Journal
JOURNAL OF NEUROSCIENCE
Volume 26, Issue 7, Pages 2031-2040Publisher
SOC NEUROSCIENCE
DOI: 10.1523/JNEUROSCI.4555-05.2006
Keywords
GABA; GABA(A) receptor; pre-M1 region; substituted cysteine accessibility method; electrostatic interactions; allosteric protein; ligand-gated ion channel
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Funding
- NINDS NIH HHS [NS34727] Funding Source: Medline
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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 GABA(A) receptor alpha(1) (K219, R220, K221) and beta(2) (K213, K215, R216) subunits to cysteine and expressed the mutant subunits with wild-type beta(2) or alpha(1) in Xenopus oocytes. Cysteine substitution of beta(2)R216 abolished channel gating by GABA without altering the binding of the GABA agonist [H-3] muscimol, indicating that this residue plays a key role in coupling GABA binding to gating. Tethering thiol-reactive methanethiosulfonate (MTS) reagents onto alpha(1)K219C, beta(2)K213C, and beta(2)K215C 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 alpha(1)K219C, beta(2)K213C, and beta(2)K215C, indicating that the pre-M1 regions move in response to channel activation. A positively charged MTS reagent modified beta(2)K213C and beta(2)K215C significantly faster than a negatively charged reagent, and GABA activation eliminated modification of beta(2)K215C 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.
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