Different Residues in the GABAA Receptor α1T60-α1K70 Region Mediate GABA and SR-95531 Actions

Although γ-aminobutyric acid type A receptor agonists and antagonists bind to a common site, they produce different conformational changes within the site because agonists cause channel opening and antagonists do not. We used the substituted cysteine accessibility method and two-electrode voltage c...

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Veröffentlicht in:The Journal of biological chemistry 2002-05, Vol.277 (21), p.18785
Hauptverfasser: Jessica H. Holden, Cynthia Czajkowski
Format: Artikel
Sprache:eng
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Zusammenfassung:Although γ-aminobutyric acid type A receptor agonists and antagonists bind to a common site, they produce different conformational changes within the site because agonists cause channel opening and antagonists do not. We used the substituted cysteine accessibility method and two-electrode voltage clamping to identify residues within the binding pocket that are important for mediating these different actions. Each residue from α 1 T60 to α 1 K70 was mutated to cysteine and expressed with wild-type β 2 subunits in Xenopus oocytes. Methanethiosulfonate reagents reacted with α 1 T60C, α 1 D62C, α 1 F64C, α 1 R66C, α 1 S68C, and α 1 K70C. γ-Aminobutyric acid (GABA) slowed methanethiosulfonate modification of α 1 F64C, α 1 R66C, and α 1 S68C, whereas SR-95531 slowed modification of α 1 D62C, α 1 F64C, and α 1 R66C, demonstrating that different residues are important for mediating GABA and SR-95531 actions. In addition, methanethiosulfonate reaction rates were fastest for α 1 F64C and α 1 R66C, indicating that these residues are located in an open, aqueous environment lining the core of the binding pocket. Positively charged methanethiosulfonate reagents derivatized α 1 F64C and α 1 R66C significantly faster than a negatively charged reagent, suggesting that a negative subsite important for interacting with the ammonium group of GABA exists within the binding pocket. Pentobarbital activation of the receptor increased the rate of methanethiosulfonate modification of α 1 D62C and α 1 S68C, demonstrating that parts of the binding site undergo structural rearrangements during channel gating.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M111778200