Allosteric modulators induce distinct movements at the GABA-binding site interface of the GABA-A receptor

Abstract

Benzodiazepines (BZDs) and barbiturates exert their CNS actions by binding to GABA-A receptors (GABARs). The structural mechanisms by which these drugs allosterically modulate GABAR function, to either enhance or inhibit GABA-gated current, are poorly understood. Here, we used the substituted cysteine accessibility method to examine and compare structural movements in the GABA-binding site interface triggered by a BZD positive (flurazepam), zero (flumazenil) and negative (3-carbomethoxy-4-ethyl-6,7-dimethoxy-β-carboline, DMCM) modulator as well as the barbiturate pentobarbital. Ten residues located throughout the GABA-binding site interface were individually mutated to cysteine. Wild-type and mutant α(1)β(2)γ(2) GABARs were expressed in Xenopus laevis oocytes and functionally characterized using two-electrode voltage clamp. We measured and compared the rates of modification of the introduced cysteines by sulfhydryl-reactive methanethiosulfonate (MTS) reagents in the absence and presence of BZD-site ligands and pentobarbital. Flurazepam and DMCM each accelerated the rate of reaction at α(1)R131C and slowed the rate of reaction at α(1)E122C, whereas flumazenil had no effect indicating that simple occupation of the BZD binding site is not sufficient to cause movements near these positions. Therefore, BZD-induced movements at these residues are likely associated with the ability of the BZD to modulate GABAR function (BZD efficacy). Low, modulating concentrations of pentobarbital accelerated the rate of reaction at α(1)S68C and β(2)P206C, slowed the rate of reaction at α(1)E122C and had no effect at α(1)R131C. These findings indicate that pentobarbital and BZDs induce different movements in the receptor, providing evidence that the structural mechanisms underlying their allosteric modulation of GABAR function are distinct.

Figure 1. Location of residues substituted to cysteine at the GABA binding interface of the GABAR

(A) Homology model of the α₁ (cyan), β₂ (green) and γ₂ (light grey) subunits of the GABAR with residues substituted to cysteine labeled and highlighted in red. The region in the α₁ subunit that links the BZD and GABA binding interfaces (denoted LINKER) is highlighted in dark blue. (B) Partial sequences of α₁ (cyan) and β₂ (green) GABA binding site loop regions A (β-strand 4), B (β-strand 7), C (β-strand 10), D (β-strand 2), and E (β-strand 5–6), with residues substituted to cysteine in red (and denoted with the letter C below the residue). Numbering is based on mature protein sequences. LINKER residues are in dark blue, and residues previously implicated in lining the GABA binding pocket are underlined.

Figure 2. Effects of allosteric modulators on the rate of MTSEA-Biotin modification of α₁ subunit cysteine substitutions in loop E

(A) Representative GABA evoked (~EC_20–30) currents following successive 10 second MTSEA-biotin applications (arrows) in the absence (top traces) and presence (bottom traces) of 10μM flurazepam at α₁E122Cβ₂γ₂ receptors. (B) Representative rates of reaction of MTSEA-biotin in the absence or presence of 10μM flurazepam, 100nM DMCM, or 50μM pentobarbital at α₁E122β₂γ₂mutant receptors. Allosteric modulators significantly slowed the rate of reaction at position 122. (C) Representative GABA-evoked (~EC₅₀) currents following successive MTSEA-biotin applications (arrows) in the absence (top traces) and presence (bottom traces) of 10μM flurazepam at α₁R131Cβ₂γ₂ receptors. (D) Representative rates of reaction of MTSEA-biotin in the absence and presence of 10μM flurazepam or 25μM pentobarbital at α₁R131Cβ₂γ₂ receptors. Rate experiments were preformed as described under Materials and Methods. Increases or decreases in I_GABA (panels B & D) were plotted versus cumulative time of MTS exposure. Data were normalized and fit to a single-phase exponential decay as described in Materials and Methods. As multiple concentrations of MTS were used for the rate experiments, only representative rate plots, all obtained using the same concentration of MTS reagent, are shown for graphical clarity and comparative accuracy. For comparison of averaged normalized rates, see Figure 5.Se cond-order rate constants for MTS modification of α₁ subunit cysteine substitutions are summarized in TABLE 1.

Figure 3. Effects of allosteric modulators on the rate of MTS modification of α₁ subunit cysteine substitutions in loop D

(A) Representative rates of reaction of MTSES in the absence and presence of 10μM flurazepam, 1μM DMCM, or 50μM pentobarbital at α₁S68Cβ₂γ₂ mutant receptors. (B) Representative rates of reaction of MTSEA-biotin in the absence and presence of 10μM flurazepam, 1μM DMCM, and 50μM pentobarbital at α₁T60Cβ₂γ₂ mutant receptors. Allosteric modulation by pentobarbital, but not BZD-site ligands DMCM or flurazepam, significantly slowed the rate of MTS reaction at S68C. The rate of MTS reaction at T60C was not significantly altered in the presence of any allosteric modulator tested. Data were acquired, normalized, and fit to a single-phase exponential decay as described in Fig 2 and Materials and Methods. Second-order rate constants for MTS modification of α₁ subunit cysteine substitutions are summarized in TABLE 1.

Figure 4. Effects of allosteric modulators on the rate of MTSEA-biotin modification of β₂ subunit cysteine substitutions in loop C

(A) Representative rates of reaction of MTSEA-biotin in the absence and presence of 10μM flurazepam, 100nM DMCM, and 50μM pentobarbital at α₁β₂P206Cγ₂ receptors. Of the loop C residues tested, only position 206 displayed altered rates of reaction in the presence of allosteric modulators. (B) Representative rates of reaction of MTSEA-biotin in the absence and presence of 10μM flurazepam, 1μM DMCM, and 25μM pentobarbital at α₁β₂R207Cγ₂ receptors. Modulators had no effects on the rates of MTSEA-biotin modification at position 207. Data were acquired, normalized, and fit to a single-phase exponential decay as described in Fig 2 and Materials and Methods. Second-order rate constants for MTS modification of β₂ subunit cysteine substitutions are summarized in TABLE 1.

Figure 5. Summary of the effects of allosteric modulators on second-order rate constants (k₂) of MTS reaction at α₁ and β₂ subunit cysteine substitutions

Data were normalized to control second-order rate constants (the rate measured in the absence of allosteric modulators) and represent the mean±SEM of at least 3 experiments. Statistical significance was determined using a one-way ANOVA with Dunnett’s post-test. (A) Rates of modification of cysteines on the α₁ subunit. Flurazepam and DMCM significantly slowed the rate of reaction at position α₁E122C (*p<0.05), while increasing the rate of reaction at position α₁R131C (**p<0.01). Pentobarbital, but not flurazepam or DMCM, significantly increased the rate of reaction at α₁S68C (*p<0.05). (B) Rates of modification of cysteines on the β₂ subunit. With the exception of β₂P206C, allosteric modulators did not alter the rate of reaction at any of the positions tested on the β₂ subunit. Modulatory pentobarbital significantly increased the rate of reaction at β₂P206C (**p<0.01) and, while not significant by ANOVA, false discovery rate analysis suggests that both flurazepam and DMCM speed the rate of reaction.