Structural mechanisms underlying benzodiazepine modulation of the GABA(A) receptor

Abstract

Many clinically important drugs target ligand-gated ion channels; however, the mechanisms by which these drugs modulate channel function remain elusive. Benzodiazepines (BZDs), anesthetics, and barbiturates exert their CNS actions by binding to GABA(A) receptors and modulating their function. The structural mechanisms by which BZD binding is transduced to potentiation or inhibition of GABA-induced current (I(GABA)) are essentially unknown. Here, we explored the role of the gamma(2)Q182-R197 region (Loop F/9) in the modulation of I(GABA) by positive (flurazepam, zolpidem) and negative [3-carbomethoxy-4-ethyl-6,7-dimethoxy-beta-carboline (DMCM)] BZD ligands. Each residue was individually mutated to cysteine, coexpressed with wild-type alpha(1) and beta(2) subunits in Xenopus oocytes, and analyzed using two-electrode voltage clamp. Individual mutations differentially affected BZD modulation of I(GABA). Mutations affecting positive modulation span the length of this region, whereas gamma(2)W183C at the beginning of Loop F was the only mutation that adversely affected DMCM inhibition. Radioligand binding experiments demonstrate that mutations in this region do not alter BZD binding, indicating that the observed changes in modulation result from changes in BZD efficacy. Flurazepam and zolpidem significantly slowed covalent modification of gamma(2)R197C, whereas DMCM, GABA, and the allosteric modulator pentobarbital had no effects, demonstrating that gamma(2)Loop F is a specific transducer of positive BZD modulator binding. Therefore, gamma(2)Loop F plays a key role in defining BZD efficacy and is part of the allosteric pathway allowing positive BZD modulator-induced structural changes at the BZD binding site to propagate through the protein to the channel domain.

Figure 1.

The GABA_A receptor α₁/γ₂ interface and structures of benzodiazepine binding site ligands. A, Homology model of the α₁ (red) and γ₂ (blue) subunits of the GABA_A receptor (Mercado and Czajkowski, 2006). BZD binding site Loops A-E are highlighted in yellow; Loop F is green. The dashed lines indicate the limits of the cell membrane. B, Structures of BZD binding site ligands used in this study.

Figure 2.

Cysteine mutations in γ₂Loop F affect the potentiation of I_GABA by flurazepam and zolpidem. A, Potentiation of 1 μm GABA responses by 3 μm FZM (left) and 10 μm ZPM (right) is graphed for WT and γ₂-mutant receptors. Dashed lines indicate the level of WT potentiation. Black bars indicate mutants in which the change in potentiation was significantly different from WT receptors (*p < 0.05; **p < 0.01). Data represent mean ± SD from at least three separate experiments. B, Concentration–response curves of WT and three representative mutant receptors: α₁β₂γ₂W183C, α₁β₂γ₂E189C, and α₁β₂γ₂R197C. Current responses (GABA EC_2–5) were recorded from oocytes expressing α₁, β₂, and γ₂- or γ₂- mutant subunits after treatment with increasing concentrations of FZM (left) or ZPM (right). The potentiation response ratio (y-axis) was calculated as [(I_GABA+BZD/I_GABA) − 1]. Data were fit by nonlinear regression, as described in Materials and Methods, and represent mean ± SD from at least three cells from two or more batches of oocytes. Maximum potentiation, EC₅₀ values, and Hill coefficients are presented in Table 1. Insets, The same data are plotted after normalizing to the maximum potentiation for each receptor to show differences in BZD EC₅₀.

Figure 3.

DMCM modulation and Zn²⁺ sensitivity of WT and mutant GABA_A receptors. A, Maximal inhibition of 1 μm GABA responses by DMCM (1 μm) is graphed for WT and γ₂-mutant receptors. The dashed line indicates the level of WT inhibition. The black bar for γ₂W183C indicates that the change in modulation was significantly different from WT receptors (**p < 0.01). Data represent mean ± SD from at least three separate experiments. B, Representative current traces for negative modulation of 1 μm GABA by 1 μm DMCM from oocytes injected with WT, α₁β₂γ₂W183C, and α₁β₂γ₂R197C receptors. Inhibition of GABA current was calculated as [(I_GABA+DMCM/I_GABA) − 1]. C, Representative current traces from oocytes expressing α₁β₂, α₁β₂γ₂, α₁β₂γ₂W183C, and α₁β₂γ₂R197C receptors. In each case, currents were recorded after application of 1 mm GABA in the presence or absence of 30 μm ZnCl₂. When ZnCl₂ was used, it was preapplied to the oocyte for 20 s before application of GABA.

Figure 4.

Cysteine mutations in γ₂Loop F do not significantly alter benzodiazepine binding to the GABA_A receptor. Representative radioligand binding curves depict the displacement of [³H] Ro15-1788 binding by unlabeled Ro15-1788 (top left), flurazepam (top right), zolpidem (bottom left), and DMCM (bottom right) for WT, α₁β₂γ₂W183C, α₁β₂γ₂E189C, and α₁β₂γ₂R197C receptors, where each point is the mean ± SEM of triplicate measurements. Data were fit by nonlinear regression as described in Materials and Methods. K_i values are reported in Table 2.

Figure 5.

MTS modification of γ₂Loop F mutants affects I_GABA and flurazepam potentiation. A, Structures and lengths (in angstroms) of MTS reagents used in this study. Lengths represent only the portion of the MTS reagent that covalently modifies an introduced cysteine. B, Representative current traces from α₁β₂γ₂V188C receptors recorded during application of GABA (EC₅₀) before and after treatment with 2 mm MTSEA-Biotin for 2 min. Note the increase in I_GABA after treatment. C, Representative current traces from α₁β₂γ₂R197C receptors showing FZM modulation of I_GABA before and after a 2 min treatment with 2 mm MTSEA-Biotin. I bars denote potentiation of I_GABA measured during application of 1 μm FZM in the presence of 1 μm GABA. Note the increase in potentiation after treatment. D, Changes in I_GABA (GABA EC₅₀) for WT and γ₂-mutant receptors after a 2 min, 2 mm application of MTSEA-Biotin (black bars) or MTSET (gray bars) are graphed. Percentage change is defined as follows: ([(I_{GABA after}/I_{GABA before}) − 1] × 100). Error bars represent mean ± SD of at least three independent experiments. Values significantly different from WT are indicated (*p < 0.05; **p < 0.01). E, Changes in FZM potentiation after 2 min, 2 mm MTSEA-Biotin modification of WT and γ₂-mutant receptors. The percentage change in FZM potentiation is defined as follows: ([(FZM potentiation_after/FZM potentiation_before) − 1] × 100). Values significantly different from WT are indicated (**p < 0.01).

Figure 6.

Positive BZD modulators alter the rate of MTS modification of α₁β₂γ₂R197C receptors. The rate of sulfhydryl modification of α₁β₂γ₂R197C receptors in the presence and absence of FZM, ZPM, DMCM, GABA, and PB. A, B, Representative GABA (1 μm) and GABA plus FZM (1 μm each) current traces recorded from α₁β₂γ₂R197C receptors. Arrows indicate 10 s application of 5 mm MTSET alone (A) or 5 mm MTSET plus 5 μm FZM (B). FZM potentiation (denoted by I bars) was measured before and after each MTS treatment. Similar recordings were made after application of MTSET in the presence of 1 μm ZPM, 1 μm DMCM, 100 μm GABA, 50 μm PB (low PB), and 500 μm PB (high PB) (data not shown). C, Observed increases in FZM potentiation of I_GABA were plotted versus cumulative MTSET exposure in the absence (control) and presence of DMCM, ZPM, and FZM. Data obtained from individual experiments were normalized to the potentiation measured at t = 0 and fit to single-exponential association curves. Data points are mean ± SD from at least three independent experiments. Plots of the changes in FZM potentiation after MTSET exposure in the presence of GABA and PB resemble control and are not shown for clarity (D). Second-order rate constants (k₂) for MTSET modification of α₁β₂γ₂R197C receptors in the absence (control) and presence of various BZD ligands, GABA, and PB were calculated as described in Materials and Methods. Values significantly different from the control rate are indicated (**p < 0.01).

Figure 7.

The localization of γ₂Loop F residues at the binding-channel interface. Homology model of the α₁ (red) and γ₂ (blue) subunits of the GABA_AR with γ₂ residues Arg197 and Trp183 shown space filled in orange and pink, respectively. Loop F is highlighted in green, and β-strand 9 is yellow. The general location of the BZD binding site (α/γ interface) and the GABA binding site (α/β interface) is indicated (β subunit not shown). Loops 2, 7, and M2-M3 and the pre-M1 region are labeled on only one subunit for clarity.