAGINDVSNFETFGNSMLCLFQVAIFAGWD

GMLDAIFNSKWSDCDPDKINPGTQVRGDCGNPSVGIFYFVSYILISWLIIVN

MYIVVVMEFLNIASKKKNKTLSEDDFRKFFQVWKRFDPDRTQYIDSSKLSDF

AAALDPPLFMAKPNKGQLIALDLPMAVGDRIHCLDILLAFTKRVMGQDVRME

KVVSEIESGFLLANPFKITCEPITTTLKRKQEAVSATIIQRAYKNYRLRRND

KNTSDIHMIDGDRDVHATKEGAYFDKAKEKSPIQSQI

23
Nax_Nav1.7_D3-
MLASPEPKGLVPFTKESFELIKQHIAKTHNEDHEEEDLKPTPDLEVGKKLPF

D4_linker_chimera
IYGNLSQGMVSEPLEDVDPYYYKKKNTFIVLNKNRTIFRFNAASILCTLSPF

NCIRRTTIKVLVHPFFQLFILISVLIDCVEMSLTNLPKWRPVLENTLLGIYT

FEILVKLFARGVWAGSFSFLGDPWNWLDFSVTVFEVIIRYSPLDFIPTLQTA

RTLRILKIIPLNQGLKSLVGVLIHCLKQLIGVIILTLFFLSIFSLIGMGLFM

GNLKHKCFRWPQENENETLHNRTGNPYYIRETENFYYLEGERYALLCGNRTD

AGQCPEGYVCVKAGINPDQGFTNEDSFGWALFALFRLMAQDYPEVLYHQILY

ASGKVYMIFFVVVSFLFSFYMASLFLGILAMAYEEEKQRVGEISKKIEPKFQ

QTGKELQEGNETDEAKTIQIEMKKRSPISTDTSLDVLEDATLRHKEELEKSK

KICPLYWYKFAKTFLIWNCSPCWLKLKEFVHRIIMAPFTDLFLIICIILNVC

FLTLEHYPMSKQTNTLLNIGNLVFIGIFTAEMIFKIIAMHPYGYFQVGWNIE

DSMIVFHGLIELCLANVAGMALLRLFRMLRIFKLGKYWPTFQILMWSLSNSW

VALKDLVLLLFTFIFFSAAFGMKLFGKNYEEFVCHIDKDCQLPRWHMHDFFH

SFLNVFRILCGEWVETLWDCMEVAGQSWCIPFYLMVILIGNLLVLYLFLALV

SSFSSCKDVTAEENNEAKNLQLAVARIKKGINYVLLKILCKTQNVPKDTMDH

VNEVYVKEDISDHTLSELSNTQDFLKDKEKSSGTEKNATENESQSLIPSPSV

SETVPIASGESDIENLDNKEIQSKSGDGGSKEKIKQSSSSECSTVDIAISEE

EEMFYGGERSKHLKNGCRRGSSLGQISGASKKGKIWQNIRKTCCKIVENNWE

KCFIGLVTLLSTGTLAFEDIYMDQRKTIKILLEYADMIFTYIFILEMLLKWM

AYGFKAYFSNGWYRLDFVVVIVFCLSLIGKTREELKPLISMKFLRPLRVLSQ

FERMKVVVRALIKTTLPTLNVFLVCLMIWLIFSIMGVDLFAGRFYECIDPTS

GERFPSSEVMNKSRCESLLFNESMLWENAKMNFDNVGNGELSLLQVATENGW

ITIMNSAIDSVAVNIQPHFEVNIYMYCYFINFIIFGVFLPLSMLILGGQDIF

MTEEQKKYYNAMKKLGSKKPQKPIPRPGNKLQGFIFDVVTSQAFNVIVMVLI

CFQAIAMMIDTDVQSLQMSIALYWINSIFVMLYTMECILKLIAFRCFYFTIA

WNIFDFMVVIFSITGLCLPMTVGSYLVPPSLVQLILLSRIIHMLRLGKGPKV

FHNLMLPLMLSLPALLNIILLIFLVMFIYAVFGMYNFAYVKKEAGINDVSNF

ETFGNSMLCLFQVAIFAGWDGMLDAIFNSKWSDCDPDKINPGTQVRGDCGNP

SVGIFYFVSYILISWLIIVNMYIVVVMEFLNIASKKKNKTLSEDDFRKFFQV

WKRFDPDRTQYIDSSKLSDFAAALDPPLFMAKPNKGQLIALDLPMAVGDRIH

CLDILLAFTKRVMGQDVRMEKVVSEIESGELLANPFKITCEPITTTLKRKQE

AVSATIIQRAYKNYRLRRNDKNTSDIHMIDGDRDVHATKEGAYEDKAKEKSP

IQSQI

24
Nax_Nav1.7_
MAMLPPPGPQSFVHFTKQSLALIEQRIAERKSKEPKEEKKDDDEEAPKPSSD

ultimateloop_
LEAGKQLPFIYGDIPPGMVSEPLEDLDPYYADKKTFIVLNKGKTIFRENATP

chimera
ALYMLSPFSPFFQLFILISVLIDCVFMSLTNLPKWRPVLENTLLGIYTFEIL

VKLFARGVWAGSFSFLGDPWNWLDFSVTVFEVIIRYSPLDFIPTLQTARTLR

ILKIIPLNQGLKSLVGVLIHCLKQLIGVIILTLFFLSIFSLIGMGLFMGNLK

HKCFRWPQENENETLHNRTGNPYYIRETENFYYLEGERYALLCGNRTDAGQC

PEGYVCVKAGINPDQGFTNFDSFGWALFALFRLMAQDYPEVLYHQILYASGK

VYMIFFVVVSFLFSFYMASLFLGQKELEFQQMLDRLKKEQEEAEAIAAAAAE

YTSIRRSRIMGLSESSSETSKLSSKSAKERRNRRKKKNQKKLSSGEEKGDAE

KLSKSESEDSIRRKSFHLGVEGHRRAHEKRLSTPNQSPLSIRGSLESARRSS

RTSLFSFKGRGRDIGSETEFADDEHSIFGDNESRRGSLFVPHRPQERRSSNI

SQASRSPPMLPVNGKMHSAVDCNGVVSLVDGRSALMLPNGQLLPEVIIDKAT

SDDSGTTNQIHKKRRCSSYLLSEDMLNDPNLRQRAMSRASILTNTVEELEES

RQKCPPWWYRFAHKFLIWNFTDLFLIICIILNVCFLTLEHYPMSKQTNTLLN

IGNLVFIGIFTAEMIFKIIAMHPYGYFQVGWNIFDSMIVFHGLIELCLANVA

GMALLRLFRMLRIFKLGKYWPTFQILMWSLSNSWVALKDLVLLLFTFIFFSA

AFGMKLFGKNYEEFVCHIDKDCQLPRWHMHDFFHSFLNVFRILCGEWVETLW

DCMEVAGQSWCIPFYLMVILIGNLLVLYLFLALSFSSDNLTAIEEDPDANNL

QIAVTRIKKGINYVKQTLREFILKAFSKKPKISREIRQAEDLNTKKENYISN

HTLAEMSKGHNFLKEKDKISGFGSSVDKHLMEDSDGQSFIHNPSLTVTVPIA

PGESDLENMNAEELSSDSDSEYSKVRLNRSSSSECSTVDNPLPGEGEEAEAE

PMNSDEPEACFTDGCVWRFSCCQVNIESGKGKKGKIWQNIRKTCCKIVENNW

FKCFIGLVTLLSTGTLAFEDIYMDQRKTIKILLEYADMIFTYIFILEMLLKW

MAYGFKAYFSNGWYRLDFVVVIVFCLSLIGKTREELKPLISMKFLRPLRVLS

QFERMKVVVRALIKTTLPTLNVFLVCLMIWLIFSIMGVDLFAGRFYECIDPT

SGERFPSSEVMNKSRCESLLFNESMLWENAKMNFDNVGNGFLSLLQVATENG

WITIMNSAIDSVAVNIQPHFEVNIYMYCYFINFIIFGVFLPLSMLILGGQDI

FMTEEQKKYYNAMKKLGSKKPQKPIPRPGNKLQGFIFDVVTSQAFNVIVMVL

ICFQAIAMMIDTDVQSLQMSIALYWINSIFVMLYTMECILKLIAFRCFYFTI

AWNIFDFMVVIFSITGLCLPMTVGSYLVPPSLVQLILLSRIIHMLRLGKGPK

VFHNLMLPLMLSLPALLNIILLIFLVMFIYAVFGMYNFAYVKKEAGINDVSN

FETFGNSMLCLFQVAIFAGWDGMLDAIFNSKWSDCDPDKINPGTQVRGDCGN

PSVGIFYFVSYILISWLIIVNMYIVVVESTEPLSEDDFEMFYEVWEKFDPDA

TQFIEFSKLSDFAAALDPPLLIAKPNKVQLIAMDLPMVSGDRIHCLDILFAF

TKRVLGESGEMDSLRSQMEERFMSANPSKVSYEPITTTLKRKQEDVSATVIQ

RAYRRYRLRQNVKNISSIYIKDGDRDDDLLNKKDMAFDNVNENSSPEKTDAT

SSTTSPPSYDSVTKPDKEKYEQDRTEKEDKGKDSKESKK

25
Nax_QTT
MLASPEPKGLVPFTKESFELIKQHIAKTHNEDHEEEDLKPTPDLEVGKKLPF

IYGNLSQGMVSEPLEDVDPYYYKKKNTFIVLNKNRTIFRFNAASILCTLSPF

NCIRRTTIKVLVHPFFQLFILISVLIDCVEMSLTNLPKWRPVLENTLLGIYT

FEILVKLFARGVWAGSFSFLGDPWNWLDFSVTVFEVIIRYSPLDFIPTLQTA

RTLRILKIIPLNQGLKSLVGVLIHCLKQLIGVIILTLFFLSIFSLIGMGLFM

GNLKHKCFRWPQENENETLHNRTGNPYYIRETENFYYLEGERYALLCGNRTD

AGQCPEGYVCVKAGINPDQGFTNEDSFGWALFALFRLMAQDYPEVLYHQILY

ASGKVYMIFFVVVSFLFSFYMASLFLGILAMAYEEEKQRVGEISKKIEPKFQ

QTGKELQEGNETDEAKTIQIEMKKRSPISTDTSLDVLEDATLRHKEELEKSK

KICPLYWYKFAKTFLIWNCSPCWLKLKEFVHRIIMAPFTDLFLIICIILNVC

FLTLEHYPMSKQTNTLLNIGNLVFIGIFTAEMIFKIIAMHPYGYFQVGWNIF

DSMIVFHGLIELCLANVAGMALLRLFRMLRIFKLGKYWPTFQILMWSLSNSW

VALKDLVLLLFTFIFFSAAFGMKLFGKNYEEFVCHIDKDCQLPRWHMHDFFH

SFLNVFRILCGEWVETLWDCMEVAGQSWCIPFYLMVILIGNLLVLYLQLALV

SSFSSCKDVTAEENNEAKNLQLAVARIKKGINYVLLKILCKTQNVPKDTMDH

VNEVYVKEDISDHTLSELSNTQDELKDKEKSSGTEKNATENESQSLIPSPSV

SETVPIASGESDIENLDNKEIQSKSGDGGSKEKIKQSSSSECSTVDIAISEE

EEMFYGGERSKHLKNGCRRGSSLGQISGASKKGKIWQNIRKTCCKIVENNWF

KCFIGLVTLLSTGTLAFEDIYMDQRKTIKILLEYADMIFTYIFILEMLLKWM

AYGFKAYFSNGWYRLDFVVVIVFCLSLIGKTREELKPLISMKFLRPLRVLSQ

FERMKVVVRALIKTTLPTLNVFLVCLMIWLIFSIMGVDLFAGRFYECIDPTS

GERFPSSEVMNKSRCESLLENESMLWENAKMNFDNVGNGFLSLLQVATENGW

ITIMNSAIDSVAVNIQPHFEVNIYMYCYFINFIIFGVFLPLSMLTTVIIDNF

NKHKIKLGGSNIFITVKQRKQYRRLKKLMYEDSQRPVPRPLNKLQGFIFDVV

TSQAFNVIVMVLICFQAIAMMIDTDVQSLQMSIALYWINSIFVMLYTMECIL

KLIAFRCFYFTIAWNIFDFMVVIFSITGLCLPMTVGSYLVPPSLVQLILLSR

IIHMLRLGKGPKVFHNLMLPLMLSLPALLNIILLIFLVMFIYAVFGMYNFAY

VKKEAGINDVSNFETFGNSMLCLFQVAIFAGWDGMLDAIFNSKWSDCDPDKI

NPGTQVRGDCGNPSVGIFYFVSYILISWLIIVNMYTVVVMEFLNIASKKKNK

TLSEDDFRKFFQVWKRFDPDRTQYIDSSKLSDFAAALDPPLFMAKPNKGQLI

ALDLPMAVGDRIHCLDILLAFTKRVMGQDVRMEKVVSEIESGELLANPFKIT

CEPITTTLKRKQEAVSATIIQRAYKNYRLRRNDKNTSDIHMIDGDRDVHATK

EGAYFDKAKEKSPIQSQI

26
Nax_EEE
MLASPEPKGLVPFTKESFELIKQHIAKTHNEDHEEEDLKPTPDLEVGKKLPF

IYGNLSQGMVSEPLEDVDPYYYKKKNTFIVLNKNRTIFRFNAASILCTLSPF

NCIRRTTIKVLVHPFFQLFILISVLIDCVEMSLTNLPKWRPVLENTLLGIYT

FEILVKLFARGVWAGSFSFLGDPWNWLDFSVTVFEVIIRYSPLDFIPTLQTA

RTLRILKIIPLNQGLKSLVGVLIHCLKQLIGVIILTLFFLSIFSLIGMGLFM

GNLKHKCFRWPQENENETLHNRTGNPYYIRETENFYYLEGERYALLCGNRTD

AGQCPEGYVCVKAGINPDQGFTNEDSFGWALFALFRLMAQDYPEVLYHQILY

ASGKVYMIFFVVVSFLFSFYMASLFEGILAMAYEEEKQRVGEISKKIEPKFQ

QTGKELQEGNETDEAKTIQIEMKKRSPISTDTSLDVLEDATLRHKEELEKSK

KICPLYWYKFAKTFLIWNCSPCWLKLKEFVHRIIMAPFTDLFLIICIILNVC

FLTLEHYPMSKQTNTLLNIGNLVFIGIFTAEMIFKIIAMHPYGYFQVGWNIF

DSMIVFHGLIELCLANVAGMALLRLFRMLRIFKLGKYWPTFQILMWSLSNSW

VALKDLVLLLFTFIFFSAAFGMKLFGKNYEEFVCHIDKDCQLPRWHMHDFFH

SFLNVFRILCGEWVETLWDCMEVAGQSWCIPFYLMVILIGNLLVLYLFLALV

SSFSSCKDVTAEENNEAKNLQLAVARIKKGINYVLLKILCKTQNVPKDTMDH

VNEVYVKEDISDHTLSELSNTQDFLKDKEKSSGTEKNATENESQSLIPSPSV

SETVPIASGESDIENLDNKEIQSKSGDGGSKEKIKQSSSSECSTVDIAISEE

EEMFYGGERSKHLKNGCRRGSSLGQISGASKKGKIWQNIRKTCCKIVENNWE

KCFIGLVTLLSTGTLAFEDIYMDQRKTIKILLEYADMIFTYIFILEMLLKWM

AYGFKAYFSNGWYRLDFVVVIVFCLSLIGKTREELKPLISMKFLRPLRVLSQ

FERMKVVVRALIKTTLPTLNVFLVCLMIWLIFSIMGVDLFAGRFYECIDPTS

GERFPSSEVMNKSRCESLLFNESMLWENAKMNFDNVGNGELSLLQVATENGW

ITIMNSAIDSVAVNIQPHFEVNIYMYCYFINFIIFGVFLPLSMLETVIIDNE

NKHKIKLGGSNIFITVKQRKQYRRLKKLMYEDSQRPVPRPLNKLQGFIFDVV

TSQAFNVIVMVLICFQAIAMMIDTDVQSLQMSIALYWINSIFVMLYTMECIL

KLIAFRCFYFTIAWNIFDFMVVIFSITGLCLPMTVGSYLVPPSLVQLILLSR

IIHMLRLGKGPKVFHNLMLPLMLSLPALLNIILLIFLVMFIYAVFGMYNFAY

VKKEAGINDVSNFETFGNSMLCLFQVAIFAGWDGMLDAIFNSKWSDCDPDKI

NPGTQVRGDCGNPSVGIFYFVSYILISWLIIVNMYEVVVMEFLNIASKKKNK

TLSEDDFRKFFQVWKRFDPDRTQYIDSSKLSDFAAALDPPLFMAKPNKGQLI

ALDLPMAVGDRIHCLDILLAFTKRVMGQDVRMEKVVSEIESGELLANPFKIT

CEPITTTLKRKQEAVSATIIQRAYKNYRLRRNDKNTSDIHMIDGDRDVHATK

EGAYFDKAKEKSPIQSQI

27
Human Nax
MLASPEPKGLVPFTKESFELIKQHIAKTHNEDHEEEDLKPTPDLEVGKKLPF

(FIG. 9)
IYGNLSQGMVSEPLEDVDPYYYKKKNTFIVLNKNRTIFRFNAASILCTLSPF

NCIRRTTIKVLVHPFFQLFILISVLIDCVFMSLTNLPKWRPVLENTLLGIYT

FEILVKLFARGVWAGSFSFLGDPWNWLDESVTVFEVIIRYSPLDFIPTLQTA

RTLRILKIIPLNQGLKSLVGVLIHCLKQLIGVIILTLFFLSIFSLIGMGLFM

GNLKHKCFRWPQENENETLHNRTGNPYYIRETENFYYLEGERYALLCGNRTD

AGQCPEGYVCVKAGINPDQGFTNFDSFGWALFALFRLMAQDYPEVLYHQILY

ASGKVYMIFFVVVSFLFSFYMASLFLGILAMAYEEEKQRVGEISKKIEPKFQ

QTGKELQEGNETDEAKTIQIEMKKRSPISTDTSLDVLEDATLRHKEELEKSK

KICPLYWYKFAKTFLIWNCSPCWLKLKEFVHRIIMAPFTDLFLIICIILNVC

FLTLEHYPMSKQTNTLLNIGNLVFIGIFTAEMIFKIIAMHPYGYFQVGWNIF

DSMIVFHGLIELCLANVAGMALLRLFRMLRIFKLGKYWPTFQILMWSLSNSW

VALKDLVLLLFTFIFFSAAFGMKLFGKNYEEFVCHIDKDCQLPRWHMHDFFH

SFLNVFRILCGEWVETLWDCMEVAGQSWCIPFYLMVILIGNLLVLYLFLALV

SSFSSCKDVTAEENNEAKNLQLAVARIKKGINYVLLKILCKTQNVPKDTMDH

VNEVYVKEDISDHTLSELSNTQDFLKDKEKSSGTEKNATENESQSLIPSPSV

SETVPIASGESDIENLDNKEIQSKSGDGGSKEKIKQSSSSECSTVDIAISEE

EEMFYGGERSKHLKNGCRRGSSLGQISGASKKGKIWQNIRKTCCKIVENNWF

KCFIGLVTLLSTGTLAFEDIYMDQRKTIKILLEYADMIFTYIFILEMLLKWM

AYGFKAYFSNGWYRLDFVVVIVFCLSLIGKTREELKPLISMKFLRPLRVLSQ

FERMKVVVRALIKTTLPTLNVFLVCLMIWLIFSIMGVDLFAGRFYECIDPTS

GERFPSSEVMNKSRCESLLFNESMLWENAKMNFDNVGNGFLSLLQVATENGW

ITIMNSAIDSVAVNIQPHFEVNIYMYCYFINFIIFGVFLPLSMLITVIIDNF

NKHKIKLGGSNIFITVKQRKQYRRLKKLMYEDSQRPVPRPLNKLQGFIFDVV

TSQAFNVIVMVLICFQAIAMMIDTDVQSLQMSIALYWINSIFVMLYTMECIL

KLIAFRCFYFTIAWNIFDFMVVIFSITGLCLPMTVGSYLVPPSLVQLILLSR

IIHMLRLGKGPKVFHNLMLPLMLSLPALLNIILLIFLVMFIYAVFGMYNFAY

VKKEAGINDVSNFETFGNSMLCLFQVAIFAGWDGMLDAIFNSKWSDCDPDKI

NPGTQVRGDCGNPSVGIFYFVSYILISWLIIVNMYIVVVMEFLNIASKKKNK

TLSEDDFRKFFQVWKRFDPDRTQYIDSSKLSDFAAALDPPLFMAKPNKGQLI

ALDLPMAVGDRIHCLDILLAFTKRVMGQDVRMEKVVSEIESGELLANPFKIT

CEPITTTLKRKQEAVSATIIQRAYKNYRLRRNDKNTSDIHMIDGDRDVHATK

EGAYFDKAKEKSPIQSQI

28
Human Nav 1.1
MEQTVLVPPGPDSFNFFTRESLAAIERRIAEEKAKNPKPDKKDDDENGPKPN

(FIG. 9)
SDLEAGKNLPFIYGDIPPEMVSEPLEDLDPYYINKKTFIVLNKGKAIFRESA

TSALYILTPFNPLRKIAIKILVHSLFSMLIMCTILTNCVFMTMSNPPDWTKN

VEYTFTGIYTFESLIKIIARGFCLEDFTFLRDPWNWLDFTVITFAYVTEFVD

LGNVSALRTFRVLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVFCLSV

FALIGLQLFMGNLRNKCIQWPPTNASLEEHSIEKNITVNYNGTLINETVFEF

DWKSYIQDSRYHYFLEGFLDALLCGNSSDAGQCPEGYMCVKAGRNPNYGYTS

FDTFSWAFLSLFRLMTQDFWENLYQLTLRAAGKTYMIFFVLVIFLGSFYLIN

LILAVVAMAYEEQNQATLEEAEQKEAEFQQMIEQLKKQQEAAQQAATATASE

HSREPSAAGRLSDSSSEASKLSSKSAKERRNRRKKRKQKEQSGGEEKDEDEF

QKSESEDSIRRKGFRFSIEGNRLTYEKRYSSPHQSLLSIRGSLFSPRRNSRT

SLFSFRGRAKDVGSENDFADDEHSTFEDNESRRDSLFVPRRHGERRNSNLSQ

TSRSSRMLAVFPANGKMHSTVDCNGVVSLVGGPSVPTSPVGQLLPEVIIDKP

ATDDNGTTTETEMRKRRSSSFHVSMDFLEDPSQRQRAMSIASILTNTVEELE

ESRQKCPPCWYKFSNIFLIWDCSPYWLKVKHVVNLVVMDPFVDLAITICIVL

NTLFMAMEHYPMTDHFNNVLTVGNLVFTGIFTAEMFLKIIAMDPYYYFQEGW

NIFDGFIVTLSLVELGLANVEGLSVLRSFRLLRVFKLAKSWPTLNMLIKIIG

NSVGALGNLTLVLAIIVFIFAVVGMQLFGKSYKDCVCKIASDCQLPRWHMND

FFHSFLIVFRVLCGEWIETMWDCMEVAGQAMCLTVFMMVMVIGNLVVLNLFL

ALLLSSFSADNLAATDDDNEMNNLQIAVDRMHKGVAYVKRKIYEFIQQSFIR

KQKILDEIKPLDDLNNKKDSCMSNHTAEIGKDLDYLKDVNGTTSGIGTGSSV

EKYIIDESDYMSFINNPSLTVTVPIAVGESDFENLNTEDESSESDLEESKEK

LNESSSSSEGSTVDIGAPVEEQPVVEPEETLEPEACFTEGCVQRFKCCQINV

EEGRGKQWWNLRRTCFRIVEHNWFETFIVEMILLSSGALAFEDIYIDQRKTI

KTMLEYADKVFTYIFILEMLLKWVAYGYQTYFTNAWCWLDFLIVDVSLVSLT

ANALGYSELGAIKSLRTLRALRPLRALSRFEGMRVVVNALLGAIPSIMNVLL

VCLIFWLIFSIMGVNLFAGKFYHCINTTTGDREDIEDVNNHTDCLKLIERNE

TARWKNVKVNFDNVGFGYLSLLQVATFKGWMDIMYAAVDSRNVELQPKYEES

LYMYLYFVIFIIFGSFFTLNLFIGVIIDNFNQQKKKFGGQDIFMTEEQKKYY

NAMKKLGSKKPQKPIPRPGNKFQGMVFDFVTRQVFDISIMILICLNMVTMMV

ETDDQSEYVTTILSRINLVFIVLFTGECVLKLISLRHYYFTIGWNIFDFVVV

ILSIVGMFLAELIEKYFVSPTLFRVIRLARIGRILRLIKGAKGIRTLLFALM

MSLPALFNIGLLLFLVMFIYAIFGMSNFAYVKREVGIDDMENFETEGNSMIC

LFQITTSAGWDGLLAPILNSKPPDCDPNKVNPGSSVKGDCGNPSVGIFFFVS

YIIISFLVVVNMYIAVILENFSVATEESAEPLSEDDFEMFYEVWEKFDPDAT

QFMEFEKLSQFAAALEPPLNLPQPNKLQLIAMDLPMVSGDRIHCLDILFAFT

KRVLGESGEMDALRIQMEERFMASNPSKVSYQPITTTLKRKQEEVSAVIIQR

AYRRHLLKRTVKQASFTYNKNKIKGGANLLIKEDMIIDRINENSITEKTDLT

MSTAACPPSYDRVTKPIVEKHEQEGKDEKAKGK

29
Human Nav 1.2
MAQSVLVPPGPDSFRFFTRESLAAIEQRIAEEKAKRPKQERKDEDDENGPKP

(FIG. 9)
NSDLEAGKSLPFIYGDIPPEMVSVPLEDLDPYYINKKTFIVLNKGKAISRFS

ATPALYILTPFNPIRKLAIKILVHSLFNMLIMCTILTNCVFMTMSNPPDWTK

NVEYTFTGIYTFESLIKILARGFCLEDFTFLRDPWNWLDFTVITFAYVTEFV

DLGNVSALRTFRVLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVFCLS

VFALIGLQLFMGNLRNKCLQWPPDNSSFEINITSFFNNSLDGNGTTENRTVS

IFNWDEYIEDKSHFYFLEGQNDALLCGNSSDAGQCPEGYICVKAGRNPNYGY

TSFDTFSWAFLSLFRLMTQDFWENLYQLTLRAAGKTYMIFFVLVIFLGSFYL

INLILAVVAMAYEEQNQATLEEAEQKEAEFQQMLEQLKKQQEEAQAAAAAAS

AESRDFSGAGGIGVFSESSSVASKLSSKSEKELKNRRKKKKQKEQSGEEEKN

DRVRKSESEDSIRRKGFRFSLEGSRLTYEKRFSSPHQSLLSIRGSLFSPRRN

SRASLFSFRGRAKDIGSENDFADDEHSTFEDNDSRRDSLFVPHRHGERRHSN

VSQASRASRVLPILPMNGKMHSAVDCNGVVSLVGGPSTLTSAGQLLPEGTTT

ETEIRKRRSSSYHVSMDLLEDPTSRQRAMSIASILTNTMEELEESRQKCPPC

WYKFANMCLIWDCCKPWLKVKHLVNLVVMDPFVDLAITICIVLNTLFMAMEH

YPMTEQFSSVLSVGNLVFTGIFTAEMFLKIIAMDPYYYFQEGWNIFDGFIVS

LSLMELGLANVEGLSVLRSFRLLRVFKLAKSWPTLNMLIKIIGNSVGALGNL

TLVLAIIVFIFAVVGMQLFGKSYKECVCKISNDCELPRWHMHDFFHSFLIVF

RVLCGEWIETMWDCMEVAGQTMCLTVFMMVMVIGNLVVLNLFLALLLSSFSS

DNLAATDDDNEMNNLQIAVGRMQKGIDFVKRKIREFIQKAFVRKQKALDEIK

PLEDLNNKKDSCISNHTTIEIGKDLNYLKDGNGTTSGIGSSVEKYVVDESDY

MSFINNPSLTVTVPIAVGESDFENLNTEEFSSESDMEESKEKLNATSSSEGS

TVDIGAPAEGEQPEVEPEESLEPEACFTEDCVRKFKCCQISIEEGKGKLWWN

LRKTCYKIVEHNWFETFIVEMILLSSGALAFEDIYIEQRKTIKTMLEYADKV

FTYIFILEMLLKWVAYGFQVYFTNAWCWLDFLIVDVSLVSLTANALGYSELG

AIKSLRTLRALRPLRALSRFEGMRVVVNALLGAIPSIMNVLLVCLIFWLIES

IMGVNLFAGKFYHCINYTTGEMFDVSVVNNYSECKALIESNQTARWKNVKVN

FDNVGLGYLSLLQVATFKGWMDIMYAAVDSRNVELQPKYEDNLYMYLYFVIF

IIFGSFFTLNLFIGVIIDNFNQQKKKFGGQDIFMTEEQKKYYNAMKKLGSKK

PQKPIPRPANKFQGMVFDFVTKQVFDISIMILICLNMVTMMVETDDQSQEMT

NILYWINLVFIVLFTGECVLKLISLRYYYFTIGWNIFDFVVVILSIVGMFLA

ELIEKYFVSPTLFRVIRLARIGRILRLIKGAKGIRTLLFALMMSLPALFNIG

LLLFLVMFIYAIFGMSNFAYVKREVGIDDMENFETFGNSMICLFQITTSAGW

DGLLAPILNSGPPDCDPDKDHPGSSVKGDCGNPSVGIFFFVSYIIISFLVVV

NMYIAVILENFSVATEESAEPLSEDDFEMFYEVWEKFDPDATQFIEFAKLSD

FADALDPPLLIAKPNKVQLIAMDLPMVSGDRIHCLDILFAFTKRVLGESGEM

DALRIQMEERFMASNPSKVSYEPITTTLKRKQEEVSAIIIQRAYRRYLLKQK

VKKVSSIYKKDKGKECDGTPIKEDTLIDKLNENSTPEKTDMTPSTTSPPSYD

SVTKPEKEKFEKDKSEKEDKGKDIRESKK

30
Human Nav 1.3
MAQALLVPPGPESFRLFTRESLAAIEKRAAEEKAKKPKKEQDNDDENKPKPN

(FIG. 9)
SDLEAGKNLPFIYGDIPPEMVSEPLEDLDPYYINKKTFIVMNKGKAIFRESA

TSALYILTPLNPVRKIAIKILVHSLFSMLIMCTILTNCVFMTLSNPPDWTKN

VEYTFTGIYTFESLIKILARGFCLEDFTFLRDPWNWLDESVIVMAYVTEFVS

LGNVSALRTFRVLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVFCLSV

FALIGLQLFMGNLRNKCLQWPPSDSAFETNTTSYFNGTMDSNGTFVNVTMST

FNWKDYIGDDSHFYVLDGQKDPLLCGNGSDAGQCPEGYICVKAGRNPNYGYT

SFDTFSWAFLSLFRLMTQDYWENLYQLTLRAAGKTYMIFFVLVIFLGSFYLV

NLILAVVAMAYEEQNQATLEEAEQKEAEFQQMLEQLKKQQEEAQAVAAASAA

SRDESGIGGLGELLESSSEASKLSSKSAKEWRNRRKKRRQREHLEGNNKGER

DSFPKSESEDSVKRSSFLFSMDGNRLTSDKKFCSPHQSLLSIRGSLESPRRN

SKTSIFSFRGRAKDVGSENDFADDEHSTFEDSESRRDSLFVPHRHGERRNSN

VSQASMSSRMVPGLPANGKMHSTVDCNGVVSLVGGPSALTSPTGQLPPEGTT

TETEVRKRRLSSYQISMEMLEDSSGRQRAVSIASILTNTMEELEESRQKCPP

CWYRFANVFLIWDCCDAWLKVKHLVNLIVMDPFVDLAITICIVLNTLFMAME

HYPMTEQFSSVLTVGNLVFTGIFTAEMVLKIIAMDPYYYFQEGWNIFDGIIV

SLSLMELGLSNVEGLSVLRSFRLLRVFKLAKSWPTLNMLIKIIGNSVGALGN

LTLVLAIIVFIFAVVGMQLFGKSYKECVCKINDDCTLPRWHMNDFFHSFLIV

FRVLCGEWIETMWDCMEVAGQTMCLIVFMLVMVIGNLVVLNLFLALLLSSES

SDNLAATDDDNEMNNLQIAVGRMQKGIDYVKNKMRECFQKAFFRKPKVIEIH

EGNKIDSCMSNNTGIEISKELNYLRDGNGTTSGVGTGSSVEKYVIDENDYMS

FINNPSLTVTVPIAVGESDFENLNTEEFSSESELEESKEKLNATSSSEGSTV

DVVLPREGEQAETEPEEDLKPEACFTEGCIKKFPFCQVSTEEGKGKIWWNLR

KTCYSIVEHNWFETFIVEMILLSSGALAFEDIYIEQRKTIKTMLEYADKVFT

YIFILEMLLKWVAYGFQTYFTNAWCWLDFLIVDVSLVSLVANALGYSELGAI

KSLRTLRALRPLRALSRFEGMRVVVNALVGAIPSIMNVLLVCLIFWLIFSIM

GVNLFAGKFYHCVNMTTGNMFDISDVNNLSDCQALGKQARWKNVKVNEDNVG

AGYLALLQVATFKGWMDIMYAAVDSRDVKLQPVYEENLYMYLYFVIFIIFGS

FFTLNLFIGVIIDNFNQQKKKFGGQDIFMTEEQKKYYNAMKKLGSKKPQKPI

PRPANKFQGMVFDFVTRQVEDISIMILICLNMVTMMVETDDQGKYMTLVLSR

INLVFIVLFTGEFVLKLVSLRHYYFTIGWNIFDFVVVILSIVGMFLAEMIEK

YFVSPTLFRVIRLARIGRILRLIKGAKGIRTLLFALMMSLPALFNIGLLLFL

VMFIYAIFGMSNFAYVKKEAGIDDMFNFETFGNSMICLFQITTSAGWDGLLA

PILNSAPPDCDPDTIHPGSSVKGDCGNPSVGIFFFVSYIIISFLVVVNMYIA

VILENFSVATEESAEPLSEDDFEMFYEVWEKFDPDATQFIEFSKLSDFAAAL

DPPLLIAKPNKVQLIAMDLPMVSGDRIHCLDILFAFTKRVLGESGEMDALRI

QMEDRFMASNPSKVSYEPITTTLKRKQEEVSAAIIQRNFRCYLLKQRLKNIS

SNYNKEAIKGRIDLPIKQDMIIDKLNGNSTPEKTDGSSSTTSPPSYDSVTKP

DKEKFEKDKPEKESKGKEVRENQK

31
Human Nav 1.4
MARPSLCTLVPLGPECLRPFTRESLAAIEQRAVEEEARLQRNKQMEIEEPER

(FIG. 9)
KPRSDLEAGKNLPMIYGDPPPEVIGIPLEDLDPYYSNKKTFIVLNKGKAIFR

FSATPALYLLSPFSVVRRGAIKVLIHALFSMFIMITILTNCVFMTMSDPPPW

SKNVEYTFTGIYTFESLIKILARGFCVDDFTFLRDPWNWLDFSVIMMAYLTE

FVDLGNISALRTFRVLRALKTITVIPGLKTIVGALIQSVKKLSDVMILTVFC

LSVFALVGLQLFMGNLRQKCVRWPPPENDTNTTWYSNDTWYGNDTWYGNEMW

YGNDSWYANDTWNSHASWATNDTFDWDAYISDEGNFYFLEGSNDALLCGNSS

DAGHCPEGYECIKTGRNPNYGYTSYDTFSWAFLALFRLMTQDYWENLFQLTL

RAAGKTYMIFFVVIIFLGSFYLINLILAVVAMAYAEQNEATLAEDKEKEEEF

QQMLEKFKKHQEELEKAKAAQALEGGEADGDPAHGKDCNGSLDTSQGEKGAP

RQSSSGDSGISDAMEELEEAHQKCPPWWYKCAHKVLIWNCCAPWLKFKNIIH

LIVMDPFVDLGITICIVLNTLFMAMEHYPMTEHFDNVLTVGNLVFTGIFTAE

MVLKLIAMDPYEYFQQGWNIFDSIIVTLSLVELGLANVQGLSVLRSFRLLRV

FKLAKSWPTLNMLIKIIGNSVGALGNLTLVLAIIVFIFAVVGMQLFGKSYKE

CVCKIALDCNLPRWHMHDFFHSFLIVFRILCGEWIETMWDCMEVAGQAMCLT

VFLMVMVIGNLVVLNLFLALLLSSFSADSLAASDEDGEMNNLQIAIGRIKLG

IGFAKAFLLGLLHGKILSPKDIMLSLGEADGAGEAGEAGETAPEDEKKEPPE

EDLKKDNHILNHMGLADGPPSSLELDHLNFINNPYLTIQVPIASEESDLEMP

TEEETDTFSEPEDSKKPWPCLYVDISQGRGKKWWTLRRACFKIVEHNWFETF

IVFMILLSSGALAFEDIYIEQRRVIRTILEYADKVFTYIFIMEMLLKWVAYG

FKVYFTNAWCWLDFLIVDVSIISLVANWLGYSELGPIKSLRTLRALRPLRAL

SRFEGMRVVVNALLGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGKFYYCINT

TTSERFDISEVNNKSECESLMHTGQVRWLNVKVNYDNVGLGYLSLLQVATFK

GWMDIMYAAVDSREKEEQPQYEVNLYMYLYFVIFIIFGSFFTLNLFIGVIID

NFNQQKKKLGGKDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPQNKIQGMVYD

LVTKQAFDITIMILICLNMVTMMVETDNQSQLKVDILYNINMIFIIIFTGEC

VLKMLALRQYYFTVGWNIFDFVVVILSIVGLALSDLIQKYFVSPTLFRVIRL

ARIGRVLRLIRGAKGIRTLLFALMMSLPALFNIGLLLFLVMFIYSIFGMSNF

AYVKKESGIDDMFNFETFGNSIICLFEITTSAGWDGLLNPILNSGPPDCDPN

LENPGTSVKGDCGNPSIGICFFCSYIIISFLIVVNMYIAIILENENVATEES

SEPLGEDDFEMFYETWEKFDPDATQFIAYSRLSDFVDTLQEPLRIAKPNKIK

LITLDLPMVPGDKIHCLDILFALTKEVLGDSGEMDALKQTMEEKFMAANPSK

VSYEPITTTLKRKHEEVCAIKIQRAYRRHLLQRSMKQASYMYRHSHDGSGDD

APEKEGLLANTMSKMYGHENGNSSSPSPEEKGEAGDAGPTMGLMPISPSDTA

WPPAPPPGQTVRPGVKESLV

32
Human Nav 1.5
MANFLLPRGTSSFRRFTRESLAAIEKRMAEKQARGSTTLQESREGLPEEEAP

(FIG. 9)
RPQLDLQASKKLPDLYGNPPQELIGEPLEDLDPFYSTQKTFIVLNKGKTIFR

FSATNALYVLSPFHPIRRAAVKILVHSLENMLIMCTILTNCVFMAQHDPPPW

TKYVEYTFTAIYTFESLVKILARGFCLHAFTFLRDPWNWLDESVIIMAYTTE

FVDLGNVSALRTFRVLRALKTISVISGLKTIVGALIQSVKKLADVMVLTVFC

LSVFALIGLQLFMGNLRHKCVRNFTALNGTNGSVEADGLVWESLDLYLSDPE

NYLLKNGTSDVLLCGNSSDAGTCPEGYRCLKAGENPDHGYTSFDSFAWAFLA

LFRLMTQDCWERLYQQTLRSAGKIYMIFFMLVIFLGSFYLVNLILAVVAMAY

EEQNQATIAETEEKEKRFQEAMEMLKKEHEALTIRGVDTVSRSSLEMSPLAP

VNSHERRSKRRKRMSSGTEECGEDRLPKSDSEDGPRAMNHLSLTRGLSRTSM

KPRSSRGSIFTFRRRDLGSEADFADDENSTAGESESHHTSLLVPWPLRRTSA

QGQPSPGTSAPGHALHGKKNSTVDCNGVVSLLGAGDPEATSPGSHLLRPVML

EHPPDTTTPSEEPGGPQMLTSQAPCVDGFEEPGARQRALSAVSVLTSALEEL

EESRHKCPPCWNRLAQRYLIWECCPLWMSIKQGVKLVVMDPFTDLTITMCIV

LNTLFMALEHYNMTSEFEEMLQVGNLVFTGIFTAEMTFKIIALDPYYYFQQG

WNIFDSIIVILSLMELGLSRMSNLSVLRSFRLLRVFKLAKSWPTLNTLIKII

GNSVGALGNLTLVLAIIVFIFAVVGMQLFGKNYSELRDSDSGLLPRWHMMDE

FHAFLIIFRILCGEWIETMWDCMEVSGQSLCLLVFLLVMVIGNLVVLNLFLA

LLLSSFSADNLTAPDEDREMNNLQLALARIQRGLRFVKRTTWDFCCGLLRQR

PQKPAALAAQGQLPSCIATPYSPPPPETEKVPPTRKETRFEEGEQPGQGTPG

DPEPVCVPIAVAESDTDDQEEDEENSLGTEEESSKQQESQPVSGGPEAPPDS

RTWSQVSATASSEAEASASQADWRQQWKAEPQAPGCGETPEDSCSEGSTADM

TNTAELLEQIPDLGQDVKDPEDCFTEGCVRRCPCCAVDTTQAPGKVWWRLRK

TCYHIVEHSWFETFIIFMILLSSGALAFEDIYLEERKTIKVLLEYADKMFTY

VFVLEMLLKWVAYGFKKYFTNAWCWLDFLIVDVSLVSLVANTLGFAEMGPIK

SLRTLRALRPLRALSRFEGMRVVVNALVGAIPSIMNVLLVCLIFWLIFSIMG

VNLFAGKFGRCINQTEGDLPLNYTIVNNKSQCESLNLTGELYWTKVKVNEDN

VGAGYLALLQVATFKGWMDIMYAAVDSRGYEEQPQWEYNLYMYIYFVIFIIF

GSFFTLNLFIGVIIDNFNQQKKKLGGQDIFMTEEQKKYYNAMKKLGSKKPQK

PIPRPLNKYQGFIFDIVTKQAFDVTIMFLICLNMVTMMVETDDQSPEKINIL

AKINLLEVAIFTGECIVKLAALRHYYFTNSWNIFDFVVVILSIVGTVLSDII

QKYFFSPTLFRVIRLARIGRILRLIRGAKGIRTLLFALMMSLPALFNIGLLL

FLVMFIYSIFGMANFAYVKWEAGIDDMFNFQTFANSMLCLFQITTSAGWDGL

LSPILNTGPPYCDPTLPNSNGSRGDCGSPAVGILFFTTYIIISFLIVVNMYI

AIILENFSVATEESTEPLSEDDFDMFYEIWEKFDPEATQFIEYSVLSDFADA

LSEPLRIAKPNQISLINMDLPMVSGDRIHCMDILFAFTKRVLGESGEMDALK

IQMEEKFMAANPSKISYEPITTTLRRKHEEVSAMVIQRAFRRHLLQRSLKHA

SFLFRQQAGSGLSEEDAPEREGLIAYVMSENFSRPLGPPSSSSISSTSFPPS

YDSVTRATSDNLQVRGSDYSHSEDLADFPPSPDRDRESIV

33
Human Nav 1.6
MAARLLAPPGPDSFKPFTPESLANIERRIAESKLKKPPKADGSHREDDEDSK

(FIG. 9)
PKPNSDLEAGKSLPFIYGDIPQGLVAVPLEDFDPYYLTQKTFVVLNRGKTLF

RFSATPALYILSPFNLIRRIAIKILIHSVFSMIIMCTILTNCVFMTFSNPPD

WSKNVEYTFTGIYTFESLVKIIARGFCIDGFTFLRDPWNWLDFSVIMMAYIT

EFVNLGNVSALRTFRVLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVE

CLSVFALIGLQLFMGNLRNKCVVWPINFNESYLENGTKGEDWEEYINNKTNF

YTVPGMLEPLLCGNSSDAGQCPEGYQCMKAGRNPNYGYTSFDTFSWAFLALF

RLMTQDYWENLYQLTLRAAGKTYMIFFVLVIFVGSFYLVNLILAVVAMAYEE

QNQATLEEAEQKEAEFKAMLEQLKKQQEEAQAAAMATSAGTVSEDAIEEEGE

EGGGSPRSSSEISKLSSKSAKERRNRRKKRKQKELSEGEEKGDPEKVFKSES

EDGMRRKAFRLPDNRIGRKESIMNQSLLSIPGSPFLSRHNSKSSIFSFRGPG

RFRDPGSENEFADDEHSTVEESEGRRDSLFIPIRARERRSSYSGYSGYSQGS

RSSRIFPSLRRSVKRNSTVDCNGVVSLIGGPGSHIGGRLLPEATTEVEIKKK

GPGSLLVSMDQLASYGRKDRINSIMSVVTNTLVEELEESQRKCPPCWYKFAN

TFLIWECHPYWIKLKEIVNLIVMDPFVDLAITICIVLNTLFMAMEHHPMTPQ

FEHVLAVGNLVFTGIFTAEMFLKLIAMDPYYYFQEGWNIFDGFIVSLSLMEL

SLADVEGLSVLRSFRLLRVFKLAKSWPTLNMLIKIIGNSVGALGNLTLVLAI

IVFIFAVVGMQLFGKSYKECVCKINQDCELPRWHMHDFFHSFLIVERVLCGE

WIETMWDCMEVAGQAMCLIVFMMVMVIGNLVVLNLFLALLLSSFSADNLAAT

DDDGEMNNLQISVIRIKKGVAWTKLKVHAFMQAHFKQREADEVKPLDELYEK

KANCIANHTGADIHRNGDFQKNGNGTTSGIGSSVEKYIIDEDHMSFINNPNL

TVRVPIAVGESDFENLNTEDVSSESDPEGSKDKLDDTSSSEGSTIDIKPEVE

EVPVEQPEEYLDPDACFTEGCVQRFKCCQVNIEEGLGKSWWILRKTCFLIVE

HNWFETFIIFMILLSSGALAFEDIYIEQRKTIRTILEYADKVETYIFILEML

LKWTAYGFVKFFTNAWCWLDFLIVAVSLVSLIANALGYSELGAIKSLRTLRA

LRPLRALSRFEGMRVVVNALVGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGK

YHYCFNETSEIRFEIEDVNNKTECEKLMEGNNTEIRWKNVKINFDNVGAGYL

ALLQVATFKGWMDIMYAAVDSRKPDEQPKYEDNIYMYIYFVIFIIFGSFFTL

NLFIGVIIDNFNQQKKKFGGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPL

NKIQGIVFDFVTQQAFDIVIMMLICLNMVTMMVETDTQSKQMENILYWINLV

FVIFFTCECVLKMFALRHYYFTIGWNIFDFVVVILSIVGMFLADIIEKYFVS

PTLFRVIRLARIGRILRLIKGAKGIRTLLFALMMSLPALFNIGLLLFLVMFI

FSIFGMSNFAYVKHEAGIDDMFNFETFGNSMICLFQITTSAGWDGLLLPILN

RPPDCSLDKEHPGSGFKGDCGNPSVGIFFFVSYIIISFLIVVNMYIAIILEN

FSVATEESADPLSEDDFETFYEIWEKFDPDATQFIEYCKLADFADALEHPLR

VPKPNTIELIAMDLPMVSGDRIHCLDILFAFTKRVLGDSGELDILRQQMEER

FVASNPSKVSYEPITTTLRRKQEEVSAVVLQRAYRGHLARRGFICKKTTSNK

LENGGTHREKKESTPSTASLPSYDSVTKPEKEKQQRAEEGRRERAKRQKEVR

ESKC

34
Human Nav 1.7
MAMLPPPGPQSFVHFTKQSLALIEQRIAERKSKEPKEEKKDDDEEAPKPSSD

(FIG. 9)
LEAGKQLPFIYGDIPPGMVSEPLEDLDPYYADKKTFIVLNKGKTIFRFNATP

ALYMLSPFSPLRRISIKILVHSLESMLIMCTILTNCIFMTMNNPPDWTKNVE

YTFTGIYTFESLVKILARGFCVGEFTFLRDPWNWLDFVVIVFAYLTEFVNLG

NVSALRTFRVLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVFCLSVFA

LIGLQLFMGNLKHKCFRNSLENNETLESIMNTLESEEDFRKYFYYLEGSKDA

LLCGESTDSGQCPEGYTCVKIGRNPDYGYTSFDTFSWAFLALFRLMTQDYWE

NLYQQTLRAAGKTYMIFFVVVIFLGSFYLINLILAVVAMAYEEQNQANIEEA

KQKELEFQQMLDRLKKEQEEAEAIAAAAAEYTSIRRSRIMGLSESSSETSKL

SSKSAKERRNRRKKKNQKKLSSGEEKGDAEKLSKSESEDSIRRKSFHLGVEG

HRRAHEKRLSTPNQSPLSIRGSLESARRSSRTSLFSFKGRGRDIGSETEFAD

DEHSIFGDNESRRGSLFVPHRPQERRSSNISQASRSPPMLPVNGKMHSAVDC

NGVVSLVDGRSALMLPNGQLLPEVIIDKATSDDSGTTNQIHKKRRCSSYLLS

EDMLNDPNLRQRAMSRASILTNTVEELEESRQKCPPWWYRFAHKFLIWNCSP

YWIKFKKCIYFIVMDPFVDLAITICIVLNTLFMAMEHHPMTEEFKNVLAIGN

LVFTGIFAAEMVLKLIAMDPYEYFQVGWNIFDSLIVTLSLVELFLADVEGLS

VLRSFRLLRVFKLAKSWPTLNMLIKIIGNSVGALGNLTLVLAIIVFIFAVVG

MQLFGKSYKECVCKINDDCTLPRWHMNDFFHSFLIVERVLCGEWIETMWDCM

EVAGQAMCLIVYMMVMVIGNLVVLNLFLALLLSSFSSDNLTAIEEDPDANNL

QIAVTRIKKGINYVKQTLREFILKAFSKKPKISREIRQAEDLNTKKENYISN

HTLAEMSKGHNFLKEKDKISGFGSSVDKHLMEDSDGQSFIHNPSLTVTVPIA

PGESDLENMNAEELSSDSDSEYSKVRLNRSSSSECSTVDNPLPGEGEEAEAE

PMNSDEPEACFTDGCVWRFSCCQVNIESGKGKIWWNIRKTCYKIVEHSWFES

FIVLMILLSSGALAFEDIYIERKKTIKIILEYADKIFTYIFILEMLLKWIAY

GYKTYFTNAWCWLDFLIVDVSLVTLVANTLGYSDLGPIKSLRTLRALRPLRA

LSRFEGMRVVVNALIGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGKFYECIN

TTDGSRFPASQVPNRSECFALMNVSQNVRWKNLKVNFDNVGLGYLSLLQVAT

FKGWTIIMYAAVDSVNVDKQPKYEYSLYMYIYFVVFIIFGSFFTLNLFIGVI

IDNFNQQKKKLGGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPGNKIQGCI

FDLVTNQAFDISIMVLICLNMVTMMVEKEGQSQHMTEVLYWINVVFIILFTG

ECVLKLISLRHYYFTVGWNIFDFVVVIISIVGMFLADLIETYFVSPTLFRVI

RLARIGRILRLVKGAKGIRTLLFALMMSLPALFNIGLLLFLVMFIYAIFGMS

NFAYVKKEDGINDMFNFETFGNSMICLFQITTSAGWDGLLAPILNSKPPDCD

PKKVHPGSSVEGDCGNPSVGIFYFVSYIIISFLVVVNMYIAVILENFSVATE

ESTEPLSEDDFEMFYEVWEKFDPDATQFIEFSKLSDFAAALDPPLLIAKPNK

VQLIAMDLPMVSGDRIHCLDILFAFTKRVLGESGEMDSLRSQMEERFMSANP

SKVSYEPITTTLKRKQEDVSATVIQRAYRRYRLRQNVKNISSIYIKDGDRDD

DLLNKKDMAFDNVNENSSPEKTDATSSTTSPPSYDSVTKPDKEKYEQDRTEK

EDKGKDSKESKK

35
Human Nav 1.8
MEFPIGSLETNNFRRFTPESLVEIEKQIAAKQGTKKAREKHREQKDQEEKPR

(FIG. 9)
PQLDLKACNQLPKFYGELPAELIGEPLEDLDPFYSTHRTFMVLNKGRTISRF

SATRALWLFSPENLIRRTAIKVSVHSWFSLFITVTILVNCVCMTRTDLPEKI

EYVFTVIYTFEALIKILARGFCLNEFTYLRDPWNWLDFSVITLAYVGTAIDL

RGISGLRTFRVLRALKTVSVIPGLKVIVGALIHSVKKLADVTILTIFCLSVF

ALVGLQLFKGNLKNKCVKNDMAVNETTNYSSHRKPDIYINKRGTSDPLLCGN

GSDSGHCPDGYICLKTSDNPDFNYTSFDSFAWAFLSLFRLMTQDSWERLYQQ

TLRTSGKIYMIFFVLVIFLGSFYLVNLILAVVTMAYEEQNQATTDEIEAKEK

KFQEALEMLRKEQEVLAALGIDTTSLHSHNGSPLTSKNASERRHRIKPRVSE

GSTEDNKSPRSDPYNQRRMSFLGLASGKRRASHGSVFHFRSPGRDISLPEGV

TDDGVFPGDHESHRGSLLLGGGAGQQGPLPRSPLPQPSNPDSRHGEDEHQPP

PTSELAPGAVDVSAFDAGQKKTFLSAEYLDEPFRAQRAMSVVSIITSVLEEL

EESEQKCPPCLTSLSQKYLIWDCCPMWVKLKTILFGLVTDPFAELTITLCIV

VNTIFMAMEHHGMSPTFEAMLQIGNIVFTIFFTAEMVFKIIAFDPYYYFQKK

WNIFDCIIVTVSLLELGVAKKGSLSVLRSFRLLRVFKLAKSWPTLNTLIKII

GNSVGALGNLTIILAIIVFVFALVGKQLLGENYRNNRKNISAPHEDWPRWHM

HDFFHSFLIVFRILCGEWIENMWACMEVGQKSICLILFLTVMVLGNLVVLNL

FIALLLNSFSADNLTAPEDDGEVNNLQVALARIQVFGHRTKQALCSFFSRSC

PFPQPKAEPELVVKLPLSSSKAENHIAANTARGSSGGLQAPRGPRDEHSDFI

ANPTVWVSVPIAEGESDLDDLEDDGGEDAQSFQQEVIPKGQQEQLQQVERCG

DHLTPRSPGTGTSSEDLAPSLGETWKDESVPQVPAEGVDDTSSSEGSTVDCL

DPEEILRKIPELADDLEEPDDCFTEGCIRHCPCCKLDTTKSPWDVGWQVRKT

CYRIVEHSWFESFIIFMILLSSGSLAFEDYYLDQKPTVKALLEYTDRVFTFI

FVFEMLLKWVAYGFKKYFTNAWCWLDFLIVNISLISLTAKILEYSEVAPIKA

LRTLRALRPLRALSRFEGMRVVVDALVGAIPSIMNVLLVCLIFWLIFSIMGV

NLFAGKFWRCINYTDGEFSLVPLSIVNNKSDCKIQNSTGSFFWVNVKVNEDN

VAMGYLALLQVATFKGWMDIMYAAVDSREVNMQPKWEDNVYMYLYFVIFIIF

GGFFTLNLFVGVIIDNFNQQKKKLGGQDIFMTEEQKKYYNAMKKLGSKKPQK

PIPRPLNKFQGFVFDIVTRQAFDITIMVLICLNMITMMVETDDQSEEKTKIL

GKINQFFVAVFTGECVMKMFALRQYYFTNGWNVFDFIVVVLSIASLIFSAIL

KSLQSYFSPTLERVIRLARIGRILRLIRAAKGIRTLLFALMMSLPALENIGL

LLFLVMFIYSIFGMSSFPHVRWEAGIDDMFNFQTFANSMLCLFQITTSAGWD

GLLSPILNTGPPYCDPNLPNSNGTRGDCGSPAVGIIFFTTYIIISFLIMVNM

YIAVILENENVATEESTEPLSEDDFDMFYETWEKFDPEATQFITFSALSDFA

DTLSGPLRIPKPNRNILIQMDLPLVPGDKIHCLDILFAFTKNVLGESGELDS

LKANMEEKFMATNLSKSSYEPIATTLRWKQEDISATVIQKAYRSYVLHRSMA

LSNTPCVPRAEEEAASLPDEGFVAFTANENCVLPDKSETASATSFPPSYESV

TRGLSDRVNMRTSSSIQNEDEATSMELIAPGP

36
Human Nav 1.9
MDDRCYPVIFPDERNFRPFTSDSLAAIEKRIAIQKEKKKSKDQTGEVPQPRP

(FIG. 9)
QLDLKASRKLPKLYGDIPRELIGKPLEDLDPFYRNHKTFMVLNRKRTIYRES

AKHALFIFGPFNSIRSLAIRVSVHSLFSMFIIGTVIINCVFMATGPAKNSNS

NNTDIAECVFTGIYIFEALIKILARGFILDEFSFLRDPWNWLDSIVIGIAIV

SYIPGITIKLLPLRTFRVFRALKAISVVSRLKVIVGALLRSVKKLVNVIILT

FFCLSIFALVGQQLFMGSLNLKCISRDCKNISNPEAYDHCFEKKENSPEFKM

CGIWMGNSACSIQYECKHTKINPDYNYTNFDNFGWSFLAMFRLMTQDSWEKL

YQQTLRTTGLYSVFFFIVVIFLGSFYLINLTLAVVTMAYEEQNKNVAAEIEA

KEKMFQEAQQLLKEEKEALVAMGIDRSSLTSLETSYFTPKKRKLFGNKKRKS

FFLRESGKDQPPGSDSDEDCQKKPQLLEQTKRLSQNLSLDHFDEHGDPLQRQ

RALSAVSILTITMKEQEKSQEPCLPCGENLASKYLVWNCCPQWLCVKKVLRT

VMTDPFTELAITICIIINTVFLAMEHHKMEASFEKMLNIGNLVFTSIFIAEM

CLKIIALDPYHYFRRGWNIFDSIVALLSFADVMNCVLQKRSWPFLRSFRVLR

VFKLAKSWPTLNTLIKIIGNSVGALGSLTVVLVIVIFIFSVVGMQLFGRSEN

SQKSPKLCNPTGPTVSCLRHWHMGDFWHSFLVVFRILCGEWIENMWECMQEA

NASSSLCVIVFILITVIGKLVVLNLFIALLLNSESNEERNGNLEGEARKTKV

QLALDRFRRAFCFVRHTLEHFCHKWCRKQNLPQQKEVAGGCAAQSKDIIPLV

MEMKRGSETQEELGILTSVPKTLGVRHDWTWLAPLAEEEDDVEFSGEDNAQR

ITQPEPEQQAYELHQENKKPTSQRVQSVEIDMFSEDEPHLTIQDPRKKSDVT

SILSECSTIDLQDGFGWLPEMVPKKQPERCLPKGFGCCFPCCSVDKRKPPWV

IWWNLRKTCYQIVKHSWFESFIIFVILLSSGALIFEDVHLENQPKIQELLNC

TDIIFTHIFILEMVLKWVAFGFGKYFTSAWCCLDFIIVIVSVTTLINLMELK

SFRTLRALRPLRALSQFEGMKVVVNALIGAIPAILNVLLVCLIFWLVFCILG

VYFFSGKFGKCINGTDSVINYTIITNKSQCESGNFSWINQKVNFDNVGNAYL

ALLQVATFKGWMDIIYAAVDSTEKEQQPEFESNSLGYIYFVVFIIFGSFFTL

NLFIGVIIDNFNQQQKKLGGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPL

NKCQGLVEDIVTSQIFDIIIISLIILNMISMMAESYNQPKAMKSILDHLNWV

FVVIFTLECLIKIFALRQYYFTNGWNLFDCVVVLLSIVSTMISTLENQEHIP

FPPTLFRIVRLARIGRILRLVRAARGIRTLLFALMMSLPSLFNIGLLLFLIM

FIYAILGMNWFSKVNPESGIDDIFNFKTFASSMLCLFQISTSAGWDSLLSPM

LRSKESCNSSSENCHLPGIATSYFVSYIIISFLIVVNMYIAVILENENTATE

ESEDPLGEDDFDIFYEVWEKFDPEATQFIKYSALSDFADALPEPLRVAKPNK

YQFLVMDLPMVSEDRLHCMDILFAFTARVLGGSDGLDSMKAMMEEKFMEANP

LKKLYEPIVTTTKRKEEERGAAIIQKAFRKYMMKVTKGDQGDQNDLENGPHS

PLQTLCNGDLSSFGVAKGKVHCD

37
Human Nax 1204-
GGSNIFITVKQRKQYRRLKKLMYEDSQRPVPRPLNK

1239

(FIG. 14B)

38
Human Nav 1.1
GGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPGNK

1494-1529

(FIG. 14B)

39
Human Nav 1.2
GGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPANK

1484-1519

(FIG. 14B)

40
Human Nav 1.3
GGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPANK

1479-1514

(FIG. 14B)

41
Human Nav 1.4
GGKDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPQNK

1206-1241

(FIG. 14B)

42
Human Nav 1.5
GGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPLNK

1481-1516

(FIG. 14B)

43
Human Nav 1.6
GGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPLNK

1475-1510

(FIG. 14B)

44
Human Nav 1.7
GGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPGNK

1468-1503

(FIG. 14B)

45
Human Nav 1.8
GGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPLNK

1429-1464

(FIG. 14B)

46
Human Nav 1.9
GGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPLNK

1319-1354

(FIG. 14B)

47
Na_x_D1_S6 (FIG.
GKVYMIFFVVVSFLFSFYMASLFLGILAMAY

20)

48
Na_v1.1_D1_S6
GKTYMIFFVLVIFLGSFYLINLILAVVAMAY

(FIG. 20)

49
Na_v1.2_D1_S6
GKTYMIFFVLVIFLGSFYLINLILAVVAMAY

(FIG. 20)

50
Na_v1.3_D1_S6
GKTYMIFFVLVIFLGSFYLVNLILAVVAMAY

(FIG. 20)

51
Na_v1.4_D1_S6
GKTYMIFFVVIIFLGSFYLINLILAVVAMAY

(FIG. 20)

52
Na_y1.5_D1_S6
GKIYMIFFMLVIFLGSFYLVNLILAVVAMAY

(FIG. 20)

53
Na_v1.6_D1_S6
GKTYMIFFVLVIFVGSFYLVNLILAVVAMAY

(FIG. 20)

54
Na_v1.7_D1_S6
GKTYMIFFVVVIFLGSFYLINLILAVVAMAY

(FIG. 20)

55
Na_v1.8_D1_S6
GKIYMIFFVLVIFLGSFYLVNLILAVVTMAY

(FIG. 20)

56
Na_v1.9_D1_S6
GLYSVFFFIVVIFLGSFYLINLTLAVVTMAY

(FIG. 20)

57
Na_x_D2_S6 (FIG.
QSWCIPFYLMVILIGNLLVLYLFLALVSSF

20)

58
Na_v1.1_D2_S6
QAMCLTVFMMVMVIGNLVVLNLFLALLLSSF

(FIG. 20)

59
Na_v1.2_D2_S6
QTMCLTVFMMVMVIGNLVVLNLFLALLLSSF

(FIG. 20)

60
Na_v1.3_D2_S6
QTMCLIVFMLVMVIGNLVVLNLFLALLLSSF

(FIG. 20)

61
Na_v1.4_D2_S6
QAMCLTVFLMVMVIGNLVVLNLFLALLLSSF

(FIG. 20)

62
Na_v1.5_D2_S6
QSLCLLVFLLVMVIGNLVVLNLFLALLLSSF

(FIG. 20)

63
Na_v1.6_D2_S6
QAMCLIVFMMVMVIGNLVVLNLFLALLLSSF

(FIG. 20)

64
Na_v1.7_D2_S6
QAMCLIVYMMVMVIGNLVVLNLFLALLLSSF

(FIG. 20)

65
Na_v1.8_D2_S6
KSICLILFLTVMVLGNLVVLNLFIALLLNSF

(FIG. 20)

66
Na_v1.9_D2_S6
SSLCVIVFILITVIGKLVVLNLFIALLLNSF

(FIG. 20)

67
Na_x_D3_S6 (FIG.
NIYMYCYFINFIIFGVFLPLSMLITVIIDNF

20)

68
Na_v1.1_D3_S6
SLYMYLYFVIFIIFGSFFTLNLFIGVIIDNF

(FIG. 20)

69
Na_v1.2_D3_S6
NLYMYLYFVIFIIFGSFFTLNLFIGVIIDNF

(FIG. 20)

70
Na_v1.3_D3_S6
NLYMYLYFVIFIIFGSFFTLNLFIGVIIDNF

(FIG. 20)

71
Na_v1.4_D3_S6
NLYMYLYFVIFIIFGSFFTLNLFIGVIIDNE

(FIG. 20)

72
Na_v1.5_D3_S6
NLYMYIYFVIFIIFGSFFTLNLFIGVIIDNF

(FIG. 20)

73
Na_v1.6_D3_S6
NIYMYIYFVIFIIFGSFFTLNLFIGVIIDNF

(FIG. 20)

74
Na_v1.7_D3_S6
SLYMYIYFVVFIIFGSFFTLNLFIGVIIDNE

(FIG. 20)

75
Na_v1.8_D3_S6
NVYMYLYFVIFIIFGGFFTLNLFVGVIIDNF

(FIG. 20)

76
Na_v1.9_D3_S6
NSLGYIYFVVFIIFGSFFTLNLFIGVIIDNF

(FIG. 20)

77
Na_x_D4_S6
PSVGIFYFVSYILISWLIIVNMYIVVVMEFL

(FIG. 20)

78
Na_v1.1_D4_S6
PSVGIFFFVSYIIISFLVVVNMYIAVILENF

(FIG. 20)

79
Na_v1.2_D4_S6
PSVGIFFFVSYIIISFLVVVNMYIAVILENF

(FIG. 20)

80
Na_v1.3_D4_S6
PSVGIFFFVSYIIISFLVVVNMYIAVILENF

(FIG. 20)

81
Na_v1.4_D4_S6
PSIGICFFCSYIIISFLIVVNMYIAIILENE

(FIG. 20)

82
Na_v1.5_D4_S6
PAVGILFFTTYIIISFLIVVNMYIAIILENF

(FIG. 20)

83
Na_v1.6_D4_S6
PSVGIFFFVSYIIISFLIVVNMYIAIILENF

(FIG. 20)

84
Na_v1.7_D4_S6
PSVGIFYFVSYIIISFLVVVNMYIAVILENF

(FIG. 20)

85
Na_v1.8_D4_S6
PAVGIIFFTTYIIISFLIMVNMYIAVILENF

(FIG. 20)

86
Na_v1.9_D4_S6
PGIATSYFVSYIIISFLIVVNMYIAVILENF

(FIG. 20)

87
Na_x VSLD1
PTLQTARTLRILKIIP

88
Na_x VSLD2
ALLRLFRMLRIFKLGK

89
Na_x VSLD3
KPLISMKFLRPLRVLSQ

90
Na_x VSLD4
QLILLSRIIHMLRLGKGPKVFH

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.

	Number	Date	Country
Parent	PCT/US2022/031302	May 2022	WO
Child	18515382		US

Mutant Human Nax Proteins and Screening Methods

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

International Classifications

Abstract

Description

Claims

CROSS-REFERENCE TO RELATED APPLICATIONS

Provisional Applications (1)

Continuations (1)