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 The invention relates generally to receptor proteins and to DNA and RNA molecules encoding therefor. In particular, the invention relates to a variant human α7 subunit in which there is a substitution of the valine-274 position of the wild-type human α7 subunit. The invention also relates to DNA and RNA molecules that encode the variant human α7 subunit, as well as to methods of using the variant subunit to identify compounds that interact with it.
 This background considers the variant α7 subunit as it relates to the nicotinic acetylcholine receptor (nAChR). The nAChR is comprised of transmembrane polypeptide subunits that form a cation-selective ion channel gated by acetylcholine (ACh) and other ligands. The hydrophobic transmembrane 2 (“TM-2”) region from each subunit is believed to form the wall of the ion channel.
 Two of the more prominent nAChRs in brain are those containing α4 subunits and those containing α7 subunits (Sargent (1993)
 A splice variant involving the TM-2 region of the α7 subunit has been detected in bovine chromaffin cells (García-Guzmán et al. (1995)
 Compared to the chick α7 wild-type (“c-α7WT”) nAChR, c-α7V251T (also referred to as α7-4) retained high calcium permeability but desensitized slowly, and was 180-fold more sensitive to ACh. In addition, the c-α7V251T nAChR responded to dihydro-β-erythroidine (“DHβE”), normally an nAChR antagonist at α7 and other wild-type InAChR, as if it were an agonist (Galzi et al. (1992) Nature 359:500-505; Bertrand et al. (1993)
 Although the chick α7 nAChR is pharmacologically similar to the mammalian of α7 nAChR, there are significant differences. For example, 1,1-dimethyl-4-phenylpiperazinium (“DMPP”) is a very weak partial agonist in the chick (α7 nAChR, but is a highly efficacious agonist at the human α7 nAChR (Peng et al. (1994)
 The present invention relates to a variant human α7 subunit in which valine-274 has been changed in analogy with the corresponding chick receptor variant. This variant is analogous to the chick α7V251T variant with regard to the relative position of the amino acid substitution in the TM-2 region. However, the variant human α7 subunit exhibits unexpectedly different pharmacological and electrophysiological characteristics.
 The α7 subunit combines with itself and may combine with other subunits to create various nicotinic acetylcholine receptors. The possibility of combination with yet other proteins, which may or may not be identified as components of other classes of receptor, is not necessarily excluded.
 Accordingly, in one embodiment, a DNA molecule or fragments thereof is provided, wherein the DNA molecule encodes a variant human α7 subunit in which the valine-274 has been replaced.
 In another embodiment, a recombinant vector comprising such a DNA molecule,or fragments thereof, is provided.
 In another embodiment, the subject invention is directed to a variant human α7 subunit in which the valine-274 has been replaced.
 In still other embodiments, the invention is directed to messenger RNA encoded by the DNA, recombinant host cells transformed or transfected with vectors comprising the DNA or fragments thereof and methods of producing recombinant polypeptides for the treatment of neurodegenerative processes, enzymatic function, affective disorders and immunofunction, using such cells.
 In another embodiment, compounds such as antagonists are provided,as well as antisense polynucleotides, which are useful in treating conditions such as neurodegenerative processes, enzymatic function, affective disorders and immunofunction. Methods of treating individuals using these compounds and antisense polynucleotides also are provided.
 In yet another embodiment, methods and reagents are provided for detecting the α7 variant.
 In yet another embodiment, the invention is directed to a method of expressing the human α7 subunit variant in a cell to produce the resultant α7 variant.
 In a further embodiment, the invention is directed to a method of identifying compounds that modulate the subunit or receptors containing the subunit and to a method of identifying cytoprotective or other therapeutic compounds using such cells.
 These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.
 The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology, electrophysiology, and pharmacology, that are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,
 All patents, patent applications and publications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.
 As used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural references unless the content clearly dictates otherwise. Thus, for example, reference to “an amplification primer” includes two or more such primers, reference to “a receptor subunit” includes more than one such subunit, and the like.
 A. Definitions
 In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.
 The term “AChR” intends a receptor for the neurotransmitter acetylcholine (“ACh”). AChRs are broadly subclassified as nicotinic or muscarinic. These types differ in their pharmacology, structures, and signal transduction mechanisms.
 The term “nAChR” intends a nicotinic acetylcholine receptor. Although nAChRs of various subunit structures are best known in muscle cells, neurons, and chromaffin cells, they are not necessarily excluded from other cells types (e.g., glial cells, mast cells, blood cells, fibroblasts, etc.).
 The term “nAChR subunit” intends a proteinaceous molecule which can combine with other such molecules in the formation of a nAChR. For example, the muscle nAChR is believed to be a pentamer comprised of four types of transmembrane subunit: two α1 subunits, one β1 subunit, one δ subunit and one γ or ε subunit depending upon the nAChR form. Neuronal nAChR analogously are also thought to be pentameric and comprised of related but different subunits. At present, eight neuronal α subunits (α2-α9) and three neuronal 5 subunits (β2-β4) have been isolated. Some neuronal nAChRs appear to require at least one a subunit and at least one β subunit for a functional complex (i.e., ion channel response to ACh or other agonists). Some subunits, however, may self-assemble to form “homooligomeric” nAChR, as in the case of α7 nAChR in
 The term “wild-type” (abbreviated “WT”) intends the typical, usual or most common form as it occurs in nature. The human wild-type α7 nAChR as used herein was described in Doucette-Stamm et al. (1993) Drug Dev. Res. 30: 252-256. An abbreviation of the form “α7XnnnO” intends an α7 subunit in which the amino acid X, located at position nnn relative to the wild type sequence, has been replaced by amino acid O. Thus, for example, the chick α7V251T subunit indicates the chick α7 subunit in which the valine located at position 251 in the wild type receptor has been replaced by a threonine.
 A “nicotinic cholinergic agonist” is a compound that binds to and activates a nicotinic acetylcholine receptor. By “activates” is intended the elicitation of one or more pharmacological, physiological, or electrophysiological responses. Such a response includes, but is not limited to, cell membrane depolarization and increased permeability to Ca
 A “nicotinic cholinergic antagonist” is a substance that binds to a nicotinic acetylcholine receptor and prevents agonists from activating the receptor. Pure antagonists do not activate the receptor, but some substances may have mixed agonist and antagonist properties. Nicotinic cholinergic channel blockers block the ability of agonists to elicit current flow through the nicotinic acetylcholine receptor channel, but do so by blocking the channel rather than by preventing agonists from binding to and activating the receptor.
 A “nicotinic cholinergic modulator” intends a substance that influences the activity of the nicotinic acetylcholine receptor through interaction at one or more sites other than the classic agonist binding site. The modulator may itself increase or decrease receptor activity, or may influence agonist activity (for example, potentiating responses) without itself eliciting an overt change in channel current. A single substance can have different properties at different nicotinic acetylcholine receptor subtypes, for example, being an agonist at one receptor and antagonist at another, or an antagonist at one and a channel blocker at another.
 By “nAChR regulator” is intended a substance that may act as an agonist, antagonist, channel blocker or modulator.
 The term “polynucleotide” as used herein means a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, the term includes double- and single-stranded DNA, as well as double- and single-stranded RNA. It also includes modifications, such as by methylation and/or by capping, and unmodified forms of the polynucleotide.
 The term “variant” is used to refer to an oligonucleotide sequence which differs from the related wild-type sequence in one or more nucleotides. Such a variant oligonucleotide is expressed as a protein variant which, as used herein, indicates a polypeptide sequence that differs from the wild-type polypeptide in the substitution, insertion or deletion of one or more amino acids. The variant polypeptide differs in primary structure (amino acid sequence), but may or may not differ significantly in secondary or tertiary structure or in function relative to the wild-type.
 The term “mutant” generally refers to an organism or a cell displaying a new genetic character or phenotype as the result of change in its gene or chromosome. In some instances, however, “mutant” may be used in reference to a variant protein or oligonucleotide and “mutation” may refer to the change underlying the variant.
 “Polypeptide” and “protein” are used interchangeably herein and indicate a molecular chain of amino acids linked through peptide bonds. The terms do not refer to a specific length of the product. Thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. The terms include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. In addition, protein fragments, analogs, mutated or variant proteins, fusion proteins and the like are included within the meaning of polypeptide.
 A “functionally conservative mutation” as used herein intends a change in a polynucleotide encoding a derivative polypeptide in which the activity is not substantially altered compared to that of the polypeptide from which the derivative is made. Such derivatives may have, for example, amino acid insertions, deletions, or substitutions in the relevant molecule that do not substantially affect its properties. For example, the derivative can include conservative amino acid substitutions, such as substitutions which preserve the general charge, hydrophobicity/hydrophilicity, side chain moiety, and/or stearic bulk of the amino acid substituted, for example, Gly/Ala, Val/Ile/Leu, Asp/Glu, Lys/Arg, Asn/Gln, Thr/Ser, and Phe/Trp/Tyr.
 By the term “structurally conservative mutant” is intended a polynucleotide containing changes in the nucleic acid sequence but encoding a polypeptide having the same amino acid sequence as the polypeptide encoded by the polynucleotide from which the degenerate variant is derived. This can occur because a specific amino acid may be encoded by more than one “codon,” or sequence of three nucleotides. “Recombinant host cells,” “host cells,” “cells,” “cell lines,” “cell cultures,” and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities refer to cells which can be, or have been, used as recipients for recombinant vectors or other transfer DNA, immaterial of the method by which the DNA is introduced into the cell or the subsequent disposition of the cell. The terms include the progeny of the original cell which has been transfected. Cells in primary culture as well as cells such as oocytes also can be used as recipients.
 A “vector” is a replicon in which another polynucleotide segment is attached, such as to bring about the replication and/or expression of the attached segment. The term includes expression vectors, cloning vectors, and the like.
 A “coding sequence” is a polynucleotide sequence that is transcribed into mRNA and/or translated into a polypeptide. The boundaries of the coding sequence are determined by a translation start codon at the 5′-terminus and a translation stop codon at the 3′-terminus. A coding sequence can include, but is not limited to, mRNA, cDNA, and recombinant polynucleotide sequences. Variants or analogs may be prepared by the deletion of a portion of the coding sequence, by insertion of a sequence, and/or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are well known to those skilled in the art. See, e.g., Sambrook et al., supra;
 “Operably linked” refers to a situation wherein the components described are in a relationship permitting them to function in their intended manner. Thus, for example, a control sequence “operably linked” to a coding sequence is ligated in such a manner that expression of the coding sequence is achieved under conditions compatible with the control sequences. A coding sequence may be operably linked to control sequences that direct the transcription of the polynucleotide whereby said polynucleotide is expressed in a host cell.
 The term “transfection” refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion, or the molecular form of the polynucleotide that is inserted. The insertion of a polynucleotide per se and the insertion of a plasmid or vector comprised of the exogenous polynucleotide are included. The exogenous polynucleotide may be directly transcribed and translated by the cell, maintained as a nonintegrated vector, for example, a plasmid, or alternatively, may be stably integrated into the host genome. “Transfection” generally is used in reference to a eukaryotic cell while the term “transformation” is used to refer to the insertion of a polynucleotide into a prokaryotic cell. “Transformation” of a eukaryotic cell also may refer to the formation of a cancerous or tumorigenic state.
 The term “isolated,” when referring to a polynucleotide or a polypeptide, intends that the indicated molecule is present in the substantial absence of other similar biological macromolecules. The term “isolated” as used herein means that at least 75 wt. %, more preferably at least 85 wt. %, more preferably still at least 95 wt. %, and most preferably at least 98 wt. % of a composition is the isolated polynucleotide or polypeptide. An “isolated polynucleotide” that encodes a particular polypeptide refers to a polynucleotide that is substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, the molecule may include functionally and/or structurally conservative mutations as defined herein.
 A “test sample” as used herein intends a component of an individual's body which is a source of the α7 subunit. These test samples include biological samples which can be evaluated by the methods of the present invention described herein and include body fluids such as whole blood, tissues and cell preparations.
 The following single-letter amino acid abbreviations are used throughout the text:
Alanine A Arginine R Asparagine N Aspartic acid D Cysteine C Glutamine Q Glutamic acid E Glycine G Histidine H Isoleucine I Leucine L Lysine K Methionine M Phenylalanine F Proline P Serine S Threonine T Tryptophan W Tyrosine Y Valine V
 B. General Methods
 A variant human α7 subunit, a polynucleotide encoding the variant subunit, and methods of making the variant subunit are provided herein. The invention includes not only the variant subunit but also methods for screening compounds using the variant subunit and cells expressing the variant subunit. Further, polynucleotides and antibodies which can be used in methods for detection of the variant subunit, as well as the reagents useful in these methods, are provided. Compounds and polynucleotides useful in regulating the variant and its expression also are provided as disclosed hereinbelow.
 In one preferred embodiment, the polynucleotide encodes a human α7 subunit variant in which the valine-274 of the wild-type α7 subunit has been replaced. Preferably, the polynucleotide encodes a human α7 subunit in which the valine-274 has been replaced by a threonine, or a conservative substitution for the threonine, e.g., serine.
 The human α7 variant nAChR exhibits both similar and unexpectedly different properties relative to other structurally related nAChRs. For example, as with the chick α7V251T variant, the human α7V274T variant's responses to cholinergic agonists decay slowly compared to the human wild-type α7 nAChR responses. In addition, human α7V274T is about two orders of magnitude more sensitive to cholinergic receptor agonists such as nicotine and ACh compared to the wild-type.
 The human and chick receptor variants differ pharmacologically, for example, in that human α7V274T is weakly activated by dihydro-f-erythroidene (DHDE) while chick α7V251T is strongly activated (66%;
 DNA encoding the variant human α7 subunit can be derived from genomic or cDNA, prepared by synthesis, or by a combination of techniques. The DNA can then be used to express the variant human α7 subunit or as a template for the preparation of RNA using methods well known in the art (see, Sambrook et al., supra)
 One method for obtaining the desired DNA involves isolating cDNA encoding the wild-type human α7 nAChR subunit as described by Doucette-Stamm et al. (1993), supra. The wild-type cDNA thus obtained is then modified and amplified using the polymerase chain reaction (“PCR”) and mutated primer sequences to obtain the DNA encoding the human α7 variant nAChR subunit. More particularly, PCR employs short oligonucleotide primers (generally 10-20 nucleotides in length) that match opposite ends of a desired sequence within the wild-type DNA molecule. The sequence between the primers need not be known. The initial template can be either RNA or DNA. If RNA is used, it is first reverse transcribed to cDNA. The cDNA is then denatured, using well known techniques such as heat, and appropriate oligonucleotide primers are added in molar excess.
 Primers bearing the mutation will hybridize to the wild-type polynucleotide at a temperature slightly below that of the wild-type primer-polynucleotide duplex. The primer can be made specific by keeping primer length and base composition within relatively narrow limits, and by keeping the mutant base or bases centrally located (Zoler et al. (1983)
 Alternatively, the wild-type DNA may be obtained from an appropriate DNA library. DNA libraries may be probed using the procedure described by Grunstein et al. (1975)
 Alternatively still, the α7 variant could be generated using an RT-PCR (reverse transcriptase-polymerase chain reaction) approach starting with human RNA. For example, single-stranded cDNA is synthesized from human RNA (approx. 1.5 μg) as the template using standard reverse transcriptase procedures. Next, the cDNA is amplified in two segments and the mutation is introduced using PCR and two pairs of primers. For example, the internal primers are designed to carry the codon for threonine (T) or other desired change in place of the wild-type valine (V) at position 274 (see also Example 1 p. 28 and
 Synthetic oligonucleotides may be prepared using an automated oligonucleotide synthesizer such as that described by Warner (1984)
 Once produced, the DNA may then be incorporated into a cloning vector or an expression vector for replication in a suitable host cell. Vector construction employs methods known in the art. Generally, site-specific DNA cleavage is performed by treating with suitable restriction enzymes under conditions which generally are specified by the manufacturer of these commercially available enzymes. After incubation with the restriction enzyme, protein is removed by extraction and the DNA recovered by precipitation. The cleaved fragments may be separated using, for example, polyacrylamide or agarose gel electrophoresis methods, according to methods known by those of skill in the art.
 Sticky end cleavage fragments may be blunt ended using
 Ligations are performed using standard buffer and temperature conditions using T4 DNA ligase and ATP. Alternatively, restriction enzyme digestion of unwanted fragments can be used to prevent ligation.
 Standard vector constructions generally include specific antibiotic resistance elements. Ligation mixtures are transformed into a suitable host, and successful transformants selected by antibiotic resistance or other markers. Plasmids from the transformants can then be prepared according to methods known to those in the art usually following a chloramphenicol amplification as reported by Clewell et al. (1972)
 Host cells are genetically engineered with the vectors of this invention which may be a cloning vector or an expression vector. The vector may be in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants/transfectants or amplifying the subunit-encoding polynucleotide. The culture conditions, such as temperature, pH and the like, generally are similar to those previously used with the host cell selected for expression, and will be apparent to those of skill in the art.
 Both prokaryotic and eukaryotic host cells may be used for expression of desired coding sequences when appropriate control sequences that are compatible with the designated host are used. For example, among prokaryotic hosts,
 Eukaryotic hosts include yeast and mammalian cells in culture systems.
 Mammalian cell lines available as hosts for expression are known in the art and are available from depositories such as the American Type Culture Collection. These include but are not limited to HeLa cells, human embryonic kidney (HEK) cells, Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, and others. Suitable promoters for mammalian cells also are known in the art and include viral promoters such as that from Simian Virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus (ADV), bovine papilloma virus (BPV) and cytomegalovirus (CMV). Mammalian cells also may require terminator sequences and poly A addition sequences; enhancer sequences which increase expression also may be included, and sequences which cause amplification of the gene also may be desirable. These sequences are known in the art. Vectors suitable for replication in mammalian cells may include viral replicons, or sequences which ensure integration of the appropriate sequences encoding the variant α7 nAChR subunit into the host genome. An example of such a mammalian expression system is described in Gopalakrishnan et al. (1995),
 Other eukaryotic systems are also known, as are methods for introducing polynucleotides into such systems, such as amphibian cells using methods described in Briggs et al. (1995)
 The baculovirus expression system can be used to generate high levels of recombinant proteins in insect host cells. This system allows for high level of protein expression, while post-translationally processing the protein in a manner similar to mammalian cells. These expression systems use viral promoters that are activated following baculovirus infection to drive expression of cloned genes in the insect cells (O'Reilly et al. (1992) Baculovirus Expression Vectors: A Laboratory Manual, IRL/Oxford University Press).
 Transfection may be by any known method for introducing polynucleotides into a host cell, including packaging the polynucleotide in a virus and transducing a host cell with the virus, by direct uptake of the polynucleotide by the host cell, and the like, which methods are known to those skilled in the art. The transfection procedures selected depend upon the host to be transfected and are determined by the rountineer.
 The expression of the variant receptor subunit may be detected by use of a radioligand selective for the receptor. For example, for the nicotinic cholinergic receptor, such a ligand may be [
 The variant nAChR polypeptide is recovered and purified from recombinant host cell cultures expressing the same by known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography or lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
 The human α7 variant polypeptide, or fragments thereof, of the present invention also may be synthesized by conventional techniques known in the art, for example, by chemical synthesis such as solid phase peptide synthesis. In general, these methods employ either solid or solution phase synthesis methods. See, e.g., J. M. Stewart and J. D. Young,
 In one preferred system, either the DNA or the RNA derived therefrom, both of which encode the desired variant human α7 subunit, may be expressed by direct injection into a cell, such as a
 Receptors expressed in a recombinant host cell may be used to identify compounds that modulate nAChR activity. In this regard, the specificity of the binding of a compound showing affinity for the receptor is demonstrated by measuring the affinity of the compound for cells expressing the receptor or membranes from these cells. This may be done by measuring specific binding of labeled (e.g., radioactive) compound to the cells, cell membranes or isolated receptor, or by measuring the ability of the compound to displace the specific binding of a standard labeled ligand. Expression of variant receptors and screening for compounds that bind to, or inhibit the binding of labeled ligand to these cells or membranes provides a method for rapid selection of compounds with high affinity for the receptor. These compounds may be agonists, antagonists or modulators of the receptor.
 Expressed receptors also may be used to screen for compounds that modulate nicotinic acetylcholine receptor activity. One method for identifying compounds that modulate nAChR activity, comprises providing a cell that expresses a variant human α7 nicotinic acetylcholine receptor (nAChR) polypeptide having an amino acid substitution at position valine-274 of the wild-type human α7 nAChR polypeptide, combining a test compound with the cell and measuring the effect of the test compound on the variant receptor activity. The cell may be a bacterial cell, a mammalian cell, a yeast cell, an amphibian cell or any other cell expressing the receptor. Preferably, the cell is a mammalian cell or an amphibian cell. Thus, for example, a test compound is evaluated for its ability to elicit an appropriate response, e.g., the stimulation of transmembrane current flow, for its ability to inhibit the response to a cholinergic agonist, or for its ability to modulate the response to an agonist or antagonist.
 In addition, expressed receptors may be used to screen compounds that exhibit a cytoprotective effect. Abnormal activation of membrane channels is a potential cause of neurodegenerative disease. In this regard, a number of inherited human disorders are accompanied by neuronal degeneration (Adams et al. (1989) Degenerative Disease of the Nervous System, in
 Additionally, the α7 variant can be used to screen for compounds useful in treating disorders such as alterations in sensory gating, immunofunction and neuropathic pain, e.g., pain associated with cancerous conditions, post herpatic neuralgia, diabetic neuropathy and osteoarthritis. Further, the α7 variant could be used to treat or to kill cancerous cells.
 Accordingly, nicotinic drugs are considered potential therapeutic agents in several neurodegenerative disorders including, without limitation, Alzheimer's disease, Down's syndrome, kuru, Parkinson's disease, multiple system atrophy, neuropathic pain, immune function, schizophrenia and the like. Activation of the wild type α7 nAChR appears to elicit cytoprotective properties (e.g., reduced cell lysis, see Donnelly-Roberts et al. (1996), supra. However, it is not yet finally established whether a full agonist or partial agonist is preferable, nor, if the latter, what type of partial agonist is best (e.g., one that stabilizes the open and desensitized states or one that stabilizes the open and resting states of the receptor). The variant α7 nAChR can be used to evaluate these questions, and to select among ligands for specific types of partial agonists or specific types of antagonists. That is because this variant α7 nAChR conducts current in the desensitized as well as the open states, unlike the wild type receptor that conducts only in the open state (see Bertrand and Changeux (1995), Sem.
 Thus, α7 nAChR ligand pharmacology can be defined in novel ways through the use of the human variant nAChR subunit. Substances could be antagonists at the wild type α7 nAChR due to their ability to stabilize the non-conducting desensitized state, or due to other mechanisms such as stabilizing the resting state or blocking the ion channel. Similar mechanisms could contribute to partial agonism at the wild type α7 nAChR. The ability of a ligand to stabilize the desensitized state could be evaluated by comparing the ligand's potency and efficacy at the variant α7 nAchR (e.g., human α7V274T) to its potency and efficacy at the wild-type α7 nAChR. The interaction of compounds with the nAChR can be identified using several methods, including, but not limited to, electrophysiologic measurement of transmembrane current flow or electrical potential, measurement of the fluorescence of potential- or ion-sensitive dyes, or measurement of radioactive ion flux (e.g.
 In addition to screening test compounds, the expressed variant α7 subunit may be used to investigate mechanisms of cytotoxicity and cytoprotection. The evidence that activation of α7 nAChR is cytoprotective comes from the finding that nAChR agonists elicit cytoprotection in cells expressing the wild-type α7 nAChR subunit and that this cytoprotection is inhibited by selective α7 antagonists (for example see Donnelly-Roberts et al., supra). The mechanism is unknown but may involve the stimulation of Ca
 Cytoprotective or cytotoxic compounds that interact with the variant DAChR may be identified using several methods. One such method comprises providing a cell that expresses a variant human α7 subunit having an amino acid substitution at position valine-274 of the wild-type human α7 nAChR polypeptide, combining a test compound with the cell, and monitoring the cell for an indicator of cytotoxicity. If it is necessary to control spontaneous action of the variant nlAChR subunit, it may be stably expressed in a recombinant mammalian cell line under the control of an inducible promoter, e.g., the LacSwitch system which is inducible by isopropylthiogalactoside (“IPTG”) . Expression of the variant α7 subunit would be maintained at a low level until induction by the addition of IPTG. Alternatively, with or without an inducible promoter, the transfected cells could be cultured in the presence of an α7 blocker, such as methyllycaconitine (“MLA”) or mecamylamine, that would prevent or reduce cytotoxic action. Both blockers are reversible, permitting one to measure the effect of test compound on α7 nAChR function after the blocker is washed out.
 Cytoprotective compounds can be identified by their ability to reduce cell death while cytotoxic compounds can be identified by their ability to promote cell death. That these effects are mediated by the α7 subunit, variant or wild type, can be identified by the ability of an α7 blocker to prevent the effect. Cell death, or cytotoxicity, can be monitored by a variety of techniques including but not limited to measurement of cell number or density in the culture, of cell growth rate (e.g. incorporation of labeled nucleotide or amino acid), or of cell integrity for example by uptake of a dye (e.g. trypan blue is excluded by healthy cells, or by inclusion of MTT by healthy cells), or by the release of a cytoplasmic constituent such as lactate dehydrogenase (LDH). Cytoprotective agents may also be screened for their ability to antagonize a variant nAChR to a greater extent than a wild-type nAChR, or for their ability to augment the decay rate of variant nAChR compared to the wild-type nAChR, using methods described in the examples provided below.
 In addition, the DNA, or RNA derived therefrom, can be used to design oligonucleotide probes for DNAs that express variant subunits. As used herein, the term “probe” refers to a structure comprised of a polynucleotide, as defined above, which contains a nucleic acid sequence complementary to a nucleic acid sequence present in a target polynucleotide. The polynucleotide regions of probes may be composed of DNA, and/or RNA, and/or synthetic nucleotide analogs. Such probes could be useful in in vitro hybridization assays to distinguish of α7 variant from wild-type message, with the proviso that it may be difficult to design a method capable of making such a distinction given the small difference in coding between variant and wild-type. Alternatively, a PCR-based assay could be used to amplify the sample RNA or DNA for sequence analysis.
 Furthermore, the α7 subunit or fragment(s) therof can be used to prepare monoclonal antibodies using techniques that are well known in the art. The variant α7 subunit or relevant fragments can be obtained using the recombinant technology outlined below, i.e., a recombinant cell that expresses the subunit or fragments can be cultured to produce quantities of the subunit or fragment that can be recovered and isolated. Alternatively, the variant α7 subunit or fragment(s) thereof can be synthesized using conventional polypeptide synthetic techniques as known in the art. Monoclonal antibodies that display specificity and selectivity for the variant α7 subunit can be labeled with a measurable and detectable moiety, e.g., a fluorescent moiety, radiolabels, enzymes, chemiluminescent labels and the like, and used in in vitro assays. It is theorized that such antibodies could be used to identify variant α7 subunits for immunodiagnostic purposes. For example, antibodies have been generated to detect amyloid β1-40 v. 1-42 in brain tissue, (T. Wisniewski et al. (1996)
 Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
 Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.
 Acetylcholine chloride (“ACh”), collagenase Type 1A, d-tubocurarine chloride (“dTC”), gentamicin and mecamylamine hydrochloride (“MEC”), were obtained from Sigma Chemical Company (St. Louis, Mo., U.S.A.). Dihydro-β-erythroidine hydrobromide (IIDHBEII), and methyllycaconitine citrate (“MLA”) were obtained from Research Biochemicals International (Natick, Massachusetts, U.S.A.). Tricaine (3-aminobenzoic acid ethyl ester methanesulfonate; Finquel) was obtained from Argent Chemical Laboratories (Fisheries Chemical Division, Redmond, Wash., U.S.A.).
 Preparation of Human α7 Wild-Tyle cDNA
 The human α7 subunit cDNA reported by Doucette-Stamm et al. (1993), supra, was modified to include the complete human signal peptide (MRCSPGGVWLALAASLLHVSLQGEF (SEQ ID NO:______)) reported by Elliott et al. (1993)
 Expression of α7 nAChR in Xenopus laevis Oocytes and Measurement of Functional Characteristics
 The preparation of Xenopus laevis oocytes, injection with receptor RNA or DNA, and measurement of α7 nAChR responses using two-electrode voltage-clamp followed procedures described previously for the wild-type human α7 nAChR (Briggs et al. (1995), supra) except that atropine was not routinely present in the bathing solution. Oocytes were maintained at 17-18° C. in normal Barth's solution (90 mM NaCl, 1 mM KCl, 0.66 mM NaNO
 Human α7V274T responses, unlike human α7WT responses, tended to increase significantly during the experiments. Therefore, experimental trials were bracketed, before and after, by control applications of 10 μM ACh in the same oocyte. All responses were normalized to the ACh responses in order to account for changes in sensitivity within the experiment and for variability in receptor expression among oocytes.
 To generate the variant α7V274T in an expression vector, the wild-type α7 subunit gene was digested with EcoR V and Kpn I restriction enzymes and the digested segment was replaced with a mutant PCR product by ligation using the procedures described below.
 The strategy, diagrammed in
 The longer 5′ fragment (X-5′) was generated using the forward external primer 5′-GTTTGGGTCCTGGTCTTACG-3′ (SEQ ID NO:______) and the reverse internal primer (X-3′) 5′-GCAGCATGAAGGTGGTAAGAGAG-3′ (X-3′) (SEQ ID NO:______) bearing the mutation. The shorter 3′ fragment was generated using the forward internal primer (Y-5′) 5′-CTCTCTTACCACCTTCATGCTGC-3′ (SEQ ID NO:______), also bearing the mutation, and the reverse external primer (Y-3′) 5′-GTACTGCAGCACGATCACCG-3′ (SEQ ID NO:______). The conditions for PCR consisted of 100 ng input α7 DNA, 2× Pfu buffer, 100 ng of each primer pair and 0.625 U Pfu enzyme (Stratagene, La Jolla, Calif.). Reactions were carried out in a Perkin-Elmer 9600 for 20 cycles at 95° C. for 24 seconds, 60° C. for 22 seconds then 72° C. for 78 seconds.
 In the second PCR step (B), these two fragments were reassembled using the external primers. The sequence was reamplified and a longer DNA fragment bearing the desired mutation was generated.
 In the next step (C), the product of step (B) was digested with KpnI and EcoRV, gel-purified, and ligated into the wild-type human α7 cDNA previously digested with KpnI and EcoRV. Dideoxy sequencing of the final cDNA showed the presence of the desired mutation and that no other mutation had been introduced during the PCR process.
 Responses to various agonist concentrations were measured using human α7V274T nAChR subunits expressed from the DNA prepared in Example 1 that was injected into
 Human α7V274T and human α7 wild-type responses to EC
 Human α7V274T responses activated and decayed slowly compared to the human α7 wild-type responses. Similarly, the analogous chick mutant nAChR activated and decayed more slowly in response to ACh (Galzi et al. (1992), supra.
 nAChR antagonists such as dihydro-β-erythroidine (DH:E), d-tubocurarine and hexamethonium, have been found to activate responses at chick α7 TM-2 nAChR variants when these compounds were applied as agonists (Bertrand et al. (1992), supra). This, together with data from single-channel recording, has suggested (a) that the variant nAChRs conduct in the receptor-desensitized state and (b) that wild-type nAChR antagonists act by stabilizing the desensitized state (Bertrand et al. (1995), supra).
 At the human α7V274T nAChR, DHβE (10 μM) also activated agonist-like inward current responses (see
TABLE 1 Effects of Cholinergic Antagonists at the Human α7V274T Mutant and α7 Wild-type nAChR Ligand nAChR (μM) % Activation % Inhibition 10 μM ACh 1 μM ACh 10 μM ACh α7V274T DHβE (10) 4 ± 1 69 ± 5 52 ± 6 (4)* (4) d-TC (1) −2 ± 1 99 ± 1 97 ± 3 (4) (4) MLA (0.01) −4 ± 2 103 ± 1 95 ± 3 (7)* (4) MEC (10) −1.9 ± 0.2 101 ± 1 53 ± 2 (4) ATROP (2) 0.1 ± 0.1 28 ± 7 13 ± 5 (4) (5)* % of % of % of 10 mM ACh 200 μM ACh 10 mM ACh α7WT DHβE (10) −0.2 ± 0.1 41 ± 10 23 ± 2 (5) (4)* d-TC (1) −0.1 ± 0.1 28 ± 2 25 ± 3 (5) MLA (0.01) −0.2 ± 0.4 100 ± 0.5 99 ± 0.4 (3) (4) MEC (10) −0.3 ± 0.2 82 ± 1 85 ± 3 (3) ATROP (2) 0.2 ± 0.5 4 ± 3 12 ± 3 (3) (3) (3)*
 Furthermore, at human α7V274T this was not a general property of nAChR antagonists. Both the α7-selective antagonist methyllycaconitine (MLA; 10 nM) and the nonselective nAChR antagonist mecamylamine (MEC; 10 μM) elicited the opposite effect, small inverse agonist-like outward currents ranging in amplitude from 0.9% to 12.4% of the maximal inward current response to ACh, as shown in
 d-Tubocurarine (d-TC; 1 μM) also did not elicit agonist-like inward currents, but did elicit small outward currents (3-5% of the maximal inward current response to ACh) in two of four human α7V274T oocytes. The outward current responses may be due to stabilization of the resting (closed) state or to channel blockade of spontaneously open nAChR. At the human α7 wild-type nAChR under similar conditions, neither DHβE (10 μM), MLA (10 nM), MEC (10 μM) nor d-TC (1 μM) elicited any significant inward or outward current response (Table 1). The muscarinic antagonist atropine (2 μM) alone had little effect at either nAChR.
 The above compounds also were evaluated as antagonists of the response to ACh at both human α7V274T and human α7 wild-type nAChRs. For each nAChR, two concentrations of ACh were used: one near the EC
 DHβE (10 μM) inhibited maximal ACh responses less strongly than it inhibited EC
 Thus, the human variant α7V274T nAChR is similar to the analogous chick α7V251T nAChR in its increased sensitivity to agonist activation and apparent slower rate of activation and desensitization. The receptors differ, however, in that DHβE (10 μM) activated the human α7V274T inward current only weakly, compared to a 66% agonist-like effect at chick α7V251T, and in that d-TC did not activate inward currents at the h-α7V274T compared to the full response at chick α7L247T nAChR (Galzi et al. (1992), supra; Bertrand et al. (1993), supra) . Thus, there is a difference in the effects of these sequence modifications on α7 nAChR function which difference is unexpected in view of the information known regarding chick α7V274T.
 The current versus voltage relationship of human α7V274T variant nAChR responses to 10 μM ACh was measured in oocytes under two-electrode voltage clamp as described by Briggs et al. (1995)
 The human α7 wild-type and α7V274T mutant nAChR were transfected into the human embryonic kidney cell line, HEK-293 using the eukaryotic expression vector, pRc/CMV (Invitrogen, San Diego, Calif.) which contains the promoter sequences from the human cytomegalovirus for high level constitutive expression and contains the neomycin resistance gene for selection of geneticin-resistant stable cell lines. The cDNAs were transfected using lipofectamine (GIBCO) as described in (Gopalakrishnan et al. (1995)
 The human α7V274T variant bears homology to the C. elegans deg-3 (u662) spontaneous mutation which appears to be cytotoxic through a mechanism that is inhibited by nicotinic antagonists (Treinin and Chalfie (1995)
 Cytotoxicity clearly could limit the ability of cells to express the α7V274T variant at high levels. To circumvent this, transfected cells are grown in the presence of a reversible nicotinic antagonist or channel blocker, such as methyllycaconitine or mecamylamine. Such substances would prevent cytotoxicity by blocking the receptor or channel, but could be removed shortly before using the cells in further experiments.
 Alternatively, for example, the human α7 wild type or variant is transfected using an inducible expression system such that expression of the α7 subunit is repressed until an inducer is added. The potential advantage of such an inducible system is that it can eliminate the cytotoxic effects of the expressed protein, for example the human α7V274T variant, that is observed when a constitutive expression system such as the pRcCMV is employed.
 One of the expression vectors that is used is the LacSwitch system (Stratagene) that uses the elements of lactose operon to control gene expression. With the LacSwitch system, basal expression is very low in the repressed state and once stably transfected in cell lines, this system permits rapid induction within 4-8 hours in presence of the inducing agent, IPTG. The system employs a eukaryotic Lac-repressor-expressing vector (p3′SS) and a eukaryotic lac-operator containing vector (pOPRSVI-CAT) into which the α7 subunit construct will be inserted by cloning. Antibiotic selection is attained via the hygromycin-resistance gene in p3′SS and via the neomycin-resistance gene in POPRSVI-CAT vector. After transfection of HEK-293 or other cells, the selection of stable cell lines is achieved by the presence of both hygromycin and geneticin. Once stable cell lines are isolated, expression of the α7 subunit will be caused by the addition of the inducing agent, IPTG. In the absence of IPTG, transcription is blocked by the binding of the Lac repressor protein to the operator in pOPRSVI-CAT vector. IPTG decreases the binding affinity of the Lac repressor protein to the operator thereby triggering transcription and expression of the inserted α7 subunit gene. The choice of such a system permits the direct evaluation of the role of the mutant α7 nAChR in mediating cell death in vitro.
 In Vitro Assessment of Cytotoxicity in Mammalian Cell Lines: To determine whether the human α7V274T variant mediates cytotoxicity, cell damage can be assessed following transient expression of the cDNA in HEK-293 cells by a number of methods, for example: (i) staining the cells with Trypan blue (4%) for 5 minutes and assessing the ability of viable cells to exclude the dye; (ii) measuring the levels of the cytosolic enzyme lactate dehydrogenase (LDH) released into the medium, as an index of cell lysis (e.g., Donnelly-Roberts et al. (1996)
 Diagnostic application: The presence of the α7V274T variant in humans could be determined in a non-invasive manner, for example using the polymerase chain reaction (PCR) and genomic DNA isolated from blood samples following standard methodology. Alternatively, if RNA is isolated, then reverse transciptase-PCR (“RT-PCR”) can be utilized to detect the α7 variant. The PCR reaction, for example, could use 100 ng of the DNA in a standard 50 μl PCR reaction with the appropriate synthetic primers. For example, the external primers used in the synthesis of the α7 variant (X-5′ and Y-3′) would allow one to amplify the region of interest. The primers would be chosen to generate a distinct size fragment encompassing the sequence transmembrane segment 2, in which the V274T substitution takes place. Following amplification, the nucleotide sequence of the message is determined. The presence of the variant can be an indication of cellular disease, such as, neurodegeneration, or other forms of cytotoxicity.
 Thus, a method of detecting target polynucleotides of human variant α7 subunit in a test sample comprises (a) contacting a target polynucleotide of human variant α7 subunit with at least one human variant α7 subunit-specific polynucleotide (probe) or complement thereof; and (b) detecting the presence of the target polynucleotide and probe complex in the test sample. Another method for detecting cDNA of human variant α7 subunit mRNA in a test sample comprises (a) performing reverse transcription in order to produce cDNA; (b) amplifying the cDNA obtained from step (a); and (c) detecting the presence of the human variant α7 subunit in the test sample. Alternatively, sampled DNA or cDNA prepared from RNA by RT-PCR, can be amplified using appropriate primers (for example, X-5′ and Y-3′) to allow detection of the variant by nucleotide sequence analysis. The detection step (c) comprises utilizing a detectable moiety capable of generating a measurable signal.
 A purified polynucleotide or fragment thereof derived from human variant α7 subunit capable of selectively hybridizing to the nucleic acid of human variant α7 subunit can be utilized in these methods, wherein said polynucleotide is SEQUENCE ID NO:______ or a fragment thereof. The purified polynucleotide can be produced by recombinant techniques.
 A polypeptide encoded by human variant α7 subunit also is useful for diagnostic applications. The polypeptide is derived from SEQUENCE ID NO:______ or fragments thereof. Further, the polypeptide can be produced by recombinant or synthetic techniques known in the art.
 A monoclonal antibody which specifically binds to human variant α7 subunit also can be utilized in these methods. The human variant α7 subunit comprises an amino acid sequence SEQUENCE ID NO:______ or fragments thereof.
 A method for detecting human variant α7 subunit in a test sample can comprise (a) contacting said test sample with an antibody or fragment thereof which specifically binds to human variant α7 subunit for a time and under conditions sufficient for the formation of resultant complexes; and (b) detecting said resultant complexes containing said antibody, wherein said antibody specifically binds to human variant α7 subunit SEQUENCE ID NO:______ or fragments thereof.
 Treatment application: Spontaneous mutation of human α7 valine-274 to threonine and related mutation could result in or hasten death of those cells expressing the protein. At least two types of treatment could be undertaken: (i) pharmacoogical intervention, for example, administration of a selective α7 antagonist such as methyllycaconitine or another compound with improved blood-brain barrier penetration; or, (ii) antisense oligonucleotide therapy to block the synthesis of the protein (e.g., see Albert and Morris (1994) Antisense knockouts: molecular scalpels for the dissection of signal transduction.
 Antisense technology can be used to reduce gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5′ coding portion of the polynucleotide sequence, which encodes for the polypeptide of the present invention, is used to design an antisense RNA oligonucleotide of from 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription thereby preventing transcription and the production of the human variant α7 subunit polypeptide. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of an mRNA molecule into the human variant α7 subunit polypeptide. Antisense oligonucleotides act with greater efficacy when modified to contain artificial internucleotide linkages which render the molecule resistant to nucleolytic cleavage. Such artificial internucleotide linkages include but are not limited to methylphospnate, phosphorothiolate and phosphoroamydate internucleotide linkages.
 Research and drug discovery application: Antisense oligonucleotides also would be of value in determining α7 wild-type and V274T functions, and mechanisms of cytotoxicity in general. For example, one method of evaluating the contribution of α7V274T to cytotoxicity, cytoprotection, or other cellular processes would be to determine whether specific blockade of its synthesis blocks such processes. This differs in approach from the use of a receptor antagonist, which may or may not block all effects of the protein. Additionally, in drug discovery this approach could be useful in evaluating whether the effect of the drug is mediated by the α7V274T variant. A similar approach could be used to evaluate the contribution of other variants or the wild-type subunit itself. In control experiments, the corresponding α7 sense and missense oligonucleotides 5′-CGAGCCCATGAGGTGTAGCC (SEQUENCE ID NO:______) and 5′-CCAGGCATTCGGAGCTTGCC (SEQUENCE ID NO:______), respectively, are used. The missense oligonucleotide is a randomized sequence maintaining the proportion of GC content in the antisense oligonucleotide, and did not match known sequences in the GenBank® database.
 Thus, polynucleotides that encode novel subunit and their antisense variants of the human α7 nAChR can be used in a variety of ways as detailed herein. Although preferred embodiments of the subject invention have been described in some detail, it is understood that obvious variations can be made without departing from the spirit and the scope of the invention as defined by the appended claims.