Allosteric Coupling in Homomeric Cys-loop Receptors

描述(申请人提供)：Cys-loop超家族的神经递质受体在中枢和外周神经系统中调节快速突触传递。它们的基本任务是将神经递质的结合转导到打开固有的离子通道，但实现这一转导的机制尚不清楚。这一建议旨在描述同源Cys-loop受体转导机制的两个基本方面：(1)激动剂激活所需的激动剂结合部位和结合孔偶联区域的数量；(2)激动剂结合到通道门控的结构基础。这些研究利用了同聚体受体相对结构简单的优点，使用了一种新的方法来产生具有指定数量的完整结合部位和偶联区域的受体，并建立在我们最近证明的相互依赖的环网功能上偶联配体结合区域和孔域的基础上。这些实验结合了哺乳动物细胞嵌合和非嵌合受体的表达，单通道电流幅度和停留时间的测量，快速应用激动剂后离子电流的测量，定点突变，以及受体的结构建模。所开发的方法将允许在所有类型的Cys-loop受体中解决转导机制，而总体见解将促进我们对突触后受体在健康、疾病和治疗药物存在时如何发挥作用的理解。 层级描述：Cys-loop受体在神经系统中从一个细胞传递到另一个细胞，与多种疾病有关，如肌无力、多动症、癫痫和尼古丁成瘾。它们也是临床上使用的药物的靶点，如肌肉松弛药、抗焦虑药和抗惊厥药。了解Cys-loop受体如何在分子水平上发挥作用，对于开发副作用较少的治疗策略和药物至关重要。

DESCRIPTION (provided by applicant): Neurotransmitter receptors of the Cys-loop superfamily mediate fast synaptic transmission throughout the central and peripheral nervous systems. Their essential task is to transduce binding of neurotransmitter into opening of an intrinsic ion channel, yet the mechanism by which this transduction is achieved remains unknown. This proposal aims to delineate two fundamental facets of the transduction mechanism in homomeric Cys-loop receptors: (1) the number of agonist binding sites and binding-pore coupling regions required for activation by the agonist and (2) the structural basis for coupling agonist binding to channel gating. The proposed studies take advantage of the relative structural simplicity of homomeric receptors, employ a novel method for generating receptors with prescribed numbers of intact binding sites and coupling regions, and build on our recent demonstration that a network of interdependent loops functionally couples ligand binding and pore domains. The experiments combine expression of chimeric and non-chimeric receptors in mammalian cells, measurements of single channel current amplitudes and dwell times, measurements of ionic currents following rapid application of agonist, site-directed mutagenesis, and structural modeling of receptors. The approaches developed will allow the transduction mechanism to be addressed in all types of Cys-loop receptors, while the overall insights will advance our understanding of how post-synaptic receptors function in health, disease and in the presence of therapeutic drugs. Lay description: Cys-loop receptors relay signals from cell to cell throughout the nervous system, and are implicated in a wide variety of diseases, such as myasthenia, hyperekplexia, epilepsy, and nicotine addiction. They are also targets for drugs used clinically, such as muscle relaxants, anxiolytics, and anticonvulsants. Knowledge of how Cys-loop receptors operate at the molecular level is essential to developing therapeutic strategies and drugs with fewer side effects.