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5-HT3 Receptor MX Helix Contributes to Receptor Function.

Published version
Peer-reviewed

Type

Article

Change log

Authors

Mocatta, James 
Mesoy, Susanne M 
Dougherty, Dennis A  ORCID logo  https://orcid.org/0000-0003-1464-2461

Abstract

5-HT3 receptors are members of the family of pentameric ligand-gated ion channels. Each subunit has an extracellular, transmembrane, and intracellular domain. Only part of the intracellular domain structure has been solved, revealing it contains two α-helical segments; one, the MA helix, is an extension of M4, while the other, the MX helix, is formed from residues located close to the end of M3. This MX helix is in distinct locations in open and closed receptor structures, suggesting it may play a role in function. Here, we explore this hypothesis using functional responses of Ala-substituted mutant receptors expressed in HEK293 cells. The data show altering many of the MX residues results in a small decrease in EC50 (up to 5-fold), although in one (H232A) this is increased. Radiolabeled ligand binding on selected mutants showed no change in binding affinity, indicating an effect on gating and not binding. In addition, five mutations (P316A, V317A, P318A, D319A, and H323A) initially resulted in nonfunctional receptors, but the function could be rescued by coexpression with a chaperone protein, suggesting a likely role in assembly or folding. Examination of previously obtained MD simulation data shows that the extent of MX encompassed by membrane lipids differs considerably in the open and closed structures, suggesting that lipid-protein interactions in this region could have a major effect on channel opening propensity. We conclude that the MX helix can modulate the function of the receptor and propose that its interactions with membrane lipids play a major role in this.

Description

Funder: AstraZeneca

Keywords

Cys-loop receptor, binding site, mutagenesis, noncanonical amino acid, nonsense suppression, Amino Acid Sequence, HEK293 Cells, Humans, Membrane Lipids, Receptors, Serotonin, 5-HT3, Serotonin

Journal Title

ACS Chem Neurosci

Conference Name

Journal ISSN

1948-7193
1948-7193

Volume Title

13

Publisher

American Chemical Society (ACS)
Sponsorship
Medical Research Council (MR/L021676/1)