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

Tim G. Hales, James I. Dunlop, Tarek Z. Deeb, Jane E. Carland, Stephen P. Kelley, Jeremy J. Lambert, John A. Peters

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    Homomeric 5-hydroxytryptamine type 3A receptors (5-HT3ARs) have a single channel conductance (?) 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 ? 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 ? to 36.5 ± 1.0 pS. The 0' residue makes the most substantial contribution to ? of the 5-HT3AR. Replacement of 0'Arg by aspartate, glutamate (a7 nAChR subunit MA 0'), or glutamine (ß2 subunit MA 0') increases ? to the resolvable range (>6 pS). By contrast, replacement of 0'Arg by phenylalanine (a4 subunit MA 0') reduced ? to 416 ± 107 fS. In reciprocal experiments with a4ß2 nAChRs (? = 31.3 ± 0.8 pS), replacement of MA 0' residues by arginine in a4ß2(Q443R) and a4(F588R)ß2 reduced ? slightly. By contrast, the ? of double mutant a4(F588R)ß2(Q443R) was halved. The MA -4' and 4' residues also influenced ? of 5-HT3ARs. Replacement of nAChR a4 or ß2 MA 4' residues by arginine made current density negligible. By contrast, replacement of both -4' residues by arginine produced functional nAChRs with substantially reduced ? (11.4 ± 0.5 pS). Homology models of the 5-HT3A and a4ß2 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. Previous SectionNext SectionThere are five families of vertebrate Cys loop receptors as follows: the nicotinic acetylcholine receptor (nAChR),3 the 5-hydroxytryptamine type 3 receptor (5-HT3R), the zinc-activated ion channel, the ?-aminobutyric acid type A receptor, and the strychnine-sensitive glycine receptor (1, 2). Structural analysis by cryo-EM of tubular crystals prepared from the Torpedo marmorata electric organ revealed that five subunits combine in nAChRs, forming a rosette around the central ion channel (3). The second transmembrane (M2) domain of each subunit participates in lining the channel pore, and collectively they present a hydrophobic constriction adjacent to what is traditionally believed to be the rate-limiting portion of the ion conduction pathway that controls single channel conductance (?) and ionic selectivity (2, 4). The homomeric 5-HT3A receptor is unique among Cys loop receptors, having a ? below the resolution of single channel recording, estimated by variance analysis to be in the femtosiemen range. The incorporation of the 5-HT3B subunit into human heteromeric 5-HT3A/B receptors increases ? to 16 pS, enabling direct observation of events by single channel recording from outside-out patches (5). The use of chimeric 5-HT3A-5-HT3B constructs and site-directed mutagenesis revealed a critical role of three arginine residues within the MA helix of the M3-M4 cytoplasmic loop in determining ? (6, 7). This has prompted speculation that the MA helix may participate in the control of ? in other Cys loop receptors (4, 7, 8). In support of this interpretation, cryo-EM analysis revealed portals within the Torpedo nAChR formed, in part, by the MA helices of adjacent subunits that may participate in the ion conduction pathway (9). In this study we investigated the influence of the 5-HT3A subunit's MA helix Arg-432 (-4'), Arg-436 (0'), and Arg-440 (4') residues in the control of ?. We investigated the effect of introducing arginine into the equivalent locations within the nAChR a4 and ß2 subunits. Our data confirm the critical role of MA -4', 0', and 4' residues in controlling ? of 5-HT3A receptors and support the hypothesis that the MA helix also forms part of the ion conduction pathway of nAChRs. Our functional data provide support for the existence of cytoplasmic portals depicted in the 4 Å structural model of the nAChR (10).
    Original languageEnglish
    Pages (from-to)8062-8071
    Number of pages10
    JournalJournal of Biological Chemistry
    Issue number12
    Publication statusPublished - Mar 2006


    • Cytoplasm metabolism
    • Receptors
    • Nicotinic chemistry
    • Serotonin chemistry


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