title: High-affinity epibatidine binding of functional, human alpha7-nicotinic acetylcholine receptors stably and heterologously expressed de novo in human SH-EP1 cells.
abstract: Human nicotinic acetylcholine receptor (nAChR) alpha7 subunits were stably and heterologously expressed in native nAChR-null SH-EP1 human epithelial cells. Immunofluorescence staining shows alpha7 subunit protein expression in virtually every transfected cell. Microautoradiographic analysis identifies 125I-labeled alpha-bungarotoxin (I-Bgt) binding sites corresponding to human alpha7 (halpha7)-nAChRs on the surface of most cells. I-Bgt binds to halpha7-nAChRs in membrane fractions with a typical apparent K(D) value of approximately 5 nM and B(max) value of approximately 1 pmol/mg membrane protein, and 62% of these sites are expressed on the cell surface. Function of heterologously expressed halpha7-nAChRs is evident as rapid, transient inward current responses to (-)-nicotine. Nicotine treatment of transfected cells produces dose- and time-dependent increases (up to approximately 100%) in numbers of I-Bgt binding sites. Epibatidine is a useful ligand for studies of nAChRs containing alpha3 or alpha4 subunits (K(D) values of about 100 or 10 pM, respectively). halpha7-nAChRs expressed in transfected SH-EP1 cells also exhibit picomolar affinity binding of 3H-labeled epibatidine (K(D) value of approximately 0.6 nM). Studies of several forms of native or heterologously expressed rat or human alpha7-nAChRs confirm high-affinity and mutually exclusive interaction with both epibatidine and alpha-bungarotoxin. Rank order potencies for drugs acting to compete for binding of either radioligand are similar (methyllycaconitine > dimethylphenyl-piperazinium > nicotine approximately cytisine > carbamylcholine approximately D-tubocurarine). These results demonstrate that transfected SH-EP1 cells are excellent models for studies of heterologously expressed, human alpha7-nAChRs that exhibit ligand binding and functional properties like native alpha7-nAChRs and that epibatdine is useful as a probe for human alpha7-nAChRs.
authors.affiliation: Division of Neurobiology, Barrow Neurological Institute, 350 West Thomas Rd., Phoenix, AZ 85013, USA.
authors.forename: Jian-Hong
authors.initials: JH
authors.lastname: Peng
authors.forename: John D
authors.initials: JD
authors.lastname: Fryer
authors.forename: Raymond S
authors.initials: RS
authors.lastname: Hurst
authors.forename: Katherine M
authors.initials: KM
authors.lastname: Schroeder
authors.forename: Andrew A
authors.initials: AA
authors.lastname: George
authors.forename: Steven
authors.initials: S
authors.lastname: Morrissy
authors.forename: Vincent E
authors.initials: VE
authors.lastname: Groppi
authors.forename: Sherry S
authors.initials: SS
authors.lastname: Leonard
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Lukas
mesh_terms: D001345:Autoradiography; D015152:Blotting, Northern; D015153:Blotting, Western; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000378:metabolism*; D002038:Bungarotoxins / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D008564:Membrane Potentials / Q000187:drug effects; D009538:Nicotine / Q000494:pharmacology; D018722:Nicotinic Agonists / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010957:Plasmids / Q000235:genetics; D011725:Pyridines / Q000378:metabolism*; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification; D011978:Receptors, Nicotinic / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D064569:alpha7 Nicotinic Acetylcholine Receptor
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D002038:Bungarotoxins; C579167:Chrna7 protein, human; C579165:Chrna7 protein, rat; D018076:DNA, Complementary; D018722:Nicotinic Agonists; D011725:Pyridines; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor; D012313:RNA; D009538:Nicotine; C082748:epibatidine