Multiple Activity Patterns of Acetylcholine Receptors

描述（由申请人提供）：肌肉乙酰胆碱受体（AChR）是一种离子通道，介导神经-肌肉突触的传递。在结合两个递质分子后，AChR迅速（并且极有可能）从封闭通道(C)转换为开放通道(O)构象。随着长时间暴露于激动剂，achr也采用灭活（脱敏）构象。我们试图了解构成结合、门控和脱敏反应的分子事件的动力学。迄今为止的研究结果表明，变构门控构象变化是异步的，在c - o异构化过程中，蛋白质胞外结构域的残基比膜结构域的残基提前移动。如果可以可视化，我们假设这种构象变化将表现为几个刚体域的前后运动的惊人序列，而不是C和O构象之间的平滑过渡。对蛋白质的扰动（例如引起慢通道先天性肌无力综合征的突变）通过改变这种布朗构象“波”的传播改变门控，从而改变突触功能。我们将使用单分子电生理学和动力学（ph值）分析来探测构成门控反应过渡态的短暂中间体的性质。我们的具体目标是i)扩展ph值的映射，ii)测量五个AChR亚基之间的同步程度，iii)探索刚体门控域的离散性，iv)量化通道打开速度限制的温度依赖性，v)扩展我们对过渡态的理论分析。我们还建议使用类似的方法来研究递质结合和脱敏反应的中间状态。这些结果将阐明AChR的动态机制，并将为药物、毒素、细胞扰动和致病突变改变离子通道功能的机制提供基本见解。它们也将为合理的蛋白质工程提供基础。

DESCRIPTION (provided by applicant): The muscle acetylcholine receptor (AChR) is an ion channel that mediates transmission at the nerve-muscle synapse. After binding two transmitter molecules, the AChR switches rapidly (and with high probability) from a closed-channel (C) to an open-channel (O) conformation. With prolonged exposure to agonist, AChRs also adopt inactivated (desensitized) conformations. We seek to understand the dynamics of the molecular events that constitute the binding, gating and desensitization reactions. Results to date suggest that the allosteric gating conformational change is asynchronous, with residues in the extracellular domain of the protein moving in advance of those in the membrane domain during the C-to-O isomerization. Could it be visualized, we hypothesize that this conformational change would appear as a staggering sequence of back and forth motions of a few rigid body domains rather than as a smooth transition between the C and O conformations. Perturbations to the protein (for example mutations that cause the disease slow-channel congenital myasthenic syndromes) alter gating, and, hence, synaptic function, by changing the propagation of this Brownian conformational 'wave'. We will use single-molecule electrophysiology and kinetic (phi-value) analysis to probe the properties of the brief intermediates that constitute the transition state of the gating reaction. Our specific aims are to i) extend the map of phi-values, ii) measure the degree of synchrony between the five AChR subunits, iii) explore the discreteness of the rigid body gating domains, iv) quantify the temperature dependence of the channel-opening speed limit, and v) extend our theoretical analyses of the transition state. We also propose to use similar approaches to study the intermediate states of the transmitter binding and desensitization reactions. The results will illuminate the dynamic machinery of the AChR, and will provide fundamental insight into the mechanisms by which drugs, toxins, cellular perturbations and disease-causing mutations modify ion channel function. They will also serve as the basis for rational protein engineering.