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