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Impact of intracellular domain flexibility upon properties of activated human 5-HT(3) receptors*

BACKGROUND AND PURPOSE: It has been proposed that arginine residues lining the intracellular portals of the homomeric 5-HT(3)A receptor cause electrostatic repulsion of cation flow, accounting for a single-channel conductance substantially lower than that of the 5-HT(3)AB heteromer. However, compari...

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Detalles Bibliográficos
Autores principales: Kozuska, J L, Paulsen, I M, Belfield, W J, Martin, I L, Cole, D J, Holt, A, Dunn, S M J
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley & Sons Ltd 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3966743/
https://www.ncbi.nlm.nih.gov/pubmed/24283776
http://dx.doi.org/10.1111/bph.12536
Descripción
Sumario:BACKGROUND AND PURPOSE: It has been proposed that arginine residues lining the intracellular portals of the homomeric 5-HT(3)A receptor cause electrostatic repulsion of cation flow, accounting for a single-channel conductance substantially lower than that of the 5-HT(3)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. EXPERIMENTAL APPROACH: Mutations were introduced into the portal region of the human 5-HT(3)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. KEY RESULTS: Single-channel conductance was increased substantially, often to wild-type heteromeric receptor values, in most 5-HT(3)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. EC(50) 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 AND IMPLICATIONS: These data support a role for intracellular salt bridges in maintaining the quaternary structure of the 5-HT(3) receptor and suggest a role for the intracellular domain in allosteric modulation of cooperativity and agonist efficacy. LINKED ARTICLE: This article is commented on by Vardy and Kenakin, pp. 1614–1616 of volume 171 issue 7. To view this commentary visit http://dx.doi.org/10.1111/bph.12550.