Structure and Dynamics of the GABA Binding Pocket: A Narrowing Cleft that Constricts during Activation

Abstract

Photo-affinity labeling and mutagenesis studies have identified several amino acids that may contribute to the ligand binding domains of ligand-gated ion channels. These types of studies, however, only generate a one-dimensional, static description of binding site structure. In this study, we used the substituted cysteine accessibility method not only to identify binding pocket residues but also to elicit information about binding site dynamics and structure. Residues surrounding the putative loop C ligand binding domain of the GABA A receptor (␤ 2 V199 to ␤ 2 S209) were individually mutated to cysteine, and the mutant subunits were coexpressed with wild-type ␣ 1 subunits in Xenopus oocytes. N-biotinylaminoethyl methanethiosulfonate (MTSEAbiotin) reacts with cysteines introduced at positions G203, S204, Y205, P206, R207, and S209. This accessibility pattern is not consistent with either an ␣-helix or ␤-strand. Instead, G203-S209 seems to form a water-accessible extended coil, whereas V199-T202 appears to buried in the protein or membrane. Coapplication of either GABA or the competitive antagonist SR-95531 significantly slows MTSEA-biotin modification of cysteines introduced at positions S204, Y205, R207, and S209, demonstrating that these residues line and face into the GABA binding pocket. MTSEA-biotin reaction rates reveal a steep accessibility gradient from G203-S209 and suggests that the binding pocket is a deep narrowing cleft. Pentobarbital activation of the receptor significantly slows MTSEA-biotin modification of cysteines at S204, R207, and S209, suggesting that the binding site may constrict during gating.

Charged Residues in the α1and β2Pre-M1 Regions Involved in GABAAReceptor Activation

“…Residues mutated to cysteine are marked with a "C" above the residue. Residues located in the GABA binding site, ␤ 2 Y205 and ␤ 2 R207 (Amin and Weiss, 1993;Wagner and Czajkowski, 2001), and the binding sites of other LGICs (Kao et al, 1984;Mishina et al, 1985;Dennis et al, 1988;Ruiz-Gomez et al, 1990;Galzi et al, 1991a,b;Vandenberg et al, 1992a,b;Rajendra et al, 1995) are marked with an asterisk. Elements of secondary structure are indicated below the sequences by the arrow (␤-strand 10) and rectangle (␣ helix, M1 transmembrane region).…”

Charged Residues in the β2 Subunit Involved in GABAA Receptor Activation

“…Mutations that alter agonist efficacy will reduce the maximal current elicited by a saturating concentration of partial agonist (20). We used the low efficacy GABA A -R agonist P4S (7,11,17) to investigate the effects of these mutations. A comparison of the saturating P4S responses of the wild-type ␣ 1 ␤ 2 ␥ 2s GABA A -R ( Fig.…”

“…These differences in intradomain interactions between ␣ and ␤ subunits may reflect local dissimilarities in structure or an asymmetry in the propagation of conformational change within the individual subunits. A possible origin for such differences could be the asymmetric nature of the agonist-binding site itself, which is believed to constrict upon binding GABA (7). The constriction of this cleft between adjacent subunit polypeptides implies that the adjacent domains of the ␣ and ␤ subunits are essentially pulled in opposite directions, thereby resulting in an asymmetric change in subunit structure.…”

“…Each GABA A -R is composed of five subunits arranged around a central ion-conducting pore (4), with each subunit consisting of a large intracellular domain, four transmembrane domains (TM1-TM4), and a larger N-terminal extracellular domain (2). Experimental evidence suggests that the GABA-binding site lies within an asymmetric pocket formed at the interface between the ␣ and ␤ receptor subunits (5)(6)(7)(8). The channel "gate" in the GABA A -R is believed to be formed by charged residues in the TM1-TM2 loop (9 -11).…”

A Single M1 Residue in the β2 Subunit Alters Channel Gating of GABAA Receptor in Anesthetic Modulation and Direct Activation

“…Amino acids that participate in ligand binding have been identified by photoaffinity labeling and sequencing, as well as by site-directed mutagenesis (17)(18)(19)(20)(21)(22)(23). Loops of amino acids that form an acetylcholine-binding pocket were identified in the ␣ subunit of nAChR (24,25), and homologous residues involved in GABA binding were identified in ␤ subunits of GABAR (26,27). Additional amino acids involved in the GABA-binding site in the ␣ subunit were homologous to residues involved in binding acetylcholine in the ␥ subunit of nAChR (21).…”