Expression system for alpha2alpha5beta2 type nicotinic acetylcholine receptor and method of use thereof

Abstract

Disclosed herein are isolated recombinant cells for the expression of α2α5β2-type nicotinic acetylcholine receptors (nAChRs) and methods of use thereof.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 178,813, filed April 23, 2021, which is incorporated by reference in its entirety.

[0002] FIELD OF THEINVENTION The present invention relates herein to isolated recombinant cells for the expression of α2α5β2-type nicotinic acetylcholine receptors (nAChRs) and methods of use thereof.

[0003] (Reference to electronically submitted sequence listing) This application contains a Sequence Listing, which has been submitted electronically in ASCII format and is incorporated by reference in its entirety. The ASCII copy was created on Feb. 28, 2022, is named PRD4128WOPCT1_SL.txt, and is 24,576 bytes in size.

[0004] BACKGROUND OF THEINVENTION Nicotinic acetylcholine receptors (nAChRs) are involved in several neurological and psychiatric conditions, including nicotine addiction, pain, and psychotic disorders (Gotti et al.,Neuronal nicotinic receptors: from structure to pathology,Prog Neurobiol.2004 Dec;74(6):363-96). Nicotine addiction associated with smoking is the main cause of lung cancer. Genome-wide association studies show that the nucleotide polymorphism (SNP) rs16969968, which codes for a change in the sequence of the α5 subunit of the nAChR (D398N), increases susceptibility to nicotine addiction. The habenular-peduncular tract, where α5 nAChRs are particularly enriched, is an important neural circuit regulating nicotine intake. For these reasons, α5 nAChRs have been proposed as a valuable drug target (Fowler et al.,Habenular nicotinic receptors containing the α5* subunit controls nicotine intake, Nature. 2011 Mar 31;471(7340):597-601; Maskos, The nicotinic receptor alpha5 coding polymorphism rs16969968 as a major target in disease: Functional dissection and remaining challenges, Journal of Neurochemistry. 2020:154-241-250). However, identification of compounds that modulate α5-containing nAChRs (such as α2α5β2-type nAChRs) has not been possible because this receptor is not functionally expressed in recombinant cell lines commonly used for drug discovery.

[0005] There remains a need to develop cell lines that robustly express the α5 subunit of the nAChR and can be used for drug discovery.

[0006] BRIEF SUMMARY OF THEINVENTION Provided herein is an isolated recombinant cell comprising: a) a heterologous nucleic acid encoding an α5 subunit of the nicotinic acetylcholine receptor (nAChR); and b) a heterologous nucleic acid encoding a chaperone protein selected from the transmembrane inner ear expressed protein TMIE and the protein FAM163B.

[0007] In one embodiment of the isolated recombinant cell provided above, the cell further comprises c) a heterologous nucleic acid encoding the α2 subunit of the nAChR, d) a heterologous nucleic acid encoding the β2 subunit of the nAChR, e) a heterologous nucleic acid encoding diamine acetyltransferase 1 (SAT1), and f) a heterologous nucleic acid encoding choline O-acetyltransferase (CHAT), wherein the α2, α5 and β2 subunits of the nAChR form an α2α5β2 type nAChR.

[0008] In a further embodiment of the isolated recombinant cell, the recombinant cell is a mammalian cell.

[0009] In further embodiments of the isolated recombinant cell, the mammalian cell is selected from the group consisting of human embryonic kidney 293T (HEK293T) cells, HEK293F cells, HeLa cells, Chinese hamster ovary (CHO) cells, NIH3T3 cells, MCF-7 cells, Hep G2 cells, baby hamster kidney (BHK) cells, and Cos7 cells.

[0010] In a further embodiment of the isolated recombinant cell, the α2 subunit of the α2α5β2-type nAChR comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:1.

[0011] In a further embodiment of the isolated recombinant cell, the α5 subunit of the α2α5β2 type nAChR comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:2.

[0012] In a further embodiment of the isolated recombinant cell, the β2 subunit of the α2α5β2-type nAChR comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:3.

[0013] In a further embodiment of the isolated recombinant cell, the TMIE comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:4.

[0014] In a further embodiment of the isolated recombinant cell, FAM163B comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:5.

[0015] In a further embodiment of the isolated recombinant cell, SAT1 comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:6.

[0016] In a further embodiment of the isolated recombinant cell, the CHAT comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:7.

[0017] Further provided herein is a method for identifying an agonist, antagonist or positive allosteric modulator of an α5-containing nAChR, comprising the steps of: a) contacting an isolated recombinant cell according to any one of claims 1 to 11 with an agent; and b) determining the activity of the α5-containing nAChR of the isolated recombinant cell, wherein if the agent enhances the activity of the α5-containing nAChR compared to the activity of the α5-containing nAChR when the isolated recombinant cell is not contacted with the agent, the agent is identified as an agonist or positive allosteric modulator (PAM), and if the agent decreases the activity of the α5-containing nAChR, the agent is identified as an antagonist.

[0018] Further provided herein is a method for identifying an agonist, antagonist or positive allosteric regulator of α2α5β2 nAChR, comprising the steps of: a) contacting an isolated recombinant cell according to any one of claims 2 to 11 with an active substance; and b) determining the activity of α2α5β2 nAChR of the isolated recombinant cell, wherein if the active substance enhances the activity of α2α5β2 nAChR compared to the activity of α2α5β2 nAChR when the isolated recombinant cell has not been contacted with the active substance, the active substance is identified as an agonist or positive allosteric regulator (PAM), and if the active substance decreases the activity of α2α5β2 nAChR, the active substance is identified as an antagonist.

[0019] In one embodiment of this method, step b) comprises determining calcium flux in the isolated recombinant cell, and if the agent enhances calcium flux compared to calcium flux when the isolated recombinant cell is not contacted with the agent, the agent is identified as an agonist.

[0020] In a further embodiment of this method, step b) comprises determining calcium flux and nicotine-induced calcium flux of the isolated recombinant cell, and if the agent does not enhance calcium flux and enhances nicotine-induced calcium flux compared to the calcium flux and nicotine-induced calcium flux when the isolated recombinant cell is not contacted with the agent, the agent is identified as a PAM.

[0021] In yet a further embodiment of this method, step b) comprises determining the nicotine-induced calcium flux of the isolated recombinant cell, and if the agent reduces the nicotine-induced calcium flux compared to the nicotine-induced calcium flux when the isolated recombinant cell is not contacted with the agent, the agent is identified as an antagonist.

[0022] In yet a further embodiment of this method, the isolated recombinant cells are incubated at about 25° C.-35° C. for about 20-50 hours prior to being contacted with the agent.

[0023] In yet a further embodiment of this method, the agent is a small molecule or a peptide.

[0024] Further provided herein is a kit comprising (i) the isolated recombinant cell provided above, and (ii) instructions for use. [Brief description of the drawings]

[0025] [Figure 1] FIG. 1 is a graph showing functional expression of α2β2 and α2α5β2 nAChRs when co-expressed with different chaperone proteins. [Diagram 2] Concentration-response curves for nicotine-induced activation of α2β2 and α2α5β2 nAChRs when co-expressed with different chaperone proteins.

[0026] DETAILED DESCRIPTION OF THE PRESENT EMBODIMENT Various publications, articles and patents are cited or described in the "Background" and throughout the specification, and each of these references is incorporated herein by reference in its entirety. Any discussion of documents, operations, materials, devices, articles and the like which is included in the specification is for the purpose of providing a context for the invention. Such discussion is not an admission that any or all of these items constitute part of the prior art to any invention disclosed or claimed.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise defined, certain terms used herein have the meanings set forth herein.

[0028] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise.

[0029] Unless otherwise stated, any numerical values, such as concentrations or concentration ranges, described herein should be understood in all cases as being modified by the term "about". Thus, numerical values ​​generally include ±10% of the described value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of numerical ranges expressly includes all possible subranges, all individual numerical values ​​within the range, including integers and fractions of values ​​within the range, unless the context clearly indicates otherwise.

[0030] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0031] As used herein, it will be understood that the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," or "containing," or any other variation thereof, are intended to be non-exclusive or non-limiting, implying the inclusion of a stated integer or group of integers, but not excluding other integers or groups of integers. For example, a composition, mixture, process, method, article, or device that includes a list of elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherently present in such composition, mixture, process, method, article, or device. Further, unless expressly stated to the contrary, "or" refers to an inclusive "or" and not an exclusive "or." For example, condition A or B is satisfied by one of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0032] As used herein, the connective term "and / or" between multiple listed elements is understood to encompass both individual and combined options. For example, when two elements are connected by "and / or," the first option refers to the first element being applicable without the second element. The second option refers to the second element being applicable without the first element. The third option refers to the first and second elements being applicable together. Any one of these options is understood to be within the meaning and thus meets the requirements of the term "and / or" as used herein. The simultaneous applicability of two or more of the options is also understood to be within the meaning and thus meets the requirements of the term "and / or."

[0033] As used herein, the term "consists of," or variations such as "consist of" or "consisting of," as used throughout the specification and claims, includes any recited integer or group of integers, but indicates that no additional integer or group of integers is added to the specified method, structure, or composition.

[0034] As used herein, the term "consists essentially of," or variations such as "consist essentially of" or "consisting essentially of," as used throughout the specification and claims, refers to the inclusion of any recited integer or group of integers, optionally including any recited integer or group of integers that do not materially change the basic or novel characteristics of the specified method, structure, or composition. See MPEP §2 111.03.

[0035] It should also be understood that terms such as "about," "approximately," "generally," and "substantially," used herein when referring to dimensions or features of preferred inventive components, indicate that the described dimensions / features are not precise boundaries or parameters, but do not exclude minor variations therefrom that are functionally the same or similar, as would be understood by one of ordinary skill in the art. At a minimum, such references involving numerical parameters include variations that do not change the least significant digit using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.).

[0036] The term "identical" or percent "identity," in the context of two or more nucleic acid or polypeptide sequences, means the percentage of identical or similar sequences, as determined using one of the following sequence comparison algorithms or by visual inspection: It refers to two or more sequences or subsequences that have the same or a certain percentage of the same amino acid residues or nucleotides when compared and aligned to maximize the correspondence. For sequence comparison, typically one sequence serves as a reference sequence to which test sequences are compared. When using sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence to the reference sequence based on the designated program parameters.

[0037] Optimal alignment of sequences for comparison can be determined, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally Current Protocols in Molecular Biology, FMAusubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 This can be done by the method of the Supplement (Ausubel).

[0038] Examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match when aligned with words of the same length in database sequences or meet some positive threshold score T, where T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds to initiate searches to find longer HSPs that contain them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.

[0039] For nucleotide sequences, the parameters M (reward score for a pair of matching residues, always >0) and N (penalty score for mismatching residues, always <0) are used to calculate the cumulative score. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score falls off its maximum achieved value by an amount X, when the accumulation of one or more negative scoring residue alignments causes the cumulative score to fall below zero, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults wordlength (W)=11, expectation (E)=10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992)).

[0040] In addition to calculating the percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide sequences or two amino acid sequences would occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the minimum sum probability in the comparison between the test nucleic acid and the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0041] A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, e.g., the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0042] As used herein, the term "polynucleotide" is also referred to interchangeably as "nucleic acid molecule," "nucleotide," or "nucleic acid," and refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules containing DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single-stranded and double-stranded regions. In addition, "polynucleotide" refers to triple-stranded regions that include RNA or DNA, or both RNA and DNA. The term polynucleotide also includes DNA or RNA that contain one or more modified bases, and DNA or RNA with backbones modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases, such as inosine. A variety of modifications can be made to DNA and RNA. Thus, "polynucleotide" includes chemically, enzymatically, or metabolically modified forms of polynucleotides typically found in nature, as well as chemical forms having characteristics of viral and cellular DNA and RNA. "Polynucleotide" also includes relatively short nucleic acid strands, often referred to as oligonucleotides.

[0043] As used herein, the term "peptide", "polypeptide", or "protein" can refer to a molecule composed of amino acids and can be recognized as a protein by those skilled in the art. Conventional one-letter or three-letter codes for amino acid residues are used herein. The terms "peptide", "polypeptide", and "protein" can be used interchangeably herein to refer to a polymer of amino acids of any length. The polymer can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. The term also encompasses amino acid polymers that are naturally modified or modified by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. The definition also includes, for example, polypeptides containing one or more analogs of an amino acid (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art.

[0044] The peptide sequences described herein are written according to the usual convention with the N-terminal region of the peptide on the left and the C-terminal region on the right, although isomeric forms of amino acids are known, unless expressly indicated otherwise, it is the L-form of the amino acid that is shown.

[0045] Isolated recombinant cells and methods for making recombinant cells The invention disclosed herein is based, at least in part, on the unexpected discovery that co-expressing certain chaperone proteins in cells with α5-containing nicotinic acetylcholine receptors (nAChRs), such as α2α5β2-type nAChRs, produces cells that highly express α5-containing nAChRs, making the cells useful for drug discovery. Provided herein are recombinant cells expressing the α5 subunit of nAChRs and methods for producing isolated recombinant cells for expression of the α5 subunit of nAChRs. Also provided herein are recombinant cells expressing the α2α5β2-type nAChRs and methods for producing isolated recombinant cells for expression of the α2α5β2-type nAChRs.

[0046] As used herein, the terms "α2α5β2 type nicotinic acetylcholine receptor", "α2α5β2 type nAChR", "alpha2 alpha5 beta2 type nicotinic acetylcholine receptor" and "alpha2 alpha5 beta2 type nAChR" are used interchangeably and refer to the α2α5β2 type nicotinic acetylcholine receptor protein, preferably the human α2α5β2 type nAChR, which is a member of the cholinergic receptor protein family.

[0047] The α2α5β2 nAChR is a ligand-gated ion channel composed of α2, α5, and β2 subunits. The α2 subunit is encoded by gene CHRNA2 (NM_000742), the α5 subunit is encoded by gene CHRNA5 (NM_000745), and the β2 subunit is encoded by gene CHRNB2 (NM_000748). When expressed together, the subunits associate to form the α2α5β2-type nAChR, which is particularly concentrated in the optic lobe in chickens and in the habenulo-interpeduncular system in mammals (see, e.g., Balestra et al., Chick Optic Lobe Contains a Developmentally Regulated α2α5β2 Nicotinic Receptor Subtype, MOL 58:300-311, 2000 / 1 / 841787 and Salas et al, “Nicotinic Receptors in the Habenulo-Interpeduncular System Are Necessary for Nicotine Withdrawal in Mice, J. Neurosci. 29:3014-18, 2009).

[0048] "Recombinant cell" refers to one or more individual cells and recombinant cell lines in which the cells heterologously express a protein(s). As used herein, "heterologous expression" of a protein in a cell refers to modifying a cell to express a protein by introducing an exogenous nucleic acid into the cell, e.g., an exogenous nucleic acid encoding the protein to be expressed. "Heterologous nucleic acid" refers to a nucleic acid exogenous to the cell that is introduced into the cell. In some embodiments, the heterologous nucleic acid is DNA. In some embodiments, the heterologous nucleic acid is RNA. Heterologous expression of a protein in a cell can be achieved using a variety of methods. For example, an expression vector may be introduced into the cell that includes a nucleic acid encoding a protein operably linked to a nucleic acid encoding a promoter (e.g., a constitutive promoter) capable of driving expression of the protein.

[0049] As used herein, the term "expression" refers to the biosynthesis of a gene product. The term includes transcription of a gene into RNA. The term also includes translation of RNA into one or more polypeptides, and further includes all naturally occurring post-transcriptional and post-translational modifications.

[0050] In a general aspect, the present invention relates to a method for making or generating a cell expressing an α5-containing nAChR (such as an α2α5β2-type nAChR) useful for drug discovery. In one embodiment, the method comprises introducing into a cell a nucleic acid encoding an α5 subunit of the nAChR and a nucleic acid encoding a chaperone protein selected from the transmembrane inner ear expressed protein TMIE and protein FAM163B. The nucleic acid(s) encoding the α5 subunit of the nAChR and the chaperone protein (i.e., TMIE or FAM163B) can be present in an expression vector (e.g., in a single expression vector or in separate expression vectors). In some embodiments, the nucleic acid encoding the α5 subunit of the nAChR and the chaperone protein (i.e., TMIE or FAM163B) are operably linked to a promoter capable of driving expression of the respective proteins. In some other embodiments, the nucleic acids encoding the α5 subunit of the nAChR and the chaperone protein (i.e., TMIE or FAM163B) are each operably linked to a promoter capable of driving expression of the respective protein. In some embodiments, the promoter is a constitutive promoter.

[0051] In one aspect, the present invention relates to a method for producing or generating a cell expressing an α2α5β2 nAChR useful for drug discovery. In one embodiment, the method comprises introducing into the cell a nucleic acid encoding an α2 subunit of the α2α5β2 nAChR, a nucleic acid encoding an α5 subunit of the α2α5β2 nAChR, a nucleic acid encoding a β2 subunit of the α2α5β2 nAChR, a nucleic acid encoding diamine acetyltransferase 1 (SAT1), a nucleic acid encoding choline O-acetyltransferase (CHAT), and one or both of a nucleic acid encoding TMIE and a nucleic acid encoding FAM163B. The nucleic acid(s) encoding one selected from the α2, α5 and β2 subunits of the α2α5β2 nAChR, SAT1, CHAT, and TMIE and FAM163B can be present in an expression vector (e.g., in a single expression vector or in separate expression vectors). In some embodiments, the nucleic acids encoding the α2, α5 and β2 subunits of the α2α5β2 nAChR, SAT1, CHAT, and one or both of TMIE and FAM163B are operably linked to a promoter capable of driving the expression of the respective proteins. In some other embodiments, the nucleic acids encoding the α2, α5 and β2 subunits of the α2α5β2 nAChR, SAT1, CHAT, and one or both of TMIE and FAM163B are each operably linked to a promoter capable of driving the expression of the respective proteins. In some embodiments, the promoter is a constitutive promoter.

[0052] In another aspect, the method includes introducing into a cell one or both of a nucleic acid encoding the α2 subunit of the α2α5β2 type nAChR, a nucleic acid encoding the α5 subunit of the α2α5β2 type nAChR, a nucleic acid encoding the β2 subunit of the α2α5β2 type nAChR, a nucleic acid encoding SAT1, a nucleic acid encoding CHAT, and a nucleic acid encoding TMIE and a nucleic acid encoding FAM163B, and the cell produced by the method expresses increased levels of the α2α5β2 type nAChR compared to the same cell without the nucleic acid encoding the α2, α5, β2 subunits of the α2α5β2 type nAChR, and the nucleic acid encoding SAT1, CHAT, TMIE or FAM163B.

[0053] In another aspect, the invention relates to a cell genetically modified to express TMIE or FAM163B, wherein the genetically modified cell expresses at least one protein at an increased level compared to the expression of the same protein in a non-modified cell under the same (or substantially the same) conditions. In one embodiment, the cell is genetically modified to express one or both selected from SAT1, CHAT, and TMIE and FAM163B, wherein the genetically modified cell expresses one or both selected from SAT1, CHAT, and TMIE and FAM163B at an increased level compared to the expression of one or both selected from SAT1, CHAT, and TMIE and FAM163B, respectively, in a non-modified cell under the same (or substantially the same) conditions.

[0054] In another aspect, the present invention relates to a cell genetically modified to express the α2, α5 and β2 subunits of the α2α5β2 nAChR, SAT1, CHAT, and TMIE or FAM163B, wherein the genetically modified cell expresses these proteins at increased levels compared to the expression of the same proteins in a non-modified cell under the same (or substantially the same) conditions. In one embodiment, the cell is genetically modified to express the α2, α5 and β2 subunits of the α2α5β2 nAChR, SAT1, CHAT, TMIE, FAM163B, wherein the genetically modified cell expresses these proteins at increased levels compared to the expression of the same proteins in a non-modified cell under the same (or substantially the same) conditions.

[0055] In another aspect, the present invention relates to an isolated recombinant cell comprising at least one expression vector selected from the group consisting of an expression vector comprising a nucleic acid sequence encoding SAT1, an expression vector comprising a nucleic acid sequence encoding CHAT, and an expression vector comprising a nucleic acid sequence encoding TMIE or FAM163B. In one embodiment, the present invention relates to an isolated recombinant cell comprising at least one expression vector selected from the group consisting of an expression vector comprising a nucleic acid sequence encoding SAT1, an expression vector comprising a nucleic acid sequence encoding CHAT, an expression vector comprising a nucleic acid sequence encoding TMIE, and an expression vector comprising a nucleic acid sequence encoding FAM163B.

[0056] In one aspect, the present invention relates to an isolated recombinant cell comprising a heterologous nucleic acid encoding an α5 subunit of a nAChR and a heterologous nucleic acid encoding a chaperone protein selected from a transmembrane inner ear expressed protein (TMIE) and protein FAM163B. In one embodiment, the heterologous nucleic acid is introduced into the recombinant cell in the form of an expression vector. In one embodiment, the isolated recombinant cell disclosed herein comprises an expression vector comprising a nucleic acid encoding an α5 subunit of a nAChR and an expression vector comprising a nucleic acid encoding a chaperone protein selected from TMIE and FAM163B. In one embodiment, the isolated recombinant cell disclosed herein comprises an expression vector comprising a nucleic acid encoding an α5 subunit of a nAChR, an expression vector comprising a nucleic acid encoding a TMIE, and an expression vector comprising a nucleic acid encoding FAM163B.

[0057] In a further aspect, the present invention relates to an isolated recombinant cell comprising a heterologous nucleic acid encoding the α2 subunit of the nAChR, a heterologous nucleic acid encoding the α5 subunit of the nAChR, a heterologous nucleic acid encoding the β2 subunit of the nAChR, a heterologous nucleic acid encoding diamine acetyltransferase 1 (SAT1), a heterologous nucleic acid encoding choline O-acetyltransferase (CHAT), and a heterologous nucleic acid encoding TMIE and / or FAM163B, wherein the α2, α5 and β2 subunits form an α2α5β2 type nAChR. In one embodiment, the heterologous nucleic acid is introduced into the recombinant cell in the form of an expression vector. In one embodiment, an isolated recombinant cell disclosed herein comprises an expression vector comprising a nucleic acid encoding an α2 subunit of a nAChR, an expression vector comprising a nucleic acid encoding an α5 subunit of a nAChR, an expression vector comprising a nucleic acid encoding a β2 subunit of a nAChR, an expression vector comprising a nucleic acid encoding SAT1, an expression vector comprising a nucleic acid encoding CHAT, and an expression vector comprising a nucleic acid encoding TMIE or FAM163B. In one embodiment, an isolated recombinant cell disclosed herein comprises an expression vector comprising a nucleic acid encoding an α2 subunit of a nAChR, an expression vector comprising a nucleic acid encoding an α5 subunit of a nAChR, an expression vector comprising a nucleic acid encoding a β2 subunit of a nAChR, an expression vector comprising a nucleic acid encoding SAT1, an expression vector comprising a nucleic acid encoding CHAT, an expression vector comprising a nucleic acid encoding a TMIE, and an expression vector comprising a nucleic acid encoding FAM163B.

[0058] In any one of the embodiments described herein, the nucleic acids encoding any one or more of the α2, α5 and β2 subunit nAChR, SAT1, CHAT, TMIE and FAM163B can be present in a single expression vector or in separate expression vectors.

[0059] In one embodiment, the α2 subunit of the nAChR comprises an amino acid sequence having at least 85%, or at least 90%, or at least 95%, e.g., 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:1.

[0060] SEQ ID NO:1: MGPSCPVFLSFTKLSLWWLLLTPAGGEEAKRPPPRAPGDPLSSPSPTALPQGGSHTETEDRLFKHLFRGYNRWARPVPNTSDVVIVRFGLSIAQLIDVDEKNQMMTTNVWLKQEWSDYKLRWNPTDFGNITS LRVPSEMIWIPDIVLYNNADGEFAVTHMTKAHLFSTGTVHWVPPAIYKSSCSIDVTFFPFDQQNCKMKFGSWTYDKAKIDLEQMEQTVDLKDYWESGEWAIVNATGTYNSKKYDCCAEIYPDVTYAFVIRRL PLFYTINLIIPCLLISCLTVLVFYLPSDCGEKITLCISVLLSLTVFLLLITEIIPSTSLVIPLIGEYLLFTMIFVTLSIVITVFVLNVHHRSPSTHTMPHWVRGALLGCVPRWLLMNRPPPPVELCHPLRLK LSPSYHWLESNVDAEEREVVVEEEDRWACAGHVAPSVGTLCSHGHLHSGASGPKAEALLQEGELLLSPHMQKALEGVHYIADHLRSEDADSSVKEDWKYVAMVIDRIFLWLFIIVCFLGTIGLFLPPFLAGMI

[0061] In one embodiment, the α5 subunit of the nAChR comprises an amino acid sequence having at least 85%, or at least 90%, or at least 95%, e.g., 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:2.

[0062] SEQ ID NO:2 MAARGSGPRALRLLLLVQLVAGRCGLAGAAGGAQRGLSEPSSIAKHEDSLLKDLFQDYERWVRPVEHLNDKIKIKFGLAISQLVDVDEKNQLMTTNVWLKQEWIDVKLRWNPDDYGG IKVIRVPSDSVWTPDIVLFDNADGRFEGSTKTVIRYNGTVTWTPPANYKSSCTIDVTFFPFDLQNCSMKFGSWTYDGSQVDIILEDQDVDKRDFFDNGEWEIVSATGSKGNRTDSC CWYPYVTYSFVIKRLPLFYTLFLIIPCIGLSFLTVLVFYLPSNEGEKICLCTSVLVSLTVFLLVIEEIIPSSSKVIPLIGEYLVFTMIFVTLSIMVTVFAINIHHRSSSTHNAMAPL VRKIFLHTLPKLLCMRSHVDRYFTQKEETESGSGPKSSRNTLEAALDSIRYITRHIMKENDVREVVEDWKFIAQVLDRMFLWTFLFVSIVGSLGLFVPVIYKWANILIPVHIGNANK

[0063] In one embodiment, the β2 subunit of the nAChR comprises an amino acid sequence having at least 85%, or at least 90%, or at least 95%, e.g., 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:3.

[0064] SEQ ID NO:3 MARRCGPVALLLGGFGLLRLCSGVWGTDTEERLVEHLLDPSRYNKLIRPATNGSELVTVQLMVSLAQLISVHEREQIMTTNVWLTQEWEDYRLTWKPEEFDNMKKVRLPSKHIWLPDVVLYNNADG MYEVSFYSNAVVSYDGSIFWLPPAIYKSACKIEVKHPFFDQQNCTMKFRSWTYDRTEIDLVLKSEVASLDDFTPSGEWDIVALPGRRNENPDDSTYVDITYDFIIRRKPLFYTINLIIPCVLITSL AILVFYLPSDCGEKMTLCISVLLALTVFLLLISKIVPPTSLDVPLVGKYLMFTMVLVTFSIVTSVCVLNVHHRSPTTHHTMAPWVKVVFLEKLPALLFMQQPRHHCARQRLRLRRRQREREGAGAL FFREAPGADSCTCFVNRASVQGLAGAFGAEPAPVAGPGRSGEPCGCGLREAVDGVRFIADHMRSEDDDQSVSEDWKYVAMVIDRLFLWIFVFVCVFGTIGMFLQPLFQNYTTTTFLHSDHSAPSSK

[0065] In one embodiment, the TMIE comprises an amino acid sequence having at least 85%, or at least 90%, or at least 95%, such as 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 4. Alternatively, the TMIE comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, or at least 90%, or at least 95%, such as 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 4 and has protein chaperone properties, including the property of enhancing expression of the α5 subunit of the nAChR.

[0066] SEQ ID NO:4 MAGWPGAGPLCVLGGAALGVCLAGVAGQLVEPSTAPPKPKPPPLTKETVVFWDMRLWHVVGIFSLFVLSIIITLCCVFNCRVPRTRKEIEARYLQRKAAKMYTDKLETVPPLNELTEVPGEDKKKKKKKKKDSVDTVAIKVEEDEKNEAKKKKKGEK

[0067] In one embodiment, FAM163B comprises an amino acid sequence having at least 85%, or at least 90%, or at least 95%, such as 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 5. Alternatively, FAM163B comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, or at least 90%, or at least 95%, such as 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 5 and has protein chaperone properties, including the property of enhancing expression of the α5 subunit of nAChR.

[0068] SEQ ID NO:5 MTAGTVVITGGILATVILLCIIAVLCYCRLQYYCCKKDESEEDEEEPDFAVHSHLPPLHSNRNLVLTNGPALYPTASTSFSQKSPQARALCRSCSHCEPPTFFLQEPPEEEEDVLNGGERVLYKSVSQEDVELPPGGFGGLQALNPNRLSAMREAFARSRSISTDV

[0069] In one embodiment, SAT1 comprises an amino acid sequence having at least 85%, or at least 90%, or at least 95%, such as 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 6. Alternatively, SAT1 comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, or at least 90%, or at least 95%, such as 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 6 and has protein chaperone properties, including the property of enhancing expression of the α2 and β2 subunits of nAChR.

[0070] SEQ ID NO:6 MAKFVIRPATAADCSDILRLIKELAKYEYMEEQVILTEKDLLEDGFGEHPFYHCLVAEVPKEHWTPEGHSIVGFAMYYFTYDPWIGKLLYLEDFFVMSDYRGFGIGSEILKNLSQVAMRCRCSSMHFLVAEWNEPSINFYKRRGASDLSSEEGWRLFKIDKEYLLKMATEE

[0071] In one embodiment, CHAT comprises an amino acid sequence having at least 85%, or at least 90%, or at least 95%, such as 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 7. Alternatively, CHAT comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, or at least 90%, or at least 95%, such as 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 7 and has protein chaperone properties, including the property of enhancing expression of the α2 and β2 subunits of nAChR.

[0072] SEQ ID NO:7 MAAKTPSSEESGLPKLPVPPLQQTLATYLQCMRHLVSEEQFRKSQAIVQQFGAPGGLGETLQQKLLERQEKTANWVSEYWLNDMYLNNRLALPVNSSPAVIFARQHFPGTDDQLRFAASLISGVLSYKALLDSHSIPTDCAKGQLSGQPLCMKQYYGLFSSYRLPGHTQDTLVAQNSSIMPEPEHVIVACCNQFFVLDVVINFRRLSEGDLFTQLRKIVKMASNEDERLPPIGLLTSDGRSEWAEARTVLVKDSTNRDSLDMIERCICLVCLDAPGGVELSDTHRALQLLHGGGYSKNGANRWYDKSLQFVVGRDGTCGVVCEHSPFDGIVLVQCTEHLLKHMTQSSRKLIRADSVSELPAPRRLRWKCSPEIQGHLASSAEKLQRIVKNLDFIVYKFDNYGKTFIKKQKCSPDAFIQVALQLAFYRLHRRLVPTYESASIRRFQEGRVDNIRSATPEALAFVRAVTDHKAAVPASEKLLLLKDAIRAQTAYTVMAITGMAIDNHLLALRELARAMCKELPEMFMDETYLMSNRFVLSTSQVPTTTEMFCCYGPVVPNGYGACYNPQPETILFCISSFHSCKETSSSKFAKAVEESLIDMRDLCSLLPPTESKPLATKEKATRPSQGHQP

[0073] To prepare the recombinant cells disclosed herein, any suitable means for introducing heterologous nucleic acid into cells can be used herein, such as DNA transfection (e.g., via a DNA vector) and RNA transduction. In one embodiment, the heterologous nucleic acid introduced into the cells to generate the recombinant cell is prepared by using a vector, preferably an expression vector. The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted. Another type of vector is a viral vector, into which additional DNA segments can be inserted. An expression vector is a vector capable of directing the expression of a gene to which it is operably linked. An expression vector, as used herein, comprises a nucleic acid encoding a protein sequence in a form suitable for expression of the nucleic acid in a host cell. Thus, an expression vector is selected based on the host cell to be used for expression and can comprise one or more control sequences, such as a promoter, operably linked to the nucleic acid sequence to be expressed. When used in reference to an expression vector, "operably linked" is intended to refer to a nucleotide sequence of interest being linked to a control sequence in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). It will be understood by those skilled in the art that the design of the expression vector can depend on factors such as the choice of the host cell to be transformed, the level of expression of protein desired and the intended use of the vector.

[0074] In view of the present disclosure, any vector known to those skilled in the art can be used, such as a plasmid, cosmid, phage vector or viral vector. In one embodiment, the vector is an expression vector, such as a plasmid. The vector can include any element for establishing the conventional functions of an expression vector, such as a promoter, a ribosome binding element, a terminator, an enhancer, a selection marker, and an origin of replication. The promoter can be a constitutive, inducible, or reconfigurable promoter. Numerous expression vectors capable of delivering nucleic acids to cells are known in the art and can be used herein. Conventional cloning techniques or artificial gene synthesis methods can be used to generate expression vectors according to embodiments of the present invention.

[0075] In view of the present disclosure, any cell known to one of skill in the art can be used for recombinant expression of α2, α5, β2 subunits of nAChR, SAT1, CHAT, TMIE and FAM163B. In one embodiment, the recombinant cell is a mammalian cell. Suitable mammalian cells may be selected from human embryonic kidney 293T (HEK293T) cells, HEK293F cells, HeLa cells, Chinese hamster ovary (CHO) cells, NIH 3T3 cells, MCF-7 cells, Hep G2 cells, baby hamster kidney (BHK) cells and Cos7 cells.

[0076] Methods for identifying agonists, antagonists, or positive allosteric modulators of alpha2alpha5beta2 nAChRs Further provided herein are methods for identifying agonists, antagonists, or positive allosteric modulators (PAMs) of α2α5β2 nAChRs. PAMs are compounds that bind at sites on the protein surface other than the active site, thus altering the conformation of the protein binding site.

[0077] In one embodiment, the method includes culturing an isolated recombinant cell disclosed herein under conditions in which the recombinant cell grows, contacting the recombinant cell with an agent, and determining whether the agent is an agonist, antagonist, or PAM of the α2α5β2 nAChR, where an agonist or PAM enhances the activity of the α2α5β2 nAChR and an antagonist decreases the activity of the α2α5β2 nAChR compared to the activity of the α2α5β2 nAChR in a recombinant cell that has not been contacted with the agent. As used herein, an agonist refers to a molecule / compound / peptide that acts to enhance the function of the α2α5β2 nAChR. As used herein, a PAM refers to a molecule / compound / peptide that enhances the effect of the response of the α2α5β2 nAChR to a ligand without directly activating the receptor. As used herein, the terms "enhance," "enhanced," "increase," or "increased," when used in reference to α2α5β2 nAChR activity, refer to increased signaling through the receptor relative to the corresponding signaling observed in cells not administered an agonist or PAM. As used herein, an antagonist refers to a molecule / compound / peptide that acts to block, reduce, or attenuate the function of α2α5β2 nAChR. In certain embodiments, the agent is a small molecule or peptide.

[0078] In one embodiment, a FLIPR assay is used to identify agonists of α2α5β2-type nAChR-mediated calcium flux. In this assay, recombinant cells are incubated with a calcium-sensitive dye (such as Ca5), exposed to a test compound, and subjected to FLIPR. TETRA Calcium flux is imaged by

[0079] In one embodiment, a FLIPR assay is used to identify antagonists of nicotine-induced α2α5β2 nAChR-mediated calcium flux. In this assay, recombinant cells are incubated with a calcium-sensitive dye (such as Ca5). Using a double-addition protocol, recombinant cells are exposed to a test compound for a first period of time and nicotine (e.g., EC 80 (1 μM). Calcium flux was then measured using FLIPR TETRA As shown in Figure 2, antagonists of α2α5β2 nAChRs reduce calcium flux.

[0080] In one embodiment, a FLIPR assay is used to identify compounds that positively modulate or enhance nicotine-induced α2α5β2 nAChR-mediated calcium flux. In this assay, recombinant cells are incubated with a calcium-sensitive dye (such as Ca5). Using a double-addition protocol, recombinant cells are exposed to a test compound for a first period of time and nicotine (e.g., EC 80 (1 μM). Calcium flux was then measured using FLIPR TETRA If a test compound enhances nicotine-induced α2α5β2 nAChR-mediated calcium flux but does not enhance calcium flux as an agonist (determined as described above), the test compound is called a positive allosteric modulator (PAM) or enhancer of α2α5β2 nAChR.

[0081] In a preferred embodiment, the cells are incubated for about 20-50 hours at about 25-35° C. prior to the FLIPR assay or other assay for measuring α2α5β2 nAChR activity. In some embodiments, the cells are incubated at about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., about 30° C., about 31° C., about 32° C., about 33° C., about 34° C., or about 35° C. for about 20 hours, about 25 hours, about 30 hours, about 35 hours, about 40 hours, about 45 hours, or about 50 hours prior to the assay.

[0082] Expression Systems and Kits Further provided herein are expression systems and kits comprising the isolated recombinant cells. The expression systems and kits may further comprise instructions for use. EXAMPLES

[0083] Those skilled in the art will appreciate that changes could be made to the embodiments described above without departing from the broad inventive concept. It is understood therefore that the invention is not limited to the particular embodiments disclosed, but is intended to cover modifications within the spirit and scope of the invention as defined herein.

[0084] [Table 1]

[0085] cell line FreeStyle 293F cell line (ThermoFisher Scientific catalog number R79007)

[0086] Vector constructs pcDNA3.1-α2 pcDNA3.1-α5 pcDNA3.1-β2 pcDNA3.1-SAT1 pcDNA3.1-CHAT pcDNA3.1-TMIE pcDNA3.1-FAM163B

[0087] Culture medium ·FreeStyle 293 Expression Medium

[0088] Transfection Reagents ·FreeStyle MAX Reagent

[0089] planting medium ·DMEM 10% FBS 1% pen-strep

[0090] Compound dilution / Ca5 buffer 1mM Mg 2+ and 2 mM Ca 2+ HEPES-buffered saline solution (500 mL) supplemented with

[0091] Calcium 5 (Ca5) dye (Molecular Devices) · Calcium dye was diluted 25x in Hank's Balanced Salt Solution. The dye was diluted 1x in HEPES assay buffer containing 2mM CaCl2 and 1mM MgCl2.

[0092] Methods and Procedures Day 1 cell preparation 1 x 10 cells by centrifuging and resuspending in fresh pre-warmed medium 6 Prepare a cell suspension of 100 cells / mL; · Prepare the following transfection mix in 50 mL of OptiPro SFM medium while shaking 1900 mL of cells; · Add 2.5mL of FreeStyle MAX reagent to 47.5mL of OptiPro SFM per empty 50mL tube; In a second 50 mL tube, add 2.5 mg of total DNA (α2, α5, β2, SAT1, CHAT and TMIE or FAM163B in a ratio of 3:3:3:3:1:2) in OptiPro SFM to a final volume of 50 mL and vortex gently; Transfer the diluted DNA to the FreeStyle MAX reagent, immediately and gently mix the solution, and incubate at room temperature for 10 minutes; · Transfer 100mL of FreeStyle MAX / DNA transfection mix to the cells and homogenize the culture; Incubate for 1 hour at 37°C with 8% CO2; Pour the cells into a sterile 500mL tube; Spin at 1000 rpm for 7 minutes; Remove the supernatant; · Cells are resuspended in freezing medium, aliquoted into cryotubes and frozen at -80°C.

[0093] Day 2 Seeding the cells Ethanol vials for decontamination; · Pour the cells into a 50mL conical tube; · Slowly add 10mL of warmed plating medium dropwise; If a pellet is present, gently pipette up and down several times to detach it from the pellet; Spin at 1000RPM for 7 minutes, aspirate supernatant and resuspend in 25mL inoculum; 35,000 cells / well in 35 μL (1.0 × 10 6 cells / mL); Incubate the plates at 37°C for 24 hours. Then transfer the plates to 30°C in a humidified atmosphere with 5% CO2 for 24-48 hours.

[0094] Day 3 FLIPR assay Wash the plate in the plate washer with compound dilution buffer (4 washes x 100 μL / wash), leaving 25 μL in each well; · Add 25 μL of 2×Ca5 dye to each well; Incubate at room temperature for 1 hour; Wash the plate in the plate washer with compound dilution buffer (4 washes x 100 μL / wash), leaving 50 μL in each well; · Transfer plate to FLIPR for compound addition.

[0095] Functional expression of α2β2 and α2α5β2 nAChRs Human α2 and β2 subunits of α2β2 nAChR were co-transfected with the indicated combinations of cDNA (vector alone; SAT1+CHAT; SAT1+CHAT+TMIE or SAT1+CHAT+FAM163B) into HEK293T cells and incubated overnight at 37°C followed by 24–48 h at 30°C. Transfected cells were incubated with Ca5 dye for 1 h at room temperature followed by E max Stimulation with nicotine (33 μM). Nicotine-induced Ca 2+ The signals are shown graphically in Figure 1 (left). As shown, co-transfection with SAT1 and CHAT enhances α2β2 nAChR function (i.e., nicotine-induced calcium flux), whereas co-transfection with TMIE or FAM163B has no effect on α2β2 nAChR function.

[0096] Separately, human α2, α5, and β2 subunits of α2α5β2-type nAChR were co-transfected with the indicated combinations of cDNA (vector alone; SAT1+CHAT; SAT1+CHAT+TMIE or SAT1+CHAT+FAM163B) into HEK293T cells and incubated overnight at 37°C followed by 24-48 h at 30°C. Transfected cells were incubated with Ca5 dye for 1 h at room temperature followed by E max Stimulation with nicotine (33 μM). Nicotine-induced Ca 2+ The signals are shown graphically in Figure 1 (right). As shown, co-transfection of TMIE or FAM163B (in the presence of SAT1 and CHAT) dramatically enhances α2β2α5 nAChR function (i.e., nicotine-induced calcium flux).

[0097] Concentration-response curves of antagonists for nicotine-induced activity of α2β2 and α2α5β2 nAChRs Human nAChR subunits α2 and β2 subunits with or without α5 were co-transfected with the indicated combinations of cDNAs (SAT1+CHAT; SAT1+CHAT+TMIE or SAT1+CHAT+FAM163B) into HEK293T cells. After overnight incubation at 37°C, cells were incubated at 30°C for an additional 24-48 hours. Cells were then incubated with Ca5 dye for 1 hour at room temperature and stimulated with various concentrations of nicotine. FLIPR TETRA Nicotine-induced calcium flux was recorded using an imaging device. FLIPR signals were averaged and plotted as a percentage of the maximum response for each transfection. As shown in Figure 2, co-transfection with FAM163B or TMIE shifted the nicotine potency to the left at both α2β2 and α2β2α5 nAChRs.

Claims

1. 1. An isolated recombinant cell comprising: a) a heterologous nucleic acid encoding the α5 subunit of the nicotinic acetylcholine receptor (nAChR); b) a heterologous nucleic acid encoding a chaperone protein selected from the transmembrane inner ear expressed protein TMIE and the protein FAM163B; 2. An isolated recombinant cell comprising:

2. c) a heterologous nucleic acid encoding the α2 subunit of the nAChR; d) a heterologous nucleic acid encoding the β2 subunit of the nAChR; e) a heterologous nucleic acid encoding diamine acetyltransferase 1 (SAT1); f) a heterologous nucleic acid encoding choline O-acetyltransferase (CHAT); Further comprising: The isolated recombinant cell of claim 1 , wherein the α2, α5 and β2 subunits of nAChR form an α2α5β2 type nAChR.

3. The isolated recombinant cell of claim 1 , wherein the recombinant cell is a mammalian cell.

4. 4. The isolated recombinant cell of claim 3, wherein the mammalian cell is selected from the group consisting of a human embryonic kidney 293T (HEK293T) cell, a HEK293F cell, a HeLa cell, a Chinese hamster ovary (CHO) cell, a NIH3T3 cell, an MCF-7 cell, a Hep G2 cell, a baby hamster kidney (BHK) cell, and a Cos7 cell.

5. The isolated recombinant cell of claim 1 , wherein the α2 subunit of the α2α5β2 nAChR comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

1.

6. The isolated recombinant cell of claim 1, wherein the α5 subunit of the α2α5β2 nAChR comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

2.

7. The isolated recombinant cell of claim 1, wherein the β2 subunit of the α2α5β2 nAChR comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

3.

8. The isolated recombinant cell of claim 1 , wherein the TMIE comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

4.

9. 2. The isolated recombinant cell of claim 1, wherein the FAM163B comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

5.

10. 3. The isolated recombinant cell of claim 2, wherein the SAT1 comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

6.

11. 3. The isolated recombinant cell of claim 2, wherein the CHAT comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

7.

12. 1. A method for identifying an agonist, antagonist, or positive allosteric modulator of an α5-containing nAChR, comprising: a) contacting the isolated recombinant cell of claim 1 with an agent; b) determining the activity of the α5-containing nAChR of the isolated recombinant cell; wherein if the agent enhances the activity of the α5-containing nAChR compared to the activity of the α5-containing nAChR when the isolated recombinant cell is not contacted with the agent, the agent is identified as an agonist or positive allosteric modulator (PAM), and if the agent decreases the activity of the α5-containing nAChR, the agent is identified as an antagonist.

13. 1. A method for identifying an agonist, antagonist, or positive allosteric modulator of an α2α5β2 nAChR, comprising: a) contacting the isolated recombinant cell of claim 2 with an agent; b) determining the activity of the α2α5β2 nAChR of the isolated recombinant cell; wherein if the agent enhances the activity of the α2α5β2 nAChR compared to the activity of the α2α5β2 nAChR when the isolated recombinant cell is not contacted with the agent, the agent is identified as an agonist or positive allosteric modulator (PAM), and if the agent decreases the activity of the α2α5β2 nAChR, the agent is identified as an antagonist.

14. 14. The method of claim 13, wherein step b) comprises determining calcium flux of the isolated recombinant cell, and if the agent enhances the calcium flux compared to the calcium flux when the isolated recombinant cell is not contacted with the agent, the agent is identified as an agonist.

15. The method of claim 13, wherein step b) comprises determining calcium flux and nicotine-induced calcium flux of the isolated recombinant cell, and if the agent does not enhance calcium flux and enhances the nicotine-induced calcium flux compared to the calcium flux and nicotine-induced calcium flux when the isolated recombinant cell is not contacted with the agent, the agent is identified as a PAM.

16. 14. The method of claim 13, wherein step b) comprises determining a nicotine-induced calcium flux of the isolated recombinant cell, and if the agent reduces the nicotine-induced calcium flux compared to the nicotine-induced calcium flux when the isolated recombinant cell is not contacted with the agent, the agent is identified as an antagonist.

17. 14. The method of claim 13, wherein the isolated recombinant cells are incubated at about 25° C. to 35° C. for about 20 to 50 hours prior to contacting with the agent.

18. The method of claim 13 , wherein the agent is a small molecule or a peptide.

19. 13. A kit comprising: (i) the isolated recombinant cell of claim 1; and (ii) instructions for use.

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