High-Affinity Epibatidine Binding Of Functional Human Î±7-Nicotinic Acetylcholine Receptors Stably And Heterologously Expressed De Novo In Human Sh-Ep1 Cells
Human nicotinic acetylcholine receptor (nAChR) Î±7 subunits were stably and heterologously expressed in native nAChR-null SH-EP1 human epithelial cells. Immune-fluorescence staining shows Î±7 subunit protein expression in virtually every transfected cell. Microautoradiographic analysis identifies 125I-labeled Î±-bungarotoxin (I-Bgt) binding sites corresponding to human Î±7 (hÎ±7)-nAChRs on the surface of most cells. I-Bgt binds to hÎ±7-nAChRs in membrane fractions with a typical apparent KD value of âˆ¼5 nM and Bmax value of âˆ¼1 pmol/mg membrane protein, and 62% of these sites are expressed on the cell surface. Function of heterologously expressed hÎ±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 âˆ¼100%) in numbers of I-Bgt binding sites. Epibatidine is a useful ligand for studies of nAChRs containing Î±3 or Î±4 subunits (KD values of about 100 or 10 pM, respectively). hÎ±7-nAChRs expressed in transfected SH-EP1 cells also exhibit picomolar affinity binding of 3H-labeled epibatidine (KD value of âˆ¼0.6 nM). Studies of several forms of native or heterologously expressed rat or human Î±7-nAChRs confirm high-affinity and mutually exclusive interaction with both epibatidine and Î±-bungarotoxin. Rank order potencies for drugs acting to compete for binding of either radioligand are similar (methyllycaconitine > dimethyl-phenyl-piperazinium > nicotine âˆ¼ cytisine > carbamylcholine âˆ¼ d-tubocurarine). These results demonstrate that transfected SH-EP1 cells are excellent models for studies of heterologously expressed, human Î±7-nAChRs that exhibit ligand binding and functional properties like native Î±7-nAChRs and that epibatdine is useful as a probe for human Î±7-nAChRs. Copyright Â© 2005 by The American Society for Pharmacology and Experimental Therapeutics.
Journal of Pharmacology and Experimental Therapeutics
Digital Object Identifier (DOI)
Peng, Jian Hong; Fryer, John D.; Hurst, Raymond S.; Schroeder, Katherine M.; George, Andrew A.; Morrissy, Steven; Groppi, Vincent E.; Leonard, Sherry S.; and Lukas, Ronald J., "High-Affinity Epibatidine Binding Of Functional Human Î±7-Nicotinic Acetylcholine Receptors Stably And Heterologously Expressed De Novo In Human Sh-Ep1 Cells" (2005). Neurobiology. 73.