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

描述(申请人提供)：递质门控离子通道的Cys-loop家族的成员是五聚体膜蛋白，它们对递质的结合做出反应，通过构象变化导致门的打开和对激动剂的亲和力增加。肌型烟碱型乙酰胆碱受体(NAChR)由于其大的单通道电导、高信噪比和相对简单的动力学特性，被用作Cys-loop离子通道研究的原型。在nAChR中，两个递质结合位点位于受体的胞外区，位于a和d亚基之间，以及a和e之间。第五位(B)的亚基不直接参与递质结合。这一提议的重点是非递质结合的b亚基。我们将使用分子生物学、电生理和动力学分析相结合的方法来确定门控运动的能量学和时序：i)第二跨膜区域，ii)细胞外区域，以及iii)包含第二和第三跨膜区域与TM1前区域之间的连接子的区域。这项工作将阐明b亚基参与导致通道激活的构象变化，并提供对非递质结合亚基中分子运动来源的洞察。拟议工作的完成将增加我们对递质门控离子通道如何发挥作用，以及非递质结合亚基如何塑造受体功能的理解。 公共卫生相关性：烟碱型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。五聚体蛋白含有位于亚基-亚基界面的两个递质结合位点。本项目旨在阐明第五亚单位在导致通道门控的结构运动中的作用，该亚基不参与递质结合。

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.