WO1998028331A2 - A variant human alpha-7 acetylcholine receptor subunit, and methods of production and use thereof - Google Patents
A variant human alpha-7 acetylcholine receptor subunit, and methods of production and use thereof Download PDFInfo
- Publication number
- WO1998028331A2 WO1998028331A2 PCT/US1997/023405 US9723405W WO9828331A2 WO 1998028331 A2 WO1998028331 A2 WO 1998028331A2 US 9723405 W US9723405 W US 9723405W WO 9828331 A2 WO9828331 A2 WO 9828331A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- human
- nachr
- variant
- subunit
- cell
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the invention relates generally to receptor proteins and to DNA and RNA molecules encoding therefor.
- the invention relates to a variant human ⁇ 7 subunit in which there is a substitution of the valine-274 position of the wild-type human ⁇ 7 subunit.
- the invention also relates to DNA and RNA molecules that encode the variant human 7 subunit, as well as to methods of using the variant subunit to identify compounds that interact with it.
- nAChR nicotinic acetylcholine receptor
- the nAChR is comprised of transmembrane polypeptide subunits that form a cation-selective ion channel gated by acetylcholine (ACh) and other ligands.
- ACh acetylcholine
- TM-2 hydrophobic transmembrane M2
- nAChRs Two of the more prominent nAChRs in brain are those containing ⁇ 4 subunits and those containing ⁇ 7 subunits (Sargent (1993) Annu. Rev. Neurosci. 16:403-443; Court et al.
- a splice variant involving the TM-2 region of the ⁇ 7 subunit has been detected in bovine chromaffin cells (Garcia-Guzman et al. (1995) Eur. J. Neurosci. 7:647-655), and a naturally-occurring mutation of a protein homologous to the ⁇ 7 subunit found in Caenorhabditis elegans, leads to neurodegeneration (Treinin et al. (1995) Neuron 14:871-877).
- the latter is a single amino acid mutation in the TM-2 region similar to the chick ⁇ 7 valine-251 to threonine ("c- ⁇ 7V251T") mutation, one of several mutations artificially introduced into the chick ⁇ 7 subunit to facilitate the study of ⁇ 7 nAChR structure and subunit function (Bertrand et al. (1995) Sem. Neurosci. 7:75-90).
- c- ⁇ 7V251T (also referred to as ⁇ 7-4) retained high calcium permeability but desensitized slowly, and was 180-fold more 5 sensitive to ACh.
- c- ⁇ 7V251T nAChR responded to dihydro- ⁇ -erythroidine ("DH ⁇ E"), normally an nAChR antagonist at ⁇ 7 and other wild-type nAChR, as if it were an agonist (Galzi et al. (1992) Nature 359:500-505; Bertrand et al. (1993) Proc. Natl. Acad. Sci. USA 90:6971-6975).
- DH ⁇ E dihydro- ⁇ -erythroidine
- chick ⁇ 7 nAChR is pharmacologically similar to the mammalian cc7 5 nAChR, there are significant differences.
- DMPP l,l-dimethyl-4-phenylpiperazinium
- DMPP is a very weak partial agonist in the chick ⁇ 7 nAChR, but is a highly efficacious agonist at the human ⁇ 7 nAChR (Peng et al. (1994) Mol. Pharmacol. 45:546-554).
- the present invention relates to a variant human ⁇ 7 subunit in which valine-274 has been changed in analogy with the corresponding chick receptor variant.
- This variant is 5 analogous to the chick ⁇ 7V25 IT variant with regard to the relative position of the amino acid substitution in the TM-2 region.
- the variant human ⁇ 7 subunit exhibits unexpectedly different pharmacological and electrophysiological characteristics.
- the ⁇ 7 subunit combines with itself and may combine with other subunits to create various nicotinic acetylcholine receptors.
- a DNA molecule is provided, wherein the DNA molecule encodes a variant human ⁇ 7 subunit in which the valine-274 has been replaced.
- a recombinant vector comprising such a DNA molecule is 5 provided.
- the subject invention is directed to a human ⁇ 7 subunit variant in which the valine-274 has been replaced.
- the invention is directed to messenger RNA encoded by the DNA, recombinant host cells transformed or transfected with vectors comprising the DNA or 5 fragments thereof and methods of producing recombinant polypeptides for the treatment of neurodegenerative processes, enzymatic function, affective disorders and immunofunction, using such cells.
- compounds such as antagonists are provided, as well as antisense polynucleotides, which are useful in treating conditions such as neurodegenerative 0 processes, enzymatic disfunction, affective disorders and immunofunction. Methods of treating individuals using these compounds and antisense polynucleotides also are provided.
- methods and reagents are provided for detecting the ⁇ 7 variant.
- the invention is directed to a method of expressing the 5 human ⁇ 7 subunit variant in a cell to produce the resultant ⁇ 7 variant.
- 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 compounds using such cells.
- Figure 1 depicts the strategy for generating the human ⁇ 7V274T AChR mutant DNA using polymerase chain reaction.
- Figures 2A-2C show the nucleotide sequence (SEQ ID NO: 1 ) of the human ⁇ 7 cDNA containing the V274T mutation. The threonine mutation is shown in bold and the restriction sites EcoRV and Kpnl are shown underlined. Also shown is the deduced amino acid sequence (SEQ ID NO: 2) of the human ⁇ 7V274T subunit variant derived from the cDNA. The V274T alteration is underlined.
- Figure 3 graphically compares the concentration-response relationships for ACh
- Figure 4 graphically depicts the activation by ACh and decay rate of the human 5 ⁇ 7 V274T response compared to that of the human ⁇ 7 WT nAChR.
- Figure 5 graphically depicts the responses of human ⁇ 7V274T to nAChR antagonists wherein MEC is mecamylamine (10 M), MLA is methyllycaconitine (10 nM), and DH ⁇ E is dihydro- ⁇ -erythroidine (10 M).
- MEC mecamylamine
- MLA is methyllycaconitine
- DH ⁇ E dihydro- ⁇ -erythroidine
- Figure 6 graphically depicts the current versus voltage relationship of responses to 10 ⁇ M ACh of the human ⁇ 7V274T expressed in Xenopus laevis oocytes, wherein the circles represent responses measured in modified Barth's solution containing 10 mM Ba 2+ (90 mM NaCI, 1 mM KC1, 0.66 mM NaNO 3 , 10 mM BaCl 2 , 2.4 mM NaHCO 3 , 2.5 mM sodium 0 pyruvate, and 10 mM Na-HEPES buffer, final pH 7.55) to prevent activation of
- Figure 7 graphically depicts the specific binding of [ 125 I] ⁇ -Bungarotoxin, an al nAChR-selective ligand, to an HEK-293 clone transfected with variant human ⁇ 7V274T.
- 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.
- 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 (for example, glial cells, mast cells, blood 0 cells, fibroblasts, etc.).
- nAChR subunit intends a proteinaceous molecule which can combine with other such molecules in the formation of a nAChR.
- the muscle nAChR is believed to be a pentamer comprised of four types of transmembrane subunit: two ⁇ l subunits, one ⁇ l subunit, one ⁇ subunit and one ⁇ or ⁇ subunit depending upon the nAChR 5 form.
- Neuronal nAChR analogously are also thought to be pentameric and comprised of related but different subunits. At present, eight neuronal ⁇ subunits ( ⁇ 2- ⁇ 9) and three neuronal ⁇ subunits ( ⁇ 2- ⁇ 4) have been isolated.
- nAChR neuronal nAChR appear to require at least one ⁇ subunit and at least one ⁇ subunit for a functional complex (i.e., ion channel response to ACh or other agonists). Some subunits, however, may self-assemble to form o "homooligomeric" nAChR, as in the case of ⁇ 7 nAChR in Xenopus oocytes and in transfected mammalian cells. Although the combination of nAChR subunits with subunits related to other types of receptor (for example, other classes of ligand-gated ion channel) has not been demonstrated, it is within the scope of the present invention that such combinations are possible.
- wild-type intends the typical, usual or most common form as it occurs in nature.
- the human wild-type ⁇ 7 nAChR as used herein was described in Doucette-Stamm et al. (1993) Drug Dev. Res. 30: 252-256.
- An abbreviation of the form “ ⁇ 7XnnnO” intends an ⁇ 7 subunit in which the amino acid X, located at position nnn relative to the wild-type sequence, has been replaced by amino acid O.
- the chick cx7V251T o nAChR indicates the chick ⁇ 7 nAChR 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, 5 but is not limited to, cell membrane depolarization and increased permeability to Ca 2+ and other cations.
- 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 5 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” is a substance that influence 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 o 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.
- nAChR regulator is intended a substance that may act as an agonist, antagonist, 5 channel blocker or modulator.
- 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 o and/or by capping, and unmodified forms of the polynucleotide.
- variant is used to refer to an oligonucleotide sequence which is differs from the related wild-type sequence in one or more nucleotides.
- 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 5 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.
- 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, o 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 5 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 5 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.
- 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 0 substituted, for example, Gly/Ala, Val/Ile/Leu, Asp/Glu, Lys/Arg, Asn/Gln, Thr/Ser, and Phe Trp/Tyr.
- 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 0 substituted, for example, Gly/Ala, Val/Ile/Leu, Asp/Glu, Lys/Arg, Asn/Gln, Thr/Ser, and Phe Trp/Tyr.
- structural conservative mutant 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 5 is derived. This can occur because a specific amino acid may be encoded by more than one "codon,” or sequence of three nucleotides, within the polynucleotide.
- Recombinant host cells refer to cells which can be, or have been, used as recipients for recombinant vectors or o 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 5 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 rr RNA 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 o 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.
- operably linked refers to a situation wherein the components described are in a relationship permitting them to function in their intended manner.
- 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 5 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.
- 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 o 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. 5 "Transformation" of a eukaryotic cell also may refer to the formation of a cancerous or tumorigenic state.
- 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.
- isolated as used herein means that at least 75 wt.%, more o 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 5 defined herein.
- a variant human ⁇ 7 subunit, a polynucleotide encoding the variant subunit, and methods of making the variant subunit are provided herein.
- the invention includes not only 5 the variant subunit but also methods for screening compounds using the variant subunit, cells expressing the variant subunit.
- the polynucleotide encodes a human ⁇ 7 subunit variant in which the valine-274 of the wild-type ⁇ 7 subunit has been replaced.
- the polynucleotide encodes a human ⁇ 7 subunit in which the valine-274 has been replaced by a o threonine, or a conservative substitution for the threonine, for example, serine.
- the human ⁇ 7 variant nAChR exhibits both similar and unexpectedly different properties relative to other structurally related nAChRs.
- the human ⁇ 7V274T responses to cholinergic agonists decay slowly compared to the human wild-type ⁇ 7 nAChR responses.
- human ⁇ 7V274T is 5 about two orders of magnitude more sensitive to chohnergic receptor agonists such as nicotine and ACh compared to the wild-type.
- human and chick receptor variants differ pharmacologically, for example, in that human ⁇ 7V274T is weakly activated by dihydro- ⁇ -erythroidene (DH ⁇ E) while chick cx7V251T is strongly activated ( Figure 5 and Galzi et al. (1992). Mutations in the channel o domain of a neuronal nicotinic receptor convert ion selectivity from cationic to anionic. Nature
- d-tubocurarine is a potent antagonist of human ⁇ 7V274T compared to an activator of the related chick ⁇ 7L247T mutant (Bertrand et al. (1992) Unconventional pharmacology of a neuronal nicotinic receptor mutated in the channel domain. Proc. Natl. Acad. Sci. (U.S. A.) 89: 1261-1265).
- the human and chick ⁇ 7 receptor variants 5 are electrophysiologically different as well.
- the chick ⁇ 7V25 IT nAChR does not exhibit inward current rectification (Galzi et al.
- PCR polymerase chain reaction
- 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) Meth. Enzymol. 100:468).
- Primer extension is effected using DNA polymerase in the presence of deoxynucleotide triphosphates or nucleotide analogs.
- the resulting product includes the respective primers at their 5'-termini, covalently linked to the newly synthesized complements of the original strands.
- the replicated molecule is again denatured, hybridized with primers, and so on, until the product is sufficiently amplified.
- PCR methods are described in for example, U.S. Patent Nos. 4,965,188; 4,800,159; 4,683,202; 4,683,195; incorporated herein by reference in their entireties.
- the product of the PCR is cloned and the clones containing the mutated DNA, derived by segregation of the primer extended strand, selected. Selection can be accomplished using the mutant primer as a hybridization probe.
- 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) Proc. Natl. Acad. Sci. USA 73:3961.
- the ⁇ 7V274T variant could be generated using an RT-PCR (reverse transcriptase-polymerase chain reaction) approach starting with human RNA.
- RT-PCR reverse transcriptase-polymerase chain reaction
- single- stranded cDNA is synthesized from human RNA (approx. 1.5 g) as the template using standard reverse transcriptase procedures.
- the cDNA is amplified in two segments and the mutation is introduced using PCR and two pairs of primers.
- 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 Figure 1).
- T threonine
- V wild-type valine
- PCR reactions are combined using the 3 and 5 end primers to re-amplify the full length coding sequence of ⁇ 7V274T. This is but one example of the generation of a7V274T from a human brain template.
- Synthetic oligonucleotides may be prepared using an automated oligonucleotide synthesizer such as that described by Warner (1984) DNA 3:401. If desired, the synthetic strands may be labelled with 32 P by treatment with polynucleotide kinase in the presence of 2 P-ATP, using standard conditions for the reaction. DNA sequences including those isolated from genomic or cDNA libraries, may be modified by known methods which include site- directed mutagenesis as described by Zoller (1982) Nucleic Acids Res. 10:6487.
- the DNA to be modified is packaged into phage as a single stranded sequence, and converted to a 5 double stranded DNA with DNA polymerase using, as a primer, a synthetic oligonucleotide complementary to the portion of the DNA to be modified, and having the desired modification included in its own sequence.
- Culture of the transformed bacteria, which contain replications of each strand of the phage are plated in agar to obtain plaques. Theoretically, 50% of the new plaques contain phage having the mutated sequence, and the remaining 50% have the original l o sequence.
- Rephcates of the plaques are hybridized to labelled synthetic probe at temperatures and conditions suitable for hybridization with the correct strand, but not with the unmodified sequence.
- the sequences which have been identified by hybridization are recovered and cloned. Alternatively, it may be necessary to identify clones by sequence analysis if there is difficulty in distinguishing the small difference variant from wild-type by hybridization. In any combination of the transformed bacteria, which contain
- 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 0 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 E. coli DNA polymerase 1 5 (Klenow) in the presence of the appropriate deoxynucleotide triphosphates (dNTPs) present in the mixture. Treatment with SI nuclease also may be used, resulting in the hydrolysis of any single stranded DNA portions.
- E. coli DNA polymerase 1 5 Klenow
- dNTPs deoxynucleotide triphosphates
- Ligations are performed using standard buffer and temperature conditions using T4 DNA ligase and ATP. Alternatively, restriction enzyme digestion of unwanted fragments can 0 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 5 amplification as reported by Clewell et al. ( 1972) J. Bacteriol. 110:667 may be added. The DNA is isolated and analyzed usually by restriction enzyme analysis and/or sequencing. Sequencing may be by the well-known dideoxy method of Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463) as further described by Messing et al. (1981) Nucleic Acid Res.
- 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 0 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 5 coding sequences when appropriate control sequences that are compatible with the designated host are used.
- appropriate control sequences that are compatible with the designated host are used.
- Escherichia coli is frequently used.
- expression control sequences for prokaryotes include but are not limited to promoters, optionally containing operator portions, and ribosome binding sites.
- Transfer vectors compatible with prokaryotic hosts can be derived from, for example, the plasmid o pBR322 that contains operons conferring ampiciUin and tetracycline resistance, and the various pUC vectors, that also contain sequences conferring antibiotic resistance markers. These markers may be used to obtain successful transformants by selection.
- prokaryotic control sequences include but are not limited to the lactose operon system (Chang et al. (1977) Nature 198:1056), the tryptophan operon system (reported by Goeddel et al. 5 (1980) Nucleic Acid Res. 8:4057) and the lambda-derived PI promoter and N gene ribosome binding site (Shimatake et al. (1981) Nature 292:128) and the hybrid Tac promoter (De Boer et al. (1983) Proc. Natl. Acad. Sci. USA 292: 128) derived from sequences of the trp and lac UV5 promoters.
- lactose operon system Chang et al. (1977) Nature 198:1056
- tryptophan operon system Reported by Goeddel et al. 5 (1980) Nucleic Acid Res. 8:4057
- the lambda-derived PI promoter and N gene ribosome binding site Shi
- coli prokaryotic hosts
- other prokaryotic hosts such as strains of Bacillus or Pseudomonas may be used if desired.
- o Eukaryotic hosts include yeast and mammalian cells in culture systems. Pichia pastoris, Saccharomyces cerevisiae and S. carlsbergensis are commonly used yeast hosts.
- Yeast-compatible vectors carry markers that permit selection of successful transformants by conferring protrophy to auxotrophic mutants or resistance to heavy metals on wild-type strains.
- Yeast compatible vectors may employ the 2- ⁇ origin of replication (Broach et al. (1983) Meth. 5 Enzymol.
- control sequences for yeast vectors are known in the art and include but are not hmited to promoters for the synthesis of glycolytic enzymes, including the promoter for 3-phosphoglycerate kinase. See, for example, Hess et al. (1968) J. Adv. Enzyme Reg. 7:149, Holland et al. (1978) Biochemistry 17:4900 and Hitzeman (1980) J. Biol. Chem. 255:2073. 5
- some useful control systems are those that comprise the glyceraldehyde-
- GAPDH 3-phosphate dehydrogenase
- ADH alcohol dehydrogenase
- terminators also derived from GAPDH
- leader sequences from yeast alpha factor.
- the transcriptional regulatory region and the transcriptional initiation region which are operably linked may be such that they are not 0 naturally associated in the wild-type organism.
- Mammahan cell Unes avaUable as hosts for expression are known in the art and are available from depositories such as the American Type Culture CoUection. These include but are not limited to HeLa cells, human embryonic kidney (HEK) ceUs, Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, and others. Suitable promoters for 5 mammalian ceUs 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 pap loma virus (BPV), cytomegalovirus (CMV).
- Simian Virus 40 SV40
- Rous sarcoma virus RSV
- ADV adenovirus
- BCV bovine pap loma virus
- CMV cytomegalovirus
- Mammatian 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 o 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 ⁇ 7 nAChR subunit into the host genome.
- An example of a mammalian expression system for nAChRs is described in Gopalakrishnan et al. (1995) Stable expression and pharmacological properties of the human ⁇ 7 nicotinic acetylcholine receptor. Eur. J. 5 Pharmacol. -Mol. Pharmacol. 290: 237-246.
- 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) Neuropharmacol. 34:583-590, insect cells using methods described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), and the like.
- 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.
- 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 5 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.
- a radioligand selective for the receptor for example, for the nicotinic cholinergic receptor, such a ligand may be [ 125 I] ⁇ -bungarotoxin.
- any radioligand binding technique known in the art may be used to detect the receptor subunit (see, for example, Winzor et al. (1995) Quantitative o Characterization of Ligand Binding, Wiley-Liss, Inc., NY).
- 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 5 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.
- HPLC high performance liquid chromatography
- the human ⁇ 7 variant polypeptide, or fragments thereof, of the present invention may also be synthesized by conventional techniques known in the art, for example, by chemical o synthesis such as solid phase peptide synthesis. In general, these methods employ either solid or solution phase synthesis methods. See, for example, J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, IL (1984) and G. Barany and R. B. Merrifield, The Peptides: Analysis, Synthesis, Biology, editors E. Gross and J. Meienhofer, Vol. 2, Academic Press, New York, (1980), pp. 3-254, for solid phase peptide 5 synthesis techniques; and M. Bodansky, Principles of Peptide Synthesis, Springer- Verlag,
- either the DNA or the RNA derived therefrom, both of which encode the desired variant human ⁇ 7 nAChR subunit may be expressed by direct injection into 0 a cell, such as zXenopus laevis oocyte.
- a cell such as zXenopus laevis oocyte.
- the functionality of the human ⁇ 7 nAChR subunit variant encoded by the DNA or the inRNA can be evaluated as follows (see Dascal (1987) CRC Crit. Rev. Biochem. 22:317-387).
- a variant-encoding polynucleotide is injected into an oocyte for translation into a functional receptor subunit.
- the function of the expressed variant human ⁇ 7 nAChR can be assessed in the oocyte by a variety of 5 electrophysiological techniques including intracellular voltage recording, two-electrode voltage clamp, patch clamp methods, and the like.
- the cation-conducting channel intrinsic to the nAChR opens in response to ACh or other nicotinic cholinergic agonists, permitting the flow of transmembrane current. This current can be monitored directly by voltage clamp techniques or indirectly by intracellular voltage recording, wherein changes in membrane potential due to the induced current are measured.
- 5 Receptors expressed in a recombinant host cell may be used to identify compounds that modulate nAChR activity.
- 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 (for example, radioactive) compound to the cells, cell o membranes or isolated receptor, or by measuring the ability of the compound to displace the specific binding of a standard labeled ligand.
- labeled for example, radioactive
- Expression of variant receptors and screening for compounds that bind to, or inhibits 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 or antagonists for the receptor.
- Expressed receptors may also be used to screen for compounds that modulate nicotinic acetylcholine receptor activity, i.e, nicotinic cholinergic agonists or antagonists.
- One method for identifying compounds that modulate nAChR activity comprises providing a cell that expresses a variant human ⁇ 7 nicotinic acetylcholine receptor (nAChR) polypeptide having an amino acid substitution at position valine-274 of the wild-type human ⁇ 7 nAChR polypeptide, o combining a test compound with the cell and measuring the effect of the test compound on the variant receptor activity.
- nAChR nicotinic acetylcholine receptor
- the cell may be a bacterial cell, a mammalian cell, a yeast cell, an amphibian cell or any other cell expressing the receptor.
- the cell is a mammalian cell or an amphibian cell.
- a test compound is evaluated for its ability to elicit an appropriate response, for example, the stimulation of transmembrane current flow, or 5 for its ability to inhibit the response to a cholinergic agonist.
- 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.
- a number of inherited human disorders are accompanied by neuronal degeneration (Adams et al. (1989) Degenerative Disease of the 0 Nervous System, in Principles of Neurology, McGraw-Hill, NY, pp. 921-967).
- Many model systems have been used to study the causes of these diseases. For example, mutations in proteins that have extensive sequence similarity to proteins that contribute to the amiloride- sensitive sodium ion channel have been associated with vacuolated neurodegeneration in the nematode C. elegans (Canessa et al.
- a so-called "gain-of-function" mutation in the deg-3 protein of C. elegans causes vacuolated degeneration of a small set of neurons (Treinin et al. (1995), supra). Studies of this mutation suggested to these investigators that mutation in neuronal acetylcholine receptors may lead to death of specific neuronal populations.
- the ⁇ 7 variant can be used to screen for compounds useful in treating 5 disorders such as alterations in sensory gating, immunofunction and neuropathic pain, for example, pain associated with cancerous conditions, post herpatic neuralgia, diabetic neuropathy and oseoarthritis. Further, the ⁇ 7 vaiant could be used to treat or to kill cancerous cells.
- nicotinic drugs are considered potential therapeutic agents in several 0 neurodegenerative disorders including, without limitation, Alzheimer's disease, Down's syndrome, kuru, Parkinson's disease, multiple system atrophy, neuropathic pain and the like, in which they may be useful in slowing cell death.
- Activation of the wild type ⁇ 7 nAChR appears to elicit cytoprotective properties (for example, reduced cell lysis, see Donnelly- Roberts et al. (1996) In vitro neuroprotective properties of the novel cholinergic channel 5 activator (ChCA), ABT-418. Brain Res. 719: 36-44).
- a full agonist or partial agonist is preferable, nor, if the latter, what type of partial agonist is best (for example, one that stabilizes the open and desensitized states or one that stabilizes the open and resting states of the receptor).
- This variant ⁇ 7 nAChR can be used to evaluate these questions, and to select among ligands for specific types of partial agonists or o specific types of antagonists. That is because this variant ⁇ 7 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) Nicotinic receptor: An allosteric protein specialized for intercellular communication.
- ⁇ 7 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 ⁇ 7 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 ⁇ 7 nAChR.
- the ability of a 5 ligand to stabihze the desensitized state could be evaluated by comparing the ligand s potency and efficacy at the variant ⁇ 7 nAChR (for example, human ⁇ 7V274T) to its potency and efficacy at the wild-type ⁇ 7 nAChR.
- variant ⁇ 7 nAChR for example, human ⁇ 7V274T
- 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 (for example ⁇ Na + 0 r 5 "Rb + ) and a variety of ⁇ 7 nAChR expression systems, for example transfected mammalian cells in culture or injected amphibian cells.
- This novel definition of ⁇ 7 nAChR pharmacology coupled with measures of ⁇ 7 ligand effect on cell or animal functioning could be critical to the development of novel therapeutics.
- a ligand that stabilizes the desensitized state of the ⁇ 7 nAChR subunit (partial agonist or antagonist) is o preferable for cytoprotection.
- the type of ligand more or less useful in other nicotinic applications such as cognition, memory, anxiety, attention, sensory gating (psychoses and schizophrenia), etc. could be evaluated using variant 7 nAChR subunits alone or in combination with other receptor subunits.
- the expressed variant ⁇ 7 subunit may be used 5 to investigate mechanisms of cytotoxicity and cytoprotection.
- the evidence that activation of ⁇ 7 nAChR is cytoprotective comes from the finding that nAChR agonists elicit cytoprotection in cells expressing the 7 nAChR subunit and that this cytoprotection is inhibited by selective ⁇ 7 antagonists (for example see Donnelly-Roberts et al., supra).
- the mechanism is unknown but may involve the stimulation of Ca 2+ influx. If so, then the increased Ca 2+ influx mediated o by the variant ⁇ 7 nAChR, due to maintained Ca 2+ permeability with prolonged current activation, may augment the cytoprotection.
- the ⁇ 7 nAChR subunit may also function 5 through mechanisms dependent upon a change in receptor state (for example, from resting to desensitized conformation), which may influence its interaction with other proteins, but not necessarily dependent upon a change in ion flux or electrical potential.
- the variant ⁇ 7 nAChR subunit would be critical in determining such mechanisms as it would allow one to identify ligands that favor different receptor states, and as it provides a tool for manipulating nAChR o channel current independently from nAChR conformation and ligand binding.
- Cytoprotective or cytotoxic compounds that interact with the variant nAChR may be identified using several methods.
- One such method comprises providing a cell that expresses a variant human ⁇ 7 nAChR subunit having an amino acid substitution at position valine-274 of the wild-type human ⁇ 7 nAChR polypeptide, combining a test compound with the cell, and 5 monitoring the cell for an indicator of cytotoxicity. If it is necessary to control spontaneous action of the variant nAChR subunit, it may be stably expressed in a recombinant mammalian cell line under the control of an inducible promoter, for example, the LacSwitch system which is inducible by isopropylthiogalactoside ("LPTG").
- LPTG lacSwitch system which is inducible by isopropylthiogalactoside
- ⁇ 7 nAChR subunit Expression of the variant ⁇ 7 nAChR subunit would be maintained at a low level until induction by the addition of IPTG.
- the transfected cells could be cultured in the presence of an ⁇ 7 nAChR blocker, such as methyllycaconitine ("MLA”) or mecamylamine, that would prevent or reduce cytotoxic action.
- MVA methyllycaconitine
- mecamylamine methyllycaconitine
- Both blockers are reversible, permitting one to measure the effect of test compound on al 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.
- ⁇ 7 nAChR subunit, variant or wild type can be identified by the ability of an al nAChR 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 (for example incorporation of labeled nucleotide or amino acid), or of cell integrity for example by uptake of a dye (for example trypan blue is excluded by healthy cells) or by the release of a cytoplasmic constituent such as lactate dehydrogenase (LDH).
- LDH lactate dehydrogenase
- 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.
- the DNA, or RNA derived therefrom can be used to design oligonucleotide probes for nAChR DNAs that express variant subunits.
- 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 al 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.
- a PCR-based assay could be used to amplify the sample RNA or DNA for sequence analysis.
- the variant nAChR subunit can be used to prepare polyclonal or monoclonal antibodies using techniques that are well known in the art.
- the variant nAChR 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.
- the variant nAChR subunit or fragment thereof can be synthesized using conventional polypeptide synthetic techniques as provided below.
- Monoclonal antibodies that display specificity and selectivity for the variant nAChR subunit can be labeled with a measurable and detectable moiety, for example, a fluorescent moiety, radiolabels, enzymes, chemiluminscent labels and the like, and used in in vitro, or in situ immunofluorescent assays, or the like.
- the antibodies can be used to identify variant nAChR subunit for immunodiagnostic purposes.
- Tables 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. In preparing the examples, efforts have been made to ensure accuracy with respect to numbers used (for example, 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, Missouri, U.S.A.).
- Dihydro- ⁇ -erythroidine hydrobromide (“DHBE”), 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, Washington, U.S.A.).
- This oligonucleotide contains an Xho I restriction site (italics) and an ATG initiation codon (bold) followed by the next 28 codons of the human al nAChR subunit cDNA sequence. It encodes the complete signal peptide and extends to the Hind III site (underlined) present in the al nAChR subunit cDNA. The Xho I and Hind HI sites were flanked with additional nucleotides to make them internal within the molecule. Additionally, the reverse complement of this oligonucleotide was synthesized.
- the oligonucleotides were annealed together, digested with Xho I and Hind III, and then ligated into a pBluescript vector containing the human al subunit cDNA previously digested with Xho I and Hind III. This created a new cDNA encoding a full length human al nAChR subunit. The sequence of the new cDNA was confirmed by dideoxy sequencing.
- the cDNA was excised from pBluescript with Xho I and Not I, the 5' overhangs were filled-in with Klenow polymerase, linked with Bst XI adapters, digested with Bst XI, and ligated into the Bst XI site of the pRcCMV vector (Invitrogen).
- the orientation of the insert in the expression vector was determined by restriction analysis with enzymes cutting the al nAChR subunit cDNA at asymmetrical positions.
- Oocytes were maintained at 17-18°C in normal Barth's solution (90 mM NaCI, 1 mM KCl, 0.66 mM NaNO 3 , 0.74 mM CaCl 2 , 0.82 mM MgCl 2 , 2.4 mM NaHCO 3 , 2.5 mM sodium pyruvate, and 10 mM Na N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) ("HEPES”) buffer, final pH 7.55) containing 100 g/ml gentamicin. Responses were measured at a holding potential of -60 mV in modified Barth's solution containing 10 mM BaCl 2 and lacking CaCl 2 and MgCl 2 .
- HEPES mM Na N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)
- Human ⁇ 7V274T responses unlike human ⁇ 7WT responses, tended to increase significantly during the experiments. Therefore, experimental trials were bracketed, before and after, by control applications of 10 M ACh in the same oocyte. All responses were normalized to the 10 M ACh responses in order to account for changes in sensitivity within the experiment and for variability in receptor expression among oocytes.
- Example 1 Preparation of Human ⁇ 7V274T cDNA
- the wild-type al nAChR 5 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 Figure 1, used two PCR steps followed by digesting with restriction enzymes to produce a mutated fragment of the wild-type al nAChR subunit cDNA o and subcloning the mutated fragment into the wild-type human al cDNA.
- A two DNA fragments carrying the desired mutation were generated by PCR using appropriate primers.
- the mutated nucleotide was incorporated in the reverse primer (X-3') for the longer fragment and in the forward primer (Y-5') for the shorter fragment.
- the two external primers (X-5' and Y-3') were chosen so that the final PCR product would contain EcoRV and Kpnl 5 restriction sites.
- the longer 5' fragment was generated using the forward external primer 5'-GTTTGGGTCCTGGTCTTACG-3' (SEQ ID NO:5) and the reverse internal primer 5'-GCAGCATGAAGGTGGTAAGAGAG-3' (SEQ ID NO:6) bearing the mutation.
- the shorter 3' fragment was generated using the forward internal primer o 5'-CTCTCTTACCACCTTCATGCTGC-3' (SEQ ID NO:7), also bearing the mutation, and the reverse external primer 5'-GTACTGCAGCACGATCACCG-3' (SEQ ID NO:8).
- the conditions for PCR consisted of 100 ng input al DNA, 2X Pfu buffer, 100 ng of each primer pair and 0.625 U Pfu enzyme (Stratagene, La Jolla, CA). 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 5 seconds.
- step (C) the product of step (B) was digested with Kpnl and EcoRV, o gel-purified, and ligated into the wild-type human al cDNA previously digested with Kpnl 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.
- Figures 2A-2C shows the nucleotide sequence (SEQ ID NO:l) of the human ⁇ 7V274T cDNA mutant.
- the amino acid sequence of the human ⁇ 7V274T variant (SEQ ID NO:2) is 5 also shown in Figures 2A-2C.
- GTS-21 was a full agonist at the human ⁇ 7V274T nAChR with an EC 50 value of 4.3 ⁇ 0.3 ⁇ M and a Hill coefficient of 1.5 ⁇ 0.1, in stark contrast to its weak partial agonist effect at the human al wild type nAChR.
- ABT-089 also was more potent and efficacious at the human 0C7V274T nAChR, with an EC 50 values of 28 ⁇ 3 ⁇ M and a Hill coefficient of 2.3 ⁇ 0.4, but it was a partial agonist with an efficacy of 40 ⁇ 1%.
- Human ⁇ 7V274T and human ⁇ 7WT responses to EC50 concentrations of ACh were matched for similar amplitude and are shown synchronized to the beginning of ACh application and adjusted for equivalent baseline holding current (see Figure 4).
- ACh was applied to human ⁇ 7V274T for 10 sec and to human ⁇ 7WT for 2.5 sec. Brief spike-like tics near the beginning and end of the human ⁇ 7WT trace are electrical artifacts marking the opening and closing of the agonist-application valve.
- Human ⁇ 7V274T responses activated and decayed slowly compared to the human ⁇ 7WT responses.
- the analogous chick mutant nAChR activated and decayed more slowly in response to ACh (Galzi et al. (1992), supra).
- nAChR antagonists such as dihydro- ⁇ -erythroidine (DH ⁇ E), rf-tubocurarine and hexamethonium, have been found to activate responses at chick al 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 variants 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).
- DH ⁇ E 10 ⁇ M also activated agonist-like inward current responses (see Figure 5). However, these responses were small, ranging from 2.8% to 6.9% of the response to 10 ⁇ M ACh (Table 1) unlike the homologous chick ⁇ 7V251T nAChR where 10 ⁇ M DHbE elicited a response 66% as large as the ACh response (Bertrand et al. (1993) Mutations at two distinct sites within the channel domain M2 alter calcium permeability of neuronal a7 nicotinic receptor. Proc. Natl. Acad. Sci (U.S ⁇ .) 90:6971-6975).
- DH ⁇ E dihydro- ⁇ -erythroidine
- d-TC rf-tubocurarine
- MLA methyllycaconitine
- MEC mecamylamine
- ATROP atropine
- d-Tubocurarine (1 ⁇ M) 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 al V274T oocytes.
- the outward current responses may be due to stabilization of the resting (closed) state or to channel blockade of spontaneously open nAChR.
- MLA (10 nM) mecamylamine (10 ⁇ M) nor d-tubocurarine (1 ⁇ M) elicited any significant inward or outward current response (Table 1).
- the muscarinic antagonist atropine (2 ⁇ M) alone had little effect at either nAChR.
- the above compounds also were evaluated as antagonists of the response to ACh at both human CX7V274T and human ⁇ 7WT nAChRs.
- two concentrations of ACh were used: one near the EC 50 value (1 ⁇ M for ⁇ 7V274T and 200 ⁇ M for ⁇ 7WT) and one near the maximal response level (10 ⁇ M for ⁇ 7V274T and 10 mM for ⁇ 7WT).
- Data are shown in Table 1.
- DH ⁇ E (10 ⁇ M), d-tubocurarine (1 ⁇ M), MLA (10 nM), and mecamylamine (10 ⁇ M) acted as antagonists at both nAChRs.
- the al selective antagonist MLA was particularly potent, as expected, blocking human ⁇ 7V274T as well as human ⁇ 7WT at a concentration of 10 nM.
- mecamylamine (10 ⁇ M), DH ⁇ E (10 ⁇ M) and ⁇ f-tubocurarine (1 ⁇ M) each appeared to inhibit human ⁇ 7V274T more than human ⁇ 7WT.
- Atropine (2 ⁇ M) inhibited the human ⁇ 7V274T response to 1 ⁇ M ACh by 28%, but had little effect on the human ⁇ 7WT response to 200 ⁇ M ACh.
- Some oocytes have endogenous muscarinic receptors activated by low-micromolar concentrations of ACh (Kusano etal.
- ACh responses at both human ⁇ 7V274T and human ⁇ 7WT (see Table 1). Mecamylamine (10 ⁇ M) also inhibited the maximal ACh response less strongly than the EC50 ACh response at the human ⁇ 7V274T nAChR, but not at the human ⁇ 7WT nAChR where mecamylamine inhibited both concentrations of ACh similarly. The lesser inhibitions at the higher concentrations of ACh may reflect competitive antagonist-agonist interactions.
- the human variant 7V274T nAChR is similar to the analogous chick ⁇ 7V251T nAChR in its increased sensitivity to agonist activation and apparent slower rate of activation and desensitization.
- the receptors differ, however, in that DH ⁇ E (10 ⁇ M) activated the human cx7V274T inward current only weakly, compared to a 66% agonist-like effect at chick ⁇ 7V251T, and in that d-tubocurarine did not activate inward currents at the h- ⁇ 7V274T compared to the full response at chick ⁇ 7L247T nAChR (Galzi et al. (1992), supra; Bertrand et al. (1993), supra).
- DH ⁇ E (10 ⁇ M) activated the human cx7V274T inward current only weakly, compared to a 66% agonist-like effect at chick ⁇ 7V251T
- the human al wild-type (WT) and ⁇ 7V274T mutant nAChR are transfected into the human embryonic kidney cell line, HEK-293 using the eukaryotic expression vector, pRc/CMV (Invitrogen, San Diego, CA) 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 are transfected using lipofectamine (GIBCO) as described in (Gopalakrishnan et al. (1995) Eur. J. Pharmacol.
- elegans deg-3 spontaneous mutation which appears to be cytotoxic through a mechanism that is inhibited by nicotinic antagonists (Treinin and Chalfie (1995) A mutated acetylcholine receptor subunit causes neuronal degeneration in C. elegans. Neuron 14: 871-877).
- the human ⁇ 7V274T 5 variant nAChR response is longer lasting than wild-type responses ( Figure 4) but, like chick ⁇ 7V251T variant nAChR, probably has high Ca 2+ permeability so that activation of the receptor may, under some conditions, lead to excessive Ca 2+ influx and thereby cell death.
- human ⁇ 7V274T variant nAChR may be prone to prolonged spontaneous opening because oocytes that have expressed ⁇ 7V274T for 3 days or 0 longer were 10-100 fold leakier electrically than were oocytes expressing human al wild-type nAChR.
- human ⁇ 7V274T and related variant nAChR may be cytotoxic in the presence and even in the absence of agonist. Spontaneous expression of such a variant could interfere with normal al nAChR function, induce premature cell death, or interfere with synapse formation. Such effects could underlie some forms of neurodegenerative diseases or other 5 disorders involving derangement of chohnergic function.
- Cytotoxicity clearly could limit the ability of cells to express the ⁇ 7V274T variant at high levels.
- 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 o removed shortly before using the cells in further experiments.
- the human al wild type or variant is transfected using an inducible expression system such that expression of the al subunit is repressed until an inducer is added.
- an inducible system is that it can eliminate the cytotoxic effects of the expressed protein, for example the human ⁇ 7V274T variant, that is observed when a 5 constitutive expression system such as the pRcCMV is employed.
- LacS witch system (Stratagene) that uses the elements of lactose operon to control gene expression.
- LacS witch 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 o system employs a eukaryotic Lac-repressor-expressing vector (p3'SS) and a eukaryotic lac- operator containing vector (pOPRSVI-CAT) into which the ⁇ 7 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.
- the selection of stable cell lines is achieved by the presence of both 5 hygromycin and geneticin.
- expression of the al subunit will be caused by the addition of the inducing agent, IPTG.
- IPTG in the absence of IPTG, transcription is blocked by the binding of the Lac repressor protein to the operator in pOPRSVI-CAT vector.
- LPTG decreases the binding affinity of the Lac repressor protein to the operator thereby triggering transcription and expression of the inserted al subunit gene.
- the choice of such a system permits the direct evaluation of the role of the mutant al nAChR in mediating cell death in vitro.
- 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 low-density hypothalamic hormone
- uptake of neutral red dye or uptake and conversion of the tetrazolium MTT as an index of viability for example, Little et al. (1996) Br. J. Dermatol. 134: 199-207; D Souza et al. (1996) J. Neurosci. Res. 43: 289-298; Malcolm et al. (1996) J. Neurochem. 66: 2350-2360
- uptake and binding of propidium iodide to nucleic acids for example, Wrobel et al. (1996) J.
- RNA reverse tran scri tase-PCR
- RT-PCR reverse tran scri tase-PCR
- the external primers used in the synthesis of the al variant 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.
- 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.
- a method of detecting target polynucleotides of human variant al subunit in a test sample comprises (a) contacting a target polynucleotide of human variant al subunit with at least one human variant al subunit-specific polynucleotide probe or complement thereof; and (b) detecting the presence of the target polynucleotide and probe complex in the test 5 sample.
- Another method for detecting cDNA of human al subunit mRNA in a test sample comprises (a) performing reverse transcription in order to product cDNA; (b) amplifying the cDNA obtained from step (a); and (c) detecting the presence of the human variant al subunit in the test sample.
- sampled DNA or cDNA prepared fom RNA by RT-PCR can be amplified using appropriate primers (for example, X-5' and Y-3') to allow detection of the o 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 al subunit capable of selectively hybridizing to the nucleic acid of human variant al subunit can be utilized in these methods, wherein said polynucleotide has a sequence comprising SEQ ID 5 NO: 1 or a portion thereof.
- the purified polynucleotide can be produced by recombinant techniques.
- a polypeptide encoded by human variant al subunit also is useful for diagnostic applications.
- the polypeptide which has an amino acid sequence comprising SEQ ID NO:2 or a portion thereof, can be produced by recombinant or synthetic techniques.
- a monoclonal antibody which specifically binds to human variant al subunit can also be utilized in these methods.
- the human variant al subunit comprises the amino acid sequence of SEQ ID NO: 2 or a portion thereof.
- a method for detecting human variant al subunit in a test sample can comprise (a) contacting said test sample with an antibody or fragment thereof which specifically binds to 5 human variant ⁇ 7 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 al subunit of SEQ ID NO:2 or a fragment thereof.
- Treatment application Spontaneous mutation of human al valine-274 to threonine and o related mutation could result in or hasten death of those cells expressing the protein.
- At least two types of treatment could be undertaken: (i) administration of a selective al 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 (for example, see Albert and Morris (1994), Antisense knockouts: molecular scalpels for the dissection of signal 5 transduction. Trends in Pharmacological Sciences 15: 250-254); or (iii) as a reagent to kill cells such as cancer cells.
- the antisense oligonucleotide 5'-GGCTACACCTCATGGGCTCG (SEQ ID NO:9) may be used.
- this oligonucleotide or others would block synthesis of any al subunit protein, including wild type, but still would be of use where the variant and not the wild type is expressed, or where knockout of the wild-type is less detrimental than continued expression of the variant.
- the efficacy of this anti- 5 sense would be demonstrated in vitro and, further, the antisense would be valuable as a research tool eo evaluat al subunit function.
- 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.
- the 5' coding portion of the polynucleotide 0 sequence which encodes for the polypeptide of the present invention, is used to design an antisense RNA ohgonucleotide 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 al subunit polypeptide.
- the antisense RNA ohgonucleotide hybridizes to the mRNA in vivo and blocks translation of an 5 mRNA molecule into the human variant al subunit polypeptide.
- Antisense ohgonucleotides 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, methylphosphate, phosphorothiolate and phosphoroamydate internucleotide linkages.
- Antisense oligonucleotides also would be of value in determining al wild-type and V274T functions, and mechanisms of cytotoxicity in general.
- one method of evaluating the contribution of ⁇ 7V274T to cytotoxicity, cytoprotection, or other cellular processes would be to determine whether specific blockade of its synthesis blocks process. This differs in approach from the use of a receptor antagonist, 5 which may or may not block all effects of the protein. AdditionaUy, in drug discovery this approach could be useful in evaluating whether the effect of the drug is mediated by the ⁇ 7V274T variant. A similar approach could be used to evaluate the contribution of other variants or the wild-type subunit itself.
- the corresponding al sense and missense ohgonucleotides 5'-CGAGCCCATGAGGTGTAGCC (SEQ ID NO: 10) and o 5'-CCAGGCATTCGGAGCTTGCC (SEQ ID NO: 1 1), 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.
- polynucleotides that encode novel subunit and their antisense variants of the human ⁇ 7 nAChR can be used in a variety of ways as detailed herein.
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP97954155A EP0946726A2 (en) | 1996-12-20 | 1997-12-22 | A variant human alpha-7 acetylcholine receptor subunit, and methods of production and use thereof |
CA002275180A CA2275180A1 (en) | 1996-12-20 | 1997-12-22 | A variant human alpha-7 acetylcholine receptor subunit, and methods of production and use thereof |
JP52893398A JP2001506135A (en) | 1996-12-20 | 1997-12-22 | Mutated human α7 acetylcholine receptor subunit, and method for producing and using the same |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/771,737 | 1996-12-20 | ||
US08/771,737 US6323000B2 (en) | 1996-12-20 | 1996-12-20 | Variant human α7 acetylcholine receptor subunit, and methods of production and uses thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
WO1998028331A2 true WO1998028331A2 (en) | 1998-07-02 |
WO1998028331A3 WO1998028331A3 (en) | 1998-11-05 |
Family
ID=25092816
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1997/023405 WO1998028331A2 (en) | 1996-12-20 | 1997-12-22 | A variant human alpha-7 acetylcholine receptor subunit, and methods of production and use thereof |
Country Status (5)
Country | Link |
---|---|
US (2) | US6323000B2 (en) |
EP (1) | EP0946726A2 (en) |
JP (1) | JP2001506135A (en) |
CA (1) | CA2275180A1 (en) |
WO (1) | WO1998028331A2 (en) |
Cited By (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000073431A2 (en) * | 1999-05-27 | 2000-12-07 | Pharmacia & Upjohn Company | Methods and compositions for measuring ion channel conductance |
WO2002014351A2 (en) * | 2000-08-14 | 2002-02-21 | Ortho-Mcneil Pharmaceutical, Inc. | α7 NICOTINIC RECEPTOR PEPTIDES AS LIGANDS FOR β AMYLOID P EPTIDES |
US6693172B1 (en) | 1999-05-27 | 2004-02-17 | Pharmacia And Upjohn Company | Double mutant alpha-7 nicotinic acetylcholine receptor |
WO2004074245A2 (en) * | 2003-02-14 | 2004-09-02 | Memory Pharmaceuticals Corporation | Monkey alpha-7 nicotinic acetylcholine receptor and methods of use thereof |
US7358057B2 (en) | 2002-05-09 | 2008-04-15 | Memory Pharmaceuticals Corporation | QM-7 and QT-6 cells transfected with mutant cell surface expressed channel receptors and assays using the transfected cells |
US7696325B2 (en) | 1999-03-10 | 2010-04-13 | Chugai Seiyaku Kabushiki Kaisha | Polypeptide inducing apoptosis |
US8034903B2 (en) | 2000-10-20 | 2011-10-11 | Chugai Seiyaku Kabushiki Kaisha | Degraded TPO agonist antibody |
US9670269B2 (en) | 2006-03-31 | 2017-06-06 | Chugai Seiyaku Kabushiki Kaisha | Methods of modifying antibodies for purification of bispecific antibodies |
US9777066B2 (en) | 2005-06-10 | 2017-10-03 | Chugai Seiyaku Kabushiki Kaisha | Pharmaceutical compositions containing sc(Fv)2 |
US10011858B2 (en) | 2005-03-31 | 2018-07-03 | Chugai Seiyaku Kabushiki Kaisha | Methods for producing polypeptides by regulating polypeptide association |
US11124576B2 (en) | 2013-09-27 | 2021-09-21 | Chungai Seiyaku Kabushiki Kaisha | Method for producing polypeptide heteromultimer |
US11649262B2 (en) | 2015-12-28 | 2023-05-16 | Chugai Seiyaku Kabushiki Kaisha | Method for promoting efficiency of purification of Fc region-containing polypeptide |
Families Citing this family (23)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1308447C (en) * | 2000-10-20 | 2007-04-04 | 中外制药株式会社 | Degraded agonist antibody |
DE60324700D1 (en) * | 2002-10-11 | 2008-12-24 | Chugai Pharmaceutical Co Ltd | CELL TOD INDUCTIVE ACTIVE SUBSTANCE |
ATE378048T1 (en) * | 2002-12-06 | 2007-11-15 | The Feinstein Inst Medical Res | INHIBITION OF INFLAMMATION USING ALPHA-7 RECEPTOR-LINKING CHOLINERGIC AGONISTS |
US7238715B2 (en) * | 2002-12-06 | 2007-07-03 | The Feinstein Institute For Medical Research | Treatment of pancreatitis using alpha 7 receptor-binding cholinergic agonists |
JP2004279086A (en) * | 2003-03-13 | 2004-10-07 | Konica Minolta Holdings Inc | Radiation image conversion panel and method for manufacturing it |
EP1616880A4 (en) * | 2003-03-13 | 2006-07-26 | Chugai Pharmaceutical Co Ltd | Ligand having agonistic activity to mutated receptor |
WO2004087763A1 (en) * | 2003-03-31 | 2004-10-14 | Chugai Seiyaku Kabushiki Kaisha | Modified antibody against cd22 and utilization thereof |
WO2004111233A1 (en) * | 2003-06-11 | 2004-12-23 | Chugai Seiyaku Kabushiki Kaisha | Process for producing antibody |
WO2005056602A1 (en) * | 2003-12-12 | 2005-06-23 | Chugai Seiyaku Kabushiki Kaisha | Method of screening modified antibody having agonistic activity |
AU2004297109A1 (en) * | 2003-12-12 | 2005-06-23 | Chugai Seiyaku Kabushiki Kaisha | Cell death inducing agent |
TW200530269A (en) * | 2003-12-12 | 2005-09-16 | Chugai Pharmaceutical Co Ltd | Anti-Mpl antibodies |
US20050170360A1 (en) * | 2004-01-30 | 2005-08-04 | Papke Roger L. | Variant neuronal nicotinic alpha-7 receptor and methods of use |
JP4799405B2 (en) * | 2004-04-09 | 2011-10-26 | 中外製薬株式会社 | Cell death inducer |
EP1870458B1 (en) | 2005-03-31 | 2018-05-09 | Chugai Seiyaku Kabushiki Kaisha | sc(Fv)2 STRUCTURAL ISOMERS |
US20090022687A1 (en) * | 2005-05-18 | 2009-01-22 | Chugai Seiyaku Kabushiki Kaisha | Novel Pharmaceuticals That Use Anti-HLA Antibodies |
EP3348639A3 (en) * | 2005-06-10 | 2018-10-31 | Chugai Seiyaku Kabushiki Kaisha | Sc(fv)2 site-directed mutant |
CN101237890A (en) * | 2005-06-10 | 2008-08-06 | 中外制药株式会社 | Stabilizer for protein preparation comprising meglumine and use thereof |
US7879913B2 (en) * | 2005-06-28 | 2011-02-01 | Catholic Healthcare West | Iptakalim hydrochloride for decreasing nicotine use |
US8316104B2 (en) | 2005-11-15 | 2012-11-20 | California Institute Of Technology | Method and apparatus for collaborative system |
CA2657951A1 (en) * | 2006-06-14 | 2007-12-21 | Chugai Seiyaku Kabushiki Kaisha | Agents for promoting the growth of hematopoietic stem cells |
AU2007273507A1 (en) * | 2006-07-13 | 2008-01-17 | Chugai Seiyaku Kabushiki Kaisha | Cell death inducer |
CL2008000719A1 (en) * | 2007-03-12 | 2008-09-05 | Univ Tokushima Chugai Seiyaku | THERAPEUTIC AGENT FOR CANCER RESISTANT TO CHEMOTHERAPEUTIC AGENTS THAT UNDERSTAND AN ANTIBODY THAT RECOGNIZES IT CLASS I AS ACTIVE INGREDIENT; PHARMACEUTICAL COMPOSITION THAT INCLUDES SUCH ANTIBODY; AND METHOD TO TREAT CANCER RESISTANT TO |
EP2344636B1 (en) * | 2008-10-09 | 2017-12-06 | Howard Hughes Medical Institute | Novel chimeric ligand-gated ion channels and methods of use thereof |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1994020617A2 (en) * | 1993-03-08 | 1994-09-15 | The Salk Institute Biotechnology/Industrial Associates, Incorporated | Human neuronal nicotinic acetylcholine receptor compositions and methods employing same |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP3149133B2 (en) * | 1989-10-27 | 2001-03-26 | ザ ソーク インスティチュート フォア バイオロジカル スタディーズ | Glutamate receptor compositions and methods |
-
1996
- 1996-12-20 US US08/771,737 patent/US6323000B2/en not_active Expired - Fee Related
-
1997
- 1997-12-22 WO PCT/US1997/023405 patent/WO1998028331A2/en active Application Filing
- 1997-12-22 EP EP97954155A patent/EP0946726A2/en not_active Withdrawn
- 1997-12-22 JP JP52893398A patent/JP2001506135A/en active Pending
- 1997-12-22 CA CA002275180A patent/CA2275180A1/en not_active Abandoned
-
2001
- 2001-09-18 US US09/954,936 patent/US6683157B2/en not_active Expired - Fee Related
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1994020617A2 (en) * | 1993-03-08 | 1994-09-15 | The Salk Institute Biotechnology/Industrial Associates, Incorporated | Human neuronal nicotinic acetylcholine receptor compositions and methods employing same |
Non-Patent Citations (5)
Cited By (19)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7696325B2 (en) | 1999-03-10 | 2010-04-13 | Chugai Seiyaku Kabushiki Kaisha | Polypeptide inducing apoptosis |
WO2000073431A3 (en) * | 1999-05-27 | 2001-05-03 | Upjohn Co | Methods and compositions for measuring ion channel conductance |
US6693172B1 (en) | 1999-05-27 | 2004-02-17 | Pharmacia And Upjohn Company | Double mutant alpha-7 nicotinic acetylcholine receptor |
US7247706B2 (en) | 1999-05-27 | 2007-07-24 | Pharmacia & Upjohn Company | Double mutant alpha-7 nicotinic acetylcholine receptors |
WO2000073431A2 (en) * | 1999-05-27 | 2000-12-07 | Pharmacia & Upjohn Company | Methods and compositions for measuring ion channel conductance |
WO2002014351A2 (en) * | 2000-08-14 | 2002-02-21 | Ortho-Mcneil Pharmaceutical, Inc. | α7 NICOTINIC RECEPTOR PEPTIDES AS LIGANDS FOR β AMYLOID P EPTIDES |
WO2002014351A3 (en) * | 2000-08-14 | 2003-08-14 | Ortho Mcneil Pharm Inc | α7 NICOTINIC RECEPTOR PEPTIDES AS LIGANDS FOR β AMYLOID P EPTIDES |
US8586039B2 (en) | 2000-10-20 | 2013-11-19 | Chugai Seiyaku Kabushiki Kaisha | Degraded TPO agonist antibody |
US8034903B2 (en) | 2000-10-20 | 2011-10-11 | Chugai Seiyaku Kabushiki Kaisha | Degraded TPO agonist antibody |
US7358057B2 (en) | 2002-05-09 | 2008-04-15 | Memory Pharmaceuticals Corporation | QM-7 and QT-6 cells transfected with mutant cell surface expressed channel receptors and assays using the transfected cells |
WO2004074245A3 (en) * | 2003-02-14 | 2004-10-14 | Memory Pharm Corp | Monkey alpha-7 nicotinic acetylcholine receptor and methods of use thereof |
WO2004074245A2 (en) * | 2003-02-14 | 2004-09-02 | Memory Pharmaceuticals Corporation | Monkey alpha-7 nicotinic acetylcholine receptor and methods of use thereof |
US10011858B2 (en) | 2005-03-31 | 2018-07-03 | Chugai Seiyaku Kabushiki Kaisha | Methods for producing polypeptides by regulating polypeptide association |
US11168344B2 (en) | 2005-03-31 | 2021-11-09 | Chugai Seiyaku Kabushiki Kaisha | Methods for producing polypeptides by regulating polypeptide association |
US9777066B2 (en) | 2005-06-10 | 2017-10-03 | Chugai Seiyaku Kabushiki Kaisha | Pharmaceutical compositions containing sc(Fv)2 |
US9670269B2 (en) | 2006-03-31 | 2017-06-06 | Chugai Seiyaku Kabushiki Kaisha | Methods of modifying antibodies for purification of bispecific antibodies |
US10934344B2 (en) | 2006-03-31 | 2021-03-02 | Chugai Seiyaku Kabushiki Kaisha | Methods of modifying antibodies for purification of bispecific antibodies |
US11124576B2 (en) | 2013-09-27 | 2021-09-21 | Chungai Seiyaku Kabushiki Kaisha | Method for producing polypeptide heteromultimer |
US11649262B2 (en) | 2015-12-28 | 2023-05-16 | Chugai Seiyaku Kabushiki Kaisha | Method for promoting efficiency of purification of Fc region-containing polypeptide |
Also Published As
Publication number | Publication date |
---|---|
CA2275180A1 (en) | 1998-07-02 |
US6683157B2 (en) | 2004-01-27 |
JP2001506135A (en) | 2001-05-15 |
EP0946726A2 (en) | 1999-10-06 |
US20030073161A1 (en) | 2003-04-17 |
US20010006796A1 (en) | 2001-07-05 |
US6323000B2 (en) | 2001-11-27 |
WO1998028331A3 (en) | 1998-11-05 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6683157B2 (en) | Variant human α7 acetylcholine receptor subunit, and methods of production and use thereof | |
McIntosh et al. | Analogs of α-conotoxin MII are selective for α6-containing nicotinic acetylcholine receptors | |
US5910582A (en) | Human neuronal nicotinic acetylcholine receptor compositions and methods employing same | |
EP0714437A1 (en) | Dna encoding inward rectifier, g-protein activated, mammalian, potassium kga channel and uses thereof | |
US6100046A (en) | Methods of identifying modulators of alpha9, a novel acetylcholine-gated ion channel receptor subunit | |
Werkman et al. | Charybdotoxin, dendrotoxin and mast cell degranulating peptide block the voltage-activated K+ current of fibroblast cells stably transfected with NGK1 (Kvl. 2) K+ channel complementary DNA | |
AU704927B2 (en) | Human neuronal nicotinic acetylcholine receptor compositions and methods employing same | |
US6096513A (en) | Polynucleotides encoding KAPPA opiod receptors | |
US6391561B1 (en) | Protein that enhances expression of potassium channels on cell surfaces and nucleic acids that encode the same | |
US6130058A (en) | Human NMDA R2A receptor subunit and isoforms of the human NMDA-R1 receptor subunit and encoding cDNAs | |
US7229779B2 (en) | Variant human α7 acetylcholine receptor subunit, and methods of production and use thereof | |
US6214581B1 (en) | Nucleic acids encoding a functional human purinoreceptor P2X3 and P2X6, and methods of production and use thereof | |
WO1999055901A2 (en) | Screening assay for identifying human purinoreceptor ligands | |
WO2005074641A2 (en) | Variant neuronal nicotinic alpha-7 receptor and methods of use | |
EP1268545B1 (en) | Potassium channel protein kcnq5, a target for diseases of central nervous system and cardiovascular system | |
MXPA99005856A (en) | A variant human alpha-7 acetylcholine receptor subunit, and methods of production and use thereof | |
US7563595B2 (en) | Nucleic acids encoding functional splice variants of the α7 nicotinic acetylcholine receptor subunit and methods for producing the encoded proteins | |
CA2339871A1 (en) | Nucleic acids encoding a functional human purinoreceptor p2x2 and methods of producing and use thereof | |
EP1045903A1 (en) | Nucleic acids encoding a functional human purinoreceptor p2x3 and methods of production and use thereof | |
WO1999011784A1 (en) | Proton gated ion channel proteins | |
WO1999061606A1 (en) | MAMMALIAN gb2 GABAB RECEPTORS | |
Cohen | Allosteric regulation of adenosine receptors | |
JPH0770191A (en) | Receptor protein, its production and use thereof | |
MXPA00006974A (en) | Nucleic acids encoding a functional human purinoreceptor p2x3 | |
MXPA01001855A (en) | Nucleic acids encoding a functional human purinoreceptor p2x2 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A2 Designated state(s): CA JP MX |
|
AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
ENP | Entry into the national phase |
Ref document number: 2275180 Country of ref document: CA Ref country code: CA Ref document number: 2275180 Kind code of ref document: A Format of ref document f/p: F |
|
ENP | Entry into the national phase |
Ref country code: JP Ref document number: 1998 528933 Kind code of ref document: A Format of ref document f/p: F |
|
WWE | Wipo information: entry into national phase |
Ref document number: PA/a/1999/005856 Country of ref document: MX |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1997954155 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 1997954155 Country of ref document: EP |