Agonist-, Antagonist-, and Benzodiazepine-Induced Structural Changes in the 1Met -Leu Region of the GABAA Receptor

The structural basis by which agonists, antagonists, and allosteric modulators exert their distinct actions on ligand-gated ion channels is poorly understood. We used the substituted cysteine accessibility method to probe the structure of the GABAA receptor in the presence of ligands that elicit different pharmacological effects. Residues in the 1Met -Leu region of the GABA binding site were individually mutated to cysteine and expressed with wild-type 2 and 2 subunits in Xenopus laevis oocytes. Using electrophysiology, we determined the rates of reaction of N-biotinaminoethyl methanethiosulfonate (MTSEA-biotin) with the introduced cysteines in the resting (unliganded) state and compared them with rates determined in the presence of GABA (agonist), 4-[6-imino-3-(4methoxyphenyl)pyridazin-1-yl]butanoic acid hydrobromide (SR-95531; antagonist), pentobarbital (allosteric modulator), and flurazepam (allosteric modulator). 1N115C, 1L117C, 1T129C, and 1R131C are predicted to line the GABA binding pocket because MTSEA-biotin modification of these residues decreased the amount of current elicited by GABA, and the rates/extents of modification were decreased both by GABA and SR-95531. Reaction rates of some substituted cysteines were different depending on the ligand, indicating that barbiturateand GABA-induced channel gating, antagonist binding, and benzodiazepine modulation induce specific structural rearrangements. Chemical reactivity of 1E122C was decreased by either GABA or pentobarbital but was unaltered by SR95531 binding, whereas 1L127C reactivity was decreased by agonist and antagonist binding but not affected by pentobarbital. Furthermore, 1E122C, 1L127C, and 1R131C changed accessibility in response to flurazepam, providing structural evidence that residues in and near the GABA binding site move in response to benzodiazepine modulation. GABAA receptors are ligand-gated ion channels (LGICs) that mediate fast inhibitory synaptic neurotransmission in the brain and are allosterically modulated by a variety of compounds, including benzodiazepines (BZDs), barbiturates, anesthetics, and neuroactive steroids (Akabas, 2004). Identifying conformational movements that occur in the receptor in response to binding of these different classes of compounds is critical for understanding the molecular mechanisms underlying their therapeutic actions. According to allosteric theory, modulators bind to a site on the protein that is distinct from the agonist binding site and exert their effects by initiating an allosteric transition in the protein that indirectly modifies the conformation of the binding site (Changeux and Edelstein, 1998). In this study, we used the substituted cysteine accessibility method to monitor the accessibility of residues in the 1Met -Leu region of the GABA binding site in the presence of GABAergic ligands that elicit distinct pharmacological effects. In particular, we were interested in determining whether BZD binding induces structural rearrangements in or near the GABA binding site. In addition, we were interested in investigating the mechanisms underlying how orthosteric agonist binding triggers channel opening and orthosteric antagonist binding does not. The GABAA receptor belongs to the Cys-loop superfamily of LGICs, which includes the nicotinic acetylcholine (nACh), This work was supported by a Pharmaceutical Research and Manufacturers of America Foundation Predoctoral Fellowship (to J.H.K.), National Institute of Neurological Disorders and Stroke grant NS34727 (to C.C.), and National Alliance for Research on Schizophrenia and Affective Disorder Award (to C.C.). Portions of this work were previously presented at the Annual Meeting of the Society for Neuroscience 2001 in San Diego, CA and 2003 in New Orleans, LA. 1 Current affiliation: Laboratory of Membrane Biochemistry & Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.106.028662. ABBREVIATIONS: LGIC, ligand-gated ion channel; BZD, benzodiazepine; nACh, nicotinic acetylcholine; 5-HT3, serotonin-type-3; AChBP, acetylcholine binding protein; SR-95531, 4-[6-imino-3-(4-methoxyphenyl)pyridazin-1-yl]butanoic acid hydrobromide; MTSES, methanethiosulfonate ethylsulfonate; MTSEA, methanethiosulfonate ethylammonium; LBD, ligand binding domain. 0026-895X/07/7102-483–493$20.00 MOLECULAR PHARMACOLOGY Vol. 71, No. 2 Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics 28662/3173742 Mol Pharmacol 71:483–493, 2007 Printed in U.S.A.