title: Structural determinants of Ca2+ permeability and conduction in the human 5-hydroxytryptamine type 3A receptor.
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 PCa/PCs 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 by 5-HT3A receptors. We introduced a D293A mutation into the 5-HT3A(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 PCa/PCs 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 PCa/PCs 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 PCa/PCs and additionally directly demonstrated a depression of single channel conductance by Ca2+.
authors.affiliation: Neurosciences Institute, Division of Pathology and Neuroscience, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom.
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Livesey
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Cooper
authors.forename: Tarek Z
authors.initials: TZ
authors.lastname: Deeb
authors.forename: Jane E
authors.initials: JE
authors.lastname: Carland
authors.forename: Janna
authors.initials: J
authors.lastname: Kozuska
authors.forename: Tim G
authors.initials: TG
authors.lastname: Hales
authors.forename: Jeremy J
authors.initials: JJ
authors.lastname: Lambert
authors.forename: John A
authors.initials: JA
authors.lastname: Peters
mesh_terms: D019943:Amino Acid Substitution; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000502:physiology*; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology; D009154:Mutation; D009474:Neurons / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000502:physiology; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D044406:Receptors, Serotonin, 5-HT3 / Q000235:genetics / Q000378:metabolism*
chemical_list: D021122:Protein Subunits; D044406:Receptors, Serotonin, 5-HT3; D002118:Calcium