Closed-state Cross-linking of Adjacent

The pore structural changes associated with Cys-loop receptor gating are currently the subject of considerable interest. Several functional approaches have shown that surface exposure of pore-lining side chains does not change significantly during activation. However, a disulfide trapping study on 1T6 C 1T6 C -aminobutyric acid type A (GABAA) receptors (GABAARs), which showed that adjacent subunits cross-link in the open state only, concluded that channel gating is mediated by subunits contra-rotating through a summed angle of 120o. Such a large rotation is not easily reconciled with other evidence. The present study initially sought to investigate an observation that appeared inconsistent with the rotation model: namely that 1T6 C 1T6 C GABAARs expressed in HEK293 cells form 6 cysteine-mediated disulfide bonds in the closed state. On the basis of electrophysiological and Western blotting experiments, we conclude that adjacent T6 C subunits dimerise efficiently in the closed state via cross-links between their respective 6 cysteines and that this locks the channels closed. This questions the subunit contra-rotation model of activation and raises the question of how the closed state cross-links form. We propose that subunit 6 cysteines move into sufficiently close proximity for disulfide formation via relatively large amplitude random thermal motions that appear to be a unique feature of subunits. Because dimerized channels are locked closed, we conclude either that the spontaneous subunit movements or asymmetries in the movements of adjacent subunits during activation are essential for pore opening. Our results identify a novel feature of GABAAR gating that may be important for understanding its activation mechanism.