High-affinity epibatidine binding of functional, human α7-nicotinic acetylcholine receptors stably and heterologously expressed de novo in human SH-EP1 cells

Human nicotinic acetylcholine receptor (nAChR) α7 subunits were stably and heterologously expressed in native nAChR-null SH-EP1 human epithelial cells. Immune-fluorescence 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 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 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 (K_D 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 (K_D 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 > dimethyl-phenyl-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.