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、钙激活和电压激活的钾 (BK) 通道发挥多种生理作用 重要作用，是几乎每个器官系统疾病的创新药物靶标，并且具有 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）通过电压和配体以及药物调节。 BK 频道是该频道的独特成员 钾通道家族，具有异常大的单通道电导和双重激活的特点 通过两个生理信号，膜电压和细胞内游离钙。各种实验 有证据表明 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，激活过程中 BK 通道孔的打开和关闭必须是 受其他机制控制。最近确定了完整 BK 通道的 3D 结构 近原子分辨率的加州海兔为理解这些提供了新的结构基础 机制。这些结构不仅证实了束交叉门的缺乏，而且表明了新颖的 深部氨基酸之间状态依赖性相互作用介导的BK通道激活机制 孔径和选择性过滤区域。我们假设 BK 通道激活门位于 选择性过滤器和/或深孔。我们在检验这一假设方面取得了进展，建立了 确定激活和选择性过滤失活之间关系的方法并分析 BK通道孔残基的结构-功能关系。随着新近获得的结构信息和 我们开发的新工具，包括串联串联亚基结构，以限制突变 四聚体通道内的各个 BK 通道亚基，我们现在准备确定孔门 定位和中心通道孔门控机制。我们建议采取以下三项具体行动： 旨在阐明 BK 通道的孔控机制：1）确定 BK 通道的性质和机制 BK 通道中的选择性滤波器选通； 2）确定深孔残基的作用及其相互作用 BK通道选通； 3) 通过确定状态来定义激活门和失活门的位置 选择性过滤器和深孔对 Cys 修饰试剂的可及性相关。总体而言，建议 研究旨在系统地研究 BK 通道的中心孔控机制。这 拟议研究的结果将加深我们对 BK 通道分子机制的理解 通过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。