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The Journal of Neuroscience, February 15, 2006, 26(7):2031-2040; doi:10.1523/JNEUROSCI.4555-05.2006

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Cellular/Molecular
Charged Residues in the ₁ and ₂ Pre-M1 Regions Involved in GABA_A Receptor Activation

Jose Mercado and Cynthia Czajkowski

Department of Physiology and Neuroscience Training Program, University of Wisconsin–Madison, Madison, Wisconsin 53706

Correspondence should be addressed to Dr. Cynthia Czajkowski, University of Wisconsin–Madison, 601 Science Drive, Madison, WI 53711. Email: czajkowski{at}physiology.wisc.edu

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 ₁ (K219, R220, K221) and ₂ (K213, K215, R216) subunits to cysteine and expressed the mutant subunits with wild-type ₂ or ₁ in Xenopus oocytes. Cysteine substitution of ₂R216 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 ₁K219C, ₂K213C, and ₂K215C 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 ₁K219C, ₂K213C, and ₂K215C, indicating that the pre-M1 regions move in response to channel activation. A positively charged MTS reagent modified ₂K213C and ₂K215C significantly faster than a negatively charged reagent, and GABA activation eliminated modification of ₂K215C 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.

Key words: GABA; GABA_A receptor; pre-M1 region; substituted cysteine accessibility method; electrostatic interactions; allosteric protein; ligand-gated ion channel

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Received Oct. 24, 2005; revised Jan. 5, 2005; accepted Jan. 6, 2006.

Correspondence should be addressed to Dr. Cynthia Czajkowski, University of Wisconsin–Madison, 601 Science Drive, Madison, WI 53711. Email: czajkowski{at}physiology.wisc.edu

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