Newell J Glen, McDevitt Ross A, Czajkowski Cynthia
Department of Physiology, University of Wisconsin, Madison, Wisconsin 53706, USA.
J Neurosci. 2004 Dec 15;24(50):11226-35. doi: 10.1523/JNEUROSCI.3746-04.2004.
Protein movements underlying ligand-gated ion channel activation are poorly understood. The binding of agonist initiates a series of conformational movements that ultimately lead to the opening of the ion channel pore. Although little is known about local movements within the GABA-binding site, a recent structural model of the GABA(A) receptor (GABA(A)R) ligand-binding domain predicts that beta2Glu155 is a key residue for direct interactions with the neurotransmitter (Cromer et al., 2002). To elucidate the role of the beta2Ile154-Asp163 region in GABA(A)R activation, each residue was individually mutated to cysteine and coexpressed with wild-type alpha1 subunits in Xenopus laevis oocytes. Seven mutations increased the GABA EC50 value (8- to 3400-fold), whereas three mutations (E155C, S156C, and G158C) also significantly increased the 2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl) pyridazinium (SR-95531) K(I) value. GABA, SR-95531, and pentobarbital slowed N-biotinylaminoethyl methanethiosulfonate modification of T160C and D163C, indicating that beta2Thr160 and beta2Asp163 are located in or near the GABA-binding site and that this region undergoes structural rearrangements during channel gating. Cysteine substitution of beta2Glu155 resulted in spontaneously open GABA(A)Rs and differentially decreased the GABA, piperidine-4-sulfonic acid (partial agonist), and SR-95531 sensitivities, indicating that the mutation perturbs ligand binding as well as channel gating. Tethering thiol-reactive groups onto beta2E155C closed the spontaneously open channels, suggesting that beta2Glu155 is a control element involved in coupling ligand binding to channel gating. Structural modeling suggests that the beta2 Ile154-Asp163 region is a protein hinge that forms a network of interconnections that couples binding site movements to the cascade of events leading to channel opening.
配体门控离子通道激活背后的蛋白质运动目前了解甚少。激动剂的结合引发了一系列构象变化，最终导致离子通道孔的开放。尽管对γ-氨基丁酸（GABA）结合位点内的局部运动知之甚少，但最近γ-氨基丁酸A受体（GABAAR）配体结合域的结构模型预测，β2Glu155是与神经递质直接相互作用的关键残基（克罗默等人，2002年）。为了阐明β2Ile154 - Asp163区域在GABAAR激活中的作用，每个残基都被单独突变为半胱氨酸，并与野生型α1亚基在非洲爪蟾卵母细胞中共表达。七个突变增加了GABA的半数有效浓度（EC50）值（8至3400倍），而三个突变（E155C、S156C和G158C）也显著增加了2 -（3 - 羧丙基）- 3 - 氨基 - 6 -（4 - 甲氧基苯基）哒嗪鎓（SR - 95531）的抑制常数（KI）值。GABA、SR - 95531和戊巴比妥减缓了T160C和D163C的N - 生物素基氨基乙基甲硫基磺酸盐修饰，表明β2Thr160和β2Asp163位于GABA结合位点内或附近，并且该区域在通道门控过程中经历结构重排。β2Glu155的半胱氨酸取代导致GABAAR自发开放，并不同程度地降低了GABA、哌啶 - 4 - 磺酸（部分激动剂）和SR - 95531的敏感性，表明该突变扰乱了配体结合以及通道门控。将硫醇反应性基团连接到β2E155C上可关闭自发开放的通道，表明β2Glu155是参与将配体结合与通道门控偶联的控制元件。结构建模表明，β2Ile154 - Asp163区域是一个蛋白质铰链，形成了一个相互连接的网络，将结合位点的运动与导致通道开放的一系列事件偶联起来。
