Disulphide trapping of the GABA(A) receptor reveals the importance of the coupling interface in the action of benzodiazepines.

Although the functional effects of benzodiazepines (BZDs) on GABA(A) receptors have been well characterized, the structural mechanism by which these modulators alter activation of the receptor by GABA is still undefined. We used disulphide trapping between engineered cysteines to probe BZD-induced conformational changes within the γ₂ subunit and at the α₁/γ₂ coupling interface (Loops 2, 7 and 9) of α₁β₂γ₂ GABA(A) receptors. Crosslinking γ₂ Loop 9 to γ₂β-strand 9 (via γ₂ S195C/F203C and γ₂ S187C/L206C) significantly decreased maximum potentiation by flurazepam, suggesting that modulation of GABA-induced current (I(GABA)) by flurazepam involves movements of γ₂ Loop 9 relative to γ₂β-strand 9. In contrast, tethering γ₂β-strand 9 to the γ₂ pre-M1 region (via γ₂S202C/S230C) significantly enhanced potentiation by both flurazepam and zolpidem, indicating γ₂S202C/S230C trapped the receptor in a more favourable conformation for positive modulation by BZDs. Intersubunit disulphide bonds formed at the α/γ coupling interface between α₁ Loop 2 and γ₂Loop 9 (α₁D56C/γ₂L198C) prevented flurazepam and zolpidem from efficiently modulating I(GABA) . Disulphide trapping α₁ Loop 2 (α₁D56C) to γ₂β-strand 1 (γ₂P64C) decreased maximal I(GABA) as well as flurazepam potentiation. None of the disulphide bonds affected the ability of the negative modulator, 3-carbomethoxy-4-ethyl-6,7-dimethoxy-β-carboline (DMCM), to inhibit I(GABA) . Positive modulation of GABA(A) receptors by BZDs requires reorganization of the loops in the α₁/γ₂ coupling interface. BZD-induced movements at the α/γ coupling interface likely synergize with rearrangements induced by GABA binding at the β/α subunit interfaces to enhance channel activation by GABA.