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A Single Mutation in The Outer Lipid-Facing Helix of a Pentameric Ligand-Gated Ion Channel Affects Channel Function through a Radially-Propagating Mechanism

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Alessandro Crnjar, Susanne M. Mesoy, Sarah C. R. Lummis, Carla Molteni

Original languageEnglish
Article number644720
Pages (from-to)155
JournalFrontiers in Molecular Biosciences
Volume8
DOIs
Accepted/In press22 Feb 2021
Published30 Apr 2021

Bibliographical note

Funding Information: AC and CM are grateful for computational support from the UK high performance computing service ARCHER, for which access was obtained via the UKCP consortium and funded by EPSRC grant EP/P022472/1; they also acknowledge the UK Materials and Molecular Modeling Hub for computational resources, which is partially funded by EPSRC grants EP/P020194/1 and EP/T022213/1. For the experimental work, SM was supported by an AstraZeneca studentship. SL was supported by MRC grant MR L021676. Publisher Copyright: © Copyright © 2021 Crnjar, Mesoy, Lummis and Molteni. Copyright: Copyright 2021 Elsevier B.V., All rights reserved.

King's Authors

Abstract

Pentameric ligand-gated ion channels (pLGICs) mediate fast synaptic transmission and are crucial drug targets. Their gating mechanism is triggered by ligand binding in the extracellular domain that culminates in the opening of a hydrophobic gate in the transmembrane domain. This domain is made of four α-helices (M1 to M4). Recently the outer lipid-facing helix (M4) has been shown to be key to receptor function, however its role in channel opening is still poorly understood. It could act through its neighboring helices (M1/M3), or via the M4 tip interacting with the pivotal Cys-loop in the extracellular domain. Mutation of a single M4 tyrosine (Y441) to alanine renders one pLGIC—the 5-HT3A receptor—unable to function despite robust ligand binding. Using Y441A as a proxy for M4 function, we here predict likely paths of Y441 action using molecular dynamics, and test these predictions with functional assays of mutant receptors in HEK cells and Xenopus oocytes using fluorescent membrane potential sensitive dye and two-electrode voltage clamp respectively. We show that Y441 does not act via the M4 tip or Cys-loop, but instead connects radially through M1 to a residue near the ion channel hydrophobic gate on the pore-lining helix M2. This demonstrates the active role of the M4 helix in channel opening.

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