Mobility of the M2 segments of the transmembrane domain in muscle type nicotinic acetylcholine receptors

Date

2011-08

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Abstract

Ionotropic receptors such as the ligand gated ion channels mediate fast synaptic transmissions of impulses in the central nervous system. The Cys loop superfamily includes ligand gated ion channels that act as receptors for neurotransmitters acetylcholine (nACh), gamma aminobutyric acid (GABAA), glycine (Gly) and serotonin (5-HT3A). The muscle type nicotinic acetylcholine receptor assembles from five homologous subunits to form a heteropentamer. All subunits share a three domain topology and structure (1) An extracellular domain with two antiparallel ?-sheets housing the ligand binding site (2) A transmembrane domain that crosses the membrane with four distinct ?-helical segments (M1-M4) (3) and a long loop between M3 and M4 forms the intracellular domain. The channel is lined by five M2 segments contributed by individual subunits. This receptor has a known subunit arrangement of ????? in the clockwise direction when viewed from the extracellular side. Disulfide trapping between engineered pairs of cysteines is a method to assess the separation of the involved cysteines as well as give us a measure of extent of proximity and flexibility of the region containing the residues. We monitored inter-subunit disulfide bond formation between Cys pairs in different M2 segments in muscle nAChR expressed in Xenopus laevis oocytes by two electrode voltage clamp experiments. Chemically, cystine bond formation can be reversed by the reducing agent dithiothreitol (DTT). We investigated ?M2 Cys mutants in the ?C192S-C193S background with and without the corresponding ?M2 Cys mutants to determine which positions in M2 can form disulfide bonds and compared it to previously published studies in the homologous GABAA receptor. This study will help to understand the thermal mobility of the M2 segment and how different subunits move relative to one another during gating transitions.

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Keywords

Muscle type acetylcholine receptor, Mobility of transmembrane segment, Electrophysiology, Disulfide crosslinking

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