Expression systems for alpha6-containing nicotinic acetylcholine receptors and methods of use thereof

Abstract

Disclosed herein are isolated recombinant cells for the expression of α6-containing nicotinic acetylcholine receptors (nAChRs) and methods of their use.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Application No. 63 / 309,092, filed February 11, 2022, which is incorporated by reference herein in its entirety.

[0002] FIELD OF THEINVENTION The present invention relates to isolated recombinant cells for the expression of α6-containing nicotinic acetylcholine receptors (nAChRs) and methods of use thereof.

[0003] (Reference to electronically submitted sequence listing) This application has been submitted electronically in ST.26 format and contains a Sequence Listing, which is incorporated herein by reference in its entirety. The ST.26 copy was created on January 26, 2023, is named JAB7137WOPCT1_SL.XML, and is 16,108 bytes in size. [Background technology]

[0004] Major depression is characterized by debilitating symptoms including hopelessness and anhedonia. Strong evidence exists linking the ventral tegmental area to nucleus accumbens dopamine system (mesolimbic dopamine system) with reward-related, hedonic, and motivated behavior. At present, no therapeutic interventions are available that specifically target dopamine neurons.

[0005] Acetylcholine (ACh) plays a major role in regulating dopamine neurons. ACh binds to two major receptors, the ionotropic nicotinic (nAChR) and the metabotropic muscarinic (mAChR). nAChRs are nonselective cation channels that depolarize dopamine neurons and allow calcium to enter the cells. This depolarization results in the generation of action potentials at high frequencies, called burst firing (>20 Hz). It is this burst firing of dopamine neurons that encodes the physiological role of the mesolimbic dopamine system with respect to the behaviors mentioned above. Thus, modulating nAChRs may provide a therapeutic intervention for mood disorders.

[0006] nAChRs are membrane-bound complexes assembled from five subunits, each consisting of a large extracellular N-terminal domain (NTD), a transmembrane domain (TMD) consisting of four transmembrane α-helices (TM1-TM4) connected by intracellular and extracellular loops containing a large second intracellular loop (ICL), and a short extracellular C-terminus. Thus, the pentameric nAChR complex contains three structural entities: an extracellular domain containing an orthosteric site, a transmembrane domain containing an ion channel, and an intracellular domain, each assembled from the NTD, TMD, and ICL of five subunits.

[0007] nAChRs containing the nAChR subunit α6 (CHRNA6, Accession Number: NM_004198) are selectively expressed in dopamine neurons, providing a therapeutic target for modulation of the mesolimbic dopamine system to treat mood disorders. However, drug development efforts have been hampered by the inability to express α6-containing nAChRs in recombinant systems typically used for such screening.

[0008] There remains a need to develop recombinant cell lines that robustly express α6-containing nAChRs and can be used for drug development. Summary of the Invention [Means for solving the problem]

[0009] Provided herein is an isolated recombinant cell comprising: a) a heterologous nucleic acid encoding the α6 subunit of nAChR; b) a heterologous nucleic acid encoding BARP; c) a heterologous nucleic acid encoding SULT2B1; d) a heterologous nucleic acid encoding LAMP5; e) a heterologous nucleic acid encoding CHAT; and f) a heterologous nucleic acid encoding NACHO.

[0010] In one embodiment of the isolated recombinant cell, the isolated recombinant cell further comprises g) a heterologous nucleic acid encoding the β2 subunit of the nAChR, and h) a heterologous nucleic acid encoding the β3 subunit of the nAChR, wherein the α6, β2, and β3 subunits of the nAChR form the α6β2β3 nAChR.

[0011] In a further embodiment of the isolated recombinant cell, the α6 subunit of the nAChR is an α6 / 3 chimera in which the entire or partial sequence of the second intracellular loop (ICL) of the α6 subunit is replaced by the corresponding sequence of the second ICL of the α3 subunit of the nAChR, and the α6 / 3 chimera and the β2 and β3 subunits of the nAChR form a chimeric α6 / 3β2β3 nAChR.

[0012] In still further embodiments of the isolated recombinant cell, the recombinant cell is a mammalian cell.

[0013] In still 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, NIH 3T3 cells, MCF-7 cells, Hep G2 cells, baby hamster kidney (BHK) cells, and Cos7 cells.

[0014] In still further embodiments of the isolated recombinant cell, the α6 subunit of the nAChR comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:1.

[0015] In still further embodiments of the isolated recombinant cell, the β2 subunit of the nAChR comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:6.

[0016] In still further embodiments of the isolated recombinant cell, the β3 subunit of the nAChR comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:7.

[0017] In yet a further embodiment of the isolated recombinant cell, the β3 subunit of the nAChR, the second ICL of the α6 subunit comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:2, and the second ICL of the α3 subunit comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:4.

[0018] In still further embodiments of the isolated recombinant cell, the α6 / 3 chimera comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:5.

[0019] In still further embodiments of the isolated recombinant cell, the BARP comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:8.

[0020] In still further embodiments of the isolated recombinant cell, SULT2B1 comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:9.

[0021] In still further embodiments of the isolated recombinant cell, LAMP5 comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:10.

[0022] In still further embodiments 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:11.

[0023] In still further embodiments of the isolated recombinant cell, NACHO comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:12.

[0024] Further provided herein is a method for identifying an agonist, antagonist, or positive allosteric modulator of an α6-containing nAChR, the method comprising: a) contacting an isolated recombinant cell as provided above with an agent; and b) determining the activity of the α6-containing nAChR of the isolated recombinant cell, wherein the agent is identified as an agonist or positive allosteric modulator (PAM) if the agent enhances the activity of the α6-containing nAChR, and the agent is identified as an antagonist if the agent decreases the activity of the α6-containing nAChR compared to the activity of the α6-containing nAChR when the isolated recombinant cell was not contacted with the agent.

[0025] Still further provided herein is a method for identifying an agonist, antagonist, or positive allosteric modulator of α6β2β3 nAChR, the method comprising: a) contacting the isolated recombinant cell provided above with an agent; and b) determining activity of the α6β2β3 nAChR of the isolated recombinant cell, wherein if the agent enhances activity of the α6β2β3 nAChR, the agent is identified as an agonist or positive allosteric modulator (PAM), and if the agent decreases activity of the α6β2β3 nAChR compared to activity of the α6β2β3 nAChR when the isolated recombinant cell was not contacted with the agent, the agent is identified as an antagonist.

[0026] In one embodiment of the above method, step b) comprises determining calcium flux in the isolated recombinant cell, and the agent is identified as an agonist if the agent enhances calcium flux compared to calcium flux when the isolated recombinant cell is not contacted with the agent.

[0027] In a further embodiment of the above method, step b) comprises determining calcium flux and nicotine-evoked calcium flux of the isolated recombinant cell, and the agent is identified as a PAM if the agent does not enhance calcium flux and enhances nicotine-evoked calcium flux compared to calcium flux and nicotine-evoked calcium flux when the isolated recombinant cell was not contacted with the agent.

[0028] In still further embodiments of the above method, step b) comprises determining nicotine-induced calcium flux of the isolated recombinant cell, and the agent is identified as an antagonist if the agent reduces nicotine-induced calcium flux compared to the nicotine-induced calcium flux when the isolated recombinant cell was not contacted with the agent.

[0029] In still further embodiments of the method provided above, 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.

[0030] In still further embodiments of the methods provided above, the agent is a small molecule or a peptide.

[0031] Still further provided herein is a kit comprising (i) the isolated recombinant cell provided above, and (ii) instructions for use. [Brief description of the drawings]

[0032] [Figure 1A] FIG. 13 is a graph showing functional expression of α6β2β3 nAChR when co-expressed with different combinations of chaperone proteins (BARP+SULT2B1+LAMP5+NACHO or BARP+SULT2B1+LAMP5+NACHO+CHAT) and incubated at 30° C. before testing. [Figure 1B] FIG. 13 is a graph showing functional expression of α6 / 3β2β3 nAChR when co-expressed with different combinations of chaperone proteins (BARP+SULT2B1+LAMP5+NACHO or BARP+SULT2B1+LAMP5+NACHO+CHAT) and incubated at 37° C. before testing. [Figure 1C] FIG. 13 is a graph showing functional expression of α6 / 3β2β3 nAChR when co-expressed with different combinations of chaperone proteins (BARP+SULT2B1+LAMP5+NACHO or BARP+SULT2B1+LAMP5+NACHO+CHAT) and incubated at 30° C. before testing. [Figure 2A] 1 is a plot of the FLIPR signal of recombinant cells expressing α6 / 3β2β3 after a first incubation with DHbE (a known antagonist of α6-containing nAChRs) at various concentrations and a second incubation with nicotine. [Figure 2B]1 is a plot of the FLIPR signal of recombinant cells expressing α6 / 3β2β3 after a first incubation with MLA (a known antagonist of α6-containing nAChRs) at various concentrations and a second incubation with nicotine. [Figure 2C] 1 is a plot of the FLIPR signal of recombinant cells expressing α6 / 3β2β3 after a first incubation with mecamylamine (a known antagonist of α6-containing nAChRs) at various concentrations and a second incubation with nicotine. [Figure 2D] 1 is a plot of the FLIPR signal of recombinant cells expressing α6 / 3β2β3 after a first incubation with α-conotoxin MII (a known antagonist of α6-containing nAChRs) at various concentrations and a second incubation with nicotine. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] 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. The 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 present 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.

[0034] 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.

[0035] It must 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.

[0036] 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.

[0037] 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.

[0038] 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 include the stated element or elements, but not to exclude other elements or elements other than those, and are intended to be non-exclusive or open-ended. 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 that are inherent to 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).

[0039] 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."

[0040] As used herein, the term "consists of," or variations such as "consist of" or "consisting of," as used throughout the specification and claims, is inclusive of any listed element or elements, but indicates that no additional element or elements are added to the specified method, structure, or composition.

[0041] 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.

[0042] 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 will 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.).

[0043] The term "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a certain percentage of the same amino acid residues or nucleotides when compared and aligned for maximum correspondence, as determined by one of the following sequence comparison algorithms or by visual inspection. For sequence comparison, typically one sequence serves as a reference sequence against which a test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the designated program parameters.

[0044] 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).

[0045] 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 for initiating 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.

[0046] 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 alignments of negative scoring residues 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)).

[0047] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, 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 will 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.

[0048] 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.

[0049] 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 contain 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.

[0050] 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.

[0051] 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.

[0052] 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 and α6-containing nicotinic acetylcholine receptors (nAChRs) (e.g., α6β2β3 nAChR) in cells generates cells that highly express α6-containing nAChRs, making the cells useful for drug development. Provided herein are recombinant cells expressing the α6 subunit of nAChR and methods of making isolated recombinant cells for expression of the α6 subunit of nAChR. In some embodiments, the α6 subunit may have its second ICL partially or completely replaced by the corresponding sequence of the second ICL of the α3 subunit of nAChR, which may be referred to as an α6 / 3 chimeric protein. Also provided herein are recombinant cells expressing α6β2β3 nAChR and methods of making isolated recombinant cells for expression of α6β2β3 nAChR. In some embodiments, the α6 subunit may be an α6 / 3 chimeric protein as described above, and the recombinant cell expresses an α6 / 3β2β3 nAChR.

[0053] As used herein, the terms "α6β2β3 nicotinic acetylcholine receptor", "α6β2β3 nAChR", "alpha6 beta2 beta3 nicotinic acetylcholine receptor", and "alpha6 beta2 beta3 nAChR" are used interchangeably and refer to the α6β2β3 nicotinic acetylcholine receptor protein, preferably human α6β2β3 nAChR, which is a member of the cholinergic receptor protein family.

[0054] The α6β2β3 nAChR is a ligand-gated ion channel composed of α6, β2 and β3 subunits. The α6 subunit is encoded by gene CHRNA6 (NM_004198), the β2 subunit is encoded by gene CHRNB2 (NM_000748) and the β3 subunit is encoded by gene CHRNB3 (NM_000749). When expressed together, the subunits assemble together to form the α6β2β3 nAChR.

[0055] According to the present invention, the term α6β2β3 nAChR also encompasses chimeric α6 / 3β2β3 nAChRs in which the second ICL of the α6 subunit is partially or completely replaced by the corresponding amino acid sequence of the second ICL of the α3 subunit of the nAChR. In one embodiment, a portion of the second ICL of the α6 subunit is replaced by the amino acid sequence of the second ICL of the α3 subunit of the nAChR. The α3 subunit is encoded by the gene CHRNA3 (NM_000743).

[0056] A "recombinant cell" refers to one or more individual cells and recombinant cell lines in which the cell heterologously expresses a protein. 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. A "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.

[0057] As used herein, the term "expression" refers to the biosynthesis of a gene product. The term includes the transcription of a gene into RNA. The term also includes the translation of RNA into one or more polypeptides, and further includes all naturally occurring post-transcriptional and post-translational modifications.

[0058] In a general aspect, the invention relates to methods for generating or producing cells expressing α6-containing nAChRs (eg, α6β2β3 nAChR, including the chimeric α6 / 3β2β3 nAChR described above) that are useful for drug development. In one embodiment, the method includes introducing into the cell a nucleic acid encoding an α6 subunit of a nAChR (including a chimeric α6 / 3 subunit), a nucleic acid encoding a β-anchoring and -regulatory protein (BARP; Accession No. NM_152769), a nucleic acid encoding a Sulfotransferase Family 2B Member 1 (SULT2B1; Accession No. NM_177973), a nucleic acid encoding a lysosomal-associated membrane protein 5 (LAMP5; Accession No. NM_012261), a nucleic acid encoding a choline O-acetyltransferase (CHAT; Accession No. NM_020984), and a nucleic acid encoding a transmembrane protein 35 (TMEM35; also known as NACHO; Accession No. NM_021637). The nucleic acids encoding the α6 subunit of the nAChR (including the chimeric α6 / 3 subunit) and the chaperone proteins (i.e., BARP, SULT2B1, LAMP5, CHAT, and NACHO) 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 α6 subunit of the nAChR (including the chimeric α6 / 3 subunit) and the chaperone proteins (i.e., BARP, SULT2B1, LAMP5, CHAT, and NACHO) are operably linked to a promoter capable of driving expression of the respective proteins. In some embodiments, the promoter is a constitutive promoter.

[0059] In another aspect, the method comprises introducing into a cell a nucleic acid encoding an α6 subunit of the α6β2β3 nAChR (including the chimeric α6 / 3 subunit described above), a nucleic acid encoding a β2 subunit of the α6β2β3 nAChR, a nucleic acid encoding a β3 subunit of the α6β2β3 nAChR, a nucleic acid encoding BARP, a nucleic acid encoding SULT2B1, a nucleic acid encoding LAMP5, a nucleic acid encoding CHAT, and a nucleic acid encoding NACHO, wherein the cell produced by the method expresses an increased level of the α6β2β3 nAChR (including the chimeric α6 / 3 nAChR described above) compared to the same cell not including the nucleic acids encoding BARP, SULT2B1, LAMP5, CHAT, and NACHO.

[0060] In another aspect, the invention relates to cells that have been genetically modified to express the α6 (including the chimeric α6 / 3 described above), β2, and β3 subunits of nAChR, BARP, SULT2B1, LAMP5, CHAT, and NACHO, wherein the genetically modified cells express these proteins at increased levels compared to the expression of the same proteins in unmodified cells under the same (or substantially the same) conditions.

[0061] 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 BARP, an expression vector comprising a nucleic acid sequence encoding SULT2B1, an expression vector comprising a nucleic acid sequence encoding LAMP5, an expression vector comprising a nucleic acid sequence encoding CHAT, and an expression vector comprising a nucleic acid sequence encoding NACHO.

[0062] In a further aspect, the invention relates to an isolated recombinant cell comprising a heterologous nucleic acid encoding an α6 subunit of a nAChR (including the chimeric α6 / 3 subunit of the nAChR described above), a heterologous nucleic acid encoding a β2 subunit of a nAChR, a heterologous nucleic acid encoding a β3 subunit of a nAChR, a heterologous nucleic acid encoding a BARP, a heterologous nucleic acid encoding SULT2B1, a heterologous nucleic acid encoding LAMP5, a heterologous nucleic acid encoding CHAT, and a heterologous nucleic acid encoding NACHO, wherein the α6 (including α6 / 3), β2, and β3 subunits form an α6β2β3 nAChR (or a chimeric α6 / 3β2β3 nAChR). 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 cells provided herein comprise an expression vector comprising a nucleic acid encoding the α6 subunit of a nAChR (including a nucleic acid encoding the α6 / 3 subunit described above), an expression vector comprising a nucleic acid encoding the β2 subunit of a nAChR, an expression vector comprising a nucleic acid encoding the β3 subunit of a nAChR, an expression vector comprising a nucleic acid encoding a BARP, an expression vector comprising a nucleic acid encoding SULT2B1, an expression vector comprising a nucleic acid encoding LAMP5, an expression vector comprising a nucleic acid encoding CHAT, and an expression vector comprising a nucleic acid encoding NACHO.

[0063] In any one of the embodiments described herein, the nucleic acids encoding any one or more of the α6 (including α6 / 3), β2, and β3 subunits of nAChR, BARP, SULT2B1, LAMP5, CHAT, and NACHO may be in a single expression vector or in separate expression vectors.

[0064] In one embodiment, the α6 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 (NM_004198.2 / NP_004189).

[0065] [ka]

[0066] In one embodiment, the second ICL of the α6 subunit 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.

[0067] [ka]

[0068] In one embodiment, the α3 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 (NM_000743.5 / NP_000734.2).

[0069] [ka]

[0070] In one embodiment, the second ICL of the α3 subunit 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:4.

[0071] [ka]

[0072] In one embodiment, an exemplary chimeric α6 / 3 comprises an amino acid sequence having at least 855, 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:5.

[0073] [ka]

[0074] 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: 6 (NM_000748.3 / NP_000739.1).

[0075] [ka]

[0076] In one embodiment, the β3 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: 7 (NM_000749.5 / NP_000740.1).

[0077] [ka]

[0078] In one embodiment, the BARP 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: 8 (XM_017026555.1 / XP_016882044.1). Alternatively, the BARP 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%, e.g., 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 8 and has a protein Chapelle property, including the property of enhancing expression of the α6 subunit of the nAChR (including the chimeric α6 / 3 subunit described above).

[0079] [ka]

[0080] In one embodiment, SULT2B1 comprises an amino acid sequence having at least 85%, or at least 90%, or at least 95%, for example, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 9 (NM_177973.2 / NP_814444.1). Alternatively, SULT2B1 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%, for example, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 9, and has a protein Chapelle property, including the property of enhancing expression of the α6 subunit of nAChR (including the chimeric α6 / 3 subunit described above).

[0081] [ka]

[0082] In one embodiment, LAMP5 comprises an amino acid sequence having at least 85%, or at least 90%, or at least 95%, for example 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 10 (NM_012261.4 / NP_036393.1). Alternatively, LAMP5 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%, for example 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 10, and has a protein Chapelle property, including the property of enhancing expression of the α6 subunit of nAChR (including the chimeric α6 / 3 subunit described above).

[0083] [ka]

[0084] In one embodiment, CHAT 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: 11 (NM_020984.4 / NP_066264.4). 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%, e.g., 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 11 and has a protein Chapelle property, including the property of enhancing expression of the α6 subunit of the nAChR (including the chimeric α6 / 3 subunit described above).

[0085] [ka]

[0086] In one embodiment, NACHO 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: 12 (NM_021637.3 / NP_067650.1). Alternatively, NACHO 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%, e.g., 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 12 and has a protein Chapelle property, including the property of enhancing expression of the α6 subunit of nAChR (including the chimeric α6 / 3 subunit described above).

[0087] [ka]

[0088] 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. A representative example of a 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. Expression vectors are vectors that can direct the expression of genes to which they are 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 include one or more regulatory sequences (e.g., promoters) operably linked to the nucleic acid sequence to be expressed. When used in reference to an expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to a regulatory 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). Those skilled in the art will appreciate that the design of the expression vector can depend on factors such as the choice of the host cell to be transformed and the level of expression of protein desired, as well as the intended use of the vector.

[0089] 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.

[0090] In view of the present disclosure, any cell known to one of skill in the art can be used for recombinant expression of α6 (including chimeric α6 / 3), β2, β3 subunits of nAChR, BARP, SULT2B1, LAMP5, CHAT, and NACHO. 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.

[0091] Methods for identifying agonists, antagonists, or positive allosteric modulators of α6β2β3 nAChR Further provided herein are methods for identifying agonists, antagonists, or positive allosteric modulators (PAMs) of α6-containing nAChRs (e.g., α6β2β3 nAChRs, including the chimeric α6 / 3β2β3 nAChRs described above). PAMs are compounds that bind at a site on the protein surface other than the active site, thus altering the conformation of the protein binding site.

[0092] In one embodiment, the method comprises 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 α6β2β3 nAChR (including the chimeric α6 / 3β2β3 nAChR described above), where an agonist or PAM enhances the activity of the α6β2β3 nAChR (including the chimeric α6 / 3β2β3 nAChR described above), and an antagonist reduces the activity of the α6β2β3 nAChR compared to the activity of the α6β2β3 nAChR (including the chimeric α6 / 3β2β3 nAChR described above) 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 α6β2β3 nAChR (including the chimeric α6 / 3β2β3 nAChR described above). As used herein, PAM refers to a molecule / compound / peptide that enhances the effect of the α6β2β3 nAChR (including the chimeric α6 / 3β2β3 nAChR described above) response to a ligand without directly activating the receptor. As used herein, the terms "enhance," "enhanced," "increase," or "increased," when used in reference to α6β2β3 nAChR (including the chimeric α6 / 3β2β3 nAChR described above) activity, refer to increased signaling through the receptor compared to the corresponding signaling observed in cells not administered an agonist or PAM. As used herein, antagonist refers to a molecule / compound / peptide that acts to block, reduce, or attenuate the function of α6β2β3 nAChR (including the chimeric α6 / 3β2β3 nAChR described above). In certain embodiments, the agent is a small molecule or peptide.

[0093] In one embodiment, a FLIPR assay is used to identify agonists of α6β2β3 nAChR (including the chimeric α6 / 3β2β3 nAChR described above)-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 TETRACalcium flux is imaged by

[0094] In one embodiment, a FLIPR assay is used to identify agonists of nicotine-induced α6β2β3 nAChR (including the chimeric α6 / 3β2β3 nAChR described above)-mediated calcium flux. In this assay, recombinant cells are incubated with a calcium-sensitive dye (such as Ca5). Using a dual-addition protocol, recombinant cells are exposed to a test compound for a first period of time and to nicotine (e.g., EC 80 (236 nM). Calcium flux was then measured using FLIPR TETRA The image is generated by

[0095] In one embodiment, a FLIPR assay is used to identify compounds that positively modulate or enhance nicotine-induced α6β2β3 nAChR (including the chimeric α6 / 3β2β3 nAChR described above)-mediated calcium flux. In this assay, recombinant cells are incubated with a calcium-sensitive dye (such as Ca5). Using a dual-addition protocol, recombinant cells are exposed to a test compound for a first period of time and to nicotine (e.g., EC 80 (236 nM). Calcium flux was then measured using FLIPR TETRA If a test compound enhances nicotine-induced α6β2β3 nAChR (including the chimeric α6 / 3β2β3 nAChR described above)-mediated calcium flux, but does not enhance calcium flux as an agonist (determined as described above), then the test compound is referred to as a positive allosteric modulator (PAM) or potentiator of α6β2β3 nAChR (including the chimeric α6 / 3β2β3 nAChR described above).

[0096] In preferred embodiments, the cells are incubated at about 25-35° C. for about 20-50 hours prior to FLIPR assay or other assay to measure α6β2β3 nAChR (including chimeric α6 / 3β2β3 nAChR as described above) 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.

[0097] 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

[0098] 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.

[0099] material

[0100] [Table 1]

[0101] cell line FreeStyle 293F cell line (ThermoFisher Scientific catalog number R79007) Vector constructs pcDNA3.1-α6 / 3 (α6 / 3 chimera, α6:1-338;465-505, α3:339-464) pCMV6-XL5-β2 pcDNA3.1-β3 pcDNA3.1-BARP pcDNA3.1-NACHO pcDNA3.1-SULT2B1 pcDNA3.1-LAMP5 pcDNA3.1-CHAT

[0102] Culture medium ·FreeStyle 293 Expression Medium

[0103] Transfection Reagents ·FectoPRO DNA Transfection Reagent

[0104] Seeding medium DMEM + L-glutamine + sodium pyruvate 10% FBS 1X Pen / Strep

[0105] Compound dilution / Ca5 buffer 1 mM Mg 2+ and 2 mM Ca 2+ HEPES-buffered saline solution (500 mL) supplemented with

[0106] 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.

[0107] 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 at 100 cells / mL. There is no need to readjust the cell density on the day of transfection.

[0108] Day 2 Transfection The following protocol uses 100 mL transfection as an example. With 90 mL of cells shaking, prepare the following transfection mix in 10 mL of Freestyle 293 Expression Medium: · Vortex the FectoPRO® reagent for 5 seconds, then add 75 μL of FectoPRO (0.75 uL of reagent per mL of total culture medium) to an empty 50 mL tube. In a second 50 mL tube, dilute 50 µg of DNA (0.5 µg total DNA per mL of total culture medium, all plasmids 1:1) with Freestyle 293 Expression Medium to a final volume of 10 mL. Vortex gently. Transfer the diluted DNA all at once to the pure FectoPRO® Reagent. Homogenize the solution immediately and incubate for 10 minutes at room temperature. · Transfer 10 mL of the FectoPRO® / DNA transfection mixture to the cells and homogenize the culture.

[0109] Day 3 Seeding cells Sediment the cells at 300 x G for 5 minutes, aspirate the supernatant, and resuspend in 10 mL seeding medium. Seed 25,000 cells / well in 50 μL (5×10 5 cells / mL). Incubate the plates in a humidified atmosphere at 5% CO2 and 30°C for 24 hours.

[0110] Day 4 FLPR assay Wash the plate in the plate washer using compound dilution buffer (4 washes x 100 μL / wash), leaving 25 μL in each well. Add 25 μL of 2x Ca5 dye to each well. Incubate at room temperature for 1 hour. Wash the plate in the plate washer using compound dilution buffer (4 washes x 100 μL / wash), leaving 25 μL in each well.

[0111] Antagonist · Transfer plates to FLIPR for compound addition. First addition plate - 2x antagonist (25 μL test compound). Second addition plate - 1x antagonist, 3x agonist (25 μL nicotine EC80) EC80 nicotine 236 nM (25 μL, 3x = 708 nM). Antagonist control wells were in columns 23 and 24. Positive control wells received IC100 DHBe (25 μL, 3x = 30 μM) in the first addition and negative control received buffer (25 μL). Second addition to control wells was IC100 DHBe (10 μM) and EC80 nicotine 236 nM (25 μL, 3x = 708 nM).

[0112] Agonist · Transfer plates to FLIPR for compound addition. Single addition plates of 2x agonist (25 μL). Agonist control wells are in columns 23 and 24. Positive control wells received EC100 nicotine (25 μL, 2x = 2 μM). Negative control wells received buffer + vehicle (25 μL).

[0113] Functional expression of α6β2β3 nAChR Nucleic acids encoding the human α6, β2, and β3 subunits of nAChRs were co-transfected with specific combinations of cDNAs (BARP+SULT2B1+LAMP5+NACHO or BARP+SULT2B1+LAMP5+NACHO+CHAT) in HEK293T cells and incubated overnight at 37°C followed by 24-48 hours at 30°C. Transfected cells were incubated with Ca5 dye for 1 hour at room temperature followed by E max Stimulated with nicotine (10 μM). Transfected nicotine-induced Ca 2+ The signals are shown graphically in Figure 1 A. As shown, co-transfection with BARP, SULT2B1, LAMP5, NACHO, and CHAT (shown as "+CHAT") enhances α6β2β3 nAChR function (i.e., nicotine-induced calcium flux) compared to co-transfection with BARP, SULT2B1, LAMP5, and NACHO (shown as "-CHAT").

[0114] Functional expression of α6 / 3β2β3 nAChR Nucleic acids encoding the chimeric α6 / 3 and human β2 and β3 subunits of nAChR were co-transfected with specific combinations of cDNA (BARP+SULT2B1+LAMP5+NACHO or BARP+SULT2B1+LAMP5+NACHO+CHAT) in HEK293T cells and incubated overnight at 37° C. Transfected cells were incubated with Ca5 dye for 1 hour at room temperature followed by E max Stimulated with nicotine (10 μM). Transfected nicotine-induced Ca 2+ The signals are shown graphically in Figure IB. As shown, co-transfection with BARP, SULT2B1, LAMP5, NACHO, and CHAT (indicated as "+CHAT") enhances α6 / 3β2β3 nAChR function (i.e., nicotine-induced calcium flux) compared to co-transfection with BARP, SULT2B1, LAMP5, and NACHO (indicated as "-CHAT").

[0115] Separately, transfected HEK293T cells were incubated at 30°C for 24-48 h before incubation with Ca5 dye and stimulation with nicotine. 2+ The signals are shown graphically in FIG. 1C. Here again, co-transfection with BARP, SULT2B1, LAMP5, NACHO, and CHAT (denoted as "+CHAT") enhances α6 / 3β2β3 nAChR function (i.e., nicotine-induced calcium flux) compared to co-transfection with BARP, SULT2B1, LAMP5, and NACHO (denoted as "-CHAT"). In addition, transfected cells incubated at 30° C. exhibited much higher nAChR function (i.e., nicotine-induced calcium flux) than those incubated at 37° C., allowing for a more robust screen for modulators of α6-containing nAChRs.

[0116] Concentration-response curves of antagonists to nicotine-induced activity of α6 / 3β2β3 nAChRs HEK293T cells transfected with nucleic acids encoding the α6 / 3, β2, and β3 subunits of nAChR, together with nucleic acids encoding BARP, SULT2B1, LAMP5, NACHO, and CHAT, as described above, were incubated at 30°C for 24-48 hours. The transfected cells were then incubated with Ca5 dye for 1 hour at room temperature and then stimulated using a two-addition protocol (first addition: 3 min with various concentrations of antagonist (i.e., DHbE, MLA, mecamylamine, or α-conotoxin MII), second addition: 3.5 min with nicotine (EC80, 236 nm)). Nicotine-induced calcium flux was then measured using FLIPR. TETRA The final FLIPR signals were measured using an imager and averaged and plotted (Figures 2A-2D). As shown, each of the antagonists tested reduces nicotine-induced calcium flux in a dose-dependent manner.

Claims

1. 1. An isolated recombinant cell comprising: a) a heterologous nucleic acid encoding the α6 subunit of the nAChR; b) a heterologous nucleic acid encoding a BARP; c) a heterologous nucleic acid encoding SULT2B1; and d) a heterologous nucleic acid encoding LAMP5; e) a heterologous nucleic acid encoding CHAT; f) a heterologous nucleic acid encoding NACHO.

2. g) a heterologous nucleic acid encoding the β2 subunit of the nAChR; h) a heterologous nucleic acid encoding the β3 subunit of the nAChR, 2. The isolated recombinant cell of claim 1, wherein the α6, β2, and β3 subunits of the nAChR form an α6β2β3 nAChR.

3. The isolated recombinant cell of claim 1 or 2, wherein the α6 subunit of the nAChR is an α6 / 3 chimera in which the entire or partial sequence of the second intracellular loop (ICL) of the α6 subunit is replaced by the corresponding sequence of the second ICL of the α3 subunit of the nAChR, and the α6 / 3 chimera and the β2 and β3 subunits of the nAChR form a chimeric α6 / 3β2β3 nAChR.

4. The isolated recombinant cell of any one of claims 1 to 3, wherein the recombinant cell is a mammalian cell.

5. 5. The isolated recombinant cell of claim 4, 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 NIH 3T3 cell, an MCF-7 cell, a Hep G2 cell, a baby hamster kidney (BHK) cell, and a Cos7 cell.

6. The isolated recombinant cell of any one of claims 1 to 5, wherein the α6 subunit of nAChR comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

1.

7. The isolated recombinant cell of any one of claims 2 to 6, wherein the β2 subunit of nAChR comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

6.

8. The isolated recombinant cell of any one of claims 2 to 7, wherein the β3 subunit of nAChR comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

7.

9. 9. The isolated recombinant cell of claim 3, wherein the second ICL of the α6 subunit comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:2, and the second ICL of the α3 subunit comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

4.

10. The isolated recombinant cell of any one of claims 3 to 9, wherein the α6 / 3 chimera comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

5.

11. The isolated recombinant cell of any one of claims 1 to 10, wherein the BARP comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

8.

12. The isolated recombinant cell of any one of claims 1 to 11, wherein the SULT2B1 comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

9.

13. The isolated recombinant cell according to any one of claims 1 to 12, wherein the LAMP5 comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

10.

14. The isolated recombinant cell of any one of claims 1 to 13, wherein the CHAT comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

11.

15. 15. The isolated recombinant cell of any one of claims 1 to 14, wherein the NACHO comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

12.

16. 1. A method for identifying an agonist, antagonist, or positive allosteric modulator of an α6-containing nAChR, the method comprising: a) contacting the isolated recombinant cell of any one of claims 1 to 15 with an agent; b) determining the activity of the α6-containing nAChR of the isolated recombinant cell, wherein the agent is identified as an agonist or positive allosteric modulator (PAM) if the agent enhances the activity of the α6-containing nAChR, and the agent is identified as an antagonist if the agent decreases the activity of the α6-containing nAChR compared to the activity of the α6-containing nAChR when the isolated recombinant cell is not contacted with the agent.

17. 1. A method for identifying an agonist, antagonist, or positive allosteric modulator of an α6β2β3 nAChR, the method comprising: a) contacting the isolated recombinant cell of any one of claims 2 to 15 with an agent; and b) determining activity of the α6β2β3 nAChR of the isolated recombinant cell, wherein the agent is identified as an agonist or positive allosteric modulator (PAM) if the agent enhances activity of the α6β2β3 nAChR, and the agent is identified as an antagonist if the agent decreases activity of the α6β2β3 nAChR compared to activity of the α6β2β3 nAChR when the isolated recombinant cell is not contacted with the agent.

18. 18. The method of claim 17, wherein step b) comprises determining calcium flux in the isolated recombinant cell, and wherein the agent is identified as an agonist if it enhances calcium flux compared to the calcium flux when the isolated recombinant cell is not contacted with the agent.

19. 18. The method of claim 17, wherein step b) comprises determining calcium flux and nicotine-induced calcium flux of the isolated recombinant cell, and the agent is identified as a PAM 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 was not contacted with the agent.

20. 18. The method of claim 17, wherein step b) comprises determining a nicotine-induced calcium flux of the isolated recombinant cell, and the agent is identified as an antagonist 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.

21. 21. The method of any one of claims 16 to 20, 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.

22. The method of any one of claims 16 to 21, wherein the agent is a small molecule or a peptide.

23. A kit comprising: (i) an isolated recombinant cell according to any one of claims 1 to 15; and (ii) instructions for use.

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