Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 278, Issue 15, Pages 13166-13172Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M211905200
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- NINDS NIH HHS [NS 34727] Funding Source: Medline
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The GABA-binding site undergoes structural rearrangements during the transition from agonist binding to channel opening. To define possible roles of the GABA(A) receptor a, subunit Pro(174)-Asp(191) segment in these processes, we used the substituted cysteine accessibility method to characterize this region. Each residue was individually mutated to cysteine, expressed with wild-type beta(2) subunits in Xenopus laevis oocytes, and examined using two-electrode voltage clamp. Most mutations did not alter GABA EC50 values. The D183C mutation produced a 7-fold reduction in GABA sensitivity. There were no significant changes in the K, values for the competitive antagonist, SR-95531. N-Biotinylaminoethyl methanethiosulfonate modified P174C-, R176C-, S177C-, V178C-, V180C-, A181C-, D183C-, R186C- and N188C-containing receptors. The pattern of accessibility suggests that this protein segment is aqueous-exposed and adopts a random coil conformation. Both GABA and SR-95531 slowed covalent modification of V178C, V180C, and D183C, indicating that these residues may line the GABA-binding site. Further, pentobarbital-induced channel activation accelerated modification of V180C and A181C and slowed the modification of R186C, suggesting that this region of the a, subunit may act as a dynamic element during channel-gating transitions.
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