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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >A conserved salt bridge critical for GABA_A receptor function and loop C dynamics
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A conserved salt bridge critical for GABA_A receptor function and loop C dynamics

机译：保守的盐桥对GABA_A受体功能和C回路动力学至关重要

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Chemical signaling in the brain involves rapid opening and closing of ligand gated ion channels (LGICs). LGICs are allosteric membrane proteins that transition between multiple conformational states (closed, open, and desensitized) in response to ligand binding. While structural models of cys-loop LGICs have been recently developed, our understanding of the protein movements under-lyincfthese conformational transitions is limited. Neurotransmitter binding is believed to initiate an inward capping movement of the loop C region of the ligand-binding site, which ultimately triggers channel gating. Here, we identify a critical intrasubunit salt bridge between conserved charged residues (βE153, βK196) in the GABA_A receptor (GABA_AR) that is involved in regulating loop C position. Charge reversals (E153K, K196E) increased the EC_(50) for GABA and for the allosteric activators pentobarbital (PB) and propofol indicating that these residues are critical for channel activation, and charge swap (E153K-K196E) significantly rescued receptor function suggesting a functional electrostatic interaction. Mutant cycle analysis of alanine substitutions indicated that E153 and K196 are energetically coupled. By monitoring disulfide bond formation between cysteines substituted at these positions (E153C-K196C), we probed the mobility of loop C in resting and ligand-bound states. Disulfide bond formation was significantly reduced in the presence of GABA or PB suggesting that agonist activation of the GABAaR proceeds via restricting loop C mobility.
机译：大脑中的化学信号传导涉及配体门离子通道（LGIC）的快速打开和关闭。 LGIC是变构膜蛋白，可响应配体结合在多种构象状态（闭合，开放和脱敏）之间转换。虽然最近开发了半胱氨酸环LGICs的结构模型，但我们对在不足的构象转换下的蛋白质运动的理解是有限的。认为神经递质结合会启动配体结合位点的环C区的向内封盖运动，最终触发通道门控。在这里，我们确定了参与调节环C位置的GABA_A受体（GABA_AR）中保守的带电残基（βE153，βK196）之间的关键亚基内盐桥。电荷逆转（E153K，K196E）增加了GABA和变构激活剂戊巴比妥（PB）和丙泊酚的EC_（50），表明这些残基对于通道激活至关重要，而电荷交换（E153K-K196E）则可以显着挽救受体功能，从而提示功能性静电相互作用。丙氨酸取代的突变周期分析表明，E153和K196能量耦合。通过监测在这些位置取代的半胱氨酸之间的二硫键形成（E153C-K196C），我们研究了环C在静止和配体结合状态下的迁移率。在存在GABA或PB的情况下，二硫键的形成显着减少，表明GABA aR的激动剂激活是通过限制环C的迁移来进行的。

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