title: Extending the analysis of nicotinic receptor antagonists with the study of alpha6 nicotinic receptor subunit chimeras.
abstract: Heterologous expression systems have increased the feasibility of developing selective ligands to target nicotinic acetylcholine receptor (nAChR) subtypes. However, the alpha6 subunit, a component in nAChRs that mediates some of the reinforcing effects of nicotine, is not easily expressed in systems such as the Xenopus oocyte. Certain aspects of alpha6-containing receptor pharmacology have been studied by using chimeric subunits containing the alpha6 ligand-binding domain. However, these chimeras would not be sensitive to an alpha6-selective channel blocker; therefore we developed an alpha6 chimera (alpha4/6) that has the transmembrane and intracellular domains of alpha6 and the extracellular domain of alpha4. We examined the pharmacological properties of alpha4/6-containing receptors and other important nAChR subtypes, including alpha7, alpha4beta2, alpha4beta4, alpha3beta4, alpha3beta2, and alpha3beta2beta3, as well as receptors containing alpha6/3 and alpha6/4 chimeras. Our data show that the absence or presence of the beta4 subunit is an important factor for sensitivity to the ganglionic blocker mecamylamine, and that dihydro-beta-erythroidine is most effective on subtypes containing the alpha4 subunit extracellular domain. Receptors containing the alpha6/4 subunit are sensitive to alpha-conotoxin PIA, while receptors containing the reciprocal alpha4/6 chimera are insensitive. In experiments with novel antagonists of nicotine-evoked dopamine release, the alpha4/6 chimera indicated that structural rigidity was a key element of compounds that could result in selectivity for noncompetitive inhibition of alpha6-containing receptors. Our data extend the information available on prototypical nAChR antagonists, and establish the alpha4/6 chimera as a useful new tool for screening drugs as selective nAChR antagonists.
authors.affiliation: Department of Pharmacology and Therapeutics, University of Florida, College of Medicine, P.O. Box 100267, JHMHSC, 1600 SW Archer Road, Gainesville, FL 32610-0267, USA. rlpapke@ufl.edu
authors.forename: Roger L
authors.initials: RL
authors.lastname: Papke
authors.forename: Linda P
authors.initials: LP
authors.lastname: Dwoskin
authors.forename: Peter A
authors.initials: PA
authors.lastname: Crooks
authors.forename: Guangrong
authors.initials: G
authors.lastname: Zheng
authors.forename: Zhenfa
authors.initials: Z
authors.lastname: Zhang
authors.forename: J Michael
authors.initials: JM
authors.lastname: McIntosh
authors.forename: Clare
authors.initials: C
authors.lastname: Stokes
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D003627:Data Interpretation, Statistical; D004079:Dihydro-beta-Erythroidine / Q000494:pharmacology; D004594:Electrophysiology; D005260:Female; D008464:Mecamylamine / Q000494:pharmacology; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D050506:Mutant Chimeric Proteins / Q000235:genetics*; D018733:Nicotinic Antagonists / Q000494:pharmacology*; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D012313:RNA / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D011978:Receptors, Nicotinic / Q000096:biosynthesis / Q000187:drug effects* / Q000235:genetics*; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
chemical_list: D050506:Mutant Chimeric Proteins; D018733:Nicotinic Antagonists; D000644:Quaternary Ammonium Compounds; D011978:Receptors, Nicotinic; C104518:nicotinic receptor alpha6; D004079:Dihydro-beta-Erythroidine; D012313:RNA; D008464:Mecamylamine; D000109:Acetylcholine