title: Molecular determinants of (+)-tubocurarine binding at recombinant 5-hydroxytryptamine3A receptor subunits.
abstract: The 5-hydroxytryptamine type 3 (5-HT3) receptor is a transmitter-gated ion channel mediating neuronal excitation. The receptor native to neurons, or as a homopentameric assembly of 5-HT3A receptor subunits, displays a species-dependent pharmacology exemplified by a 1800-fold difference in the potency of (+)-tubocurarine [(+)-Tc] as an antagonist of the current response mediated by mouse and human receptor orthologs. Here, we attempt to identify amino acid residues involved in binding (+)-Tc by use of chimeric and mutant 5-HT3A subunits of mouse and human expressed in Xenopus laevis oocytes. Replacement of the entire extracellular N-terminal domain of the mouse 5-HT3A (m5-HT3A) subunit by that of the human ortholog and vice versa exchanged the differential potency of (+)-Tc, demonstrating the ligand binding site to be contained wholly within this region. Mutagenesis of multiple amino acid residues within a putative binding domain that exchanged nonconserved residues between mouse and human receptors shifted the apparent affinity of (+)-Tc in a reciprocal manner. The magnitude of the shift increased with the number of residues (3, 5, or 7) exchanged, with septuple mutations of m5-HT3A and human 5-HT3A subunits producing a 161-fold decrease and 53-fold increase in the apparent affinity of (+)-Tc, respectively. The effect of point mutations was generally modest, the exception being m5-HT3A D206E, which produced a 9-fold decrease in apparent affinity. We conclude that multiple amino acids within a binding loop of human and mouse 5-HT3A subunits influence the potency of (+)-Tc.
authors.affiliation: Department of Pharmacology and Neuroscience, Ninewells Hospital and Medical School, The University of Dundee, Dundee, United Kingdom.
authors.forename: A G
authors.initials: AG
authors.lastname: Hope
authors.forename: D
authors.initials: D
authors.lastname: Belelli
authors.forename: I D
authors.initials: ID
authors.lastname: Mair
authors.forename: J J
authors.initials: JJ
authors.lastname: Lambert
authors.forename: J A
authors.initials: JA
authors.lastname: Peters
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D017354:Point Mutation; D011985:Receptors, Serotonin / Q000235:genetics / Q000378:metabolism*; D044406:Receptors, Serotonin, 5-HT3; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014403:Tubocurarine / Q000378:metabolism*
chemical_list: D011985:Receptors, Serotonin; D044406:Receptors, Serotonin, 5-HT3; D011993:Recombinant Fusion Proteins; D014403:Tubocurarine