title: Impact of intracellular domain flexibility upon properties of activated human 5-HT3 receptors.
abstract: OBJECTIVE
  It has been proposed that arginine residues lining the intracellular portals of the homomeric 5-HT3 A receptor cause electrostatic repulsion of cation flow, accounting for a single-channel conductance substantially lower than that of the 5-HT3 AB heteromer. However, comparison of receptor homology models for wild-type pentamers suggests that salt bridges in the intracellular domain of the homomer may impart structural rigidity, and we hypothesized that this rigidity could account for the low conductance.
  
  
  METHODS
  Mutations were introduced into the portal region of the human 5-HT3 A homopentamer, such that putative salt bridges were broken by neutralizing anionic partners. Single-channel and whole cell currents were measured in transfected tsA201 cells and in Xenopus oocytes respectively. Computational simulations of protein flexibility facilitated comparison of wild-type and mutant receptors.
  
  
  RESULTS
  Single-channel conductance was increased substantially, often to wild-type heteromeric receptor values, in most 5-HT3 A mutants. Conversely, introduction of arginine residues to the portal region of the heteromer, conjecturally creating salt bridges, decreased conductance. Gating kinetics varied significantly between different mutant receptors. EC50 values for whole-cell responses to 5-HT remained largely unchanged, but Hill coefficients for responses to 5-HT were usually significantly smaller in mutants. Computational simulations suggested increased flexibility throughout the protein structure as a consequence of mutations in the intracellular domain.
  
  
  CONCLUSIONS
  These data support a role for intracellular salt bridges in maintaining the quaternary structure of the 5-HT3 receptor and suggest a role for the intracellular domain in allosteric modulation of cooperativity and agonist efficacy.
authors.affiliation: Department of Pharmacology, University of Alberta, Edmonton, AB, Canada.
authors.forename: J L
authors.initials: JL
authors.lastname: Kozuska
authors.forename: I M
authors.initials: IM
authors.lastname: Paulsen
authors.forename: W J
authors.initials: WJ
authors.lastname: Belfield
authors.forename: I L
authors.initials: IL
authors.lastname: Martin
authors.forename: D J
authors.initials: DJ
authors.lastname: Cole
authors.forename: A
authors.initials: A
authors.lastname: Holt
authors.forename: S M J
authors.initials: SM
authors.lastname: Dunn
mesh_terms: D000818:Animals; D002460:Cell Line; D003198:Computer Simulation; D006801:Humans; D008564:Membrane Potentials; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D044406:Receptors, Serotonin, 5-HT3 / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D058827:Serotonin 5-HT3 Receptor Agonists / Q000494:pharmacology; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014162:Transfection; D014982:Xenopus laevis
chemical_list: C509409:HTR3A protein, human; D044406:Receptors, Serotonin, 5-HT3; D058827:Serotonin 5-HT3 Receptor Agonists
keywords: 5-HT3 receptor; allostery; constrained geometric simulation; cooperativity; electrophysiology; intracellular domain; ligand-gated ion channels; single-channel conductance