Closed-state Cross-linking of Adjacent β1 Subunits in α1β1 GABAa 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 ␣1 T6C ␤1 T6C ␥-aminobutyric acid type A (GABA A ) receptors (GABA A Rs), 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 ϳ120 o . 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 ␣1 T6C ␤1 T6CGABA 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 ␤ T6C 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 GABA A R gating that may be important for understanding its activation mechanism.Cys-loop receptors mediate fast neurotransmission in the nervous system. These receptors are pentameric homomers or heteromers, with each subunit comprising four ␣-helical transmembrane domains and a large extracellular amino-terminal domain that harbors the ligand binding sites and the signature Cys-loop (1-4). Each subunit contributes its second transmembrane domain (TM2) 3 to the lining of the axial channel pore. Neurotransmitter binding initiates a conformational change at the extracellular binding sites which is propagated throughout the receptor (5) culminating in the activation of the channel gate located in either the central region (6, 7) or the intracellular region (8, 9) of the pore. There is currently a great deal of interest in understanding how the TM2 domains move to mediate channel activation.The original model, based on electron diffraction images of two-dimensional crystal arrays of Torpedo nicotinic acetylcholine receptors (nAChRs), concluded that the TM2 domains were kinked inwards to form a central pore constriction and that upon ligand binding they rotated about their long axes to open the pore (10). However, several functionally based approaches have since shown that the pattern of residue exposure in the pore does not change in a manner suggesti...

The TM2 6′ Position of GABAA Receptors Mediates Alcohol Inhibition

“…We noted that adjacent ␤ subunits are found only in ␣␤ GABA A Rs, whereas in ␣␤␥ GABA A Rs the ␥ subunit separates the two ␤ subunits . These findings are consistent with previous reports in ␣1␤1 GABA A Rs that adjacent ␤ subunits cross-link via engineered Thr6Ј cysteines in the closed state, whereas nonadjacent ␣ subunits do not cross-link (Shan et al, 2002;Yang et al, 2007). Because of the formation of spontaneous disulfide bonds, we were unable to fully characterize alcohol and MTS reagent modulation of ␣2␤2(T6ЈC) GABA A Rs.…”

“…Substitution with tryptophan at residue Thr261 (Thr6Ј) in the GABA A R ␣2 subunit enhanced alcohol potentiation (Ueno at el., 2000), suggesting that the tryptophan moiety blocked an alcohol inhibitory site and unmasked additional enhancement by alcohol. Furthermore, cross-linking studies have provided evidence that the 6Ј positions of GABA A R ␣ and ␤ subunits interact (Horenstein et al, 2001;Shan et al, 2002;Yang et al, 2007) and may contribute to a combined inhibitory site for anesthetics (Rosen et al, 2007). In this study, we used site-directed mutagenesis and MTS labeling of GABA A Rs expressed in Xenopus laevis oocytes to test the hypothesis that 6Ј position residues in both ␣2␤2-and ␣2␤2␥2-containing GABA A Rs contribute to the inhibitory effects of alcohol.…”

Ligand- and Subunit-specific Conformational Changes in the Ligand-binding Domain and the TM2-TM3 Linker of α1 β2 γ2 GABAA Receptors

“…It is also unlikely that dimers may have formed between ␤2-K24ЈC and ␣1 or ␥2 subunits due to the lack of nearby free cysteine groups on those subunits. ␤ subunit dimerization has previously been reported to inhibit receptor activation in dimeric ␣1 ␤1-T6ЈC and ␣1 ␤2-E20ЈC GABA A Rs and in trimeric ␣1 ␤1-E20ЈC ␥2 GABA A Rs (39,40,42). These studies concluded that either spontaneous ␤ subunit movements, asymmetric movements of adjacent ␤ subunits, or both were essential for channel activation.…”

Experimental determination of the vertical alignment between the second and third transmembrane segments of muscle nicotinic acetylcholine receptors

“…Disulfide trapping has been widely applied to investigate proximity and mobility of engineered Cys-pairs in both soluble and membrane proteins (Falke and Koshland 1987;Matsumura et al 1989;Careaga and Falke 1992b;Pakula and Simon 1992;Zhan et al 1994;Gruber and Capaldi 1996;Danielson et al 1997;Krovetz et al 1997;Yu et al 1999;Elling et al 2000;Horenstein et al 2001Horenstein et al , 2005Shapovalov et al 2003;Bera and Akabas 2005;Chen et al 2006;Yang et al 2007). Here, we use disulfide bond formation as a measure for proximity.…”