A State-Dependent Salt-Bridge Interaction Exists across the β/α Intersubunit Interface of the GABAA Receptor
A State-Dependent Salt-Bridge Interaction Exists across the β/α Intersubunit Interface of the GABAA Receptor
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DOI：
10.1124/mol.110.068619
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发表时间：
2011-04-01
影响因子：
3.6
通讯作者：
Wagner, David A.
中科院分区：
文献类型：
--
作者：
Laha, Kurt T.;Wagner, David A.
GABA(A) 受体是一种多亚基蛋白，可将亚基间界面处的神经递质结合转导至中央离子通道的开口。介导此操作所涉及步骤的结构组件定义不明确。大量工作集中于阐明 GABA 结合袋周围残基的具体功能和相互作用。在这里，我们探索了两个带电残基（β(2)Asp163 和 α(1)Arg120)，同源模型表明它们参与盐桥相互作用。当突变为丙氨酸时，单突变体以及双突变体都会增加 EC50-GABA，降低 GABA 结合率，并加速失活和 GABA 解结合率。双突变体循环分析表明，每个丙氨酸突变对 GABA 结合率的影响是相加且独立的。相反，在分析失活时间常数期间发现了显着的耦合能量。使用动力学模型，我们进一步证明 GABA 解结合速率尤其是强耦合的。这些数据表明，β(2)Asp163 和 α(1)Arg120 形成状态依赖性盐桥，当 GABA 与受体结合时相互作用，但当受体处于未结合状态时则不相互作用。
The GABA(A) receptor is a multisubunit protein that transduces the binding of a neurotransmitter at an intersubunit interface into the opening of a central ion channel. The structural components that mediate the steps involved in this action are poorly defined. A large amount of work has focused on clarifying the specific functions and interactions of residues believed to surround the GABA binding pocket. Here, we explored two charged residues (beta(2)Asp163 and alpha(1)Arg120), which have been suggested by homology models to participate in a salt-bridge interaction. When mutated to alanine, both single mutants, as well as the double mutant, increase EC50-GABA, decrease the GABA binding rate, and accelerate deactivation and GABA unbinding rates. Double-mutant cycle analysis demonstrates that the effects of each alanine mutation on the GABA binding rate were additive and independent. In contrast, a significant coupling energy was found during an analysis of deactivation time constants. Using kinetic modeling, we further demonstrated that the GABA unbinding rates, in particular, are strongly coupled. These data suggest that beta(2)Asp163 and alpha(1)Arg120 form a state-dependent salt bridge, interacting when GABA is bound to the receptor but not when the receptor is in the unbound state.