Title

High-Affinity Epibatidine Binding Of Functional Human α7-Nicotinic Acetylcholine Receptors Stably And Heterologously Expressed De Novo In Human Sh-Ep1 Cells

Department

neurobiology

Document Type

Article

Abstract

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 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 > 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. Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics.

Publication Date

4-1-2005

Publication Title

Journal of Pharmacology and Experimental Therapeutics

ISSN

00223565

Volume

313

Issue

1

First Page

24

Last Page

35

Digital Object Identifier (DOI)

10.1124/jpet.104.079004

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