Structural determinants of Ca2+ permeability and conduction in the human 5-hydroxytryptamine type 3A receptor

Matthew R. Livesey, Michelle A. Cooper, Tarek Z. Deeb, Jane E. Carland, Janna Kozuska, Tim G. Hales, Jeremy J. Lambert, John A. Peters

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    Abstract

    Cation-selective cysteine (Cys)-loop transmitter-gated ion channels provide an important pathway for Ca2+ entry into neurones. We examined the influence on Ca2+ permeation of amino acids located at intra-and extracellular ends of the conduction pathway of the human 5-hydroxytryptamine type 3A (5-HT3A) receptor. Mutation of cytoplasmic arginine residues 432, 436, and 440 to glutamine, aspartate, and alanine (the aligned residues of the human 5-HT3B subunit (yielding 5-HT3A(QDA)) increased P-Ca/P-Cs from 1.4 to 3.7. The effect was attributable to the removal of an electrostatic influence of the Arg-436 residue. Despite its relatively high permeability to Ca2+, the single channel conductance of the 5-HT3A(QDA) receptor was depressed in a concentration-dependent and voltage-independent manner by extracellular Ca2+. A conserved aspartate, located toward the extracellular end of the conduction pathway and known to influence ionic selectivity, contributed to the inhibitory effect of Ca2+ on macroscopic currents mediated by5-HT3A receptors. We introduced a D293A mutation into the 5HT(3A)(QDA) receptor(yielding the 5-HT3A(QDA D293A) construct) to determine whether the aspartate is required for the suppression of single channel conductance by Ca2+. The D293A mutation decreased the P-Ca/P-Cs ratio to 0.25 and reduced inwardly directed single channel conductance from 41 to 30 pS but did not prevent suppression of single channel conductance by Ca2+. The D293A mutation also reduced P-Ca/P-Cs when engineered into the wild-type 5-HT3A receptor. The data helped to identify key residues in the cytoplasmic domain (Arg-436) and extracellular vestibule (Asp-293) that markedly influence P-Ca/P-Cs and additionally directly demonstrated a depression of single channel conductance by Ca2+.

    Original languageEnglish
    Pages (from-to)19301-19313
    Number of pages13
    JournalJournal of Biological Chemistry
    Volume283
    Issue number28
    DOIs
    Publication statusPublished - 11 Jul 2008

    Keywords

    • SINGLE-CHANNEL CONDUCTANCE
    • GATED ION-CHANNEL
    • RECOMBINANT 5-HT3 RECEPTOR
    • CYS-LOOP FAMILY
    • NICOTINIC ACETYLCHOLINE
    • HEK-293 CELLS
    • SUBUNIT
    • SELECTIVITY
    • EXPRESSION
    • PERMEATION

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