Allosteric Coupling in Homomeric Cys-loop Receptors

描述（由申请人提供）：Cys-loop超家族的神经递质受体介导整个中枢和周围神经系统的快速突触传递。它们的基本任务是将神经递质结合转化为内在离子通道的打开，但这种转导的实现机制尚不清楚。本提案旨在描述同质Cys-loop受体转导机制的两个基本方面：(1)激动剂结合位点和激动剂激活所需的结合孔偶联区域的数量；(2)激动剂结合到通道门控的偶联结构基础。本研究利用了同质受体结构的相对简单性，采用了一种新的方法来产生具有规定数量的完整结合位点和偶联区域的受体，并建立在我们最近证明的相互依赖的环网络在功能上偶联配体结合和孔结构域的基础上。实验结合了嵌合和非嵌合受体在哺乳动物细胞中的表达、单通道电流振幅和停留时间的测量、激动剂快速应用后离子电流的测量、定点诱变和受体结构建模。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，而总体见解将推进我们对突触后受体如何在健康，疾病和治疗药物存在下发挥作用的理解。

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.