Abstract
The mechanisms by which the GABA and benzodiazepine (BZD) binding sites of the GABA-A receptor are allosterically coupled remain elusive. In this study, we separately monitored ligand-induced structural changes in the BZD binding site (α/γ interface) and at aligned positions in the α/β interface. α1His101 and surrounding residues were individually mutated to cysteine and expressed with wild-type β2 and γ2 subunits in Xenopus laevis oocytes. The accessibilities of introduced cysteines to modification by methanethiosulfonate ethylammonium (MTSEA)-Biotin were measured in the presence and absence of GABA-site agonists, antagonists, BZDs, and pentobarbital. The presence of flurazepam or the BZD-site antagonist flumazenil (Ro15-1788) decreased the rate of modification of α1H101C at the BZD binding site. GABA and muscimol each increased MTSEA-Biotin modification of α1H101C located at the BZD-site, gabazine (SR-95531, a GABA binding site antagonist) decreased the rate, whereas pentobarbital had no effect. Modification of α1H101C at the α/β interface was significantly slower than modification of α1H101C at the BZD site, and the presence of GABA or flurazepam had no effect on its accessibility, indicating the physicochemical environments of the α/γ and α/β interfaces are different. The data are consistent with the idea that GABA-binding site occupation by agonists causes a GABA binding cavity closure that is directly coupled to BZD binding cavity opening, and GABA-site antagonist binding causes a movement linked to BZD binding cavity closure. Pentobarbital binding/gating resulted in no observable movements in the BZD binding site near α1H101C, indicating that structural mechanisms underlying allosteric coupling between the GABA and BZD binding sites are distinct.
Footnotes
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This work was supported by National Institutes of Health, National Institute of Neurological Disorders and Stroke grant NS34727 (to C.C.).
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This work appears in the doctoral thesis of L.M.S.: Structural Elements That Govern Benzodiazepine Modulation of the GABA-A Receptor. Ph.D. thesis, University of Wisconsin-Madison, 2004. This work was previously presented in abstract form: Sharkey LM, Czajkowski C. Evidence that the two α1 subunit interfaces (α/γ and α/β) of the GABAA receptor are structurally different and play different roles in receptor function. Soc Neurosci Abstr28:40.4.
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ABBREVIATIONS: BZD, benzodiazepine; MTSEA, methanethiosulfonate ethylammonium; nAChR, nicotinic acetylcholine receptor; BCCM, methyl betacarboline-3-carboxylate; FLZM, flurazepam; GABA-A, GABA type A; PENTO, pentobarbital; Ro15-4513, ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo(1,5-α)(1,4)benzodiazepine-3-carboxylate; SR95531, gabazine (2-(3-carboxypropyl)-3-amino-6-(4 methoxyphenyl)pyridazinium bromide); Ro15-1788, flumazenil (8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a][1,4]benzodiazepine-3-carboxylic acid, ethyl ester).
- Received January 3, 2008.
- Accepted April 18, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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