Closed-state Cross-linking of Adjacent β1 Subunits in α1β1 GABA_a Receptors via Introduced 6′ Cysteines*

Abstract

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 α1^T6′Cβ1^T6′C γ-aminobutyric acid type A (GABA_A) receptors (GABA_ARs), which showed that adjacent β subunits cross-link in the open state only, concluded that channel gating is mediated by β subunits contra-rotating through a summed angle of ∼120°. 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 α1^T6′Cβ1^T6′C GABA_ARs 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 β^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 β subunit contra-rotation model of activation and raises the question of how the closed state cross-links form. We propose that β 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 β subunits. Because dimerized channels are locked closed, we conclude either that the spontaneous β subunit movements or asymmetries in the movements of adjacent β subunits during activation are essential for pore opening. Our results identify a novel feature of GABA_AR gating that may be important for understanding its activation mechanism.