title: Receptor protection studies comparing recombinant and native nicotinic receptors: Evidence for a subpopulation of mecamylamine-sensitive native alpha3beta4* nicotinic receptors.
abstract: Studies involving receptor protection have been used to define the functional involvement of specific receptor subtypes in tissues expressing multiple receptor subtypes. Previous functional studies from our laboratory demonstrate the feasibility of this approach when applied to neuronal tissues expressing multiple nicotinic acetylcholine receptors (nAChRs). In the current studies, the ability of a variety of nAChR agonists and antagonists to protect native and recombinant alpha3beta4 nAChRs from alkylation were investigated using nAChR binding techniques. Alkylation of native alpha3beta4* nAChRs from membrane preparations of bovine adrenal chromaffin cells resulted in a complete loss of specific [(3)H]epibatidine binding. This loss of binding to native nAChRs was preventable by pretreatment with the agonists, carbachol or nicotine. The partial agonist, cytisine, produced partial protection. Several nAChR antagonists were also tested for their ability to protect. Hexamethonium and decamethonium were without protective activity while mecamylamine and tubocurarine were partially effective. Addition protection studies were performed on recombinant alpha3beta4 nAChRs. As with native alpha3beta4* nAChRs, alkylation produced a complete loss of specific [(3)H]epibatidine binding to recombinant alpha3beta4 nAChRs which was preventable by pretreatment with nicotine. However, unlike native alpha3beta4* nAChRs, cytisine and mecamylamine, provide no protection for alkylation. These results highlight the differences between native alpha3beta4* nAChRs and recombinant alpha3beta4 nAChRs and support the use of protection assays to characterize native nAChR subpopulations.
authors.affiliation: Division of Pharmacology, The Ohio State University, College of Pharmacy, 500 West 12th Avenue, Columbus, 43210, USA.
authors.forename: R Benjamin
authors.initials: RB
authors.lastname: Free
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Kaser
authors.forename: R Thomas
authors.initials: RT
authors.lastname: Boyd
authors.forename: Dennis B
authors.initials: DB
authors.lastname: McKay
mesh_terms: D000478:Alkylation / Q000187:drug effects; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000493:pharmacokinetics; D002417:Cattle; D002460:Cell Line; D003545:Cysteine / Q000494:pharmacology; D004347:Drug Interactions; D006801:Humans; D008464:Mecamylamine / Q000494:pharmacology*; D018722:Nicotinic Agonists / Q000493:pharmacokinetics / Q000494:pharmacology; D018733:Nicotinic Antagonists / Q000494:pharmacology*; D011725:Pyridines / Q000493:pharmacokinetics; D011978:Receptors, Nicotinic / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D014162:Transfection / Q000379:methods; D014316:Tritium / Q000493:pharmacokinetics
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D018722:Nicotinic Agonists; D018733:Nicotinic Antagonists; D011725:Pyridines; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins; C403530:nicotinic receptor alpha3beta4; D014316:Tritium; D008464:Mecamylamine; D003545:Cysteine; C082748:epibatidine