STRUCTURAL CHANGE IN THE BETA SUBUNIT DURING GATING OF THE MUSCLE NICOTINIC RECEP

描述（由申请人提供）：递质门控离子通道的Cys环家族成员是五聚体膜蛋白，其对递质结合产生响应，构象变化导致门打开和对激动剂的亲和力增加。肌型烟碱乙酰胆碱受体（muscle-type nicotinic acetylcholine receptor，nAChR）是Cys环离子通道研究的原型，其单通道电导大，信号噪声比高，动力学性质相对简单。在nAChR中，两个递质结合位点位于受体的胞外结构域中a和d亚基以及a和e亚基之间的界面处。第五位（B）的亚基不直接参与递质结合。该建议的重点是非递质结合B亚基。我们将使用分子生物学、电生理学和动力学分析方法的组合来确定以下区域中的门控运动的能量学和时间：i）第二跨膜结构域，ii）细胞外区域，以及iii）包含第二和第三跨膜区域与前TM 1区域之间的接头的区域。这项工作将阐明参与的B亚基的构象变化，导致通道激活，并提供深入了解的来源，分子运动的非传输结合亚基。这项工作的完成将增加我们对递质门控离子通道功能的理解，以及非递质结合亚基如何塑造受体功能。 公共卫生相关性：烟碱乙酰胆碱受体介导脊椎动物神经肌肉接头的快速突触传递。五聚体蛋白含有两个位于亚基-亚基界面的递质结合位点。本项目旨在阐明第五亚基的作用，这是不参与递质结合，在结构运动导致通道门控。

DESCRIPTION (provided by applicant): Members of the Cys-loop family of transmitter-gated ion channels are pentameric membrane proteins that respond to the binding of transmitter with a conformational change resulting in the opening of a gate and an increased affinity to the agonist. The muscle-type nicotinic acetylcholine receptor (nAChR) has served as a prototype in Cys-loop ion channel research due to its large single-channel conductance resulting in a high signal-to-noise ratio, and the relatively simple kinetic properties. In the nAChR, the two transmitter binding sites are located in the extracellular domain of the receptor at the interfaces between an a and a d subunit, and an a and an e. The subunit in the fifth position (b) is not directly involved in transmitter binding. The focus of this proposal is the non-transmitter-binding b subunit. We will use a combination of molecular biological, electrophysiological, and kinetic analyses approaches to determine the energetics and timing of gating motions in: i) the second membrane-spanning domain, ii) the extracellular region, and iii) the region comprising the linker between the second and third membrane-spanning regions and the pre-TM1 region. The work will elucidate the involvement of the b subunit in the conformational changes that lead to channel activation, and provide insight into the sources of molecular motions in the non- transmitter-binding subunit. The completion of the proposed work will increase our understanding of how transmitter-gated ion channels function, and how the non-transmitter-binding subunit shapes receptor function. PUBLIC HEALTH RELEVANCE: The nicotinic acetylcholine receptor mediates fast synaptic transmission in the vertebrate neuromuscular junction. The pentameric protein contains two transmitter binding sites located at subunit-subunit interfaces. This project is aimed at elucidating the role of the fifth subunit, which is not involved in transmitter binding, in the structural motions resulting in channel gating.