Common determinants of single channel conductance within the large cytoplasmic loop of 5-hydroxytryptamine type 3 and α4β2 nicotinic acetylcholine receptors

Homomeric 5-hydroxytryptamine type 3A receptors (5-HT(3A)Rs) have a single channel conductance (gamma) 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 gamma 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-HT3B (-4'Gln, 0'Asp, and 4'Ala) residues increases gamma to 36.5 +/- 1.0 pS. The 0' residue makes the most substantial contribution to gamma of the 5-HT3AR. Replacement of 0' Arg by aspartate, glutamate (alpha(7)nAChR subunit MA 0'), or glutamine (beta(2) subunit MA 0') increases gamma to the resolvable range (>6 pS). By contrast, replacement of 0' Arg by phenylalanine (alpha(4) subunit MA 0') reduced gamma to 416 +/- 107 fS. In reciprocal experiments with alpha(4)beta(2) nAChRs (gamma=31.3 +/- 0.8 pS), replacement of MA 0' residues by arginine in alpha(4)beta(2)(Q443R) and alpha(4)(F588R) beta(2) reduced gamma slightly. By contrast, the gamma of double mutant alpha 4(F588R)beta(2)(Q443R) was halved. The MA -4' and 4' residues also influenced gamma of 5-HT(3A)Rs. Replacement of nAChR alpha(4) or beta(2) MA 4' residues by arginine made current density negligible. By contrast, replacement of both -4' residues by arginine produced functional nAChRs with substantially reduced gamma (11.4 +/- 0.5 pS). Homology models of the 5-HT3A and alpha(4)beta(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.