Closed-state cross-linking of adjacent beta1 subunits in alpha1beta1 GABAa receptors via introduced 6' cysteines.
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 alpha1(T6'C)beta1(T6'C) gamma-aminobutyric acid type A (GABA(A)) receptors (GABA(A)Rs), which showed that adjacent beta subunits cross-link in the open state only, concluded that channel gating is mediated by beta subunits contra-rotating through a summed angle of approximately 120 degrees. 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 alpha1(T6'C)beta1(T6'C) GABA(A)Rs 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 beta(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 beta subunit contra-rotation model of activation and raises the question of how the closed state cross-links form. We propose that beta 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 beta subunits. Because dimerized channels are locked closed, we conclude either that the spontaneous beta subunit movements or asymmetries in the movements of adjacent beta subunits during activation are essential for pore opening. Our results identify a novel feature of GABA(A)R gating that may be important for understanding its activation mechanism.
Yang
Zhe
Z
School of Biomedical Sciences, University of Queensland, Brisbane QLD 4072, Australia.
Webb
Timothy I
TI
Lynch
Joseph W
JW
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D003545:Cysteine / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D051381:Rats; D011963:Receptors, GABA-A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
D011963:Receptors, GABA-A; D003545:Cysteine