PT - JOURNAL ARTICLE AU - Jian-Hong Peng AU - John D. Fryer AU - Raymond S. Hurst AU - Katherine M. Schroeder AU - Andrew A. George AU - Steven Morrissy AU - Vincent E. Groppi AU - Sherry S. Leonard AU - Ronald J. Lukas TI - High-Affinity Epibatidine Binding of Functional, Human α7-Nicotinic Acetylcholine Receptors Stably and Heterologously Expressed de Novo in Human SH-EP1 Cells AID - 10.1124/jpet.104.079004 DP - 2005 Apr 01 TA - Journal of Pharmacology and Experimental Therapeutics PG - 24--35 VI - 313 IP - 1 4099 - http://jpet.aspetjournals.org/content/313/1/24.short 4100 - http://jpet.aspetjournals.org/content/313/1/24.full SO - J Pharmacol Exp Ther2005 Apr 01; 313 AB - Human nicotinic acetylcholine receptor (nAChR) α7 subunits were stably and heterologously expressed in native nAChR-null SH-EP1 human epithelial cells. Immunofluorescence staining shows α7 subunit protein expression in virtually every transfected cell. Microautoradiographic analysis identifies 125I-labeled α-bungarotoxin (I-Bgt) binding sites corresponding to human α7 (hα7)-nAChRs on the surface of most cells. I-Bgt binds to hα7-nAChRs in membrane fractions with a typical apparent KD value of ∼5 nM and Bmax value of ∼1 pmol/mg membrane protein, and 62% of these sites are expressed on the cell surface. Function of heterologously expressed hα7-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 α3 or α4 subunits (KD values of about 100 or 10 pM, respectively). hα7-nAChRs expressed in transfected SH-EP1 cells also exhibit picomolar affinity binding of 3H-labeled epibatidine (KD value of ∼0.6 nM). Studies of several forms of native or heterologously expressed rat or human α7-nAChRs confirm high-affinity and mutually exclusive interaction with both epibatidine and α-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 α7-nAChRs that exhibit ligand binding and functional properties like native α7-nAChRs and that epibatdine is useful as a probe for human α7-nAChRs. The American Society for Pharmacology and Experimental Therapeutics