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.2001 Jul 15;21(14):4977-86.
doi: 10.1523/JNEUROSCI.21-14-04977.2001.

A (beta)-strand in the (gamma)2 subunit lines the benzodiazepine binding site of the GABA A receptor: structural rearrangements detected during channel gating

Affiliations

A (beta)-strand in the (gamma)2 subunit lines the benzodiazepine binding site of the GABA A receptor: structural rearrangements detected during channel gating

J A Teissére et al. J Neurosci..

Abstract

Benzodiazepines (BZDs) exert their effects in the CNS by binding to a modulatory site on GABA(A) receptors. Individual amino acids have been implicated in BZD recognition and modulation of the GABA(A) receptor, but the secondary structure of the amino acids contributing to the BZD binding site has not been elucidated. In this report we used the substituted cysteine accessibility method to understand the structural dynamics of a region of the GABA(A) receptor implicated in BZD binding, gamma(2)Y72-gamma(2)Y83. Each residue within this region was mutated to cysteine and expressed with wild-type alpha(1) and beta(2) subunits in Xenopus oocytes. Methanethiosulfonate (MTS) reagents were used to modify covalently the engineered cysteines, and the subsequent effects on BZD modulation of the receptor were monitored functionally by two-electrode voltage clamp. We identified an alternating pattern of accessibility to sulfhydryl modification, indicating that the region gamma(2)T73-gamma(2)T81 adopts a beta-strand conformation. By monitoring the ability of BZD ligands to impede the covalent modification of accessible cysteines, we also identified two residues within this region, gamma(2)A79 and gamma(2)T81, that line the BZD binding site. Sulfhydryl modification of gamma(2)A79C or gamma(2)T81C allosterically shifts the GABA EC(50) of the receptor, suggesting that certain MTS compounds may act as tethered agonists at the BZD binding site. Last, we present structural evidence that a portion of the BZD binding site undergoes a conformational change in response to GABA binding and channel gating (opening and desensitization). These data represent an important step in understanding allosteric communication in ligand-gated ion channels.

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Figures

Fig. 1.
Fig. 1.
Aligned partial sequences of the rat GABAA receptor γ1–3 and α1subunit isoforms.Numbering reflects alignment with the mature γ2 subunit. Residues in γ1 and γ3 that are identical to γ2 residues are represented by adash. γ2F77, thecircled residue, has been implicated previously in BZD binding and modulation (Buhr et al., 1997; Sigel et al., 1998). Each amino acid in the region γ2Y72–γ2Y83 was mutated individually to cysteine and is denoted by aCabove the corresponding wild-type γ2 residue.Underlined residues in α1 (Boileau et al., 1999) and γ2 represent amino acids that are accessible to sulfhydryl-specific modification after mutation to cysteine.
Fig. 2.
Fig. 2.
GABA (A) and BZD (B) concentration–response curves of wild-type α1β2γ2 GABAAreceptors and three representative mutant receptors: α1β2γ2D75C, α1β2γ2A79C, and α1β2γ2T81C. Oocytes expressing α1, β2, and γ2 or γ-mutant subunits were treated with increasing concentrations of GABA or flurazepam (FLZM) while current responses were recorded by using two-electrode voltage clamp.A, Responses to GABA are normalized toIGABA Max.B, FLZM potentiation ofIGABA was measured with 1 μmGABA. For each mutant, FLZM potentiation is normalized to maximal potentiation. Data were fit by nonlinear regression, as described in Materials and Methods. Experiments were performed at least three times with similar results. EC50 values obtained from the curve fits are reported in Table 1.
Fig. 3.
Fig. 3.
Zn2+ sensitivity of α1β2, α1β2γ2, and α1β2γ2 mutant GABAA receptors. GABA-activated current traces were recorded from oocytes expressing α1β2, α1β2γ2, α1β2γ2F77C, or α1β2γ2W82C receptors.Bars represent 10–20 sec applications of 10 μm GABA in the presence or absence of 10 μmZnCl2. Data reflect triplicate determinations.
Fig. 4.
Fig. 4.
MTSEA-biotin effects on the γ2Y72C–γ2Y83C region.A, Representative current traces from α1β2γ2A79C receptors showing FLZM modulation ofIGABA before and after a 2 min application of 2 mm MTSEA-biotin.I-bars denote potentiation ofIGABA measured during an application of 1 μm FLZM in the presence of 1 μm GABA. Note the decrease in FLZM potentiation and the increase inIGABA after MTSEA-biotin modification (arrow).B, Changes in FLZM potentiation after MTSEA-biotin modification of αβγ (wild-type;wt) and mutant receptors. The percentage of change in FLZM potentiation after modification is defined as [((FLZM PotentiationAfter/FLZM PotentiationBefore) − 1)·100]. A negative value represents a decrease in FLZM potentiation after MTSEA-biotin reaction, and a positive value represents an increase in FLZM potentiation after MTSEA-biotin reaction.Black barsindicate mutants in which the change in potentiation was significantly different (p < 0.01) fromwtreceptor calculated by a one-way ANOVA with a Dunnett's post test. Data represent mean ± SD from at least three independent experiments. *No detectable BZD potentiation ofIGABA; **no detectable γ2 subunit expression.
Fig. 5.
Fig. 5.
Rate of sulfhydryl modification of α1β2γ2A79C and α1β2γ2T81C receptors in the presence and absence of FLZM and Ro 15-1788.A, B, Representative GABA (1 μm) and GABA plus FLZM (1 μm each) current traces recorded from α1β2γ2A79C receptors.Arrows indicate 5 sec applications of 200 μm MTSEA-biotin alone (A) or 200 μm MTSEA-biotin plus 5 μm FLZM (B). FLZM potentiation ofIGABA was measured before and after each MTS treatment.I-bars on traces show BZD-potentiated current.C, Observed decreases in FLZM potentiation ofIGABA were plotted versus cumulative MTSEA-biotin exposure in α1β2γ2A79C receptors. Data obtained from individual experiments were normalized to the potentiation measured att = 0 and fit to single-exponential decay curves (▪, MTS alone; ●, MTS + 5 μm FLZM; ■, MTS + 5 μm Ro 15-1788). Data points are mean ± SD from at least three independent experiments.D, Rate experiments were performed similarly for receptors containing γ2T81C, except that 5 sec applications of 20 μm MTSEA-biotin-CAP were used in place of MTSEA-biotin (▪, MTS alone; ●, MTS + 5 μm FLZM; ■, MTS + 5 μm Ro 15-1788). The calculated second-order rate constants for the MTS reaction are presented in Table 2.
Fig. 6.
Fig. 6.
MTS modification of α1β2γ2A79C and α1β2γ2T81C receptors increasesIGABA. Traces represent the effect of 2 min applications (arrows) of 2 mmMTSEA-biotin or 2 mm MTSEA-biotin-CAP on current evoked by 3 μm GABA in oocytes expressing receptors containing either γ2A79C (A) or γ2T81C (B) subunits. The application of 2 mmMTSEA-biotin to oocytes expressing α1β2γ2(C) or α1β2γ2T81C (B) receptors had no significant effect onIGABA.
Fig. 7.
Fig. 7.
MTSEA-biotin and MTSEA-biotin-CAP shift GABA EC50 when linked covalently to γ2A79C.A, B, GABA concentration–response curves obtained from single oocytes expressing α1β2γ2A79C receptors before (▪) and after (▾) reaction with 2 mm MTSEA-biotin (A) or before (▪) and after (♦) reaction with 2 mm MTSEA-biotin-CAP (B). The experiments were repeated two additional times with similar results.C, GABA concentration–response curves obtained from α1β2γ2A79C receptors in the absence (▪) and presence (○) of 1 μm FLZM. Data were fit by nonlinear regression, as described in Materials and Methods. Data represent mean ± SEM from three independent experiments. EC50 values obtained from the curve fits are reported in Table 3.
Fig. 8.
Fig. 8.
A, Structures and lengths (in angstroms) of the different MTS reagents used in our experiments. Lengths were measured after energy minimization (<0.5 kcal/Å) and represent only the portion of the MTS reagent that covalently modifies an introduced cysteine. Cleavage points of each MTS reagent are indicated by anarrow.B, Summary of the second-order rate constants calculated for MTS derivitization of γ2D75C-, γ2A79C-, and γ2T81C-containing receptors. Oocytes expressing mutant receptors were incubated in the presence of MTS alone (control),MTS + FLZM,MTS + Ro 15-1788, orMTS + GABA. MTS reagents used were as follows: γ2D75C, MTSEA; γ2A79C, MTSEA-biotin; γ2T81C, MTSEA-biotin-CAP. Second-order rate constants were calculated for each MTS reaction and were normalized to the rate measured in the absence of ligand (control). Displayed values are mean ± SD from at least three independent experiments. *,**Indicate values significantly different from control MTS values, withp < 0.05 andp < 0.01, respectively.
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References

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