Common determinants of single channel conductance within the large cytoplasmic loop of 5-hydroxytryptamine type 3 and α4β 2 nicotinic acetylcholine receptors

Abstract

Homomeric 5-hydroxytryptamine type 3A receptors (5-HT3ARs) have a single channel conductance (γ) below the resolution of single channel recording (966 ± 75 fS, estimated by variance analysis). By contrast, heteromeric 5-HT3A/B and nicotinic acetylcholine receptors (nAChRs) have picosiemen range γ values. In this study, single channel recordings revealed that replacement of cytoplasmic membrane-associated (MA) helix arginine 432 ( 4′), 436 (0′), and 440 (4′) residues by 5-HT 3B (4′Gln, 0′Asp, and 4′Ala) residues increases γ to 36.5 ± 1.0 pS. The 0′ residue makes the most substantial contribution to γ of the 5-HT3AR. Replacement of 0′Arg by aspartate, glutamate (α7 nAChR subunit MA 0′), or glutamine (β2 subunit MA 0′) increases γ to the resolvable range (>6 pS). By contrast, replacement of 0′Arg by phenylalanine (α4 subunit MA 0′) reduced γ to 416 ± 107 fS. In reciprocal experiments with α4β 2 nAChRs (γ 31.3 ± 0.8 pS), replacement of MA 0′ residues by arginine in α4β2(Q443R) and α4(F588R)β2 reduced γ slightly. By contrast, the γ of double mutant α4(F588R)β2(Q443R) was halved. The MA -4′ and 4′ residues also influenced γ of 5-HT3ARs. Replacement of nAChR α4 or β2 MA 4′ residues by arginine made current density negligible. By contrast, replacement of both -4′ residues by arginine produced functional nAChRs with substantially reduced γ (11.4 ± 0.5 pS). Homology models of the 5-HT3A and α4β 2 nAChRs against Torpedo nAChR revealed MA -4′, 0′, and 4′ residues within five intracellular portals. This locus may be a common determinant of ion conduction throughout the Cys loop receptor family. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.