title: Common determinants of single channel conductance within the large cytoplasmic loop of 5-hydroxytryptamine type 3 and alpha4beta2 nicotinic acetylcholine receptors.
abstract: Homomeric 5-hydroxytryptamine type 3A receptors (5-HT3ARs) 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 (alpha7 nAChR subunit MA 0'), or glutamine (beta2 subunit MA 0') increases gamma to the resolvable range (>6 pS). By contrast, replacement of 0'Arg by phenylalanine (alpha4 subunit MA 0') reduced gamma to 416 +/- 107 fS. In reciprocal experiments with alpha4beta2 nAChRs (gamma = 31.3 +/- 0.8 pS), replacement of MA 0' residues by arginine in alpha4beta2(Q443R) and alpha4(F588R)beta2 reduced gamma slightly. By contrast, the gamma of double mutant alpha4(F588R)beta2(Q443R) was halved. The MA -4' and 4' residues also influenced gamma of 5-HT3ARs. Replacement of nAChR alpha4 or beta2 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 alpha4beta2 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.
authors.affiliation: Department of Pharmacology and Physiology, George Washington University, Washington, DC 20037, USA. phmtgh@gwumc.edu
authors.forename: Tim G
authors.initials: TG
authors.lastname: Hales
authors.forename: James I
authors.initials: JI
authors.lastname: Dunlop
authors.forename: Tarek Z
authors.initials: TZ
authors.lastname: Deeb
authors.forename: Jane E
authors.initials: JE
authors.lastname: Carland
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Kelley
authors.forename: Jeremy J
authors.initials: JJ
authors.lastname: Lambert
authors.forename: John A
authors.initials: JA
authors.lastname: Peters
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D002118:Calcium / Q000737:chemistry; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism*; D004247:DNA / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004594:Electrophysiology; D006801:Humans; D007477:Ions; D008958:Models, Molecular; D015233:Models, Statistical; D008969:Molecular Sequence Data; D009154:Mutation; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats; D011978:Receptors, Nicotinic / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D012701:Serotonin / Q000737:chemistry*; D013997:Time Factors; D014101:Torpedo; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D007477:Ions; D011978:Receptors, Nicotinic; C403531:nicotinic receptor alpha4beta2; D012701:Serotonin; D004247:DNA; D001120:Arginine; D003545:Cysteine; D002118:Calcium