METHOD FOR TRANSPORTING POTASSIUM IONS FROM FRONT SIDE TO BACK SIDE OF LIPID BILAYER MEMBRANE

Abstract

An object of the present invention is to detect a specific chemical substance with high sensitivity and high precision. By using a specific lipid bilayer membrane, a chemical substance is detected with high sensitivity and high precision. Here, the specific lipid bilayer membrane comprises a chemical substance receptor, a chimeric G protein, and a potassium ion channel. The chimeric G protein comprises a chimeric Gα subunit and a Gβγ subunit complex. The chimeric Gα subunit is selected from the group consisting of Gi/olf13 (SEQ ID NO: 04), Gi/olf28 (SEQ ID NO: 05), Gi/olf94 (SEQ ID NO: 07), Gi/olf113 (SEQ ID NO: 08), Gi/olf α3-β5,C (SEQ ID NO: 12), and Gi/olfα4-β6,C (SEQ ID NO: 15).

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for transporting potassium ions from a front side of a lipid bilayer membrane to a back side thereof.

2. Description of the Background Art

G protein-coupled receptors (hereinafter, abbreviated as “GPCR”) existing in lipid bilayer membranes of cells functions in association with G proteins existing in the lipid bilayer membranes of cells. As shown in FIG. 1, when a chemical substance such as a neurotransmitter binds to a GPCR, a trimeric G protein coupled to the GPCR in a cell becomes activated to dissociate into a G_α subunit and a dimer (hereinafter, referred to as a “G_βγ subunit complex”) consisting of G_β and G_γ subunits. Then, each of those transmits various signals.

As shown in FIG. 2, binding of the G_βγ subunit complex to a G protein-coupled inwardly rectifying potassium channel (hereinafter, referred to as “GIRK”) causes the GIRK gate to be open. When the GIRK gate is opened, potassium ions existing on a front side of the lipid bilayer membrane move through the gate to a back side of the lipid bilayer membrane. The movement of the potassium ions can be detected or quantified with an ionic current measuring technique.

If the G_α subunit is a chimeric G_α subunit, the potassium ions move from the front side to the back side more efficiently in some cases. It is suggested to use, as a chemical substance sensor, a lipid bilayer membrane where a G protein comprising a chimeric G_α subunit has been expressed.

The following Non-patent Literatures 1 to 6 are relevant to the present invention.

CITATION LIST

Non-Patent Literature

• [NPL 1] Gloriam D., Fredriksson R., Schioth H., (2007) “The G protein-coupled receptor subset of the rat genome” BMC Genomics 8, 338-405
• [NPL 2] Oldham, W. M. and H. E. Hamm (2008). “Heterotrimeric G protein activation by G-protein-coupled receptors.” Nat. Rev. Mol. Cell. Biol. 9, 60-71.
• [NPL 3] Kim W. Chan, Jin-Liang Sui, Michel Vivaudou, and Diomedes E. Logothetis, (1996) “Control of channel activity through a unique amino acid residue of a G protein-gated inwardly rectifying K+ channel subunit” Proc. Natl. Acad. Sci., 93, 14193-14198
• [NPL 4] Gregory J. Digby, Pooja R. Sethi, and Nevin A. Lambert, (2008), “Differential dissociation of G protein heterotrimers” J. Physiol, 586, 3325-3335
• [NPL 5] Thomsen W., Frazer J., Unett D., (2005), “Functional assays for screening GPCR targets” Curr. Opin. Biotech., 16, 655-665
• [NPL 6] Leaney J. L., Milligan G., Tinker A., (2000) “The G Protein a Subunit Has a Key Role in Determining the Specificity of Coupling to, but Not the Activation of, G Protein-gated Inwardly Rectifying K1 Channels” J. Biol. Chem., 275, 921-929

Problems to be Solved by the Invention

An object of the present invention is to provide a method for transporting potassium ions from a front side of a lipid bilayer membrane to a back side thereof in a further highly sensitive manner in response to a chemical substance.

Solution to the Problems

The present inventors have discovered that a chimeric G protein comprising a specific chimeric G_α subunit solves the above described problem, and have established the present invention.

Thus, the present invention provides the following items 1 to 9.

Item 1:

A method for transporting potassium ions from a front side of a lipid bilayer membrane to a back side thereof, the method comprising the following steps (a) and (b):

step (a) of preparing the lipid bilayer membrane, wherein

• • the lipid bilayer membrane comprises a receptor of a chemical substance, a G protein, and a potassium ion channel,
  • the G protein comprises a chimeric G_α subunit and a G_βγ subunit complex, and
  • the chimeric G_α subunit consists of any one of G_i/olf13 (SEQ ID NO: 04), G_i/olf28 (SEQ ID NO: 05), G_i/olf94 (SEQ ID NO: 07), G_i/olf113 (SEQ ID NO: 08), G_{i/olfα3-β5,C} (SEQ ID NO: 12), or G_{i/olfα4-β6,C} (SEQ ID NO: 15); and

step (b) of supplying the chemical substance and the potassium ions to the front side to release the chimeric G_α subunit and G_βγ subunit complex, and to allow the G_βγ subunit complex to bind to the potassium ion channel, and transporting the potassium ions from the front side to the back side.

Item 2:

The method according to item 1, wherein the chemical substance is an adrenergic receptor agonist.

Item 3:

The method according to item 1, wherein the potassium ion channel is a G protein-coupled inwardly rectifying potassium ion channel.

Item 4:

A method for detecting or quantifying a chemical substance, the method comprising the following steps (c), (d), and (e):

step (c) of preparing a lipid bilayer membrane, a first liquid located on a front side of the lipid bilayer membrane, and a second liquid located on a back side of the lipid bilayer membrane, wherein

• • the lipid bilayer membrane comprises a chemical substance receptor, a G protein, and a potassium ion channel,
  • the G protein comprises a chimeric G_α subunit and a G_βγ subunit complex,
  • the chimeric G_α subunit consists of any one of G_i/olf13 (SEQ ID NO: 04), G_i/olf28 (SEQ ID NO: 05), G_i/olf94 (SEQ ID NO: 07), G_i/olf113 (SEQ ID NO: 08), G_{i/olfα3-β5,C} (SEQ ID NO: 12), or G_{i/olf α4-β6,C} (SEQ ID NO: 15), and
  • the first liquid contains potassium ions;

step (d) of supplying the chemical substance to the first liquid; and

step (e) of measuring an amount of potassium ions in at least one of the first and second liquids to detect or quantify the chemical substance based on the amount of the potassium ions.

Item 5:

The method according to item 4, wherein the chemical substance is an adrenergic receptor agonist.

Item 6:

The method according to item 4, wherein the potassium ion channel is a G protein-coupled inwardly rectifying potassium ion channel.

Item 7:

A method for detecting or quantifying a chemical substance, the method comprising the following steps (f), (g), and (h):

step (f) of preparing a lipid bilayer membrane, wherein

• • the lipid bilayer membrane comprises a chemical substance receptor, a G protein, and a potassium ion channel,
  • the G protein comprises a chimeric G_α subunit and a G_βγ subunit complex, and
  • the chimeric G_α subunit consists of any one of G_i/olf13 (SEQ ID NO: 04), G_i/olf28 (SEQ ID NO: 05), G_i/olf94 (SEQ ID NO: 07), G_i/olf113(SEQ ID NO: 08), G_{i/olfα3-β5,C} (SEQ ID NO: 12), or G_{i/olfα4-β6,C} (SEQ ID NO: 15);

step (g) of supplying a first liquid and a second liquid located respectively on a front side and a back side of the lipid bilayer membrane such that the lipid bilayer membrane is interposed between the first liquid and second liquid, wherein

• • the first liquid contains potassium ions and the chemical substance; and

step (h) of measuring an amount of potassium ions in at least one of the first and second liquids to detect or quantify the chemical substance based on the amount of the potassium ions.

Item 8:

The method according to item 7, wherein the chemical substance is an adrenergic receptor agonist.

Item 9:

The method according to item 7, wherein the potassium ion channel is a G protein-coupled inwardly-rectifying ion channel.

Advantageous Effects of the Invention

The present invention increases an amount of potassium ions transported from a front side of a lipid bilayer membrane to a back side thereof. Thus, a target chemical substance is detected with higher sensitivity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing signal transduction conducted by a trimeric G protein.

FIG. 2 is a schematic diagram showing a chemical substance sensor utilizing the function of a GIRK.

FIG. 3 is a schematic diagram showing a secondary structure of a G protein.

FIG. 4 is a schematic diagram showing a secondary structure of G_αi.

FIG. 5 schematically shows a secondary structure of G_αolf.

FIG. 6 schematically shows a secondary structure of G_i/olf5.

FIG. 7 schematically shows a secondary structure of G_i/olf13.

FIG. 8 schematically shows a secondary structure of G_i/olf28.

FIG. 9 schematically shows a secondary structure of G_i/olf45.

FIG. 10 schematically shows a secondary structure of G_i/olf94;

FIG. 11 schematically shows a secondary structure of G_i/olf113.

FIG. 12 schematically shows a secondary structure of G_i/olf156.

FIG. 13 schematically shows a secondary structure of G_i/olf195.

FIG. 14 schematically shows a secondary structure of G_i/olfα3-β5.

FIG. 15 schematically shows a secondary structure of G_{i/olfα3-β5,C}.

FIG. 16 schematically shows a secondary structure of G_i/olfα4-β6.

FIG. 17 schematically shows a secondary structure of G_{i/olfα3-β5,α4-β6}.

FIG. 18 schematically shows a secondary structure of G_{i/olfα4-β6,C}.

FIG. 19 schematically shows a secondary structure of G_{i/olfα3-β5,α4-β6,C}.

FIG. 20 illustrates a concept of a method for constructing a β1 adrenergic receptor expression plasmid.

FIG. 21 illustrates a concept of a method for constructing a Kir3.1 expression plasmid.

FIG. 22 illustrates a concept of a method for constructing a Kir3.1 (F137S) expression plasmid.

FIG. 23 illustrates a concept of a method for constructing a chimeric G protein expression plasmid.

FIG. 24 illustrates a concept of a method for constructing a chimeric G protein expression plasmid.

FIG. 25 illustrates a concept of a method for constructing a chimeric G protein expression plasmid.

FIG. 26 indicates ionic current of G_αi (C351G) protein expressing cells.

FIG. 27 indicates ionic current of G_i/olf13 protein expressing cells.

FIG. 28 indicates ionic current of wild type G_αolf protein expressing cells.

FIG. 29 shows changes in ionic current of chimeric G protein expressing cells.

FIG. 30 shows changes in ionic current of G protein expressing cells.

FIG. 31 shows changes in ionic current of G protein expressing cells.

FIG. 32 indicates an advantage of a chemical sensor of the present invention over a conventional chemical substance detection method.

FIG. 33 shows dependence of potassium ion current of chimeric G protein expressing cells over concentrations of various chemical substances.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Definition of Terms

The terms used in the present specification are defined as follows:

The term “lipid bilayer membrane” refers to a membrane forming a surface of a cell, i.e., a cell membrane.

The term “chimeric G_α subunit” refers to a G_α subunit in which a region of the original G_α subunit (e.g., G_αi) is substituted with a corresponding region of a different G_α protein (e.g., G_αolf).

The term “chimeric G protein” refers to a G protein in which the α subunit thereof is a chimeric G_α subunit.

The lipid bilayer membrane used in the present invention comprises a chemical substance receptor, a chimeric G protein, and a potassium ion channel.

An example of cells having the lipid bilayer membrane is an established cell line derived from a human cell.

(Chemical Substance Receptor)

A G protein-coupled receptor is employed as a chemical substance receptor. G protein-coupled receptors include hormone receptors, neurotransmitter receptors, pheromone receptors, olfactory receptors and gustatory receptors, as well as various orphan G protein-coupled receptors. Examples of the hormone receptors are adrenergic receptors.

(Chimeric G Protein)

A chimeric G protein comprises a chimeric G_α subunit and a G_βγ subunit complex.

In the present invention, the chimeric G_α subunit is selected from the group consisting of G_i/olf13, G_i/olf28, G_i/olf94, G_i/olf113, G_{i/olfα3-β5,C}, and G_{i/olfα4-β6,C}. Subscripts included in the names of the chimeric G_α subunits indicate G_αi protein domains in which amino acid sequences thereof are substituted with corresponding amino acid sequences of G_αolf. When these chimeric G_α subunits are used, a larger amount of potassium ions are transported from a front side of the lipid bilayer membrane to a back side thereof in response to contact with the chemical substance. The amino acid sequences of each of the chimeric G_α subunits are shown below.

(Gi/olf13)
(SEQ ID NO: 04)
MGCTLSAEDKAAVERSKMIDRNLREDGEKAAREVKLLLLGAGESGKSTIVKQMKIIHEA
GYSEEECKQYKAVVYSNTIQSIIAIIRAMGRLKIDFGDSARADDARQLFVLAGAAEEGFM
TAELAGVIKRLWKDSGVQACFNRSREYQLNDSAAYYLNDLDRIAQPNYIPTQQDVLRT
RVKTTGIVETHFTFKDLHFKMFDVGGQRSERKKWIHCFEGVTAIIFCVALSDYDLVLAE
DEEMNRMHESMKLFDSICNNKWFTDTSIILFLNKKDLFEEKIKKSPLTICYPEYAGSNTY
EEAAAYIQCQFEDLNKRKDTKEIYTHFTCATDTKNVQFVFDAVTDIIQRMHLKQYELL
(Gi/olf28)
(SEQ ID NO: 05)
MGCTLSAEDKAAVERSKMIDRNLREDGEKAAREVKLLLLGAGESGKSTIVKQMKIIHEA
GYSEEECKQYKAVVYSNTIQSIIAIIRAMGRLKIDFGDSARADDARQLFVLAGAAEEGFM
TAELAGVIKRLWKDSGVQACFNRSREYQLNDSAAYYLNDLDRIAQPNYIPTQQDVLRT
RVKTTGIVETHFTFKDLHFKMFDVGGQRSERKKWIHCFEGVTAIIFCVALSDYDLVLAE
DEEMNRMHESMKLFDSICNNKWFTDTSIILFLNKKDLFEEKIKKSPLTICYPEYAGSNTY
EEAAAYIQCQFEDLNKRKDTKEIYTHFTCAVDTENIRRVFNDCRDIIQRMHLKQYELL
(Gi/olf94)
(SEQ ID NO: 07)
MGCTLSAEDKAAVERSKMIDRNLREDGEKAAREVKLLLLGAGESGKSTIVKQMKIIHEA
GYSEEECKQYKAVVYSNTIQSIIAIIRAMGRLKIDFGDSARADDARQLFVLAGAAEEGFM
TAELAGVIKRLWKDSGVQACFNRSREYQLNDSAAYYLNDLDRIAQPNYIPTQQDVLRT
RVKTTGIVETHFTFKDLHFKMFDVGGQRSERKKWIHCFEGVTAIIFCVALSDYDLVLAE
DEEMNRMHESMKLFDSICNNKWFTDTSIILFLNKKDLFEEKVLAGKSKIEDYFPEYANY
TVPEDATPDAGEDPKVTRAKFFIRDLFLRISTATGDGKHYCYPHFTCAVDTENIRRVFND
CRDIIQRMHLKQYELL
(Gi/olf113)
(SEQ ID NO: 08)
MGCTLSAEDKAAVERSKMIDRNLREDGEKAAREVKLLLLGAGESGKSTIVKQMKIIHEA
GYSEEECKQYKAVVYSNTIQSIIAIIRAMGRLKIDFGDSARADDARQLFVLAGAAEEGFM
TAELAGVIKRLWKDSGVQACFNRSREYQLNDSAAYYLNDLDRIAQPNYIPTQQDVLRT
RVKTTGIVETHFTFKDLHFKMFDVGGQRSERKKWIHCFEGVTAIIFCVALSDYDLVLAE
DEEMNRMHESMKLFDSICNNKWLRTISIILFLNKQDMLAEKVLAGKSKIEDYFPEYANY
TVPEDATPDAGEDPKVTRAKFFIRDLFLRISTATGDGKHYCYPHFTCAVDTENIRRVFND
CRDIIQRMHLKQYELL
(Gi/olfα3-β5, C)
(SEQ ID NO: 12)
MGCTLSAEDKAAVERSKMIDRNLREDGEKAAREVKLLLLGAGESGKSTIVKQMKIIHEA
GYSEEECKQYKAVVYSNTIQSIIAIIRAMGRLKIDFGDSARADDARQLFVLAGAAEEGFM
TAELAGVIKRLWKDSGVQACFNRSREYQLNDSAAYYLNDLDRIAQPNYIPTQQDVLRT
RVKTTGIVETHFTFKDLHFKMFDVGGQRSERKKWIHCFEGVTAIIFCVALSDYDLVLAE
DEEMNRMHESMKLFDSICNNKWLRTISIILFLNKKDLFEEKIKKSPLTICYPEYAGSNTYE
EAAAYIQCQFEDLNKRKDTKEIYTHFTCATDTKNVQFVFDAVTDIIQRMHLKQYELL
(Gi/olfα4-β6, C)
(SEQ ID NO: 15)
MGCTLSAEDKAAVERSKMIDRNLREDGEKAAREVKLLLLGAGESGKSTIVKQMKIIHEA
GYSEEECKQYKAVVYSNTIQSIIAIIRAMGRLKIDFGDSARADDARQLFVLAGAAEEGFM
TAELAGVIKRLWKDSGVQACFNRSREYQLNDSAAYYLNDLDRIAQPNYIPTQQDVLRT
RVKTTGIVETHFTFKDLHFKMFDVGGQRSERKKWIHCFEGVTAIIFCVALSDYDLVLAE
DEEMNRMHESMKLFDSICNNKWFTDTSIILFLNKKDLFEEKIKKSPLTICYPEYAGSNTY
EEAAAYIQCQFEDLNTATGDGKHYCYTHFTCATDTKNVQFVFDAVTDIIQRMHLKQYE
LL

As shown in FIG. 3, a protein forming a G_α subunit comprises a domain referred to as a GTPase domain and a domain referred to as a helical domain. Both domains are linked to each other via two α-helixes referred to as linker 1 and linker 2. The GTPase domain includes five α-helixes, six β-sheets, and loops connecting those.

As shown in FIG. 7, the secondary structure of G_i/olf13 is a structure in which 13 amino acids of the C-terminal of G_αi (C351G) are substituted with corresponding amino acids of G_αolf.

As shown in FIG. 8, the secondary structure of G_i/olf28 is a structure in which the region located at the C-terminal side including β6-α5 loop of G_αi (C351G) is substituted with a corresponding region of G_αolf.

As shown in FIG. 10, the secondary structure of G_i/olf94 is a structure in which the region located at the C-terminal side including αG-α4 loop of G_αi (C351G) is substituted with a corresponding region of G_αolf.

As shown in FIG. 11, the secondary structure of G_i/olf113 is a structure in which the region located at the C-terminal side including α3-β5 loop of G_αi (C351G) is substituted with a corresponding region of G_αolf.

As shown in FIG. 15, the secondary structure of G_{i/olfα3-β5,C} is a structure in which α3-β5 loop and α5-C-terminal loop of G_αi (C351G) are substituted with corresponding loops of G_αolf.

As shown in FIG. 18, the secondary structure of G_{i/olfα4-β6,C} is a structure in which α3-β5 loop and α5-C-terminal loop of G_αi (C351G) are substituted with corresponding loops of G_αolf.

(Potassium Ion Channel)

As potassium ion channels, G protein-coupled inwardly rectifying potassium channels (GIRKs) are used. The GIRKs are classified into four subtypes, Kir3.1, Kir3.2, Kir3.3 and Kir3.4. In the present invention, preferably used is a mutated form of Kir3.1, i.e., Kir3.1 (F137S) in which phenylalanine at position 137 of Kir3.1 is substituted with serine. The reason is described below. Each of the subtypes Kir3.1 to Kir3.4 assembles with a different subtype to form a heteromultimer. In other words, a GIRK includes two types of the subtypes. However, the mutated form Kir3.1 (F137S) assembles into a homotetramer, and the tetramer constitutes a potassium ion channel. Therefore, when the mutated form Kir3.1 (F137S) is used, the single type of protein is required for forming a potassium ion channel.

The lipid bilayer membrane comprising the chemical substance receptor, the chimeric G protein and the potassium ion channel, is prepared, for example, by a method described below. An expression plasmid coding for the chemical substance receptor, an expression plasmid coding for the chimeric G protein, and an expression plasmid coding for the potassium ion channel are constructed. Each of the constructed plasmids is introduced into cells and is expressed. Specific procedure of the method is described in detail in the examples.

(Potassium Ion Transport)

A front side and a back side of the lipid bilayer membrane comprising the chemical substance receptor, the chimeric G protein and the potassium ion channel, are each in contact with appropriate buffer solutions.

When the chemical substance is supplied to the front side of the lipid bilayer membrane and binds to the chemical substance receptor, the chimeric G protein bound to the chemical substance receptor on the back side of the lipid bilayer membrane is divided into and released as a chimeric G_α subunit and a G_βγ subunit complex. Contact of the G_βγ subunit complex to the potassium ion channel leads to opening of a gate of the potassium ion channel. When the gate of the potassium ion channel is opened, potassium ions are transported from the front side to the back side of the lipid bilayer membrane.

(Detection or Quantification of Chemical Substance)

The chemical substance can be detected or quantified by using the above described method of transporting potassium ions from the front side to the back side of the lipid bilayer membrane. A change in ionic current caused by the potassium ion transport is measured to detect or quantify the chemical substance existing on the front side of the lipid bilayer membrane. More particularly, the chemical substance is detected or quantified as described below. First, potassium ions are supplied to the front side of the lipid bilayer membrane comprising the chemical substance receptor, the chimeric G protein and the potassium ion channel described above. More particularly, for example, a first liquid is supplied to the front side of the lipid bilayer membrane, and a second liquid is supplied to the back side of the lipid bilayer membrane. Thus, the lipid bilayer membrane is interposed between the first liquid and the second liquid. Generally, the first liquid and the second liquid are buffer solutions having a pH of around 7. The first liquid contains potassium ions. The second liquid may also contain potassium ions.

A target chemical substance is also supplied on the front side of the lipid bilayer membrane. More particularly, the target chemical substance is supplied to the first liquid in a state where the lipid bilayer membrane is interposed between the first liquid and the second liquid. The target chemical substance is supplied to the chemical substance receptor. Supply of the target chemical substance to the chemical substance receptor causes the subunits of the chimeric G protein to be released. This gives rise to the transport of potassium ions (contained in the first liquid) locating in the front side of the lipid bilayer membrane to the back side (the second liquid) thereof. The change in ionic current generated by the potassium ion transport is measured to detect or quantify the chemical substance on the basis of the measurement result. The above-described supply of the chemical substance may be conducted with a solution instead of the first fluid, where the solution has the same composition as that of the first fluid except for containing an additional chemical substance.

Generally, a standard curve is used to quantify a chemical substance.

(Chemical Substance)

Any chemical substance capable of functioning as an agonist of the chemical substance receptor can be used as the chemical substance, and there is no particular limitation in the chemical substance. If an adrenergic receptor is used as the chemical substance receptor, the chemical substance includes, for example, isoproterenol, dopamine, and dobutamine.

EXAMPLES

Construction of Plasmids

Methods for constructing plasmids used in the Examples and Comparative Examples are shown below.

(Construction of Adrenergic Receptor Expression Plasmid)

FIG. 20 shows a procedure to construct an adrenergic receptor expression plasmid. The gene of a β adrenergic receptor (GenBank Accession Number: J05561.1) was amplified in accordance with the following procedure. Rat heart-derived cDNA (rat heart cDNA) was amplified as two separate fragments by PCR methods. Primers used to amplify one of the fragments were primer 1 (SEQ ID NO: 17) and primer 2 (SEQ ID NO: 18). Primers used to amplify the other fragment were primer 3 (SEQ ID NO: 19) and primer 4 (SEQ ID NO: 20). Each of the obtained two fragments was ligated into a plasmid. PCRs were performed by using these two plasmids as templates. Primers used to amplify one of the plasmids were primer 5 (SEQ ID NO: 21) and primer 2 (SEQ ID NO: 18). Primers used to amplify the other plasmid were primer 3 (SEQ ID NO: 19) and primer 6 (SEQ ID NO: 22). As a result, restriction enzyme sites were added to the ends of the amplified fragments. One of the two obtained fragments was treated with EcoRI and HindIII. The other one was treated with HindIII and SalI. The treated fragments were ligated into an expression plasmid pretreated with EcoRI and SalI to obtain a β1 adrenergic receptor expression plasmid (hereinafter, referred to as “plasmid (βAR)”).

Table 1 shows sequences of the used primers.

TABLE 1
		SEQ ID
	Sequence	NO:
Primer 1	atgggcgcgggggcgctcg	17
Primer 2	gaagacgaagaggcgatccggcaccagg	18
Primer 3	cactgggcatcatcatgggtgtgttcac	19
Primer 4	ctacaccttggactcggaggagaagcc	20
Primer 5	ttcgaattcgccaccatgggcgcgggggcgct	21
Primer 6	gaagtcgacctacaccttggactcggagg	22

(Construction of Mutated Potassium Ion Channel Kir3.1 (F137S) Expression Plasmid)

The gene of a mutated potassium ion channel Kir3.1 (F137S) was constructed by partially mutating the mouse Kir3.1 gene. FIG. 21 shows a procedure to construct a mouse Kir3.1 expression plasmid. The mouse Kir3.1 gene was amplified in two separate fragments. Mouse Brain cDNA Library (Clontech Laboratories, Inc.,) was used as a template. Primers used in the first round of PCR to amplify one of the fragments were primer 7 (SEQ ID NO: 23) and primer 8 (SEQ ID NO: 24). Primers used to amplify the other fragment were primer 9 (SEQ ID NO: 25) and primer 10 (SEQ ID NO: 26). Primers used to amplify one of the fragments in the second round of PCR were primer 11 (SEQ ID NO: 27) and primer 12 (SEQ ID NO: 28). Nhe I and Cla I were used as restriction enzymes to cut out a PCR product. Primers used to amplify the other fragment were primer 13 (SEQ ID NO: 29) and primer 14 (SEQ ID NO: 30). Cla I and Not I were used as restriction enzymes to cut out a PCR product. The obtained gene fragments were ligated into an expression plasmid pretreated with Nhe I and Not I to obtain a mouse Kir3.1 expression plasmid (hereinafter, referred to as “plasmid (Kir3.1)”). Table 2 shows sequences of the used primers.

TABLE 2
	Sequence	SEQ ID NO:
Primer 7	gcgcctccgcttcgtgtttgaatctggc	23
Primer 8	gccttccaggatgacgacaacctcgaac	24
Primer 9	ccgggtgggcaacctgcgcaacagcc	25
Primer 10	gccaggctaggatagacctctcag	26
Primer 11	gcgctagcgccaccatgtctgcactccgaa	27
Primer 12	ggcatcgatcacgtggcaaattgtgagagg	28
Primer 13	gtgatcgatgccaaaagccccttctatgac	29
Primer 14	gcgcggccgcctatgtgaaacggtcagag	30

FIG. 22 shows a procedure to construct a mutated potassium ion channel Kir3.1 (F137S) expression plasmid. The Kir3.1 (F137S) gene was constructed by the following procedure. By using the plasmid (Kir3.1) as a template, the Kir3.1 gene was amplified as two fragments. These fragments were connected by performing overlap extension PCR method. Primer 11 (SEQ ID NO: 27) and primer 16 (SEQ ID NO: 32) were used to amplify one of the fragments. Then, primer 15 (SEQ ID NO: 31) and primer 14′ (SEQ ID NO: 78) were used to amplify the other fragment. The PCR products were mixed, heat denatured, and amplified by performing PCR. Primer 11 (SEQ ID NO: 27) and primer 14′ (SEQ ID NO: 78) were used for this PCR reaction. The obtained fragment was treated with Nhe I and Xho I. The treated fragment was ligated into a plasmid pretreated with Nhe I and Xho I to obtain an expression plasmid (hereinafter, referred to as “plasmid (Kir3.1 (F137S)”).

(Construction of Wild Type G Protein (G_αolf) Expression Plasmid)

RNA was isolated from mouse olfactory bulb and was then reverse transcribed by using reverse transcriptase to obtain total cDNA in mouse olfactory cells. The obtained cDNA was used as template, and the G_αolf gene was amplified by PCR. Primer 17 (SEQ ID NO: 33) and primer 18 (SEQ ID NO: 34) were used. The obtained gene fragment was ligated into a plasmid, and thereby the G_αolf gene (GenBank Accession Number: AY179168.1) was cloned. The obtained G_αolf fragment was further amplified by PCR. Primer 19 (SEQ ID NO: 35) and primer 20 (SEQ ID NO: 36) were used. As a result, restriction enzyme sites were added to the ends of the amplified fragment. The amplified fragment having the restriction enzyme sites was ligated into an expression plasmid to obtain a wild type G protein (G_αolf) expression plasmid (hereinafter, referred to as “plasmid (G_αolf”).

(Construction of G Protein (G_αi (C351G)) Expression Plasmid)

FIG. 23 shows a procedure to construct G_αi (C351G) protein expression plasmid in which cysteine at position 351 of G_αi was substituted with glycine. It has been revealed that G_αi (C351G) has the same functions as wild type G_αi, except for having resistance to pertussis toxin which is a selective inhibitor of G_αi. A procedure in which the G_αi (mouse G_il: NM-010305) gene was amplified is shown below. Mouse spleen-derived cDNA (mouse spleen cDNA) was amplified as two separate fragments by performing PCRs. Primers used to amplify one of the fragments were primer 21 (SEQ ID NO: 37) and primer 22 (SEQ ID NO: 38). Primers used to amplify the other fragment were primer 23 (SEQ ID NO: 39) and primer 24 (SEQ ID NO: 40). Each of the obtained two fragments was ligated into a plasmid. PCRs were performed by using these two plasmids as templates. Primers used to amplify one of the plasmids were primer 25 (SEQ ID NO: 41) and primer 26 (SEQ ID NO: 42). Primers used to amplify the other plasmid were primer 27 (SEQ ID NO: 43) and primer 28 (SEQ ID NO: 44). As a result, restriction enzyme sites were added to the ends of the amplified fragments. One of the two obtained fragments was treated with EcoRI and BamHI. The other one was treated with BamHI and EcoRI. The treated fragments were ligated into an expression plasmid pretreated with EcoRI to obtain a wild type G protein (G_αi) expression plasmid (hereinafter, referred to as “plasmid (G_αi)”). Table 3 shows sequences of the used primers.

TABLE 3
	Sequence	SEQ ID NO:
Primer 14′	gcctcgagctatgtgaaacggtcagag	78
Primer 15	ctctgccttcctcttctccatcgagaccga	31
Primer 16	ggtctcgatggagaagaggaaggcagagg	32
Primer 17	atggggtgtttgggcaacagcagcaagac	33
Primer 18	ggaggaggaggaggggtaggtttagg	34
Primer 19	aatgaattcgccaccatggggtgtttgggcaacag	35
Primer 20	aatgtcgactcacaagagttcgtactgcttgag	36
Primer 21	cggcagcgtgcggactagcagacct	37
Primer 22	gaacagcttcatgctctcgtgcatacgg	38
Primer 23	gctgaacgattcggcagcgtactatctg	39
Primer 24	ggtcagaactctggtcaggtccaggatg	40
Primer 25	gcgctcgagccaccatgggctgcacattgagcgct	41
Primer 26	aagtggatccactgctttgaagg	42
Primer 27	agtggatccacttatccgctc	43
Primer 28	cgcgaattcttagaagagaccaatgtcttttaggttattctttatgat	44
	gacgtctgttacagcatcgaacacgaac

(Construction of Chimeric G Protein (G_i/olf5) Expression Plasmid)

A chimeric G protein (G_i/olf5) expression plasmid (hereinafter, referred to as “plasmid (G_i/olf5)”) was constructed in accordance with a procedure same as that used for constructing the plasmid (G_ai), except for using primer 29 (SEQ ID NO: 45) instead of primer 28. The construction procedure is shown in FIG. 23.

(Construction of Chimeric G Protein (G_i/s13) Expression Plasmid)

A chimeric G protein (G_i/s13) expression plasmid (hereinafter, referred to as “plasmid (G_i/s13)”) was constructed in accordance with a procedure same as that used for constructing the plasmid (G_αi), except for using primer 30 (SEQ ID NO: 46) instead of primer 28. The construction procedure is shown in FIG. 23.

(Construction of Chimeric G Protein (G_i/olf13) Expression Plasmid)

A chimeric G protein (G_i/olf13) expression plasmid (hereinafter, referred to as “plasmid (G_i/olf13)”) was constructed in accordance with a procedure same as that used for constructing the plasmid (G_αi), except for using primer 31 (SEQ ID NO: 47) instead of primer 28. The construction procedure is shown in FIG. 23. Table 4 shows sequences of the used primers.

TABLE 4
	Sequence	SEQ ID NO:
Primer 29	cgcgaattcttagagcagctcgtattgttttaggttatt	45
	ctttatgatgacgtctgttacagcatcgaacacgaa
	c
Primer 30	cgcgaattcttagagcagctcgtattggcggagatg	46
	catgcgctggatgatgtctgttacagcatcgaacac
	gaac
Primer 31	cgcgaattcttagagcagctcgtattgcttgagatgc	47
	atgcgctggatgatgtctgttacagcatcgaacacg
	aac

(Construction of Chimeric G Protein (G_i/olf28) Expression Plasmid)

A chimeric G protein (G_i/olf28) expression plasmid (hereinafter, referred to as “plasmid (G_i/olf28)”) was constructed through overlap extension PCR by using the plasmid (G_i/olf13) as template I and the plasmid (G_αolf) as template II. As shown in FIG. 24, A-PRIMER and B-PRIMER were used as primers for the PCR using template I. By using A-PRIMER and B-PRIMER, a base sequence coding for amino acids of predetermined regions included in G_αi can be selectively amplified. One part of the obtained fragment was added to the complementary sequence of B-PRIMER to form a sticky end. C-PRIMER and D-PRIMER were used as primers for the PCR using template II. By using C-PRIMER and D-PRIMER, a base sequence coding for amino acids of predetermined regions included in G_αolf can be selectively amplified. One part of the obtained fragment was added to the complementary sequence of C-PRIMER to form a sticky end. More particularly, primer 32 (SEQ ID NO: 48) was used as A-PRIMER, and primer 33 (SEQ ID NO: 49) was used as B-PRIMER. Primer 34 (SEQ ID NO: 50) was used as C-PRIMER, and primer 35 (SEQ ID NO: 51) was used as D-PRIMER. Both PCR products were connected to each other via the sticky ends by overlap extension PCR. Here, A-PRIMER and D-PRIMER were used as primers. The obtained fragment was treated with EcoRI and SalI, and the treated fragment was ligated into an expression plasmid pretreated with EcoRI and SalI to obtain plasmid (G_i/olf128). Escherichia coli transformed with the plasmid (G_i/olf128) was cultured. The plasmid (G_i/olf28) purified from Escherichia coli was used for transfecting cells.

(Construction of Chimeric G Protein (G_i/olf45) Expression Plasmid)

A chimeric G protein (G_i/olf48) expression plasmid (hereinafter, referred to as “plasmid (G_i/olf48)”) was constructed in accordance with a procedure same as that used for constructing the plasmid (G_i/olf28), except for using primers shown in Table 5.

(Construction of Chimeric G Protein (G_i/olf94) Expression Plasmid)

A chimeric G protein (G_i/olf94) expression plasmid (hereinafter, referred to as “plasmid (G_i/olf94)”) was constructed in accordance with a procedure same as that used for constructing the plasmid (G_i/olf28), except for using primers shown in Table 5.

(Construction of Chimeric G Protein (G_i/olf113) Expression Plasmid)

A chimeric G protein (G_i/olf113) expression plasmid (hereinafter, referred to as “plasmid (G_i/olf113)”) was constructed in accordance with a procedure same as that used for constructing the plasmid (G_i/olf28), except for using primers shown in Table 5.

(Construction of Chimeric G Protein (G_i/olf156) Expression Plasmid)

A chimeric G protein (G_i/olf156) expression plasmid (hereinafter, referred to as “plasmid (G_i/olf156)”) was constructed in accordance with a procedure same as that used for constructing the plasmid (G_i/olf28), except for using primers shown in Table 5.

(Construction of Chimeric G Protein (G_i/olf195) Expression Plasmid)

A chimeric G protein (G_i/olf195) expression plasmid (hereinafter, referred to as “plasmid (G_i/olf195)”) was constructed in accordance with a procedure same as that used for constructing the plasmid (G_i/olf28), except for using primers shown in Table 5.

TABLE 5
			Sequence (5′→3′)	SEQ ID NO:
Gi/olf 38	A-PRIMER	Primer 32	cctgaattctgccaccatgggctgcacattgagcg	48
	B-PRIMER	Primer 33	cccgtggctgtattgaggtcttcaaactgacactg	49
	C-PRIMER	Primer 34	agtttgaagacctcaatacagccacgggtgatg	50
	D-PRIMER	Primer 35	accgtcgacgtcacaagagttcgtactgcttgag	51
Gi/olf 45	A-PRIMER	Primer 42	cctgaattctgccaccatgggctgcacattgagcg	58
	B-PRIMER	Primer 43	tgtgtccacggcgcacgtgaagtggg	59
	C-PRIMER	Primer 44	acgtgcgccgtggacacagagaacatccg	60
	D-PRIMER	Primer 45	accgtcgacgtcacaagagttcgtactgcttgag	61
Gi/olf 94	A-PRIMER	Primer 46	cctgaattctgccaccatgggctgcacattgagcg	62
	B-PRIMER	Primer 47	tgccaagactttttcttcgaagaggtccttcttg	63
	C-PRIMER	Primer 48	gaagaaaaagtcttggcagggaagtcaaaaatcg	64
	D-PRIMER	Primer 49	accgtcgacgtcacaagagttcgtactgcttgag	65
Gi/olf 113	A-PRIMER	Primer 50	cctgaattctgccaccatgggctgcacattgagcg	66
	B-PRIMER	Primer 51	atggttcgcaaccacttgttgttacagatgctatcg	67
	C-PRIMER	Primer 52	acaacaagtggttgcgaaccatttctatcatcc	68
	D-PRIMER	Primer 53	accgtcgacgtcacaagagttcgtactgcttgag	69
Gi/olf 156	A-PRIMER	Primer 54	cctgaattctgccaccatgggctgcacattgagcg	70
	B-PRIMER	Primer 55	acatcattaaagcagtggatccacttcttccg	71
	C-PRIMER	Primer 56	actgctttaatgatgtcactgcgatcatttacg	72
	D-PRIMER	Primer 57	accgtcgacgtcacaagagttcgtactgcttgag	73
Gi/olf 195	A-PRIMER	Primer 58	cctgaattctgccaccatgggctgcacattgagcg	74
	B-PRIMER	Primer 59	actctgcatctgaggacatcctgctgagttg	75
	C-PRIMER	Primer 60	gtcctcagatgcagagtgctgacatcagg	76
	D-PRIMER	Primer 61	accgtcgacgtcacaagagttcgtactgcttgag	77

Table 6 shows regions of amino acid sequences amplified by respective primers.

	TABLE 6
	Gαi		Gαolf		Chimeric G
	Primer	Region	Primer	Region	protein
		1-354			Gαi
	Primer 32/	1-326	Primer 34/	354-381	Gi/olf28
	Primer 33		Primer 35
	Primer 42/	1-311	Primer 44/	335-381	Gi/olf45
	Primer 43		Primer 45
	Primer 46/	1-277	Primer 48/	288-381	Gi/olf94
	Primer 47		Primer 49
	Primer 54/	1-215	Primer 56/	226-381	Gi/olf156
	Primer 55		Primer 57
	Primer 58/	1-176	Primer 60/	187-381	Gi/olf195
	Primer 59		Primer 61
	Primer 50/	1-258	Primer 52/	269-381	Gi/olf113
	Primer 51		Primer 53
				1-381	Golf

(Construction of Chimeric G Protein (G_i/olfα3-β5) Expression Plasmid)

A chimeric G protein (G_i/olfα3-β5) expression plasmid (hereinafter, referred to as “plasmid (G_i/olfα3-β5)”) was constructed through overlap extension PCR by using the plasmid (G_i/olf13) as template I and the plasmid (G_αi) as template II. As shown in FIG. 25, primer 36 (SEQ ID NO: 52) and E-PRIMER were used as primers for the PCR using template I. More particularly, primer 37 (SEQ ID NO: 53) was used as E-PRIMER. One part of the obtained fragment was added to the complementary sequence of E-PRIMER to form a sticky end. Primer 39 (SEQ ID NO: 55) and F-PRIMER were used as primers for the PCR using template II. More particularly, primer 38 (SEQ ID NO: 54) was used as F-PRIMER. One part of the obtained fragment was added to the complementary sequence of F-PRIMER to form a sticky end. Both PCR products were connected to each other via sticky ends by overlap extension PCR using primer 36 and primer 39. The obtained fragment was treated with EcoRI, and ligated into an expression plasmid pretreated with EcoRI to obtain the plasmid (G_i/olfα3-β5). Escherichia coli transformed with the plasmid (G_i/olfα3-β5) was cultured. The plasmid (G_i/olfα3-β5) purified from Escherichia coli was used for transfecting cells.

(Construction of Chimeric G Protein (G_{i/olfα3-β5,C}) Expression Plasmid)

A chimeric G protein (G_{i/olfα3-β5,C}) expression plasmid (hereinafter, referred to as “plasmid (G_{i/olfα3-β5,C})”) was constructed in accordance with a procedure same as that used for constructing the plasmid (G_olfα3-β5), except for using templates and restriction enzymes described in Table 7.

(Construction of Chimeric G Protein (G_i/olfα4-β6) Expression Plasmid)

A chimeric G protein (G_i/olfα4-β6) expression plasmid (hereinafter, referred to as “plasmid (G_i/olfα4-β6)”) was constructed in accordance with a procedure same as that used for constructing the plasmid (G_olfα3-β5), except for using templates, E-PRIMER, F-PRIMER, and a restriction enzyme described in Table 7.

(Construction of Chimeric G Protein (G_{i/olfα4-β6,C}) Expression Plasmid)

A chimeric G protein (G_{i/olfα4-β6,C}) expression plasmid (hereinafter, referred to as “plasmid (G_{i/olfα4-β6,C})”) was constructed in accordance with a procedure same as that used for constructing the plasmid (G_olfα3-β5), except for using templates, E-PRIMER, F-PRIMER, and restriction enzymes described in Table 7.

(Construction of Chimeric G Protein (G_{i/olfα3-β5,α4-β6}) Expression Plasmid)

A chimeric G protein (G_{i/olfα3-β5,α4-β6}) expression plasmid (hereinafter, referred to as “plasmid (G_{i/olfα3-β5,α4-β6})”) was constructed in accordance with a procedure same as that used for constructing the plasmid (G_olfα3-β5), except for using templates, E-PRIMER, F-PRIMER, and a restriction enzyme described in Table 7.

(Construction of Chimeric G Protein (G_{i/olfα3-β5,α4-β6,c}) Expression Plasmid)

A chimeric G protein (G_{i/olfα3-β5,α4-β6,C}) expression plasmid (hereinafter, referred to as “plasmid (G_i/olfα3-β5,α4-β6,C)”) was constructed in accordance with a procedure same as that used for constructing the plasmid (G_olfα3-β5), except for using templates, E-PRIMER, F-PRIMER, and restriction enzymes described in Table 7.

TABLE 7
Constructed					Restriction
Expression Plasmid	Template I	Template II	E-PRIMER	F-PRIMER	Enzyme
Plasmid (Gi/olfα3-β5)			Primer 37	Primer 38	EcoRI
Plasmid (Gi/olfα3-β5,C)			Primer 37	Primer 38	EcoRI/SalI
Plasmid (Gi/olfα4-β6)			Primer 40	Primer 41	EcoRI
Plasmid (Gi/olfα4-β6,C)			Primer 40	Primer 41	EcoRI/SalI
Plasmid (Gi/olfα3-β5,α4-β6)			Primer 37	Primer 38	EcoRI
Plasmid (Gi/olfα3-β5,α4-β6,C)			Primer 37	Primer 38	EcoRI/SalI

[Measurement of Electrophysiological Activities]

A cell was obtained that expressed a chimeric G protein, a potassium ion channel, and a chemical substance receptor (hereinafter, the cell is referred to as “chimeric G protein expressing cell”). An electrophysiological activity of the chimeric G protein expressing cell was measured by using patch-clamp method. The chimeric G protein expressing cell was prepared in accordance with the following procedure. First, a chimeric G protein expression plasmid, a potassium ion channel expression plasmid, and a chemical substance receptor expression plasmid were prepared. Next, the three expression plasmids were transfected into HEK293T cells, and were then expressed inside the cells.

Example 1

A chimeric G protein comprising G_i/olf13 as a chimeric G_α subunit was employed. The mutated potassium ion channel Kir3.1 (F137S) was employed as a potassium ion channel. The β1 adrenergic receptor was employed as a chemical substance receptor.

(Preparation of Chimeric G Protein Expressing Cells)

The plasmid (βAR), the plasmid Kir3.1 (F137S), and the plasmid (G_i/olf13) were expressed in HEK293T cells. The procedure for the expression is shown below. Approximately 80% confluent cultured HEK293T cells were collected, and plated on a new culture petri dish. The passage number of the used cells was not more than ten generations. DMEM (supplemented with 10% FBS (fetal bovine serum) and streptomycin) was used. After culturing the cells for 24 hours, the cells were transfected with the plasmids by using a transfection reagent. The cells were cultured for 48 hours after being transfected with the plasmids, and thereby chimeric G protein expressing cells were prepared.

(Measurement of Current Change Levels)

Membrane current of the chimeric G protein expressing cell was measured by using a patch-clamp technique. Measurements were conducted with and without supplying isoproterenol (hereinafter, referred to as “ISO”) which was a β1 adrenergic receptor agonist. Procedure for the measurements is shown below.

(Samples for Measurements)

The chimeric G protein expressing cells were plated on coverslips (3 mm×10 mm; Matsunami Glass Ind., Ltd.,) treated with PLL (poly-L-lysine) and kept still for four hours to obtain samples for the measurements.

(Glass Electrodes)

Procedure for preparing glass electrodes used for the measurements is described below. By using a glass electrode manufacturing device (“Laser Puller P-2000” manufactured by Sutter Instrument Co.,), glass pipettes having a 1 μm diameter at one end were prepared from glass tubes (outer diameter 1.5 mm, internal diameter 0.86 mm, length 100 mm). Silver/silver chloride (Ag/AgCl) electrodes were inserted in the glass pipettes. The glass pipettes were filled with buffer A. The composition of the buffer A was similar to the composition of an intracellular fluid. The composition of the buffer A is shown in Table 8

(Measurements)

Measurement operations were conducted under a microscope (“1X71” manufactured by Olympus, Inc.). A liquid circulation chamber was mounted on the silver/silver chloride (Ag/AgCl) electrode. A sample for measurement was placed in the liquid circulation chamber filled with Tyroad's buffer. At a state where a single cell was in contact with the tip of a glass electrode, negative pressure was applied in the glass electrode. The portion of a cell membrane in contact with the tip of the glass electrode was torn to form an equivalent circuit (Whole-cell mode). The electric potential difference between inside and outside a cell was kept at 0 mV by using a patch clamp amplifier (“EPC10” manufactured by HEKA Instrument Inc.). Under this mode, the Tyroad's buffer in the liquid circulation chamber was substituted with a GIRK buffer. Pulse potentials (electric potential: −50 mV; duration time: 100 mS) were applied every 10 seconds, and membrane currents obtained upon the application were measured. Table 8 shows the compositions of the Tyroad's buffer and the GIRK buffer.

Next, membrane currents obtained when isoproterenol (hereinafter, abbreviated as “ISO”) which is a β1 adrenergic receptor agonist was brought into contact with the cell were measured in accordance with the following procedure. The GIRK buffer in the liquid circulation chamber was substituted with a sample solution. Under a condition in which the electric potential difference between inside and outside a cell was kept at 0 mV, pulse potentials (electric potential: −50 mV; duration time: 100 mS) were applied every 10 seconds, and membrane currents obtained upon the application were measured. The composition of the sample solution is shown in Table 8.

	TABLE 8
	Tyroad's	GIRK		Sample
	buffer	buffer	Buffer A	solution
Sodium Chloride (mM)	140	—	—	—
Potassium Chloride (mM)	5	140	107	140
Calcium Chloride (mM)	2	2.6	1	2.6
Magnesium Chloride (mM)	1	1.2	1.2	1.2
Glucose (mM)	10	10	—	10
HEPES (mM)	10	5	5	5
EGTA (mM)			10	—
ATP (mM)	—	—	2	—
GTP (mM)	—	—	0.3	—
ISO (nM)	—	—	—	30
pH	7.4	7.4	7.2	7.2
Osmotic Pressure	290-300	290-300	290-300	290-300
(mOsm/kg)

Examples 2 to 6

Chimeric G protein expressing cells were prepared and membrane currents were measured similarly to Example 1, except for employing chimeric G proteins comprising respective G_α subunits described in Table 9 instead of the chimeric G protein comprising G_i/olf13.

	TABLE 9
	Gα subunit	SEQ ID NO:
	Example 1	Gi/olf13	4
	Example 2	Gi/olf28	5
	Example 3	Gi/olf94	7
	Example 4	Gi/olf113	8
	Example 5	Gi/olfα3-β5,C	12
	Example 6	Gi/olfα4-β6,C	15

Comparative Examples 1 to 10

Chimeric G protein expressing cells were prepared and membrane currents were measured similarly to Example 1, except for employing chimeric G proteins comprising respective G_α subunits described in Table 10 instead of the chimeric G protein comprising G_i/olf13.

	TABLE 10
	Gα subunit	SEQ ID NO:
	Comparative Example 1	Gαi (C351G)	1
	Comparative Example 2	Gαolf	2
	Comparative Example 3	Gi/olf5	3
	Comparative Example 4	Gi/olf45	6
	Comparative Example 5	Gi/olf156	9
	Comparative Example 6	Gi/olf195	10
	Comparative Example 7	Gi/olfα3-β5	11
	Comparative Example 8	Gi/olfα4-β6	13
	Comparative Example 9	Gi/olfα3-β5,α4-β6	14
	Comparative Example 10	Gi/olfα3-β5,α4-β6,C	16

Comparative Example 11

When the chimeric G protein expressing cells was prepared, the cells were prepared similarly to Example 1, except for transfecting the cells with the plasmid (G_i/olf13). Then, membrane currents of the prepared cells were measured.

The amino acid sequence of G_α subunit (G_αi (C351G) used in Comparative Example 1 is shown below. FIG. 4 schematically shows a secondary structure of (G_αi (C351G)).

(SEQ ID NO: 1)
MGCTLSAEDKAAVERSKMIDRNLREDGEKAAREVKLLLLGAGESGKSTIV
KQMKIIHEAGYSEEECKQYKAVVYSNTIQSIIAIIRAMGRLKIDFGDSAR
ADDARQLFVLAGAAEEGFMTAELAGVIKRLWKDSGVQACFNRSREYQLND
SAAYYLNDLDRIAQPNYIPTQQDVLRTRVKTTGIVETHFTFKDLHFKMFD
VGGQRSERKKWIHCFEGVTAIIFCVALSDYDLVLAEDEEMNRMHESMKLF
DSICNNKWFTDTSIILFLNKKDLFEEKIKKSPLTICYPEYAGSNTYEEAA
AYIQCQFEDLNKRKDTKEIYTHFTCATDTKNVQFVFDAVTDVIIKNNLKD
IGLF

The amino acid sequence of G_α subunit (G_αolf) used in Comparative Example 2 is shown below. FIG. 5 schematically shows a secondary structure of (G_αolf).

(SEQ ID NO: 2)
MGCLGNSSKTAEDQGVDEKERREANKKIEKQLQKERLAYKATHRLLLLGA
GESGKSTIVKQMRILHVNGFNPEEKKQKILDIRKNVKDAIVTIVSAMSTI
IPPVPLANPENQFRSDYIKSIAPITDFEYSQEFFDHVKKLWDDEGVKACF
ERSNEYQLIDCAQYFLERIDSVSLVDYTPTDQDLLRCRVLTSGIFETRFQ
VDKVNFHMFDVGGQRDERRKWIQCFNDVTAIIYVAACSSYNMVIREDNNT
NRLRESLDLFESIWNNRWLRTISIILFLNKQDMLAEKVLAGKSKIEDYFP
EYANYTVPEDATPDAGEDPKVTRAKFFIRDLFLRISTATGDGKHYCYPHF
TCAVDTENIRRVFNDCRDIIQRMHLKQYELL

The amino acid sequence of a chimeric G_α subunit (G_i/olf5) used in Comparative Example 3 is shown below. G_i/olf5 is a chimeric G_α subunit obtained by substituting five amino acids on the C-terminal side of G_αi protein with corresponding amino acids of G_αolf. FIG. 6 schematically shows a secondary structure of G_i/olf5.

(SEQ ID NO: 3)
MGCTLSAEDKAAVERSKMIDRNLREDGEKAAREVKLLLLGAGESGKSTIV
KQMKIIHEAGYSEEECKQYKAVVYSNTIQSIIAIIRAMGRLKIDFGDSAR
ADDARQLFVLAGAAEEGFMTAELAGVIKRLWKDSGVQACFNRSREYQLND
SAAYYLNDLDRIAQPNYIPTQQDVLRTRVKTTGIVETHFTFKDLHFKMFD
VGGQRSERKKWIHCFEGVTAIIFCVALSDYDLVLAEDEEMNRMHESMKLF
DSICNNKWFTDTSIILFLNKKDLFEEKIKKSPLTICYPEYAGSNTYEEAA
AYIQCQFEDLNKRKDTKEIYTHFTCATDTKNVQFVFDAVTDVIIKNNLKQ
YELL

The amino acid sequence of a chimeric G_α subunit (G_i/olf45) used in Comparative Example 4 is shown below. FIG. 9 schematically shows a secondary structure of G_i/olf45.

(SEQ ID NO: 6)
MGCTLSAEDKAAVERSKMIDRNLREDGEKAAREVKLLLLGAGESGKSTIV
KQMKIIHEAGYSEEECKQYKAVVYSNTIQSIIAIIRAMGRLKIDFGDSAR
ADDARQLFVLAGAAEEGFMTAELAGVIKRLWKDSGVQACFNRSREYQLND
SAAYYLNDLDRIAQPNYIPTQQDVLRTRVKTTGIVETHFTFKDLHFKMFD
VGGQRSERKKWIHCFEGVTAIIFCVALSDYDLVLAEDEEMNRMHESMKLF
DSICNNKWFTDTSIILFLNKKDLFEEKIKKSPLTICYPEYAGSNTYEEAA
AYIQCQFEDLNTATGDGKHYCYPHFTCAVDTENIRRVFNDCRDIIQRMHL
KQYELL

The amino acid sequence of G_α subunit (G_i/olf156) used in Comparative Example 5 is shown below. FIG. 12 schematically shows a secondary structure of G_i/olf156.

(SEQ ID NO: 9)
MGCTLSAEDKAAVERSKMIDRNLREDGEKAAREVKLLLLGAGESGKSTIV
KQMKIIHEAGYSEEECKQYKAVVYSNTIQSIIAIIRAMGRLKIDFGDSAR
ADDARQLFVLAGAAEEGFMTAELAGVIKRLWKDSGVQACFNRSREYQLND
SAAYYLNDLDRIAQPNYIPTQQDVLRTRVKTTGIVETHFTFKDLHFKMFD
VGGQRSERKKWIHCFNDVTAIIYVAACSSYNMVIREDNNTNRLRESLDLF
ESIWNNRWLRTISIILFLNKQDMLAEKVLAGKSKIEDYFPEYANYTVPED
ATPDAGEDPKVTRAKFFIRDLFLRISTATGDGKHYCYPHFTCAVDTENIR
RVFNDCRDIIQRMHLKQYELL

The amino acid sequence of a chimeric G_α subunit (G_i/olf195) used in Comparative Example 6 is shown below. FIG. 13 schematically shows a secondary structure of G_i/olf195.

(SEQ ID NO: 10)
MGCTLSAEDKAAVERSKMIDRNLREDGEKAAREVKLLLLGAGESGKSTIV
KQMKIIHEAGYSEEECKQYKAVVYSNTIQSIIAIIRAMGRLKIDFGDSAR
ADDARQLFVLAGAAEEGFMTAELAGVIKRLWKDSGVQACFNRSREYQLND
SAAYYLNDLDRIAQPNYIPTQQDVLRCRVLTSGIFETRFQVDKVNFHMFD
VGGQRDERRKWIQCFNDVTAIIYVAACSSYNMVIREDNNTNRLRESLDLF
ESIWNNRWLRTISIILFLNKQDMLAEKVLAGKSKIEDYFPEYANYTVPED
ATPDAGEDPKVTRAKFFIRDLFLRISTATGDGKHYCYPHFTCAVDTENIR
RVFNDCRDIIQRMHLKQYELL

The amino acid sequence of a chimeric G_α subunit (G_i/olfα3-β5) used in Comparative Example 7 is shown below. FIG. 14 schematically shows a secondary structure of G_i/olfα3-β5.

(SEQ ID NO: 11)
MGCTLSAEDKAAVERSKMIDRNLREDGEKAAREVKLLLLGAGESGKSTIV
KQMKIIHEAGYSEEECKQYKAVVYSNTIQSIIAIIRAMGRLKIDFGDSAR
ADDARQLFVLAGAAEEGFMTAELAGVIKRLWKDSGVQACFNRSREYQLND
SAAYYLNDLDRIAQPNYIPTQQDVLRTRVKTTGIVETHFTFKDLHFKMFD
VGGQRSERKKWIHCFEGVTAIIFCVALSDYDLVLAEDEEMNRMHESMKLF
DSICNNKWLRTISIILFLNKKDLFEEKIKKSPLTICYPEYAGSNTYEEAA
AYIQCQFEDLNKRKDTKEIYTHFTCATDTKNVQFVFDAVTDVIIKNNLKD
CGLF

The amino acid sequence of a chimeric G_α subunit (G_i/olfα4-β6) used in Comparative Example 8 is shown below. FIG. 16 schematically shows a secondary structure of G_{αi/olfα4-β6}.

(SEQ ID NO: 13)
MGCTLSAEDKAAVERSKMIDRNLREDGEKAAREVKLLLLGAGESGKSTIV
KQMKIIHEAGYSEEECKQYKAVVYSNTIQSIIAIIRAMGRLKIDFGDSAR
ADDARQLFVLAGAAEEGFMTAELAGVIKRLWKDSGVQACFNRSREYQLND
SAAYYLNDLDRIAQPNYIPTQQDVLRTRVKTTGIVETHFTFKDLHFKMFD
VGGQRSERKKWIHCFEGVTAIIFCVALSDYDLVLAEDEEMNRMHESMKLF
DSICNNKWFTDTSIILFLNKKDLFEEKIKKSPLTICYPEYAGSNTYEEAA
AYIQCQFEDLNTATGDGKHYCYTHFTCATDTKNVQFVFDAVTDVIIKNNL
KDCGLF

The amino acid sequence of a chimeric G_α subunit (G_{i/olfα3-β5,α4-β6}) used in Comparative Example 9 is shown below. FIG. 17 schematically shows a secondary structure of G_{αi/olfα3-β5,α4-β6}.

(SEQ ID NO: 14)
MGCTLSAEDKAAVERSKMIDRNLREDGEKAAREVKLLLLGAGESGKSTIV
KQMKIIHEAGYSEEECKQYKAVVYSNTIQSIIAIIRAMGRLKIDFGDSAR
ADDARQLFVLAGAAEEGFMTAELAGVIKRLWKDSGVQACFNRSREYQLND
SAAYYLNDLDRIAQPNYIPTQQDVLRTRVKTTGIVETHFTFKDLHFKMFD
VGGQRSERKKWIHCFEGVTAIIFCVALSDYDLVLAEDEEMNRMHESMKLF
DSICNNKWLRTISIILFLNKKDLFEEKIKKSPLTICYPEYAGSNTYEEAA
AYIQCQFEDLNTATGDGKHYCYTHFTCATDTKNVQFVFDAVTDVIIKNNL
KDCGLF

The amino acid sequence of a chimeric G_α subunit (G_{i/olfα3-β5,α4-β6,C}) used in Comparative Example 10 is shown below. FIG. 19 schematically shows a secondary structure of G_{i/olfα3-β5,α4-β6,C}.

(SEQ ID NO: 16)
MGCTLSAEDKAAVERSKMIDRNLREDGEKAAREVKLLLLGAGESGKSTIV
KQMKIIHEAGYSEEECKQYKAVVYSNTIQSIIAIIRAMGRLKIDFGDSAR
ADDARQLFVLAGAAEEGFMTAELAGVIKRLWKDSGVQACFNRSREYQLND
SAAYYLNDLDRIAQPNYIPTQQDVLRTRVKTTGIVETHFTFKDLHFKMFD
VGGQRSERKKWIHCFEGVTAIIFCVALSDYDLVLAEDEEMNRMHESMKLF
DSICNNKWLRTISIILFLNKKDLFEEKIKKSPLTICYPEYAGSNTYEEAA
AYIQCQFEDLNTATGDGKHYCYTHFTCATDTKNVQFVFDAVTDIIQRMHL
KQYELL

Table 11 and FIG. 30 show measurement results from Examples 1 to 4, Comparative Examples 1 to 6, and Comparative Example 11. The measurement results are shown as change levels in current density, i.e., a difference between current densities (current value/cell membrane capacity) of membrane current measured when ISO was not supplied and when ISO was supplied.

	TABLE 11
		Change Level
		in Current	Limit of
	Gα subunit	Density [pA/pF]	Error
Comparative Example 11		−26.29	3.38
Comparative Example 1	Gαi (C351G)	−9.14	3.36
Comparative Example 3	Gi/olf5	−13.37	6.05
Example 1	Gi/olf13	−110.50	20.36
Example 2	Gi/olf28	−144.91	30.42
Comparative Example 4	Gi/olf45	−83.20	17.00
Example 3	Gi/olf94	−154.64	22.14
Example 4	Gi/olf113	−135.63	15.71
Comparative Example 5	Gi/olf156	−93.32	19.35
Comparative Example 6	Gi/olf195	−93.49	8.57
Comparative Example 2	Gαolf	−22.59	3.70

Table 12 and FIG. 30 show measurement results from Example 1, Example 5, Example 6, Comparative Example 1, and Comparative Examples 7 to 10. The measurement results are shown as amounts of change in current densities, i.e., a difference between current density (current value/cell membrane capacity) of membrane current measured when ISO was not supplied and when ISO was supplied.

	TABLE 12
		Change Level	Limit
		in Current	of
	Gα subunit	Density [pA/pF]	Error
Comparative Example 1	Gαi (C351G)	−16.37	4.19
Example 1	Gi/olf13	−110.50	20.36
Comparative Example 7	Gi/olfα3-β5	−56.66	7.76
Example 5	Gi/olfα3-β5,C	−142.54	11.47
Comparative Example 8	Gi/olfα4-β6	−34.15	2.63
Example 6	Gi/olfα4-β6,C	−147.49	28.15
Comparative Example 10	Gi/olfα3-β5,α4-β6,C	−86.88	12.85
Comparative Example 9	Gi/olfα3-β5,α4-β6	−52.78	5.00

(Comparison to Camp Technique)

Chemical substance detection sensitivity according to the present invention was compared to detection sensitivity of cAMP technique, which is a commonly used chemical substance detection technique. The cAMP technique is a technique for measuring an increase in cAMP concentration in an intracellular fluid caused by an interaction between an agonist and a receptor, and quantifying the concentration of the agonist based on the measured value. More particularly, cells expressing β1 adrenergic receptor and potassium ion channel Kir3.1 (F137S) were placed in contact with a buffer solution containing ISO, and then were homogenized. The concentration of cAMP contained in the obtained homogenate liquid was measured. The concentration measurement was performed by using a measurement kit (cyclic AMP EIA kit; Assay Designs, Inc.). The kit measures cAMP concentration by utilizing competitive EIA (competitive enzyme immunoassay). A cAMP concentration obtained when the ISO concentration inside of a buffer solution was 1000 nM was represented as 100 as a standard, and cAMP concentrations at respective ISO concentrations were calculated as ratios with regard to this standard cAMP concentration. On the other hand, measurements were conducted similarly to Example 3, except that the measurements were conducted by changing the ISO concentration. A change value in current density obtained when the ISO concentration was 30 nM was represented as 100 as a standard, and change values in current density at respective ISO concentrations were calculated as ratios with regard to the standard change value. Table 13 and FIG. 32 show the results.

TABLE 13
ISO Concentration
(nM)	cAMP technique	Present Invention
0	9.56	—
0.01	2.95	—
0.03	1.78	2.34
0.1	3.00	—
0.3	4.25	19.22
1	3.21	48.01
3	—	78.00
10	9.95	—
30	—	100
100	44.66	—
300	93.82	95.94
1000	100	—
3000	—	—

The following describes what is understood from Table 13 and FIG. 32. A detectable lower limit of ISO concentration when using cAMP technique is thought to be approximately 10 nM. This is because an ISO concentration-dependent increase in the cAMP concentration was not observed upon contact with a buffer solution having an ISO concentration of 0 to 1 nM. A half maximal effective concentration (EC₅₀) of ISO, which is an index of ISO detection sensitivity, with regard to (31 adrenergic receptor was 135 nM. On the other hand, with the method according to the present invention using the chimeric a subunit G_i/olf94 protein, the lower limit of detection concentration of ISO was 0.3 nM. The change level in ionic current saturated at an ISO concentration of approximately 30 nM. In this case, the half maximal effective concentration of ISO was 1 nM with regard to β1 adrenergic receptor. With the chemical substance detection method using the chimeric G_α subunit protein of the present invention, ISO can be detected with sensitivity approximately 100 times higher than a conventional generally-used cAMP technique. It is thought that the highly sensitive detection was enabled since bindings of chemical substances to receptors have been directly transferred to ion channels via chimeric G proteins without depending on a complicated intermolecular reaction within cells.

(Detection of β Adrenergic Receptor Agonist)

Detections of β1 agonists other than isoproterenol were conducted. Measurements were conducted similarly to Example 3, except for using, in addition to ISO as chemical substances, dobutamine, which is a selective agonist of β1 adrenergic receptor, and dopamine which is an agonist of β adrenergic receptor. Table 14 and FIG. 33 show measurement results. Change values in current density observed at concentrations of 30000 nM for dopamine, 100 nM for dobutamine, and 30 nM for ISO were each represented as 100, and measurement results were described as relative values with regard to those.

	TABLE 14
	Concentration	Change Level in Current Density
	[nM]	dopamine	dobutamine	isoproterenol
	0.03	—	—	2.34
	0.3	—	0.67	19.22
	1	—	—	48.01
	3	3.07	6.80	78.01
	30	5.32	20.59	100
	300	4.11	99.15	95.94
	3000	66.61	100	—
	30000	100	—	—

With every agonist used, the change values in current density increased in association with increases in agonist concentrations. The EC₅₀ values for dopamine and dobutamine were 1000 nM and 50 nM, respectively. It is reasonably presumed that a change in current density is generated by agonists activating chimeric G proteins via chemical substance receptors and opening gates of ion channels. This is because the EC₅₀ values are different depending on the type of agonist. Therefore, agonists are not directly but indirectly activating chimeric G proteins.

Reference Example

The present inventors examined the following in advance of the present invention, and determined a design plan for the chimeric G_α subunits. G_αi (C351G), G_αolf, and G_i/olf13 were used as G_α subunits to measure ionic current generated upon contact with ISO. The measurements were conducted in accordance with a method similar to that in Example 1.

As shown in FIG. 26, when G_αi (C351G) was used as the chimeric G_α subunit and when ISO was not supplied, a current value of approximately −0.2 nA was measured immediately after a pulse wave was applied. Here, the current value being “−” (negative) indicates that potassium ions have moved from a front side to a back side of a lipid bilayer membrane (from outside to inside a cell). When ISO was supplied, the current value changed and became approximately 0.5 nA.

As shown in FIG. 27, when G_i/olf13 was used as the chimeric G_α subunit, the current value changed due to having ISO supplied and became approximately −2.5 nA. As shown in FIG. 28, when G_αolf protein was used as the chimeric G_α subunit, a current value of approximately 0.5 nA was measured similarly to the case where G_αi (C351G) was used, and change level of current value caused by an addition of ISO was small.

The present inventors derived the following conclusion from the above described measurement results. G_αi (C351G) can activate potassium ion channels but cannot bind to chemical substance receptors. G_αolf can bind to chemical substance receptors but cannot activate potassium ion channels. Thus, the current change level caused by the addition of ISO was small. On the other hand, when G_i/olf13 was used, a large current change was observed caused by the addition of ISO, since the chimeric G_α subunit can bind to chemical substance receptors via the G_αolf region at the C-terminal and the G_αi region activates potassium ion channels. Therefore, the chimeraization of the G_α subunit increases the current change level caused by the addition of ISO.

Furthermore, a current change level obtained when G_i/s13 was used as the chimeric G_α subunit was compared to a current change level obtained when G_i/olf13 was used as the chimeric G_α subunit. Table 15 and FIG. 29 show measurement results. The measurement results obtained when G_αi (C351G) and G_olf apply were used are shown on the side as references.

	TABLE 15
	Gi/olf13	Gi/s13	Gαi (C351G)	Gαolf
	Current Change	−1.36	−1.11	−0.20	−0.37
	Level [nA]
	Limit of Error	0.05	0.15	0.05	0.06

The current change level obtained when G_i/olf13 was used was larger than the current change level obtained when G_i/s13 was used. Therefore, it was determined that larger current changes can be expected when a chimera of G_αi and G_αolf were used. Hence, it was determined to create a chimeric G_α subunit in which a region generally considered to participate in the coupling with a chemical substance receptor in the amino acid sequence of G_αi was substituted with a corresponding amino acid sequence of G_αolf.

INDUSTRIAL APPLICABILITY

According to a chimeric G protein of the present invention, a detection of a chemical substance that binds to a chemical substance receptor is enabled.

Claims

1. A method for transporting potassium ions from a front side of a lipid bilayer membrane to a back side thereof, the method comprising the following steps (a) and (b): step (a) of preparing the lipid bilayer membrane, wherein the lipid bilayer membrane comprises a receptor of a chemical substance, a G protein, and a potassium ion channel,the G protein comprises a chimeric Gα subunit and a Gβγ subunit complex, andthe chimeric Gα subunit consists of any one of Gi/olf13 (SEQ ID NO: 04), Gi/olf28 (SEQ ID NO: 05), Gi/olf94 (SEQ ID NO: 07), Gi/olf113 (SEQ ID NO: 08), Gi/olfα3-β5,C (SEQ ID NO: 12), or Gi/olfα4-β6,C (SEQ ID NO: 15); andstep (b) of supplying the chemical substance and the potassium ions to the front side to release the chimeric Gα subunit and Gβγ subunit complex, and to allow the Gβγ subunit complex to bind to the potassium ion channel for transporting the potassium ions from the front side to the back side.

2. The method according to claim 1, wherein the chemical substance is an adrenergic receptor agonist.

3. The method according to claim 1, wherein the potassium ion channel is a G protein-coupled inwardly rectifying potassium ion channel.

4. A method for detecting or quantifying a chemical substance, the method comprising the following steps (c), (d), and (e): step (c) of preparing a lipid bilayer membrane, a first liquid located on a front side of the lipid bilayer membrane, and a second liquid located on a back side of the lipid bilayer membrane, wherein the lipid bilayer membrane comprises a chemical substance receptor, a G protein, and a potassium ion channel,the G protein comprises a chimeric Gα subunit and a Gβγ subunit complex,the chimeric Gα subunit consists of any one of Gi/olf13 (SEQ ID NO: 04), Gi/olf28 (SEQ ID NO: 05), Gi/olf94 (SEQ ID NO: 07), Gi/olf113 (SEQ ID NO: 08), Gi/olfα3-β5,C (SEQ ID NO: 12), or Gi/olf α4-β6,C (SEQ ID NO: 15), andthe first liquid contains potassium ions;step (d) of supplying the chemical substance to the first liquid; andstep (e) of measuring an amount of potassium ions in at least one of the first and second liquids to detect or quantify the chemical substance based on the amount of the potassium ions.

5. The method according to claim 4, wherein the chemical substance is an adrenergic receptor agonist.

6. The method according to claim 4, wherein the potassium ion channel is a G protein-coupled inwardly rectifying potassium ion channel.

7. A method for detecting or quantifying a chemical substance, the method comprising the following steps (f), (g), and (h): step (f) of preparing a lipid bilayer membrane, wherein the lipid bilayer membrane comprises a chemical substance receptor, a G protein, and a potassium ion channel,the G protein comprises a chimeric Gα subunit and a Gβγ subunit complex, andthe chimeric Gα subunit consists of any one of Gi/olf13 (SEQ ID NO: 04), Gi/olf28 (SEQ ID NO: 05), Gi/olf94 (SEQ ID NO: 07), Gi/olf113 (SEQ ID NO: 08), Gi/olfα3-β5,C (SEQ ID NO: 12), or Gi/olfα4-β6,C (SEQ ID NO: 15);step (g) of supplying a first liquid and a second liquid located respectively on a front side and a back side of the lipid bilayer membrane such that the lipid bilayer membrane is interposed between the first liquid and second liquid, wherein the first liquid contains potassium ions and the chemical substance; andstep (h) of measuring an amount of potassium ions in at least one of the first and second liquids to detect or quantify the chemical substance based on the amount of the potassium ions.

8. The method according to claim 7, wherein the chemical substance is an adrenergic receptor agonist.

9. The method according to claim 7, wherein the potassium ion channel is a G protein-coupled inwardly-rectifying ion channel.