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

BACKGROUND AND SIGNIFICANCE 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. EXPERIMENTAL APPROACH We used disulphide trapping between engineered cysteines to probe BZD‐induced conformational changes within the γ 2 subunit and at the α 1 /γ 2 coupling interface (Loops 2, 7 and 9) of α 1 β 2 γ 2 GABA A receptors. KEY RESULTS Crosslinking γ 2 Loop 9 to γ 2 β‐strand 9 (via γ 2 S195C/F203C and γ 2 S187C/L206C) significantly decreased maximum potentiation by flurazepam, suggesting that modulation of GABA‐induced current (I GABA ) by flurazepam involves movements of γ 2 Loop 9 relative to γ 2 β‐strand 9. In contrast, tethering γ 2 β‐strand 9 to the γ 2 pre‐M1 region (via γ 2 S202C/S230C) significantly enhanced potentiation by both flurazepam and zolpidem, indicating γ 2 S202C/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 α 1 Loop 2 and γ 2 Loop 9 (α 1 D56C/γ 2 L198C) prevented flurazepam and zolpidem from efficiently modulating I GABA . Disulphide trapping α 1 Loop 2 (α 1 D56C) to γ 2 β‐strand 1 (γ 2 P64C) 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 . CONCLUSIONS AND IMPLICATIONS Positive modulation of GABA A receptors by BZDs requires reorganization of the loops in the α 1 /γ 2 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.