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NEUROPHARMACOLOGY

High-Affinity Epibatidine Binding of Functional, Human 7-Nicotinic Acetylcholine Receptors Stably and Heterologously Expressed de Novo in Human SH-EP1 Cells

Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona (J.-H.P., J.D.F., K.M.S., A.A.G., S.M., R.J.L.); Pharmacia Corporation, Kalamazoo, Michigan (R.S.H., V.E.G.); and Department of Psychiatry, University of Colorado Health Sciences Center, Denver, Colorado (S.S.L.)

Human nicotinic acetylcholine receptor (nAChR) 7 subunits were stably and heterologously expressed in native nAChR-null SH-EP1 human epithelial cells. Immunofluorescence staining shows 7 subunit protein expression in virtually every transfected cell. Microautoradiographic analysis identifies ¹²⁵I-labeled -bungarotoxin (I-Bgt) binding sites corresponding to human 7 (h7)-nAChRs on the surface of most cells. I-Bgt binds to h7-nAChRs in membrane fractions with a typical apparent K_D value of 5 nM and B_max value of 1 pmol/mg membrane protein, and 62% of these sites are expressed on the cell surface. Function of heterologously expressed h7-nAChRs is evident as rapid, transient inward current responses to (–)-nicotine. Nicotine treatment of transfected cells produces dose- and time-dependent increases (up to 100%) in numbers of I-Bgt binding sites. Epibatidine is a useful ligand for studies of nAChRs containing 3 or 4 subunits (K_D values of about 100 or 10 pM, respectively). h7-nAChRs expressed in transfected SH-EP1 cells also exhibit picomolar affinity binding of ³H-labeled epibatidine (K_D value of 0.6 nM). Studies of several forms of native or heterologously expressed rat or human 7-nAChRs confirm high-affinity and mutually exclusive interaction with both epibatidine and -bungarotoxin. Rank order potencies for drugs acting to compete for binding of either radioligand are similar (methyllycaconitine > dimethylphenyl-piperazinium > nicotine cytisine > carbamylcholine d-tubocurarine). These results demonstrate that transfected SH-EP1 cells are excellent models for studies of heterologously expressed, human 7-nAChRs that exhibit ligand binding and functional properties like native 7-nAChRs and that epibatdine is useful as a probe for human 7-nAChRs.

Address correspondence to: Dr. R. J. Lukas, Division of Neurobiology, Barrow Neurological Institute, 350 West Thomas Rd., Phoenix, AZ 85013. E-mail: rlukas{at}chw.edu

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