Horenstein Jeffrey, Riegelhaupt Paul, Akabas Myles H
Departments of Physiology and Biophysics and of Neuroscience, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461, USA.
J Biol Chem. 2005 Jan 14;280(2):1573-81. doi: 10.1074/jbc.M410881200. Epub 2004 Nov 2.
The gamma-aminobutyric acid, type A (GABAA), receptor ion channel is lined by the second membrane-spanning (M2) segments from each of five homologous subunits that assemble to form the receptor. Gating presumably involves movement of the M2 segments. We assayed protein mobility near the M2 segment extracellular ends by measuring the ability of engineered cysteines to form disulfide bonds and high affinity Zn(2+)-binding sites. Disulfide bonds formed in alpha1beta1E270Cgamma2 but not in alpha1N275Cbeta1gamma2 or alpha1beta1gamma2K285C. Diazepam potentiation and Zn2+ inhibition demonstrated that expressed receptors contained a gamma subunit. Therefore, the disulfide bond in alpha1beta1E270Cgamma2 formed between non-adjacent subunits. In the homologous acetylcholine receptor 4-A resolution structure, the distance between alpha carbon atoms of 20' aligned positions in non-adjacent subunits is approximately 19 A. Because disulfide trapping involves covalent bond formation, it indicates the extent of movement but does not provide an indication of the energetics of protein deformation. Pairs of cysteines can form high affinity Zn(2+)-binding sites whose affinity depends on the energetics of forming a bidentate-binding site. The Zn2+ inhibition IC50 for alpha1beta1E270Cgamma2 was 34 nm. In contrast, it was greater than 100 microM in alpha1N275Cbeta1gamma2 and alpha1beta1gamma2K285C receptors. The high Zn2+ affinity in alpha1beta1E270Cgamma2 implies that this region in the beta subunit has a high protein mobility with a low energy barrier to translational motions that bring the positions into close proximity. The differential mobility of the extracellular ends of the beta and alpha M2 segments may have important implications for GABA-induced conformational changes during channel gating.
γ-氨基丁酸A型（GABAA）受体离子通道由五个同源亚基各自的第二个跨膜（M2）片段排列而成，这些亚基组装形成受体。门控可能涉及M2片段的移动。我们通过测量工程化半胱氨酸形成二硫键和高亲和力锌（2+）结合位点的能力，来检测M2片段细胞外末端附近的蛋白质流动性。在α1β1E270Cγ2中形成了二硫键，但在α1N275Cβ1γ2或α1β1γ2K285C中未形成。地西泮增强作用和锌（2+）抑制作用表明表达的受体含有γ亚基。因此，α1β1E270Cγ2中的二硫键是在不相邻的亚基之间形成的。在同源乙酰胆碱受体4-A分辨率结构中，不相邻亚基中20'对齐位置的α碳原子之间的距离约为19埃。由于二硫键捕获涉及共价键形成，它表明了移动的程度，但没有提供蛋白质变形能量学的指示。半胱氨酸对可以形成高亲和力锌（2+）结合位点，其亲和力取决于形成双齿结合位点的能量学。α1β1E270Cγ2的锌（2+）抑制IC50为34纳米。相比之下，在α1N275Cβ1γ2和α1β1γ2K285C受体中大于100微摩尔。α1β1E270Cγ2中高锌（2+）亲和力意味着β亚基中的该区域具有高蛋白质流动性，对使位置紧密接近的平移运动具有低能量屏障。β和α M2片段细胞外末端的差异流动性可能对通道门控期间GABA诱导的构象变化具有重要意义。
