Molecular determinants of (+)-tubocurarine binding at recombinant 5- hydroxytryptamine(3A) receptor subunits

Anthony G. Hope, Delia Belelli, Ian D. Mair, Jeremy J. Lambert, John A. Peters

Research output: Contribution to journalArticle

39 Citations (Scopus)

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-HT(3A) 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-HT(3A) subunits of mouse and human expressed in Xenopus laevis oocytes. Replacement of the entire extracellular N-terminal domain of the mouse 5- HT(3A) (m5-HT(3A)) 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 non-conserved 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-HT(3A) and human 5-HT(3A) 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-HT(3A) 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-HT(3A) subunits influence the potency of (+)-Tc.

Original languageEnglish
Pages (from-to)1037-1043
Number of pages7
JournalMolecular Pharmacology
Volume55
Issue number6
DOIs
Publication statusPublished - 1 Jan 1999

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Tubocurarine
Serotonin Receptors
Serotonin
Amino Acids
Receptors, Serotonin, 5-HT3
Xenopus laevis
Ion Channels
Point Mutation
Mutagenesis
Oocytes
Binding Sites
Pharmacology
Ligands
Neurons
Mutation

Cite this

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title = "Molecular determinants of (+)-tubocurarine binding at recombinant 5- hydroxytryptamine(3A) 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-HT(3A) 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-HT(3A) subunits of mouse and human expressed in Xenopus laevis oocytes. Replacement of the entire extracellular N-terminal domain of the mouse 5- HT(3A) (m5-HT(3A)) 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 non-conserved 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-HT(3A) and human 5-HT(3A) 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-HT(3A) 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-HT(3A) subunits influence the potency of (+)-Tc.",
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Molecular determinants of (+)-tubocurarine binding at recombinant 5- hydroxytryptamine(3A) receptor subunits. / Hope, Anthony G.; Belelli, Delia; Mair, Ian D.; Lambert, Jeremy J.; Peters, John A.

In: Molecular Pharmacology, Vol. 55, No. 6, 01.01.1999, p. 1037-1043.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Molecular determinants of (+)-tubocurarine binding at recombinant 5- hydroxytryptamine(3A) receptor subunits

AU - Hope, Anthony G.

AU - Belelli, Delia

AU - Mair, Ian D.

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AU - Peters, John A.

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N2 - 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-HT(3A) 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-HT(3A) subunits of mouse and human expressed in Xenopus laevis oocytes. Replacement of the entire extracellular N-terminal domain of the mouse 5- HT(3A) (m5-HT(3A)) 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 non-conserved 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-HT(3A) and human 5-HT(3A) 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-HT(3A) 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-HT(3A) subunits influence the potency of (+)-Tc.

AB - 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-HT(3A) 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-HT(3A) subunits of mouse and human expressed in Xenopus laevis oocytes. Replacement of the entire extracellular N-terminal domain of the mouse 5- HT(3A) (m5-HT(3A)) 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 non-conserved 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-HT(3A) and human 5-HT(3A) 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-HT(3A) 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-HT(3A) subunits influence the potency of (+)-Tc.

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DO - 10.1124/mol.55.6.1037

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SN - 0026-895X

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