Human neuronal nicotinic acetylcholine receptor compositions and methods employing same

Abstract

DNA encoding human neuronal nicotinic acetylcholine receptor alpha and beta subunits, mammalian and amphibian cells containing the DNA, methods for producing α and β subunits and isolated or substantially pure α4, α7 and β4 subunits are provided. In addition, cells that expresses these subunits singly or combination with other subunits of nicotinic acetylcholine receptors and methods using the cells are provided.

Description

FIELD OF INVENTION

[0003] DNA encoding human neuronal nicotinic acetylcholine receptor protein subunits and the encoded proteins are provided. In particular, human neuronal nicotinic acetylcholine receptor α-subunit-encoding DNA, α-subunit proteins, β-subunit-encoding DNA, β-subunit proteins, and combinations thereof are provided. Also provided are methods that use the DNA for expression of the encoded subunits.

BACKGROUND OF THE INVENTION

[0004] Ligand-gated ion channels provide a means for communication between cells of the central nervous system. These channels convert a signal (e.g., a chemical referred to as a neurotransmitter) that is released by one cell into an electrical signal that propagates along a target cell membrane. A variety of neurotransmitters and neurotransmitter receptors exist in the central and peripheral nervous systems. Five families of ligand-gated receptors, including the nicotinic acetylcholine receptors (NAChRs) of neuromuscular and neuronal origins, have been identified (Stroud et al. (1 990) Biochemistry 29:11009-11023). There is, however, little understanding of the manner in which the variety of receptors generates different responses to neurotransmitters or to other modulating ligands in different regions of the nervous system.

[0005] The nicotinic acetylcholine receptors (NAChRs) are multisubunit proteins of neuromuscular and neuronal origins. These receptors form ligand-gated ion channels that mediate synaptic transmission between nerve and muscle and between neurons upon interaction with the neurotransmitter acetylcholine (ACh). Since various nicotinic acetylcholine receptor (NAChR) subunits exist, a variety of NAChR compositions (i.e., combinations of subunits) exist. The different NAChR compositions exhibit different specificities for various ligands and are thereby pharmacologically distinguishable. Thus, the nicotinic acetylcholine receptors expressed at the vertebrate neuromuscular junction in vertebrate sympathetic ganglia and in the vertebrate central nervous system have been distinguished on the basis of the effects of various ligands that bind to different NAChR compositions. For example, the elapid α-neurotoxins that block activation of nicotinic acetylcholine receptors at the neuromuscular junction do not block activation of some neuronal nicotinic acetylcholine receptors that are expressed on several different neuron-derived cell lines.

[0006] Muscle NAChR is a glycoprotein composed of five subunits with the stoichiometry α2β(γ or ε)δ. Each of the subunits has a mass of about 50-60 kilodaltons (kd) and is encoded by a different gene. The α2β(γ or ε)δ complex forms functional receptors containing two ligand binding sites and a ligand-gated transmembrane channel. Upon interaction with a cholinergic agonist, muscle nicotinic AChRs conduct sodium ions. The influx of sodium ions rapidly short-circuits the normal ionic gradient maintained across the plasma membrane, thereby depolarizing the membrane. By reducing the potential difference across the membrane, a (Stroud et al. (1990) Biochemistry 29:11009-11023). There is, however, little understanding of the manner in which the variety of receptors generates different responses to neurotransmitters or to other modulating ligands in different regions of the nervous system.

[0007] The nicotinic acetylcholine receptors (NAChRs) are multisubunit proteins of neuromuscular and neuronal origins. These receptors form ligand-gated ion channels that mediate synaptic transmission between nerve and muscle and between neurons upon interaction with the neurotransmitter acetylcholine (ACh). Since various nicotinic acetylcholine receptor (NAChR) subunits exist, a variety of NAChR compositions (i.e., combinations of subunits) exist. The different NAChR compositions exhibit different specificities for various ligands and are thereby pharmacologically distinguishable. Thus, the nicotinic acetylcholine receptors expressed at the vertebrate neuromuscular junction in vertebrate sympathetic ganglia and in the vertebrate central nervous system have been distinguished on the basis of the effects of various ligands that bind to different NAChR compositions. For example, the elapid α-neurotoxins that block activation of nicotinic acetylcholine receptors at the neuromuscular junction do not block activation of some neuronal nicotinic acetylcholine receptors that are expressed on several different neuron-derived cell lines.

[0008] Muscle NAChR is a glycoprotein composed of five subunits with the stoichiometry α2β(γ or ε)δ. Each of the subunits has a mass of about 50-60 kilodaltons (kd) and is encoded by a different gene. The α2β(γ or ε)δ complex forms functional receptors containing two ligand binding sites and a ligand-gated transmembrane channel. Upon interaction with a cholinergic agonist, muscle nicotinic AChRs conduct sodium ions. The influx of sodium ions rapidly short-circuits the normal ionic gradient maintained across the plasma membrane, thereby depolarizing the membrane. By reducing the potential difference across the membrane, a chemical signal is transduced into an electrical signal that signals muscle contraction at the neuromuscular junction.

[0009] Functional muscle nicotinic acetylcholine receptors have been formed with αβδγ subunits, αβγ subunits, αβδ subunits, αδγ subunits or αδ subunits, but not with only one subunit (see e.g., Kurosaki et al. (1987) FEBS Lett. 214: 253-258; Camacho et al. (1993) J. Neuroscience 13:605-613). In contrast, functional neuronal AChRs (nAChRs) can be formed from α subunits alone or combinations of α and β subunits. The larger α subunit is generally believed to be the ACh-binding subunit and the lower molecular weight β subunit is generally believed to be the structural subunit, although it has not been definitively demonstrated that the β subunit does not have the ability to bind ACh. Each of the subunits which participate in the formation of a functional ion channel are, to the extent they contribute to the structure of the resulting channel, “structural” subunits, regardless of their ability (or inability) to bind ACh. Neuronal AChRs (nAChRs), which are also ligand-gated ion channels, are expressed in ganglia of the autonomic nervous system and in the central nervous system (where they mediate signal transmission), in post-synaptic locations (where they modulate transmission), and in pre- and extra-synaptic locations (where they may have additional functions).

[0010] DNA encoding NAChRs has been isolated from several sources. Based on the information available from such work, it has been evident for some time that NAChRs expressed in muscle, in autonomic ganglia, and in the central nervous system are functionally diverse. This functional diversity could be due, at least in part, to the large number of different NAChR subunits which exist. There is an incomplete understanding, however, of how (and which) NAChR subunits combine to generate unique NAChR subtypes, particularly in neuronal cells. Indeed, there is evidence that only certain NAChR subtypes may be involved in diseases such as Alzheimer's disease. Moreover, it is not clear whether NAChRs from analogous tissues or cell types are similar across species.

[0011] Accordingly, there is a need for the isolation and characterization of DNAs encoding each human neuronal NAChR subunit, recombinant cells containing such subunits and receptors prepared therefrom. In order to study the function of human neuronal AChRs and to obtain disease-specific pharmacologically active agents, there is also a need to obtain isolated (preferably purified) human neuronal nicotinic AChRs, and isolated (preferably purified) human neuronal nicotinic AChR subunits. In addition, there is also a need to develop assays to identify such pharmacologically active agents.

[0012] The availability of such DNAs, cells, receptor subunits and receptor compositions will eliminate the uncertainty of speculating as to human nNAChR structure and function based on predictions drawn from non-human nNAChR data, or human or non-human muscle or ganglia NAChR data.

[0013] Therefore, it is an object herein to isolate and characterize DNA encoding subunits of human neuronal nicotinic acetylcholine receptors. It is also an object herein to provide methods for recombinant production of human neuronal nicotinic acetylcholine receptor subunits. It is also an object herein to provide purified receptor subunits and to provide methods for screening compounds to identify compounds that modulate the activity of human neuronal AChRs.

[0014] These and other objects will become apparent to those of skill in the art upon further study of the specification and claims.

BRIEF DESCRIPTION OF THE INVENTION

[0015] In accordance with the present invention, there are provided isolated DNAs encoding novel human alpha and beta subunits of neuronal NAChRs. In particular, isolated DNA encoding human α4, α7, and β4 subunits of neuronal NAChRs are provided. Messenger RNA and polypeptides encoded by the above-described DNA are also provided.

[0016] Further in accordance with the present invention, there are provided recombinant human neuronal nicotinic AChR subunits, including α4, α7, and β4 subunits, as well as methods for the production thereof. in addition, recombinant human neuronal nicotinic acetylcholine receptors containing at least one human neuronal nicotinic AChR subunit are also provided, as well as methods for the production thereof. Further provided are recombinant neuronal nicotinic AChRs that contain a mixture of one or more NAChR subunits encoded by a host cell, and one or more nNAChR subunits encoded by heterologous DNA or RNA (i.e., DNA or RNA as described herein that has been introduced into the host cell), as well as methods for the production thereof.

[0017] Plasmids containing DNA encoding the above-described subunits are also provided. Recombinant cells containing the above-described DNA, mRNA or plasmids are also provided herein. Such cells are useful, for example, for replicating DNA, for producing human NAChR subunits and recombinant receptors, and for producing cells that express receptors containing one or more human subunits.

[0018] Also provided in accordance with the present invention are methods for identifying cells that express functional nicotinic acetylcholine receptors. Methods for identifying compounds which modulate the activity of NAChRs are also provided.

[0019] The DNA, mRNA, vectors, receptor subunits, receptor subunit combinations and cells provided herein permit production of selected neuronal nicotinic AChR receptor subtypes and specific combinations thereof, as well as antibodies to the receptor subunits. This provides a means to prepare synthetic or recombinant receptors and receptor subunits that are substantially free of contamination from many other receptor proteins whose presence can interfere with analysis of a single NAChR subtype. The availability of desired receptor subtypes makes it possible to observe the effect of a drug substance on a particular receptor subtype and to thereby perform initial in vitro screening of the drug substance in a test system that is specific for humans and specific for a human neuronal nicotinic AChR subtype.

[0020] The availability of subunit-specific antibodies makes possible the application of the technique of immunohistochemistry to monitor the distribution and expression density of various subunits (e.g., in normal vs diseased brain tissue). Such antibodies could also be employed for diagnostic and therapeutic applications.

[0021] The ability to screen drug substances in vitro to determine the effect of the drug on specific receptor compositions should permit the development and screening of receptor subtype-specific or disease-specific drugs. Also, testing of single receptor subunits or specific receptor subtype combinations with a variety of potential agonists or antagonists provides additional information with respect to the function and activity of the individual subunits and should lead to the identification and design of compounds that are capable of very specific interaction with one or more of the receptor subunits or receptor subtypes. The resulting drugs should exhibit fewer unwanted side effects than drugs identified by screening with cells that express a variety of subtypes.

[0022] Further in relation to drug development and therapeutic treatment of various disease states, the availability of DNAs encoding human nNAChR subunits enables identification of any alterations in such genes (e.g., mutations) which may correlate with the occurrence of certain disease states. In addition, the creation of animal models of such disease states becomes possible, by specifically introducing such mutations into synthetic DNA sequences which can then be introduced into laboratory animals or in vitro assay systems to determine the effects thereof.

BRIEF DESCRIPTION OF THE FIGURE

[0023] FIG. 1 presents a restriction map of two pCMV promoter-based vectors, pCMV-T7-2 and pCMV-T7-3.

DETAILED DESCRIPTION OF THE INVENTION

[0024] In accordance with the present invention, we have isolated and characterized DNAs encoding novel human alpha and beta subunits of neuronal NAChRs. Specifically, isolated DNAs encoding human α4, α7, and β4 subunits of neuronal NAChRs are described herein. Recombinant messenger RNA (mRNA) and recombinant polypeptides encoded by the above-described DNA are also provided.

[0025] As used herein, isolated (or substantially pure) as a modifier of DNA, RNA, polypeptides or proteins means that the DNA, RNA, polypeptides or proteins so designated have been separated from their in vivo cellular environments through the efforts of human beings. Thus as used herein, isolated (or substantially pure) DNA refers to DNAs purified according to standard techniques employed by those skilled in the art (see, e.g., Maniatis et al.(1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

[0026] Similarly, as used herein, “recombinant” as a modifier of DNA, RNA, polypeptides or proteins means that the DNA, RNA, polypeptides or proteins so designated have been prepared by the efforts of human beings, e.g., by cloning, recombinant expression, and the like. Thus as used herein, recombinant proteins, for example, refers to proteins produced by a recombinant host, expressing DNAs which have been added to that host through the efforts of human beings.

[0027] As used herein, a human alpha subunit gene is a gene that encodes an alpha subunit of a human neuronal nicotinic acetylcholine receptor. The alpha subunit is a subunit of the NAChR to which ACh binds. Assignment of the name “alpha” to a putative nNAChR subunit, according to Deneris et al. [Tips (1991) 12:34-40] is based on the conservation of adjacent cysteine residues in the presumed extracellular domain of the subunit that are the homologues of cysteines 192 and 193 of the Torpedo alpha subunit (see Noda et al. (1982) Nature 299:793-797). As used herein, an alpha subunit subtype refers to a human nNAChR subunit that is encoded by DNA that hybridizes under high stringency conditions to at least one of the nNAChR alpha subunit-encoding DNAs (or deposited clones) disclosed herein. An alpha subunit also binds to ACh under physiological conditions and at physiological concentrations and, in the optional presence of a beta subunit (i.e., some alpha subunits are functional alone, while others require the presence of a beta subunit), generally forms a functional AChR as assessed by methods described herein or known to those of skill in this art.

[0028] Also contemplated are alpha subunits encoded by DNAs that encode alpha subunits as defined above, but that by virtue of degeneracy of the genetic code do not necessarily hybridize to the disclosed DNA or deposited clones under specified hybridization conditions. Such subunits also form a functional receptor, as assessed by the methods described herein or known to those of skill in the art, generally with one or more beta subunit subtypes. Typically, unless an alpha subunit is encoded by RNA that arises from alternative splicing (i.e., a splice variant), alpha-encoding DNA and the alpha subunit encoded thereby share substantial sequence homology with at least one of the alpha subunit DNAs (and proteins encoded thereby) described or deposited herein. It is understood that DNA or RNA encoding a splice variant may overall share less than 90% homology with the DNA or RNA provided herein, but include regions of nearly 100% homology to a DNA fragment or deposited clone described herein, and encode an open reading frame that includes start and stop codons and encodes a functional alpha subunit.

[0029] As used herein, a splice variant refers to variant NAChR subunit-encoding nucleic acid(s) produced by differential processing of primary transcript(s) of genomic DNA, resulting in the production of more than one type of mRNA. cDNA derived from differentially processed genomic DNA will encode NAChR subunits that have regions of complete amino acid identity and regions having different amino acid sequences. Thus, the same genomic sequence can lead to the production of multiple, related mRNAs and proteins. The resulting mRNA and proteins are referred to herein as “splice variants”.

[0030] Stringency of hybridization is used herein to refer to conditions under which polynucleic acid hybrids are stable. As known to those of skill in the art, the stability of hybrids is reflected in the melting temperature (Tm) of the hybrids. Tm can be approximated by the formula:

81.5° C.−16.6(log10[Na+])+0.41(% G+C)−600/l,

[0031] where l is the length of the hybrids in nucleotides. Tm decreases approximately 1-1.5° C. with every 1% decrease in sequence homology. In general, the stability of a hybrid is a function of sodium ion concentration and temperature. Typically, the hybridization reaction is performed under conditions of lower stringency, followed by washes of varying, but higher, stringency. Reference to hybridization stringency relates to such washing conditions. Thus, as used herein:

[0032] (1) HIGH STRINGENCY refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65° C. (i.e., if a hybrid is not stable in 0.018M NaCl at 65° C., it will not be stable under high stringency conditions, as contemplated herein). High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5×Denhardt's solution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE, and 0.1% SDS at 65° C.;

[0033] (2) MODERATE STRINGENCY refers to conditions equivalent to hybridization in 50% formamide, 5×Denhardt's solution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE, 0.2% SDS, at 65° C.; and

[0034] (3) LOW STRINGENCY refers to conditions equivalent to hybridization in 10% formamide, 5×Denhardt's solution, 6×SSPE, 0.2% SDS, followed by washing in 1×SSPE, 0.2% SDS, at 50° C.

[0035] It is understood that these conditions may be duplicated using a variety of buffers and temperatures and that they are not necessarily precise.

[0036] Denhardt's solution and SSPE (see, e.g., Sambrook, Fritsch, and Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989) are well known to those of skill in the art as are other suitable hybridization buffers. For example, SSPE is pH 7.4 phosphate-buffered 0.18M NaCl. SSPE can be prepared, for example, as a 20×stock solution by dissolving 175.3 g of NaCl, 27.6 g of NaH2PO4 and 7.4 g EDTA in 800 ml of water, adjusting the pH to 7.4, and then adding water to 1 liter. Denhardt's solution (see, Denhardt (1966) Biochem. Biophys. Res. Commun. 23:641) can be prepared, for example, as a 50×stock solution by mixing 5 g Ficoll (Type 400, Pharmacia LKB Biotechnology, INC., Piscataway N.J.), 5 g of polyvinylpyrrolidone, 5 g bovine serum albumin (Fraction V; Sigma, St. Louis Mo.) water to 500 ml and filtering to remove particulate matter.

[0037] The phrase “substantial sequence homology” is used herein in reference to the nucleotide sequence of DNA, the ribonucleotide sequence of RNA, or the amino acid sequence of protein, that have slight and non-consequential sequence variations from the actual sequences disclosed herein. Species having substantial sequence homology are considered to be equivalent to the disclosed sequences and as such are within the scope of the appended claims In this regard, “slight and non-consequential sequence variations” mean that “homologous” sequences, i.e., sequences that have substantial homology with the DNA, RNA, or proteins disclosed and claimed herein, are functionally equivalent to the sequences disclosed and claimed herein. Functionally equivalent sequences will function in substantially the same manner to produce substantially the same compositions as the nucleic acid and amino acid compositions disclosed and claimed herein. In particular, functionally equivalent DNAs encode proteins that are the same as those disclosed herein or that have conservative amino acid variations, such as substitution of a non-polar residue for another non-polar residue or a charged residue for a similarly charged residue. These changes include those recognized by those of skill in the art as those that do not substantially alter the tertiary structure of the protein.

[0038] In practice, the term substantially the same sequence means that DNA or RNA encoding two proteins hybridize under conditions of high stringency and encode proteins that have the same sequence of amino acids or have changes in sequence that do not alter their structure or function. As used herein, substantially identical sequences of nucleotides share at least about 90% identity, and substantially identical amino acid sequences share more than 95% amino acid identity. It is recognized, however, that proteins (and DNA or mRNA encoding such proteins) containing less than the above-described level of homology arising as splice variants or that are modified by conservative amino acid substitutions (or substitution of degenerate codons) are contemplated to be within the scope of the present invention.

[0039] As used herein, “α4 subunit DNA” refers to DNA encoding a neuronal nicotinic acetylcholine receptor subunit of the same name. Such DNA can be characterized in a number of ways, for example

[0040] the DNA may encode the amino acid sequence set forth in SEQ. ID No. 6, or

[0041] the DNA may encode the amino acid sequence encoded by clone HnAChRα4.2, deposited under ATCC Accession No. 69239, or the 5′ nucleotides of the DNA may encode the amino acid sequence encoded by clone HnAChRα4.1, deposited under ATCC Accession No. 69152.

[0042] Presently preferred α4-encoding DNAs can be characterized as follows

[0043] the DNA may hybridize to the coding sequence set forth in SEQ. ID No. 5 (preferably to substantially the entire coding sequence thereof, i.e., nucleotides 184-2067) under high stringency conditions, or

[0044] the DNA may hybridize under high stringency conditions to the sequence (preferably to substantially the entire sequence) of the α4-encoding insert of clone HnAChRα4.2, deposited under ATCC Accession No. 69239, or

[0045] the 5′ nucleotides of the DNA may hybridize under high stringency conditions to the sequence of the α4-encoding insert of clone HnAChRα4.1, deposited under ATCC Accession No. 69152.

[0046] Especially preferred α4-encoding DNAs of the invention are characterized as follows

[0047] DNA having substantially the same nucleotide sequence as the coding region set forth in SEQ. ID No. 5 (i.e., nucleotides 184-2067 thereof), or

[0048] DNA having substantially the same nucleotide sequence as the α4-encoding insert of clone HnAChRα4.2, deposited under ATCC Accession No.69239, or

[0049] the 5′ nucleotides of the DNA have substantially the same sequence as the α4-encoding insert of clone HnAChRα4.1, deposited under ATCC Accession No. 69152.

[0050] Typically, unless an α4 subunit arises as a splice variant, α4-encoding DNA will share substantial sequence homology (i.e., greater than about 90%), with the α4 DNAs described or deposited herein. DNA or RNA encoding a splice variant may share less than 90% overall sequence homology with the DNA or RNA provided herein, but such a splice variant would include regions of nearly 100% homology to the above-described DNAs.

[0051] As used herein, “α7 subunit DNA” refers to DNA encoding a neuronal nicotinic acetylcholine receptor subunit of the same name. Such DNA can be characterized in a number of ways, for example, the nucleotides of the DNA may encode the amino acid sequence set forth in SEQ. ID No. 8. Presently preferred α7-encoding DNAs can be characterized as DNA which hybridizes under high stringency conditions to the coding sequence set forth in SEQ. ID No. 7 (preferably to substantially the entire coding sequence thereof, i.e., nucleotides 73-1581). Especially preferred α7-encoding DNAs of the invention are characterized as having substantially the same nucleotide sequence as the coding sequence set forth in SEQ. ID No. 7 (i.e., nucleotides 73-1581 thereof).

[0052] Typically, unless an α7 subunit arises as a splice variant, α7-encoding DNA will share substantial sequence homology (greater than about 90%) with the α7 DNAs described or deposited herein. DNA or RNA encoding a splice variant may share less than 90% overall sequence homology with the DNA or RNA provided herein, but such DNA would include regions of nearly 100% homology to the above-described DNA.

[0053] The α7 subunits derived from the above-described DNA are expected to bind to the neurotoxin α-bungarotoxin (α-bgtx). The activity of AChRs that contain α7 subunits should be inhibited upon interaction with α-bgtx. Amino acid residues 210 through 217, as set forth in SEQ ID No. 8, are believed to be important elements in the binding of α-bgtx (see, for example, Chargeaux et al. (1992) 13:299-301).

[0054] As used herein, a human beta subunit gene is a gene that encodes a beta subunit of a human neuronal nicotinic acetylcholine receptor. Assignment of the name “beta” to a putative nNAChR subunit, according to Deneris et al. supra, is based on the lack of adjacent cysteine residues (which are characteristic of alpha subunits). The beta subunit is frequently referred to as the structural NAChR subunit (although it is possible that beta subunits also have ACh binding properties). Combination of beta subunit(s) with appropriate alpha subunit(s) leads to the formation of a functional receptor. As used herein, a beta subunit subtype refers to a nNAChR subunit that is encoded by DNA that hybridizes under high stringency conditions to at least one of the nNAChR-encoding DNAs (or deposited clones) disclosed herein. A beta subunit forms a functional NAChR, as assessed by methods described herein or known to those of skill in this art, with appropriate alpha subunit subtype(s).

[0055] Also contemplated are beta subunits encoded by DNAs that encode beta subunits as defined above, but that by virtue of degeneracy of the genetic code do not necessarily hybridize to the disclosed DNA or deposited clones under the specified hybridization conditions. Such subunits also form functional receptors, as assessed by the methods described herein or known to those of skill in the art, in combination with appropriate alpha subunit subtype(s). Typically, unless a beta subunit is encoded by RNA that arises as a splice variant, beta-encoding DNA and the beta subunit encoded thereby share substantial sequence homology with the beta-encoding DNA and beta subunit protein described herein. It is understood that DNA or RNA encoding a splice variant may share less than 90% overall homology with the DNA or RNA provided herein, but such DNA will include regions of nearly 100% homology to the DNA described herein.

[0056] As used herein, “β4 subunit DNA” refers to DNA encoding a neuronal nicotinic acetylcholine receptor subunit of the same name. Such DNA can be characterized in a number of ways, for example, the nucleotides of the DNA may encode the amino acid sequence set forth in SEQ. ID No. 12. Presently preferred β4-encoding DNAs can be characterized as DNA which hybridizes under high stringency conditions to the coding sequence set forth in SEQ. ID No. 11 (preferably to substantially the entire coding sequence thereof, i.e., nucleotides 87-1583). Especially preferred β4-encoding DNAs of the invention are characterized as having substantially the same nucleotide sequence as set forth in SEQ. ID No. 11.

[0057] Typically, unless a β4 subunit arises as a splice variant, β4-encoding DNA will share substantial sequence homology (greater than about 90%) with the β4 DNAs described or deposited herein. DNA or RNA encoding a splice variant may share less than 90% overall sequence homology with the DNA or RNA provided herein, but such DNA would include regions of nearly 100% homology to the above-described DNA.

[0058] DNA encoding human neuronal nicotinic AChR alpha and beta subunits may be isolated by screening suitable human cDNA or human genomic libraries under suitable hybridization conditions with DNA disclosed herein (including nucleotides derived from any of SEQ ID Nos. 5, 7, 9 or 11, or with any of the deposited clones referred to herein (e.g., ATCC accession no. 69239 or 69152). Suitable libraries can be prepared from neuronal tissue samples, hippocampus tissue, or cell lines, such as the human neuroblastoma cell line IMR32 (ATCC Accession No. CCL127), and the like. The library is preferably screened with a portion of DNA including the entire subunit-encoding sequence thereof, or the library may be screened with a suitable probe.

[0059] As used herein, a probe is single-stranded DNA or RNA that has a sequence of nucleotides that includes at least 14 contiguous bases that are the same as (or the complement of) any 14 bases set forth in any of SEQ ID Nos. 1, 3, 5, 7, 9, or 11, or in the subunit encoding DNA in any of the deposited clones described herein (e.g., ATCC accession no. 69239 or 69152). Preferred regions from which to construct probes include 5′ and/or 3′ coding sequences, sequences predicted to encode transmembrane domains, sequences predicted to encode the cytoplasmic loop, signal sequences, acetylcholine (ACh) and α-bungarotoxin (α-bgtx) binding sites, and the like. Amino acids 210-220 are typically involved in ACh and α-bgtx binding. The approximate amino acid residues which comprise such regions for other preferred probes are set forth in the following table:

sub-	signal					cytoplasmic
unit	sequence	TMD*1	TMD2	TMD3	TMD4	loop
α2	1-55	264-289	297-	326-	444-	351-443
			320	350	515
α3	1-30	240-265	273-	302-	459-	327-458
			296	326	480
α4	1-33	241-269	275-	303-	593-	594-617
			289	330	618
α7	1-23	229-256	262-	290-	462-	318-461
			284	317	487
β2	1-25	234-259	267-	295-	453-	321-452
			288	320	477
β4	1-23	234-258	264-	290-	454-	320-453
			285	319	478
*TMD = transmembrane domain

[0060] Alternatively, portions of the DNA can be used as primers to amplify selected fragments in a particular library.

[0061] After screening the library, positive clones are identified by detecting a hybridization signal; the identified clones are characterized by restriction enzyme mapping and/or DNA sequence analysis, and then examined, by comparison with the sequences set forth herein or with the deposited clones described herein, to ascertain whether they include DNA encoding a complete alpha or beta subunit. If the selected clones are incomplete, they may be used to rescreen the same or a different library to obtain overlapping clones. If desired, the library can be rescreened with positive clones until overlapping clones that encode an entire alpha or beta subunit are obtained. If the library is a cDNA library, then the overlapping clones will include an open reading frame. If the library is genomic, then the overlapping clones may include exons and introns. In both instances, complete clones may be identified by comparison with the DNA and encoded proteins provided herein.

[0062] Complementary DNA clones encoding various subtypes of human nNAChR alpha and beta subunits have been isolated. Each subtype of the subunit appears to be encoded by a different gene. The DNA clones provided herein may be used to isolate genomic clones encoding each subtype and to isolate any splice variants by screening libraries prepared from different neural tissues. Nucleic acid amplification techniques, which are well known in the art, can be used to locate splice variants of human NAChR subunits. This is accomplished by employing oligonucleotides based on DNA sequences surrounding divergent sequence(s) as primers for amplifying human RNA or genomic DNA. Size and sequence determinations of the amplification products can reveal the existence of splice variants. Furthermore, isolation of human genomic DNA sequences by hybridization can yield DNA containing multiple exons, separated by introns, that correspond to different splice variants of transcripts encoding human NAChR subunits.

[0063] It has been found that not all subunit subtypes are expressed in all neural tissues or in all portions of the brain. Thus, in order to isolate cDNA encoding particular subunit subtypes or splice variants of such subtypes, it is preferable to screen libraries prepared from different neuronal or neural tissues. Preferred libraries for obtaining DNA encoding each subunit include: hippocampus to isolate human α4- and α5-encoding DNA; IMR32 to isolate human α3-, α5-, α7- and β4-encoding DNA, thalamus to isolate α2 and β2-encoding DNA; and the like.

[0064] It appears that the distribution of expression of human neuronal nicotinic AChRs differs from the distribution of such receptors in rat. For example, RNA encoding the rat α4 subunit is abundant in rat thalamus, but is not abundant in rat hippocampus (see, e.g., Wada et al. (1989) J. Comp. Neurol 284:314-335). No α4-encoding clones could be obtained, however, from a human thalamus library. Instead, human α4 clones were ultimately obtained from a human hippocampus library. Thus, the distribution of α4 nNAChR subunit in humans and rats appears to be quite different.

[0065] Rat α3 subunit appears to be a CNS-associated subunit that is abundantly expressed in the thalamus and weakly expressed in the brain stem (see, e.g., Boulter et al. (1986) Nature 319:368-374; Boulter et al. (1987) Proc. Natl. Acad. Sci. USA 84:7763-7767; and Wada et al. (1989) J. Comp. Neurol 284:314-335). In efforts to clone DNA encoding the human nicotinic AChR α3 subunit, however, several human libraries, including a thalamus library, were unsuccessfully screened. Surprisingly, clones encoding human α3 subunit were ultimately obtained from a brain stem library and from IMR32 cells that reportedly express few, if any, functional nicotinic acetylcholine receptors (see, e.g., Gotti et al. ((1986) Biochem. Biophys. Res. Commun. 137: 1141-1147, and Clementi et al. (1986) J. Neurochem. 47: 291-297).

[0066] Rat α7 subunit transcript reportedly is abundantly expressed in the hippocampus (see Seguela et al. (1993) J. Neurosci. 13:596-604). Efforts to clone DNA encoding a human α7 subunit from a human hippocampus library (1×106 recombinants) were unsuccessful. Surprisingly, clones encoding a human NAChR α7 subunit were ultimately obtained from an IMR32 cell cDNA library.

[0067] The above-described nucleotide sequences can be incorporated into vectors for further manipulation. As used herein, vector (or plasmid) refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof. Selection and use of such vehicles are well within the level of skill of the art.

[0068] An expression vector includes vectors capable of expressing DNAs that are operatively linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome. Presently preferred plasmids for expression of invention AChR subunits in eukaryotic host cells, particularly mammalian cells, include cytomegalovirus (CMV) promoter-containing vectors such as pCMV, pcDNA1, and the like.

[0069] As used herein, a promoter region refers to a segment of DNA that controls transcription of DNA to which it is operatively linked. The promoter region includes specific sequences that are sufficient for RNA polymerase recognition, binding and transcription initiation. This portion of the promoter region is referred to as the promoter. In addition, the promoter region includes sequences that modulate this recognition, binding and transcription initiation activity of RNA polymerase. These sequences may be cis acting or may be responsive to trans acting factors. Promoters, depending upon the nature of the regulation, may be constitutive or regulated. Exemplary promoters contemplated for use in the practice of the present invention include the SV40 early promoter, the cytomegalovirus (CMV) promoter, the mouse mammary tumor virus (MMTV) steroid-inducible promoter, Moloney murine leukemia virus (MMLV) promoter, and the like.

[0070] As used herein, the term “operatively linked” refers to the functional relationship of DNA with regulatory and effector sequences of nucleotides, such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences. For example, operative linkage of DNA to a promoter refers to the physical and functional relationship between the DNA and the promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA. In order to optimize expression and/or in vitro transcription, it may be necessary to remove or alter 5′ untranslated portions of the clones to remove extra, potential alternative translation initiation (i.e., start) codons or other sequences that interfere with or reduce expression, either at the level of transcription or translation. Alternatively, consensus ribosome binding sites (see, for example, Kozak (1991) J. Biol. Chem. 266:19867-19870) can be inserted immediately 5′ of the start codon to enhance expression. The desirability of (or need for) such modification may be empirically determined.

[0071] As used herein, expression refers to the process by which polynucleic acids are transcribed into mRNA and translated into peptides, polypeptides, or proteins. If the polynucleic acid is derived from genomic DNA, expression may, if an appropriate eukaryotic host cell or organism is selected, include splicing of the mRNA.

[0072] Particularly preferred vectors for transfection of mammalian cells are the pSV2dhfr expression vectors, which contain the SV40 early promoter, mouse dhfr gene, SV40 polyadenylation and splice sites and sequences necessary for maintaining the vector in bacteria, cytomegalovirus (CMV) promoter-based vectors such as pCDNA1 (Invitrogen, San Diego, Calif.), and MMTV promoter-based vectors such as pMSG (Catalog No. 27-4506-01 from Pharmacia, Piscataway, N.J.).

[0073] Full-length DNAs encoding human neuronal NAChR subunits have been inserted into vector pCMV-T7, a pUC19-based mammalian cell expression vector containing the CMV promoter/enhancer, SV40 splice/donor sites located immediately downstream of the promoter, a polylinker downstream of the splice/donor sites, followed by an SV40 polyadenylation signal. Placement of NAChR subunit DNA between the CMV promoter and SV40 polyadenylation signal provides for constitutive expression of the foreign DNA in a mammalian host cell transfected with the construct. For inducible expression of human NAChR subunit-encoding DNA in a mammalian cell, the DNA can be inserted into a plasmid such as pMSG. This plasmid contains the mouse mammary tumor virus (MMTV) promoter for steroid-inducible expression of operatively associated foreign DNA. If the host cell does not express endogenous glucocorticoid receptors required for uptake of glucorcorticoids (i.e., inducers of the MMTV promoter) into the cell, it is necessary to additionally transfect the cell with DNA encoding the glucocorticoid receptor (ATCC accession no. 67200). Full-length human DNA clones encoding human α3, α4, α7, β2 and β4 have also been subcloned into pIBI24 (International Biotechnologies, Inc., New Haven, Conn.) or pCMV-T7-2 for synthesis of in vitro transcripts.

[0074] In accordance with another embodiment of the present invention, there are provided cells containing the above-described polynucleic acids (i.e., DNA or mRNA). Such host cells as bacterial, yeast and mammalian cells can be used for replicating DNA and producing nAChR subunit(s). Methods for constructing expression vectors, preparing in vitro transcripts, transfecting DNA into mammalian cells, injecting oocytes, and performing electrophysiological and other analyses for assessing receptor expression and function as described herein are also described in PCT Application Nos. PCT/US91/02311, PCT/US91/05625 and PCT/US92/11090, and in co-pending U.S. application Ser. Nos. 07/504,455, 07/563,751 and 07/812,254. The subject matter of these applications are hereby incorporated by reference herein in their entirety.

[0075] Incorporation of cloned DNA into a suitable expression vector, transfection of eukaryotic cells with a plasmid vector or a combination of plasmid vectors, each encoding one or more distinct genes or with linear DNA, and selection of transfected cells are well known in the art (see, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press). Heterologous DNA may be introduced into host cells by any method known to those of skill in the art, such as transfection with a vector encoding the heterologous DNA by CaPO4 precipitation (see, e.g., Wigler et al. (1979) Proc. Natl. Acad. Sci. 76:1373-1376). Recombinant cells can then be cultured under conditions whereby the subunit(s) encoded by the DNA is (are) expressed. Preferred cells include mammalian cells (e.g., HEK 293, CHO and Ltk cells), yeast cells (e.g., methylotrophic yeast cells, such as Pichia pastoris), bacterial cells (e.g., Escherichia coli), and the like.

[0076] While the DNA provided herein may be expressed in any eukaryotic cell, including yeast cells (such as, for example, P. pastoris (see U.S. Pat. Nos. 4,882,279, 4,837,148, 4,929,555 and 4,855,231), Saccharomyces cerevisiae, Candida tropicalis, Hansenula polymorpha, and the like), mammalian expression systems, including commercially available systems and other such systems known to those of skill in the art, for expression of DNA encoding the human neuronal nicotinic AChR subunits provided herein are presently preferred. Xenopus oocytes are preferred for expression of RNA transcripts of the DNA.

[0077] In preferred embodiments, DNA is ligated into a vector, and introduced into suitable host cells to produce transformed cell lines that express a specific human nNAChR receptor subtype, or specific combinations of subtypes. The resulting cell lines can then be produced in quantity for reproducible quantitative analysis of the effects of drugs on receptor function. In other embodiments, mRNA may be produced by in vitro transcription of DNA encoding each subunit. This mRNA, either from a single subunit clone or from a combination of clones, can then be injected into Xenopus oocytes where the mRNA directs the synthesis of the human receptor subunits, which then form functional receptors. Alternatively, the subunit-encoding DNA can be directly injected into oocytes for expression of functional receptors. The transfected mammalian cells or injected oocytes may then be used in the methods of drug screening provided herein.

[0078] Cloned full-length DNA encoding any of the subunits of human neuronal nicotinic AChR may be introduced into a plasmid vector for expression in a eukaryotic cell. Such DNA may be genomic DNA or cDNA. Host cells may be transfected with one or a combination of plasmids, each of which encodes at least one human neuronal nicotinic AChR subunit.

[0079] Eukaryotic cells in which DNA or RNA may be introduced include any cells that are transfectable by such DNA or RNA or into which such DNA or RNA may be injected. Preferred cells are those that can be transiently or stably transfected and also express the DNA and RNA. Presently most preferred cells are those that can form recombinant or heterologous human neuronal nicotinic AChRs comprising one or more subunits encoded by the heterologous DNA. Such cells may be identified empirically or selected from among those known to be readily transfected or injected.

[0080] Exemplary cells for introducing DNA include cells of mammalian origin (e.g., COS cells, mouse L cells, Chinese hamster ovary (CHO) cells, human embryonic kidney cells, African green monkey cells and other such cells known to those of skill in the art), amphibian cells (e.g., Xenopus laevis oöcytes), yeast cells (e.g., Saccharomyces cerevisiae, Pichia pastoris), and the like. Exemplary cells for expressing injected RNA transcripts include Xenopus laevis oöcytes. Cells that are preferred for transfection of DNA are known to those of skill in the art or may be empirically identified, and include HEK 293 (which are available from ATCC under accession #CRL 1573); Ltk cells (which are available from ATCC under accession #CCL1.3); COS-7 cells (which are available from ATCC under accession #CRL 1651); and DG44 cells (dhfr CHO cells; see, e.g., Urlaub et al. (1986) Cell. Molec. Genet. 12: 555). Presently preferred cells include DG44 cells and HEK 293 cells, particularly HEK 293 cells that have been adapted for growth in suspension and that can be frozen in liquid nitrogen and then thawed and regrown. HEK 293 cells are described, for example, in U.S. Pat. No. 5,024,939 to Gorman (see, also, Stillman et al. (1985) Mol. Cell. Biol. 5:2051-2060).

[0081] DNA may be stably incorporated into cells or may be transiently introduced using methods known in the art. Stably transfected mammalian cells may be prepared by transfecting cells with an expression vector having a selectable marker gene (such as, for example, the gene for thymidine kinase, dihydrofolate reductase, neomycin resistance, and the like), and growing the transfected cells under conditions selective for cells expressing the marker gene. To produce such cells, the cells should be transfected with a sufficient concentration of subunit-encoding nucleic acids to form human neuronal nicotinic AChRs that contain the human subunits encoded by heterologous DNA. The precise amounts and ratios of DNA encoding the subunits may be empirically determined and optimized for a particular combination of subunits, cells and assay conditions. Recombinant cells that express neuronal nicotinic AChR containing subunits encoded only by the heterologous DNA or RNA are especially preferred.

[0082] Heterologous DNA may be maintained in the cell as an episomal element or may be integrated into chromosomal DNA of the cell. The resulting recombinant cells may then be cultured or subcultured (or passaged, in the case of mammalian cells) from such a culture or a subculture thereof. Methods for transfection, injection and culturing recombinant cells are known to the skilled artisan. Similarly, the human neuronal nicotinic AChR subunits may be purified using protein purification methods known to those of skill in the art. For example, antibodies or other ligands that specifically bind to one or more of the subunits may be used for affinity purification of the subunit or human neuronal nicotinic AChRs containing the subunits.

[0083] In accordance with one embodiment of the present invention, methods for producing cells that express human neuronal nicotinic AChR subunits and functional receptors are also provided. In one such method, host cells are transfected with DNA encoding at least one alpha subunit of a neuronal nicotinic acetylcholine receptor and at least one beta subunit of a neuronal nicotinic acetylcholine receptor. Using methods such as northern blot or slot blot analysis, transfected cells that contain alpha and/or beta subunit encoding DNA or RNA can be selected. Transfected cells are also analyzed to identify those that express NAChR protein. Analysis can be carried out, for example, by measuring the ability of cells to bind acetylcholine, nicotine; or a nicotine agonist, compared to the nicotine binding ability of untransfected host cells or other suitable control cells, by electrophysiologically monitoring the currents through the cell membrane in response to a nicotine agonist, and the like.

[0084] In particularly preferred aspects, eukaryotic cells which contain heterologous DNAs express such DNA and form recombinant functional neuronal nicotinic AChR(s). In more preferred aspects, recombinant neuronal nicotinic AChR activity is readily detectable because it is a type that is absent from the untransfected host cell or is of a magnitude not exhibited in the untransfected cell. Such cells that contain recombinant receptors could be prepared, for example, by causing cells transformed with DNA encoding the human neuronal nicotinic AChR α3 and β4 subunits to express the corresponding proteins. The resulting synthetic or recombinant receptor would contain only the α3 and β4 nNAChR subunits. Such a receptor would be useful for a variety of applications, e.g., as part of an assay system free of the interferences frequently present in prior art assay systems employing non-human receptors or human tissue preparations. Furthermore, testing of single receptor subunits with a variety of potential agonists or antagonists would provide additional information with respect to the function and activity of the individual subunits. Such information may lead to the identification of compounds which are capable of very specific interaction with one or more of the receptor subunits. Such specificity may prove of great value in medical application.

[0085] Thus, DNA encoding one or more human neuronal nicotinic AChR subunits may be introduced into suitable host cells (e.g., eukaryotic or prokaryotic cells) for expression of individual subunits and functional NAChRs. Preferably combinations of alpha and beta subunits may be introduced into cells: such combinations include combinations of any one or more of α1, α2, α3, α4, α5 and α7 with β2 or β4. Sequence information for α1 is presented in Biochem. Soc. Trans. (1989) 17:219-220; sequence information for α5 is presented in Proc. Natl. Acad. Sci. USA (1992) 89:1572-1576; and sequence information for α2, α3, α4, α7, β2 and β4 is presented in the Sequence Listing provided herewith. Presently preferred combinations of subunits include any one or more of α1, α2, α3 or α5 with β4; or α4 or α7 in combination with either β2 or β4. It is recognized that some of the subunits may have ion transport function in the absence of additional subunits. For example, the α7 subunit is functional in the absence of any added beta subunit.

[0086] As used herein, “α2 subunit DNA” refers to DNA that encodes a human neuronal nicotinic acetylcholine receptor subunit of the same name, and to DNA that hybridizes under conditions of high stringency to the DNA of SEQ ID No. 1, or to the DNA of deposited clone having ATCC Accession No. 68277, or to DNA that encodes the amino acid sequence set forth in SEQ ID No. 2. Typically, unless an α2 subunit arises as a splice variant, an α2 DNA shares substantial sequence homology (greater than about 90%) with the α2 DNA described herein. DNA or RNA encoding a splice variant may share less than 90% overall sequence homology with the DNA or RNA described herein, but such a splice variant would include regions of nearly 100% homology to the above-described DNA.

[0087] As used herein, “α3 subunit DNA” refers to DNA that encodes a neuronal subunit of the same name, and to DNA that hybridizes under conditions of high stringency to the DNA of SEQ ID No. 3, or to the DNA of deposited clone having ATCC Accession No. 68278, or to DNA that encodes the amino acid sequence set forth in SEQ ID No. 4. Typically, unless an α3 arises as a splice variant, an α3 DNA shares substantial sequence homology (greater than about 90%) with the α3 DNA described herein. DNA or RNA encoding a splice variant may share less than 90% overall sequence homology with the DNA or RNA provided herein, but such a splice variant would include regions of nearly 100% homology to the above described DNA.

[0088] As used herein, “α5 subunit DNA” refers to DNA that encodes a human neuronal nicotinic acetylcholine receptor subunit of the same name, as described, for example, by Chini et al. (1992) Proc. Natl. Acad. Sci. USA 89:1572-1576.

[0089] As used herein, “β2 subunit DNA” refers to DNA that encodes a neuronal subunit of the same name and, to DNA that hybridizes under conditions of high stringency to the DNA of SEQ ID No. 9, or to the DNA of deposited clone HnAChRβ2, having ATCC Accession No. 68279, or to DNA encoding the amino acid sequence set forth in SEQ ID No. 10. Typically, unless β2 subunit arises as a splice variant, a β2 DNA shares substantial sequence homology (greater than about 90%) with the β2 DNA described herein. DNA or RNA encoding a splice variant may share overall less than 90% homology with the DNA or RNA provided herein, but such a splice variant would include regions of nearly 100% homology to the above-described DNA.

[0090] In certain embodiments, eukaryotic cells with heterologous human neuronal nicotinic AChRs are produced by introducing into the cell a first composition, which contains at least one RNA transcript that is translated in the cell into a subunit of a human neuronal nicotinic AChR. In preferred embodiments, the subunits that are translated include an alpha subunit of a human neuronal nicotinic AChR. More preferably, the composition that is introduced contains an RNA transcript which encodes an alpha subunit and also contains an RNA transcript which encodes a beta subunit of a human neuronal nicotinic AChR. RNA transcripts can be obtained from cells transfected with DNAs encoding human neuronal nicotinic acetylcholine receptor subunits or by in vitro transcription of subunit-encoding DNAs. Methods for in vitro transcription of cloned DNA and injection of the resulting mRNA into eukaryotic cells are well known in the art. Amphibian oocytes are particularly preferred for expression of in vitro transcripts of the human nNAChR DNA clones provided herein. See, for example, Dascal (1989) CRC Crit. Rev. Biochem. 22:317-387, for a review of the use of Xenopus oocytes to study ion channels.

[0091] Thus, pairwise (or stepwise) introduction of DNA or RNA encoding alpha and beta subtypes into cells is possible. The resulting cells may be tested by the methods provided herein or known to those of skill in the art to detect functional AChR activity. Such testing will allow the identification of pairs of alpha and beta subunit subtypes that produce functional AChRs, as well as individual subunits that produce functional AChRs.

[0092] As used herein, activity of a human neuronal nicotinic AChR refers to any activity characteristic of an NAChR. Such activity can typically be measured by one or more in vitro methods, and frequently corresponds to an in vivo activity of a human neuronal nicotinic AChR. Such activity may be measured by any method known to those of skill in the art, such as, for example, measuring the amount of current which flows through the recombinant channel in response to a stimulus.

[0093] Methods to determine the presence and/or activity of human neuronal nicotinic AChRs include assays that measure nicotine binding, 86Rb ion-flux, Ca2+ influx, the electrophysiological response of cells, the electrophysiological response of oocytes transfected with RNA from the cells, and the like. In particular, methods are provided herein for the measurement or detection of an AChR-mediated response upon contact of cells containing the DNA or mRNA with a test compound.

[0094] As used herein, a recombinant or heterologous human neuronal nicotinic AChR refers to a receptor that contains one or more subunits encoded by heterologous DNA that has been introduced into and expressed in cells capable of expressing receptor protein. A recombinant human neuronal nicotinic AChR may also include subunits that are produced by DNA endogenous to the host cell. In certain embodiments, recombinant or heterologous human neuronal nicotinic AChR may contain only subunits that are encoded by heterologous DNA.

[0095] As used herein, heterologous or foreign DNA and RNA are used interchangeably and refer to DNA or RNA that does not occur naturally as part of the genome of the cell in which it is present or to DNA or RNA which is found in a location or locations in the genome that differ from that in which it occurs in nature. Typically, heterologous or foreign DNA and RNA refers to DNA or RNA that is not endogenous to the host cell and has been artificially introduced into the cell. Examples of heterologous DNA include DNA that encodes a human neuronal nicotinic AChR subunit, DNA that encodes RNA or proteins that mediate or alter expression of endogenous DNA by affecting transcription, translation, or other regulatable biochemical processes, and the like. The cell that expresses heterologous DNA may contain DNA encoding the same or different expression products. Heterologous DNA need not be expressed and may be integrated into the host cell genome or maintained episomally.

[0096] Recombinant receptors on recombinant eukaryotic cell surfaces may contain one or more subunits encoded by the DNA or mRNA encoding human neuronal nicotinic AChR subunits, or may contain a mixture of subunits encoded by the host cell and subunits encoded by heterologous DNA or mRNA. Recombinant receptors may be homogeneous or may be a mixture of subtypes. Mixtures of DNA or mRNA encoding receptors from various species, such as rats and humans, may also be introduced into the cells. Thus, a cell can be prepared that expresses recombinant receptors containing only α3 and β4 subunits, or any other combination of alpha and beta subunits provided herein. For example, α4 and/or α7 subunits of the present invention can be co-expressed with β2 and/or β4 receptor subunits; similarly, β4 subunits according to the present invention can be co-expressed with α2, α3, α4, α5 and/or α7 receptor subunits. As noted previously, some of the nNAChR subunits may be capable of forming functional receptors in the absence of other subunits, thus co-expression is not always required to produce functional receptors.

[0097] As used herein, a functional neuronal nicotinic AChR is a receptor that exhibits an activity of neuronal nicotinic AChRs as assessed by any in vitro or in vivo assay disclosed herein or known to those of skill in the art. Possession of any such activity that may be assessed by any method known to those of skill in the art and provided herein is sufficient to designate a receptor as functional. Methods for detecting NAChR protein and/or activity include, for example, assays that measure nicotine binding, 86Rb ion-flux, Ca2+ influx, the electrophysiofogical response of cells containing heterologous DNA or mRNA encoding one or more receptor subunit subtypes, and the like. Since all combinations of alpha and beta subunits may not form functional receptors, numerous combinations of alpha and beta subunits should be tested in order to fully characterize a particular subunit and cells which produce same. Thus, as used herein, “functional” with respect to a recombinant or heterologous human neuronal nicotinic AChR means that the receptor channel is able to provide for and regulate entry of human neuronal nicotinic AChR-permeable ions, such as, for example, Na+, K+, Ca 2+ or Ba2+, in response to a stimulus and/or bind ligands with affinity for the receptor. Preferably such human neuronal nicotinic AChR activity is distinguishable, such as by electrophysiological, pharmacological and other means known to those of skill in the art, from any endogenous nicotinic AChR activity that may be produced by the host cell.

[0098] In accordance with a particular embodiment of the present invention, recombinant human neuronal nicotinic AChR-expressing mammalian cells or oocytes can be contacted with a test compound, and the modulating effect(s) thereof can then be evaluated by comparing the AChR-mediated response in the presence and absence of test compound, or by comparing the AChR-mediated response of test cells, or control cells (i.e., cells that do not express nNAChRs), to the presence of the compound.

[0099] As used herein, a compound or signal that “modulates the activity of a neuronal nicotinic AChR” refers to a compound or signal that alters the activity of NAChR so that activity of the NAChR is different in the presence of the compound or signal than in the absence of the compound or signal. In particular, such compounds or signals include agonists and antagonists. The term agonist refers to a substance or signal, such as ACh, that activates receptor function; and the term antagonist refers to a substance that interferes with receptor function. Typically, the effect of an antagonist is observed as a blocking of activation by an agonist. Antagonists include competitive and non-competitive antagonists. A competitive antagonist (or competitive blocker) interacts with or near the site specific for the agonist (e.g., ligand or neurotransmitter) for the same or closely situated site. A non-competitive antagonist or blocker inactivates the functioning of the receptor by interacting with a site other than the site that interacts with the agonist.

[0100] As understood by those of skill in the art, assay methods for identifying compounds that modulate human neuronal nicotinic AChR activity (e.g., agonists and antagonists) generally require comparison to a control. One type of a “control” cell or “control” culture is a cell or culture that is treated substantially the same as the cell or culture exposed to the test compound, except the control culture is not exposed to test compound. For example, in methods that use voltage clamp electrophysiological procedures, the same cell can be tested in the presence and absence of test compound, by merely changing the external solution bathing the cell. Another type of “control” cell or “control” culture may be a cell or a culture of cells which are identical to the transfected cells, except the cells employed for the control culture do not express functional human neuronal nicotinic AChRs. In this situation, the response of test cell to test compound is compared to the response (or lack of response) of receptor-negative (control) cell to test compound, when cells or cultures of each type of cell are exposed to substantially the same reaction conditions in the presence of compound being assayed.

[0101] The functional recombinant human neuronal nicotinic AChR includes at least an alpha subunit, or an alpha subunit and a beta subunit of a human neuronal nicotinic AChR. Eukaryotic cells expressing these subunits have been prepared by injection of RNA transcripts and by transfection of DNA. Such cells have exhibited nicotinic AChR activity attributable to human neuronal nicotinic AChRs that contain one or more of the heterologous human neuronal nicotinic AChR subunits. For example, Xenopus laevis oocytes that had been injected with in vitro transcripts of the DNA encoding human neuronal nicotinic AChR α3 and β4 subunits exhibited AChR agonist induced currents; whereas cells that had been injected with transcripts of either the α3 or β4 subunit alone did not. In addition, HEK 293 cells that had been co-transfected with DNA encoding human neuronal NAChR α3 and β4 subunits exhibited AChR agonist-induced increases in intracellular calcium concentration, whereas control HEK 293 cells (i.e., cells that had not been transfected with α3- and β4-encoding DNA) did not exhibit any AChR agonist-induced increases in intracellular calcium concentration.

[0102] With respect to measurement of the activity of functional heterologous human neuronal nicotinic AChRs, endogenous AChR activity and, if desired, activity of AChRs that contain a mixture of endogenous host cell subunits and heterologous subunits, should, if possible, be inhibited to a significant extent by chemical, pharmacological and electrophysiological means.

DEPOSITS

[0103] The deposited clones have been deposited at the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md., U.S.A. 20852, under the terms of the Budapest Treaty on the International Recognition of Deposits of Microorganisms for Purposes of Patent Procedure and the Regulations promulgated under this Treaty. Samples of the deposited material are and will be available to industrial property offices and other persons legally entitled to receive them under the terms of the Treaty and Regulations and otherwise in compliance with the patent laws and regulations of the United States of America and all other nations or international organizations in which this application, or an application claiming priority of this application, is filed or in which any patent granted on any such application is granted. In particular, upon issuance of a U.S. patent based on this or any application claiming priority to or incorporating this application by reference thereto, all restrictions upon availability of the deposited material will be irrevocably removed.

[0104] The invention will now be described in greater detail with reference to the following non-limiting examples.

EXAMPLE 1

Isolation of DNA Encoding Human nNAChR Subunits

[0105] A. DNA Encoding a Human nNAChR β4 Subunit

[0106] Random primers were used in synthesizing cDNA from RNA isolated from the IMR32 human neuroblastoma cell line (the cells had been treated with dibutyryl cAMP and bromodeoxyuridine prior to constructing the library). The library constructed from the cDNAs was screened with a fragment of a rat nicotinic AChR β4 subunit cDNA. Hybridization was performed at 42° C. in 5×SSPE, 5×Denhardt's solution, 50% formamide, 200 μg/ml herring sperm DNA and 0.2% SDS. Washes were performed in 0.1×SSPE, 0.2% SDS at 65° C. Five clones were identified that hybridized to the probe.

[0107] The five clones were plaque-purified and characterized by restriction enzyme mapping and DNA sequence analysis. The insert DNA of one of the five clones contained the complete coding sequence of a β4 subunit of a human nicotinic AChR (see nucleotides 87-1583 of SEQ ID No. 11). The amino acid sequence deduced from the nucleotide sequence of the full-length clone has ˜81% identity with the amino acid sequence deduced from the rat nicotinic AChR β4 subunit DNA. Several regions of the deduced rat and human β4 amino acid sequences are notably dissimilar: amino acids 1-23 (the human sequence has only ˜36% identity with respect to the rat sequence), 352-416 (the human sequence has only ˜48% identity with respect to the rat sequence), and 417-492 (the human sequence has only ˜78% identity with respect to the rat sequence). Furthermore, amino acids 376-379 in the rat β4 subunit are not contained in the human β4 subunit.

[0108] B. DNA Encoding a Human nNAChR α7 Subunit

[0109] An amplified IMR32 cell cDNA library (1×106 recombinants; cells treated with dibutyryl cAMP and bromodeoxyuridine) was screened with a fragment of a rat nicotinic AChR α7 subunit cDNA. The hybridization conditions were identical to those described above for screening an IMR32 cell cDNA library with the rat β4 subunit DNA. Washes were performed in 0.2×SSPE, 0.2% SDS at 65° C. Seven positive clones were identified by hybridization to the labeled rat DNA probe. Six of the clones were plaque-purified and characterized by restriction enzyme mapping and DNA sequence analysis. One of the clones contains the complete coding sequence of a human AChR receptor α7 subunit gene (see nucleotides 73-1581 of SEQ ID No. 7).

[0110] C. DNA Encoding a Human nNAChR α4 Subunit

[0111] Random primers were used in synthesizing cDNA from RNA isolated from human hippocampus tissue. cDNAs larger than 2.0 kb were inserted into the λgt10 phage vector to create a cDNA library. Approximately 1×106 recombinants were screened with a fragment of a DNA encoding a rat nicotinic AChR α4 subunit using the same hybridization and washing conditions as described above for screening an IMR32 cell cDNA library for α7 subunit cDNAs. Three clones hybridized strongly to the probe. Two of these three clones, designated KEα4.1 and KEα4.2, have been deposited with the American Type Culture Collection (ATCC, Rockville, Md.) and assigned accession nos. 69152 and 69239, respectively.

[0112] Characterization of the plaque-purified clones revealed that one of the clones, KEα4.2, contains the complete coding sequence of a human nicotinic AChR α4 subunit gene (coding sequence of this human α4 subunit cDNA is provided as nucleotides 184-2067 in SEQ ID No. 5). Comparison of the 5′ ends of the coding sequences of the human and rat α4 subunit cDNAs reveals that the rat sequence contains an 18-nucleotide segment that is not present in the human sequence.

[0113] D. DNA Encoding Human nNAChR α2, α3, & β2 Subunits

[0114] Plasmids containing DNA that encodes and/or that can be used to isolate DNA that encodes human neuronal nicotinic acetylcholine receptor α2, α3 and β2 subunits have been deposited with the American Type Culture Collection (ATCC). The clone names and deposit accession numbers are:

Subunit	Clone Name	ATCC Accession No.
α2	HnAChRα2	68277
α3	HnACHRα3	68278
β2	HnAChRβ2	68279

[0115] In addition, DNA sequences that encode full-length α2, α3 and β2 subunits are set forth in SEQ ID Nos. 1, 3 and 9, respectively.

EXAMPLE 2

Preparation of Constructs for the Expression of Recombinant Human Neuronal Nicotinic AChR Subunits

[0116] Isolated cDNAs encoding human neuronal nicotinic AChR subunits were incorporated into vectors for use in expressing the subunits in mammalian host cells and for use in generating in vitro transcripts of the DNAs to be expressed in Xenopus oocytes. Several different vectors were utilized in preparing the constructs as follows.

[0117] A. Construct for Expression of a Human nNAChR α3 Subunit

[0118] DNA encoding a human neuronal nicotinic AChR α3 subunit was subcloned into the pCMV-T7-2 general expression vector to create pCMV-KEα3. Plasmid pCMV-T7-2 (see FIG. 1) is a pUC19-based vector that contains a CMV promoter/enhancer, SV40 splice donor/splice acceptor sites located immediately downstream of the promoter, a T7 bacteriophage RNA polymerase promoter positioned downstream of the SV40 splice sites, an SV40 polyadenylation signal downstream of the T7 promoter, and a polylinker between the T7 promoter and the polyadenylation signal. This vector thus contains all the regulatory elements required for expression of heterologous DNA in a mammalian host cell, wherein the heterologous DNA has been incorporated into the vector at the polylinker. In addition, because the T7 promoter is located just upstream of the polylinker, this plasmid can be used for synthesis of in vitro transcripts of heterologous DNA that has been subcloned into the vector at the polylinker. FIG. 1 also shows a restriction map of pCMV-T7-3. This plasmid is identical to pCMV-T7-2 except that the restriction sites in the polylinker are in the opposite order as compared to the order in which they occur in pCMV-T7-2.

[0119] A 1.7 kb SfiI (blunt-ended)/EcoRI DNA fragment containing nucleotides 27-1759 of SEQ ID No. 3 (i.e., the entire α3 subunit coding sequence plus 12 nucleotides of 5′ untranslated sequence and 206 nucleotides of 3′ untranslated sequence) was ligated to EcoRV/EcoRI-digested pCMV-T7-2 to generate pCMV-KEα3. Plasmid pCMV-KEα3 was used for expression of the α3 subunit in mammalian cells and for generating in vitro transcripts of the α3 subunit DNA.

[0120] B. Constructs for Expression of a Human nNAChR β4 Subunit

[0121] A 1.9 kb EcoRI DNA fragment containing nucleotides 1-1915 of SEQ ID No. 11 (i.e., the entire β4 subunit coding sequence plus 86 nucleotides of 5′ untranslated sequence and 332 nucleotides of 3′ untranslated sequence) was ligated to EcoRI-digested pGEM7Zf(+) (Promega Catalog #P2251; Madison, Wis.). The resulting construct, KEβ4.6/pGEM, contains the T7 bacteriophage RNA polymerase promoter in operative association with two tandem β4 subunit DNA inserts (in the same orientation) and was used in generating in vitro transcripts of the DNA.

[0122] The same 1.9 kb EcoRI DNA fragment containing nucleotides 1-1915 of SEQ ID No. 11 was ligated as a single insert to EcoRI-digested pCMV-T7-3 to generate pCMV-KEβ4. Plasmid pCMV-KEβ4 was used for expression of the β4 subunit in mammalian cells and for generating in vitro transcripts of the β4 subunit DNA.

[0123] C. Constructs for Expression of a Human nNAChR α7 Subunit

[0124] Two pCMV-T7-2-based constructs were prepared for use in recombinant expression of a human neuronal nicotinic AChR α7 subunit. The first construct, pCMV-KEα7.3, was prepared by ligating a 1.9 kb XhoI DNA fragment containing nucleotides 1-1876 of SEQ ID No. 7 (i.e., the entire α7 subunit coding sequence plus 72 nucleotides of 5′ untranslated sequence and 295 nucleotides of 3′ untranslated sequence) to SalI-digested pCMV-T7-3. The second construct, pCMV-KEα7, was prepared by replacing the 5′ untranslated sequence of the 1.9 kb XhoI α7 subunit DNA fragment described above with a consensus ribosome binding site (5′-GCCACC-3′; see Kozak (1987) Nucl. Acids Res. 15:8125-8148). The resulting modified fragment was ligated as a 1.8 kb BglII/XhoI fragment with BglII/SalI-digested pCMV-T7-2 to generate pCMV-KEα7. Thus, in pCMV-KEα7, the translation initiation codon of the coding sequence of the α7 subunit cDNA is preceded immediately by a consensus ribosome binding site.

[0125] D. Constructs for Expression of a Human nNAChR β2 Subunit

[0126] DNA fragments encoding portions of a human neuronal nicotinic AChR β2 subunit were ligated together to generate a full-length β2 subunit coding sequence contained in plasmid pIBI24 (International Biotechnologies, Inc. (IBI), New Haven, Conn.). The resulting construct, Hβ2.1F, contains nucleotides 1-2450 of SEQ ID No. 9 (i.e., the entire β2 subunit coding sequence, plus 266 nucleotides of 5′ untranslated sequence and 675 nucleotides of 3′ untranslated sequence) in operative association with the T7 promoter. Therefore, Hβ2.1F was used for synthesis of in vitro transcripts from the β2 subunit DNA.

[0127] Since the 5′ untranslated sequence of the β2 subunit DNA contains a potential alternative translation initiation codon (ATG) beginning 11 nucleotides upstream (nucleotides 256-258 in SEQ ID No. 9) of the correct translation initiation codon (nucleotides 267-269 in SEQ ID No. 9), and because the use of the upstream ATG sequence to initiate translation of the β2 DNA would result in the generation of an inoperative peptide (because the upstream ATG is not in the correct reading frame), an additional β2-encoding construct was prepared as follows. A 2.2 kb KspI/EcoRI DNA fragment containing nucleotides 262-2450 of SEQ ID No. 9 was ligated to pCMV-T7-2 in operative association with the T7 promoter of the plasmid to create pCMV-KEβ2. The β2 subunit DNA contained in pCMV-KEβ2 retains only 5 nucleotides of 5′ untranslated sequence upstream of the correct translation initiation codon.

EXAMPLE 3

Expression of Recombinant Human Nicotinic AChR in Oocytes

[0128] Xenopus oocytes were injected with in vitro transcripts prepared from constructs containing DNA encoding α3, α7, β2 and β4 subunits. Electrophysiological measurements of the oocyte transmembrane currents were made using the two-electrode voltage clamp technique (see, e.g., Stuhmer (1992) Meth. Enzymol. 207:319-339).

[0129] 1. Preparation of in vitro Transcripts

[0130] Recombinant capped transcripts of pCMV-KEα3, pCMV-KEβ2, KEβ4.6/pGEM and pCMV-KEβ4 were synthesized from linearized plasmids using the mCAP RNA Capping Kit (Cat. #200350 from Stratagene, Inc., La Jolla, Calif.). Recombinant capped transcripts of pCMV-KEα7, pCMV-KEα7.3 and Hβ2.1F were synthesized from linearized plasmids using the MEGAscript T7 in vitro transcription kit according to the capped transcript protocol provided by the manufacturer (Catalog #1334 from AMBION, Inc., Austin, Tex.). The mass of each synthesized transcript was determined by UV absorbance and the integrity of each transcript was determined by electrophoresis through an agarose gel.

[0131] 2. Electrophysiology

[0132] Xenopus oocytes were injected with either 12.5, 50 or 125 ng of human nicotinic AChR subunit transcript per oocyte. The preparation and injection of oocytes were carried out as described by Dascal (1987) in Crit. Rev. Biochem. 22:317-387. Two-to-six days following mRNA injection, the oocytes were examined using the two-electrode voltage clamp technique. The cells were bathed in Ringer's solution (115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2, 10 mM HEPES, pH 7.3) containing 1 μM atropine with or without 100 μM d-tubocurarine. Cells were voltage-clamped at −60 to −80 mV. Data were acquired with Axotape software at 2-5 Hz. The agonists acetylcholine (ACh), nicotine, and cytisine were added at concentrations ranging from 0.1 μM to 100 μM. The results of electrophysiological analyses of the oocytes are summarized in Table 1.

TABLE 1
	Number of
	oocytes
Template, ng RNA	re-		Current
injected	sponding	Agonists	Amplitude
pCMV-KEα3,	12.5	ng	0 of 8	ACh,
				Nicotine
KEβ4.6/pGEM,	12.5	ng	0 of 9	ACh,
				Nicotine
pCMV-KEα3,	12.5	ng +	4 of 5	ACh,	20-550	nA
KEβ4.6/pGEM,	12.5	ng		Nicotine
pCMV-KEα3,	12.5	ng +	3 of 4	ACh,	20-300	nA
KEβ4.6/pGEM,	12.5	ng		Cytisine,
				Nicotine
pCMV-KEα3,	125	ng +	5 of 5	ACh,	200-500	nA
and	125	ng		Nicotine,
pCMV-KEβ4,				Cytisine
pCMV-KEα3,	125	ng +	6 of 6	ACh,	100-400	nA
pCMV-KEβ4,	125	ng		Nicotine,
				Cytisine
pCMV-KEα7.3,	125	ng	3 of 15	ACh	˜20	nA
pCMV-KEα7,	125	ng	11 of 11	ACh	20-250	nA
pCMV-KEα3,	12.5	ng +	2 of 9	ACh,	<10	nA
pCMV-KE,β2,	12.5	ng		Nicotine
pCMV-KEα3,	125	ng +	0 of 9	ACh,
pCMV-KEβ2,	125	ng		Nicotine
pCMV-KEα3,	125	ng +	13 of 16	ACh (100	˜20	nA
Hβ2.1F,	125	ng		μM)	˜80	nA
				ACh (300
				μM)

[0133] a. Oocytes Injected with α3 and/or β4 Transcripts

[0134] Oocytes that had been injected with 12.5 ng of the α3 transcript or 12.5 ng of the β4 transcript did not respond to application of up to 100 μM ACh, nicotine or cytisine. Thus, it appears that these subunits do not form functional homomeric nicotinic AChR channels. By contrast, oocytes injected with 12.5 or 125 ng of the α3 transcript and 12.5 ng or 125 ng of the β4 transcript exhibited detectable inward currents in response to ACh, nicotine, and cytisine at the tested concentrations (0.1 μM to 10 μM). Some differences in the kinetics of the responses to cytisine compared to nicotine and ACh were observed. The relative potency of the agonists appeared to be cytisine>ACh>nicotine, which differs from the results of similar studies of oocytes injected with transcripts of the rat nicotinic AChR α3 and β4 subunits (see, for example, Luetje et al. (1991) J. Neurosci. 11:837-845).

[0135] The responses to ACh and nicotine were reproducibly blocked by d-tubocurarine. For example, complete blockage of the response to ACh was observed in the presence of 100 μM d-tubocurarine. The inhibition appeared to be reversible. The responses to ACh, nicotine and cytisine were also at least partially blocked by 100 nM mecamylamine.

[0136] The current response of α3-β4-injected oocytes to 10 μM ACh was also examined in terms of membrane voltage. In these experiments, voltage steps were applied to the cells in the presence of ACh. The graph of current vs. voltage appeared typical of responses observed for Na+, K+-permeable channels. For example, the zero current level (reversal potential) is less than −40 mV. The contribution of Ca++ flux to the total current can be ascertained by varying the calcium concentration in the external medium and taking multiple current measurements at different holding potentials around the reversal potential. Such studies indicate that the channel carrying the current generated in response to ACh treatment of α3-β4-injected oocytes is permeable to Na+, K+ and Ca++.

[0137] b. Oocytes Injected with α7 Subunit Transcripts

[0138] As described in Example 1, two constructs were prepared for use in expressing the human neuronal nicotinic AChR α7 subunit. Plasmid pCMV-KEα7.3 contains the α7 subunit coding sequence with 72 nucleotides of 5′ untranslated sequence upstream of the translation initiation codon. Plasmid pCMV-KEα7 contains the α7 subunit coding sequence devoid of any 5′ untranslated sequence and further contains a consensus ribosome binding site immediately upstream of the coding sequence.

[0139] Oocytes injected with 125 ng of α7 transcript synthesized from pCMV-KEα7 displayed inward currents in response to 10 or 100 μM ACh. This response was blocked by 100 μM d-tubocurarine.

[0140] Oocytes injected with 125 ng of α7 transcript synthesized from pCMV-KEα7.3 exhibited ACh-induced currents that were substantially weaker than those of oocytes injected with α7 transcript synthesized from pCMV-KEα7. These results indicate that human neuronal nicotinic AChR α7 subunit transcripts generated from α7 subunit DNA containing a ribosome binding site in place of 5′ untranslated sequence may be preferable for expression of the α7 receptor in oocytes.

[0141] C. Oocytes Injected with α3 and β2 Subunit Transcripts

[0142] As described in Example 1, two constructs were prepared for use in expressing the human neuronal nicotinic AChR β2 subunit. Plasmid Hβ2.1F contains the β2 subunit coding sequence with 266 nucleotides of 5′ untranslated sequence upstream of the translation initiation codon. Plasmid pCMV-KEβ2 contains the β2 subunit coding sequence and only 5 nucleotides of 5′ untranslated sequence upstream of the translation initiation codon.

[0143] Oocytes injected with transcripts of pCMV-KEα3 and pCMV-KEβ2 displayed no current in response to nicotinic AChR agonists. In contrast, oocytes injected with transcripts of pCMV-KEα3 and Hβ2.1F displayed ˜20 nA inward currents in response to 100 μM ACh and ˜80 nA inward currents in response to 300 μM ACh. The current response was blocked by 100 μM d-tubocurarine. These results indicate that human neuronal nicotinic AChR β2 subunit transcripts generated from β2 subunit DNA containing 5′ untranslated sequence may be preferable to transcripts generated from β2 DNA containing only a small portion of 5′ untranslated sequence for expression of the α3β2 receptors in oocytes.

EXAMPLE 4

Recombinant Expression of Human nNAChR Subunits in Mammalian Cells

[0144] Human embryonic kidney (HEK) 293 cells were transiently and stably transfected with DNA encoding human neuronal nicotinic AChR α3 and β4, or α7 subunits. Transient transfectants were analyzed for expression of nicotinic AChR using various assays, e.g., electrophysiological methods, Ca2+-sensitive fluorescent indicator-based assays and [125I]-α-bungarotoxin-binding assays.

[0145] 1. Transient Transfection of HEK Cells

[0146] Two transient transfections were performed. In one transfection, HEK cells were transiently co-transfected with DNA encoding α3 (plasmid pCMV-KEα3) and β4 (plasmid pCMV-KEβ4) subunits. In the other transfection, HEK cells were transiently transfected with DNA encoding the α7 subunit (plasmid pCMV-KEα7). In both transfections, ˜2×106 HEK cells were transiently transfected with 18 μg of the indicated plasmid(s) according to standard CaPO4 transfection procedures [Wigler et al. (1979) Proc. Natl. Acad. Sci. USA 76:1373-1376]. In addition, 2 μg of plasmid pCMVβgal (Clontech Laboratories, Palo Alto, Calif.), which contains the Escherichia coli β-galactosidase gene fused to the CMV promoter, were co-transfected as a reporter gene for monitoring the efficiency of transfection. The transfectants were analyzed for β-galactosidase expression by measurement of β-galactosidase activity [Miller (1972) Experiments in Molecular Genetics, pp.352-355, Cold Spring Harbor Press]. Transfectants can also be analyzed for β-galactosidase expression by direct staining of the product of a reaction involving β-galactosidase and the X-gal substrate [Jones (1986) EMBO 5:3133-3142].

[0147] The efficiency of transfection of HEK cells with pCMV-KEα3/pCMV-KEβ4 was typical of standard efficiencies, whereas the efficiency of transfection of HEK cells with pCMV-KEα7 was below standard levels.

[0148] 2. Stable Transfection of HEK Cells

[0149] HEK cells were transfected using the calcium phosphate transfection procedure [Current Protocols in Molecular Biology, Vol. 1, Wiley Inter-Science, Supplement 14, Unit 9.1.1-9.1.9 (1990)]. Ten-cm plates, each containing one-to-two million HEK cells were transfected with 1 ml of DNA/calcium phosphate precipitate containing 9.5 μg pCMV-KEα3, 9.5 μg pCMV-KEβ4 and 1 μg pSV2neo (as a selectable marker). After 14 days of growth in media containing 1 μg/ml G418, colonies had formed and were individually isolated by using cloning cylinders. The isolates were subjected to limiting dilution and screened to identify those that expressed the highest level of nicotinic AChR, as described below.

[0150] 3. Analysis of Transfectants

[0151] a. Fluorescent Indicator-based Assays

[0152] Activation of the ligand-gated nicotinic AChR by agonists leads to an influx of cations, including Ca++, through the receptor channel. Ca++ entry into the cell through the channel can induce release of calcium contained in intracellular stores. Monovalent cation entry into the cell through the channel can also result in an increase in cytoplasmic Ca++ levels through depolarization of the membrane and subsequent activation of voltage-dependent calcium channels. Therefore, methods of detecting transient increases in intracellular calcium concentration can be applied to the analysis of functional nicotinic AChR expression. One method for measuring intracellular calcium levels relies on calcium-sensitive fluorescent indicators.

[0153] Calcium-sensitive indicators, such as fluo-3 (Catalog No. F-1241, Molecular Probes, Inc., Eugene, Oreg.), are available as acetoxymethyl esters which are membrane permeable. When the acetoxymethyl ester form of the indicator enters a cell, the ester group is removed by cytosolic esterases, thereby trapping the free indicator in the cytosol. Interaction of the free indicator with calcium results in increased fluorescence of the indicator; therefore, an increase in the intracellular Ca2+ concentration of cells containing the indicator can be expressed directly as an increase in fluorescence. An automated fluorescence detection system for assaying nicotinic AChR has been described in commonly assigned pending U.S. patent application Ser. No. 07/812,254 and corresponding PCT Patent Application No. US92/11090.

[0154] HEK cells that were transiently or stably co-transfected with DNA encoding α3 and β4 subunits were analyzed for expression of functional recombinant nicotinic AChR using the automated fluorescent indicator-based assay. The assay procedure was as follows.

[0155] Untransfected HEK cells (or HEK cells transfected with pCMV-T7-2) and HEK cells that had been co-transfected with pCMV-KEα3 and pCMV-KEβ4 were plated in the wells of a 96-well microtiter dish and loaded with fluo-3 by incubation for 2 hours at 20° C. in a medium containing 20 μM fluo-3, 0.2% Pluronic F-127 in HBS (125 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 0.62 mM MgSO4, 6 mM glucose, 20 mM HEPES, pH 7.4). The cells were then washed with assay buffer (i.e., HBS). The antagonist d-tubocurarine was added to some of the wells at a final concentration of 10 μM. The microtiter dish was then placed into a fluorescence plate reader and the basal fluorescence of each well was measured and recorded before addition of 200 μM nicotine to the wells. The fluorescence of the wells was monitored repeatedly during a period of approximately 60 seconds following addition of nicotine.

[0156] The fluorescence of the untransfected HEK cells (or HEK cells transfected with pCMV-T7-2) did not change after addition of nicotine. In contrast, the fluorescence of the co-transfected cells, in the absence of d-tubocurarine, increased dramatically after addition of nicotine to the wells. This nicotine-stimulated increase in fluorescence was not observed in co-transfected cells that had been exposed to the antagonist d-tubocurarine. These results demonstrate that the co-transfected cells express functional recombinant AChR that are activated by nicotine and blocked by d-tubocurarine.

[0157] b. α-Bungarotoxin Binding Assays

[0158] HEK293 cells transiently transfected with pCMV-KEα7 were analyzed for [125I]-α-bungarotoxin (BgTx) binding. Whole transfected cells and membranes prepared from transfected cells were examined in these assays. Rat brain membranes were included in the assays as a positive control.

[0159] Rat brain membranes were prepared according to the method of Hampson et al. (1987) J. Neurochem 49:1209. Membranes were prepared from the HEK cells transfected with pCMV-KEα7 and HEK cells transiently transfected with plasmid pUC19 only (negative control) according to the method of Perez-Reyes et al. (1989) Nature 340:233. Whole transfected and negative control cells were obtained by spraying the tissue culture plates with phosphate-buffered saline containing 0.1% (w/v) BSA. The cells were then centrifuged at low speed, washed once, resuspended in assay buffer (118 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 20 mM HEPES, 0.1% (w/v)BSA, 0.05% (w/v) bacitracin and 0.5 mM PMSF, pH 7.5) and counted.

[0160] Specific binding of [125I]-α-BgTx to rat brain membranes was determined essentially as described by Marks et al. (1982) Molec. Pharmacol. 22:554-564, with several modifications. The membranes were washed twice in assay buffer. The assay was carried out in 12×75 mm polypropylene test tubes in a total volume of 0.5 ml assay buffer. The membranes were incubated with 10 nM [125I]-α-BgTx (New England Nuclear, Boston, Mass.) for one hour at 37° C. The assay mixtures were then centrifuged at 2300×g for 10 minutes at 4° C. The supernatant was decanted and the pellets were washed twice with 2 ml aliquots of ice-cold assay buffer. The supernatants were decanted again and the radioactivity of the pellets was measured in a γ-counter. Non-specific binding was determined in the presence of 1 μM unlabeled α-BgTx. Specific binding was determined by subtracting nonspecific binding from total binding. Specific binding of [125I]-α-BgTx to membranes prepared from transfected and negative control cells was determined as described for determining specific binding to rat brain membranes except that the assay buffer did not contain BSA, bacitracin and PMSF. Specific binding of [125I]-α-BgTx to transfected and negative control whole cells was determined basically as described for determining specific binding to rat brain membranes.

[0161] [125I]-α-BgTx binding was evaluated as a function of membrane concentration and as a function of incubation time. [125I]-α-BgTx binding to rat brain membranes increased in a linear fashion with increasing amounts of membrane (ranging between 25-500 μg). The overall signal-to-noise ratio of binding (i.e., ratio of total binding to non-specific binding) was 3:1. Although some binding of [125I]-α-BgTx to transfected cell membranes was detected, it was mostly non-specific binding and did not increase with increasing amounts of membrane. [125I]-α-BgTx binding to the transfectants and negative control cells appeared to be similar.

[0162] To monitor [125I]-α-BgTx binding to rat brain membranes and whole transfected and negative control cells, 300 μg of membrane or 500,000 cells were incubated with 1 nM or 10 nM [125I]-α-BgTx, respectively, at 37° C. for various times ranging from 0-350 min. Aliquots of assay mixture were transferred to 1.5 ml microfuge tubes at various times and centrifuged. The pellets were washed twice with assay buffer. [125I]-α-BgTx binding to rat brain membranes increased with time and was maximal after three hours. The binding profiles of the transfected and negative control cells were the same and differed from that of rat brain membranes.

EXAMPLE 5

Characterization of Cell Lines Expressing nNAChRs

[0163] Recombinant cell lines generated by transfection with DNA encoding human neuronal nicotinic AChRs, such as those described in Example 3, can be further characterized using one or more of the following methods.

[0164] A. Northern or Slot Blot Analysis for Expression of α- and/or β-subunit Encoding Messages

[0165] Total RNA is isolated from ˜1×107 cells and 10-15 μg of RNA from each cell type is used for northern or slot blot hybridization analysis. The inserts from human neuronal NAChR-encoding plasmids can be nick-translated and used as probe. In addition, the β-actin gene sequence (Cleveland et al. (1980) Cell 20:95-105) can be nick-translated and used as a control probe on duplicate filters to confirm the presence or absence of RNA on each blot and to provide a rough standard for use in quantitating differences in α- or β-specific mRNA levels between cell lines. Typical northern and slot blot hybridization and wash conditions are as follows:

[0166] hybridization in 5×SSPE, 5×Denhardt's solution, 50% formamide, at 42° C. followed by washing in 0.2×SSPE, 0.1% SDS, at 65° C.

[0167] B. Nicotine-binding Assay

[0168] Cell lines generated by transfection with human neuronal nicotinic AChR α- or α- and β-subunit-encoding DNA can be analyzed for their ability to bind nicotine, for example, as compared to control cell lines: neuronally-derived cell lines PC12 (Boulter et al., (1986), supra; ATCC #CRL1721) and IMR32 (Clementi, et al. (1986); Int. J. Neurochem. 47:291-297; ATCC #CCL127), and muscle-derived cell line BC3H1 (Patrick, et al., (1977); J. Biol. Chem. 252:2143-2153). Negative control cells (i.e., host cells from which the transfectants were prepared) are also included in the assay. The assay is conducted as follows:

[0169] Just prior to being assayed, transfected cells are removed from plates by scraping. Positive control cells used are PC12, BC3H1, and IMR32 (which had been starved for fresh media for seven days). Control cell lines are removed by rinsing in 37° C. assay buffer (50 mM Tris/HCl, 1 mM MgCl2, 2 mM CaCl2, 120 mM NaCl, 3 mM EDTA, 2 mg/ml BSA and 0.1% aprotinin at pH7.4). The cells are washed and resuspended to a concentration of 1×106/250 μl. To each plastic assay tube is added 250 μl of the cell solution, 15 nM 3H-nicotine, with or without 1 mM unlabeled nicotine, and assay buffer to make a final volume of 500 μl. The assays for the transfected cell lines are incubated for 30 min at room temperature; the assays of the positive control cells are incubated for 2 min at 1° C. After the appropriate incubation time, 450 μl aliquots of assay volume are filtered through Whatman GF/C glass fiber filters which has been pretreated by incubation in 0.05% polyethyleneimine for 24 hours at 4° C. The filters are then washed twice, with 4 ml each wash, with ice cold assay buffer. After washing, the filters are dried, added to vials containing 5 ml scintillation fluid and radioactivity is measured.

[0170] C. 86Rb Ion-flux Assay

[0171] The ability of nicotine or nicotine agonists and antagonists to mediate the influx of 86Rb into transfected and control cells has been found to provide an indication of the presence of functional AChRs on the cell surface. The 86Rb ion-flux assay is conducted as follows:

[0172] 1. The night before the experiment, cells are plated at 2×106 per well (i.e., 2 ml per well) in a 6-well polylysine-coated plate.

[0173] 2. The culture medium is decanted and the plate washed with 2 ml of assay buffer (50 mM HEPES, 260 mM sucrose, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, 5.5. mM glucose) at room temperature.

[0174] 3. The assay buffer is decanted and 1 ml of assay buffer, containing 3 μCi/ml 86Rb, with 5 mM ouabain and agonist or antagonist in a concentration to effect a maximum response, is added.

[0175] 4. The plate is incubated on ice at 1° C. for 4 min.

[0176] 5. The buffer is decanted into a waste container and each well was washed with 3 ml of assay buffer, followed by two washes of 2 ml each.

[0177] 6. The cells are lysed with 2×0.5 ml of 0.2% SDS per well and transferred to a scintillation vial containing 5 ml of scintillation fluid.

[0178] 7. The radioactivity contained in each vial is measured and the data calculated.

[0179] Positive control cells provided the following data in this assay:

	PC12	IMR32
	Maximum	Maximum
	EC50	response	EC50	response
	Agonist
	nicotine	52	μM	2.1Xa	18	μM	7.7Xa
	CCh	35	μM	3.3Xb	230	μM	7.6Xc
	cytisine	57	μM	3.6Xd	14	μM	10Xe
	Antagonist
	d-tubocurarine	0.81	μM		2.5	μM
	mecamylamine	0.42	μM		0.11	μM
	hexamethonium	ndf			22	μM
	atropine	12.5	μM		43	μM
	*CCh = carbamyicholine
	a 200 μM nicotine
	b300 μM Cch
	c3 mM CCh
	d1 mM cytisine
	e100 μM cytisine
	fnd = not determined

[0180] D. Electrophysiological Analysis of Mammalian Cells Transfected with Human Neuronal Nicotinic AChR Subunit-encoding DNA

[0181] Electrophysiological measurements may be used to assess the activity of recombinant receptors or to assess the ability of a test compound to potentiate, antagonize or otherwise modulate the magnitude and duration of the flow of cations through the ligand-gated recombinant AChR. The function of the expressed neuronal AChR can be assessed by a variety of electrophysiological techniques, including two-electrode voltage clamp and patch clamp methods. The cation-conducting channel intrinsic to the AChR opens in response to acetylcholine (ACh) or other nicotinic cholinergic agonists, permitting the flow of transmembrane current carried predominantly by sodium and potassium ions under physiological conditions. This current can be monitored directly by voltage clamp techniques. In preferred embodiments, transfected mammalian cells or injected oocytes are analyzed electrophysiologically for the presence of AChR agonist-dependent currents.

[0182] While the invention has been described in detail with reference to certain preferred embodiments thereof, it will be understood that modifications and variations are within the spirit and scope of that which is described and claimed.

SUMMARY OF SEQUENCES

[0183] Sequence ID No. 1 is a nucleotide sequence encoding an α2 subunit of human neuronal nicotinic acetylcholine receptor, and the deduced amino acid sequence thereof.

[0184] Sequence ID No. 2 is the amino acid sequence of the α2 subunit of human neuronal nicotinic acetylcholine receptor set forth in Sequence ID No. 1.

[0185] Sequence ID No. 3 is a nucleotide sequence encoding an α3 subunit of human neuronal nicotinic acetylcholine receptor, and the deduced amino acid sequence thereof.

[0186] Sequence ID No. 4 is the amino acid sequence of the α3 subunit of human neuronal nicotinic acetylcholine receptor set forth in Sequence ID No. 3.

[0187] Sequence ID No. 5 is a nucleotide sequence encoding an α4 subunit of a human neuronal nicotinic acetylcholine receptor, and the deduced amino acid sequence thereof.

[0188] Sequence ID No. 6 is the amino acid sequence of the α4 subunit of a human neuronal nicotinic acetylcholine receptor set forth in Sequence ID No. 5.

[0189] Sequence ID No. 7 is a nucleotide sequence encoding an α7 subunit of human neuronal nicotinic acetylcholine receptor, and the deduced amino acid sequence thereof.

[0190] Sequence ID No. 8 is the amino acid sequence of the α7 subunit of human neuronal nicotinic acetylcholine receptor set forth in Sequence ID No. 7.

[0191] Sequence ID No. 9 is a nucleotide sequence encoding a β2 subunit of human neuronal nicotinic acetylcholine receptor, and the deduced amino acid sequence thereof.

[0192] Sequence ID No. 10 is the amino acid sequence of the β2 subunit of human neuronal nicotinic acetylcholine receptor set forth in Sequence ID No. 9.

[0193] Sequence ID No. 11 is a nucleotide sequence encoding a β4 subunit of human neuronal nicotinic acetylcholine receptor, and the deduced amino acid sequence thereof.

[0194] Sequence ID No. 12 is the amino acid sequence of the β4 subunit of human neuronal nicotinic acetylcholine receptor set forth in Sequence ID No. 11.

Claims

1. Isolated nucleic acid, comprising a sequence of nucleotides encoding an α4, α7 or β4 subunit of a human neuronal nicotinic acetylcholine receptor.

2. Isolated DNA of claim 1, wherein the subunit is an α4 subunit.

3. Isolated DNA of claim 1, wherein the subunit is an α7 subunit.

4. Isolated DNA of claim 1, wherein the subunit is a β4 subunit.

5. Isolated cells, comprising a nucleic acid molecule of claim 1 that encodes an α subunit, wherein the cells are bacterial cells, mammalian cells or amphibian oöcytes; and the nucleic acid molecule is heterologous to the cells.

6. The cells of claim 5, further containing nucleic acid encoding a β subunit of human neuronal nicotinic acetylcholine receptor.

7. Isolated cells, comprising a nucleic acid molecule of claim 1 that encodes a β subunit, wherein the cells are bacterial cells, mammalian cells or amphibian oöcytes; and the nucleic acid molecule is heterologous to the cells.

8. The cells of claim 6, wherein the β subunit is selected from β2 or β4.

9. The cells of claim 5, wherein the cells express functional neuronal nicotinic acetylcholine receptors that contain one or more subunits encoded by the nucleic acid.

10. The cells of claim 7, wherein the cells express functional neuronal nicotinic acetylcholine receptors that contain one or more subunits encoded by the nucleic acid; and the cells further comprise nucleic acid encoding an α subunit.

11. The cells of claim 10, wherein the α subunit is selected from α1, α2, α3, α4, α5 or α7.

12. A method for identifying functional neuronal nicotinic acetylcholine receptor subunits and combinations thereof, comprising: (a) introducing a nucleic acid molecule of claim 1 that encodes an α subunit of a human neuronal nicotinic acetylcholine receptor and optionally introducing a nucleic acid molecule encoding a β subunit of a human neuronal nicotinic acetylcholine receptor into mammalian or amphibian cells; and (b) assaying for neuronal nicotinic acetylcholine receptor activity in cells of step (a).

13. A method of screening compounds to identify compounds that modulate the activity of human neuronal nicotinic acetylcholine receptors, comprising: contacting the cells of claim 5 with a test compound; determining the effect of a compound on the neuronal nicotinic acetylcholine receptor activity in cells of claim 5 compared to the effect on control cells that are substantially identical to the cells of claim 5, but do not express the receptors, or to the effect on neuronal nicotinic acetylcholine receptor activity of the cells in the absence of the compound; and identifying compounds that modulate the activity of human neuronal nicotinic acetylcholine receptors.

14. An isolated and substantially purified human neuronal nicotinic acetylcholine receptor α4 or α7 subunit.

15. A recombinant human neuronal nicotinic acetylcholine receptor, comprising one or more of the subunits of claim 14.

16. The human neuronal nicotinic acetylcholine receptor of claim 14, that comprises at least one human neuronal nicotinic acetylcholine receptor beta subunit.

17. The receptor of claim 16, wherein the β subunit is a β4 subunit.

18. An isolated and substantially purified human neuronal nicotinic acetylcholine receptor β4 subunit.

19. A recombinant human neuronal nicotinic acetylcholine receptor, comprising one or more of the subunits of claim 17 and also comprising an α subunit of a human neuronal nicotinic acetylcholine receptor.

20. A method for identifying functional neuronal nicotinic acetylcholine receptor subunits and combinations thereof, comprising: (a) introducing a nucleic acid molecule of that encodes an α subunit of a human neuronal nicotinic acetylcholine receptor and introducing a nucleic acid molecule of claim 1 encoding a β4 subunit of a human neuronal nicotinic acetylcholine receptor into mammalian or amphibian cells; and (b) assaying for neuronal nicotinic acetylcholine receptor activity in cells of step (a).

21. The method of claim 20, wherein the α subunit is selected from α1, α2, α3, α4, α5 or α7.

22. A method of screening compounds to identify compounds that modulate the activity of human neuronal nicotinic acetylcholine receptors, comprising: contacting the cells of claim 10 with a test compound; determining the effect of a compound on the neuronal nicotinic acetylcholine receptor activity in cells of claim 10 compared to the effect on control cells that are substantially identical to the cells of claim 10, but do not express the receptors, or the effect on neuronal nicotinic acetylcholine receptor activity of the cells in the absence of the compound; and identifying compounds that modulate the activity of human neuronal nicotinic acetylcholine receptors.