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 (beta(2)V199 to beta(2)S209) were individually mutated to cysteine, and the mutant subunits were coexpressed with wild-type alpha(1) subunits in Xenopus oocytes. N-biotinylaminoethyl methanethiosulfonate (MTSEA-biotin) reacts with cysteines introduced at positions G203, S204, Y205, P206, R207, and S209. This accessibility pattern is not consistent with either an alpha-helix or beta-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.

Fig. 1.

Alignment of loop C domains from different LGICs. The β₂ loop C domain of the GABA binding site is aligned with homologous domains from the benzodiazepine binding site of the GABA_A α₁ subunit, the acetylcholine binding site of the nicotinic acetylcholine receptor α₁ subunit, and the glycine binding site of the glycine receptor α₁subunit. Residues that have been predicted to be in or near the binding pocket by photo-affinity labeling or mutagenesis are shown inbold (Galzi and Changeux, 1994). Residues in β₂ that were mutated to cysteines are denoted by aCabove the wild-type residue.

Fig. 2.

GABA dose–response curves and P4S currents.A, GABA dose–response relationships for wild-type α₁β₂ receptors (●) and three representative mutants: α₁β₂-R207C (▴), α₁β₂-S201C (♦), and α₁β₂-Y205C (▾). Data were fit by nonlinear regression as described in Materials and Methods. All data points are normalized to I_max-GABA and are shown as mean responses ± SEM from four or more cells.B, Current traces recorded from oocytes expressing wild type or α₁β₂-S201C. Arrowsindicate a 5 sec application of saturating P4S (wild type, 1 mm; S201C, 10 mm) or GABA (wild type, 1 mm; S201C, 100 mm). Line breakin current trace represents 5 min wash with ND96. C, Bar graph denoting P4S efficacy of wild-type and mutant receptors asI_max-P4S/I_max-GABAwhere values given as mean ± SEM follow: α₁β₂, 0.50 ± 0.03,n = 4; α₁β₂-F200C, 0.45 ± 0.06, n = 4; α₁β₂-S201C, 0.12 ± 0.01,n = 3; and α₁β₂-R207C, 0.21 ± 0.02, n = 4. * p< 0.01 indicates values that are significantly different from wild type calculated using a one-way ANOVA with a Dunnett's post hoc test.

Fig. 3.

Effects of MTSEA-biotin on wild-type and mutant GABA_A receptors. A, Representative current traces demonstrating the effect of MTSEA-biotin treatment (2 mm, 2 min) on currents from wild-type and Y205C-containing receptors. For wild-type traces, [GABA] is 3 μm, and for Y205C traces, [GABA] is 30 mm. B, Effect of MTSEA-biotin treatment on all mutants shown as % change = ([I_{GABA-post MTSEA-biotin}/I_{GABA-pre MTSEA-biotin}] − 1) × 100. Results represent the mean ± SEM of at least three experiments. Black bars indicate that the percent change is significantly different from wild type (p < 0.01). Gray barsindicate no significant difference from wild type (p > 0.05).

Fig. 4.

Measurement of MTSEA-biotin reaction rates.A, B, Examples of traces recorded during experiments measuring the reaction rate of MTSEA-biotin with α₁β₂-R207C receptors. Downward deflections represent inward current elicited by a 5 sec application of 300 μm GABA (≈EC₅₀).Arrows indicate either 10 sec application of 200 μm MTSEA-biotin (A) or a 20 sec coapplication of MTSEA-biotin plus 1 μm SR-95531 (B). C, NormalizedI_GABA plotted as a function of cumulative time of MTSEA-biotin exposure. Single exponential curve fits illustrate the effect of various compounds on the reaction rate of MTSEA-biotin with α₁β₂-R207C receptors. Data points are normalized to the current measured at t = 0 and are presented as mean ± SEM. PB, Pentobarbital.

Fig. 5.

Summary of effect of GABA, SR-95531, and pentobarbital on the rate at which MTSEA-biotin modifies introduced cysteines. Second-order rate constants were calculated for each reaction, and for each mutant, the rates were normalized to the control rate (rate measured when no other compound is present). *p < 0.01 indicates that rate is significantly different from control rate. All data represent the mean ± SEM of at least three experiments.

Fig. 6.

Graphic summary of results. Barsindicate which residues fall into each of the following categories:Mediates K_D-GABA, mutation of this residue alters microscopic affinity for GABA; Mediates Efficacy, mutation of this residue reduces efficacy of P4S;Accessible to MTS, we can detect reaction of MTSEA-biotin with a cysteine introduced at this position; In Binding Pocket, the rate at which MTSEA-biotin reacts with a cysteine introduced at this residue is slowed by the presence of both GABA and SR-95531. Relative Rxn. Rate, The height of thebar is scaled to the log ofk₂ for each mutant with MTSEA-biotin.