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J Neurosci. 2006 Feb 15;26(7):2031-40.

Charged residues in the alpha1 and beta2 pre-M1 regions involved in GABAA receptor activation.

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  • 1Department of Physiology, Neuroscience Training Program, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.

Abstract

For Cys-loop ligand-gated ion channels (LGIC), the protein movements that couple neurotransmitter binding to channel gating are not well known. The pre-M1 region, which links the extracellular agonist-binding domain to the channel-containing transmembrane domain, is in an ideal position to transduce binding site movements to gating movements. A cluster of cationic residues in this region is observed in all LGIC subunits, and in particular, an arginine residue is absolutely conserved. We mutated charged pre-M1 residues in the GABAA receptor alpha1 (K219, R220, K221) and beta2 (K213, K215, R216) subunits to cysteine and expressed the mutant subunits with wild-type beta2 or alpha1 in Xenopus oocytes. Cysteine substitution of beta2R216 abolished channel gating by GABA without altering the binding of the GABA agonist [3H]muscimol, indicating that this residue plays a key role in coupling GABA binding to gating. Tethering thiol-reactive methanethiosulfonate (MTS) reagents onto alpha1K219C, beta2K213C, and beta2K215C increased maximal GABA-activated currents, suggesting that structural perturbations of the pre-M1 regions affect channel gating. GABA altered the rates of sulfhydryl modification of alpha1K219C, beta2K213C, and beta2K215C, indicating that the pre-M1 regions move in response to channel activation. A positively charged MTS reagent modified beta2K213C and beta2K215C significantly faster than a negatively charged reagent, and GABA activation eliminated modification of beta2K215C by the negatively charged reagent. Overall, the data indicate that the pre-M1 region is part of the structural machinery coupling GABA binding to gating and that the transduction of binding site movements to channel movements is mediated, in part, by electrostatic interactions.

PMID:
16481436
[PubMed - indexed for MEDLINE]
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