Pore Gating Mechanisms of BK Channels

BK通道的孔门机制

• 批准号: 9916769
• 负责人: Richard Aldrich
• 金额: $ 42.04万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9916769
• 关键词: Amino Acids; Aplysia; Biochemical; Biophysics; Calcium; Cardiovascular system; Coupled; Cysteine; Development; Disease; Drug Modulation; Drug Targeting; Family; Goals; Individual; Induced Mutation; Ions; Ligands; Location; Mediating; Membrane; Methods; Modeling; Modification; Molecular; Molecular Conformation; Monovalent Cations; Mutation; Nature; Neurobiology; Pathway interactions; Permeability; Physiological; Play; Potassium; Potassium Channel; Prevention; Property; Reagent; Regulation; Research; Resolution; Role; Scanning; Side; Signal Transduction; Structure; Structure-Activity Relationship; System; Testing; Work; body system; design; dimer; extracellular; innovation; insight; large-conductance calcium-activated potassium channels; member; mutant; novel; novel therapeutics; three dimensional structure; tool; voltage

Large-conductance, calcium- and voltage-activated potassium (BK) channels play a variety of physiologically important roles, are innovative drug targets for disorders of almost every organ system, and possess biophysical features that make them an ideal system for studying allosteric mechanisms of channel function (gating) by voltage and ligands and modulation by drugs. The BK channel is a unique member of the potassium channel family, characterized by exceptionally large single-channel conductance and dual activation by two physiological signals, membrane voltage and intracellular free calcium. A variety of experimental evidence indicates that BK channels lack the intracellular bundle-crossing gate that is present in many other potassium channels. Thus the opening and closing of the BK channel pore during activation must be controlled by other mechanisms. Recent determination of the 3D structure of the complete BK channel from Aplysia californica at a near-atomic resolution provides a new structural basis for understanding these mechanisms. The structures not only confirm that the lack of a bundle-crossing gate, but suggest novel mechanisms of BK channel activation mediated by state-dependent interaction among amino acids in the deep pore and selectivity filter regions. We hypothesize that the BK channel activation gate is located within the selectivity filter and/or deep-pore. We have made progress towards testing this hypothesis by establishing methods to determine the relationship between activation and selectivity filter inactivation and analyzing the structure-function relationship of BK channel pore residues. With the newly available structural information and novel tools that we have developed, including concatenated tandem subunit constructs to restrict mutations to individual BK channel subunits within the tetrameric channel, we are now poised to determine the pore gate localization and central channel pore gating mechanisms. We propose to pursue the following three specific aims to elucidate the pore-gating mechanisms of BK channels: 1) determine the properties and mechanisms of selectivity filter gating in BK channels; 2) determine the role of the deep-pore residues and their interactions in BK channel gating; and 3) define the location of the activation and inactivation gates by determining the state- dependent accessibility of the selectivity filter and deep pore to Cys-modifying reagents. Overall, the proposed research is designed to investigate systematically the central pore-gating mechanisms of BK channels. The findings from the proposed studies will deepen our understanding of molecular mechanisms of BK channel activation by voltage and calcium and facilitate development of novel therapeutic reagents targeting BK channels for the treatment or prevention of neurobiological, cardiovascular, and other types of disorders and diseases.

大电导、钙和电压激活的钾(BK)通道在多种生理状态下发挥作用 具有重要作用，是治疗几乎所有器官系统疾病的创新药物靶点，并拥有 生物物理特征使其成为研究通道功能变构机制的理想系统 (门控)通过电压和配基以及药物的调节。BK频道是 钾通道家族，具有极大的单通道电导和双激活特性 通过两种生理信号，膜电压和细胞内游离钙。各种实验性的 证据表明，BK通道缺乏许多其他通道中存在的细胞内束交叉门 钾通道。因此，在激活期间BK通道孔的打开和关闭必须 由其他机制控制。最近对完整的BK通道三维结构的测定 近原子分辨率的加利福尼亚海兔为理解这些现象提供了新的结构基础。 机制。这些结构不仅证实了缺乏横跨大门的捆绑，而且让人觉得新奇 深层氨基酸间状态依赖相互作用介导BK通道激活的机制 孔滤区和选择性滤区。我们假设BK通道激活门位于 选择性过滤器和/或深孔。我们已经在验证这一假说方面取得了进展，通过建立 方法确定活化和选择性过滤器失活之间的关系，并分析 BK通道孔道残留物的构效关系。利用最新可用的结构信息和 我们开发的新工具，包括串联的串联亚单位结构，将突变限制在 在四聚体通道内的单个BK通道亚基，我们现在准备确定孔门 局部化和中心通道气孔门控机制。我们建议进行以下三个具体的工作 旨在阐明BK通道的开孔机制：1)确定BK通道的特性和机制 BK通道中的选择性过滤门控；2)确定深孔残留物在 BK通道选通；以及3)通过确定状态- 选择性过滤器和深孔对半胱氨酸修饰剂的可及性。整体而言，建议的 这项研究旨在系统地研究BK通道的中心气孔选通机制。这个 这些研究结果将加深我们对BK通道分子机制的理解 电压和钙激活并促进针对BK的新型治疗试剂的开发 治疗或预防神经生物学、心血管和其他类型疾病的渠道 疾病。