BK(Ca) channel in heart mitochondria

心脏线粒体中的 BK(Ca) 通道

• 批准号: 8298046
• 负责人: ENRICO STEFANI
• 金额: $ 63.47万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-15 至 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8298046
• 关键词: 3' Untranslated Regions; Ablation; Address; Adult; Affinity; Agreement; Amino Acids; Animals; Antibodies; Binding; Biological; Boxing; Brain; C-terminal; Calcium; Calcium-Activated Potassium Channel; Cardiac; Cardiac Myocytes; Cell membrane; Cells; Characteristics; Data; Detection; Epitopes; Exons; Fluorescence; Genbank; Gene Structure; Genes; Goals; Gray unit of radiation dose; Heart; Heart Injuries; Heart Mitochondria; Heat shock proteins; Inner mitochondrial membrane; Investigation; Ischemia; Knock-out; Medicine; Messenger RNA; Mitochondria; Molecular; Molecular Chaperones; Molecular Structure; Molecular Target; Myocardial Infarction; Names; Outcome; Oxygen; Permeability; Play; Potassium Channel; Property; Protein Import; Protein Isoforms; Proteins; RNA Splicing; Recombinants; Reperfusion Injury; Reporting; Role; Signal Transduction; Site; Spliced Genes; Testing; Therapeutic; Translating; Translations; Variant; Vertebral column; deprivation; iberiotoxin; interdisciplinary approach; knockout animal; molecular size; paxilline; prevent; programs; protein structure; sensor; stem

DESCRIPTION (provided by applicant): The large-conductance, Ca2+-activated K+ channel from cardiac mitochondria (mitoBKCa) is thought to play a role in cardioprotection. MitoBKCa molecular size is uncertain with reported immunochemical signals at ~55 and ~125 kDa. In addition, mitoBKCa molecular identity and its mitochondrial targeting mechanisms remain unknown, while there is scarce information about its functional properties or direct evidence for their role in cardioprotection. Because cardiac mitoBKCa shares conductance, Ca2+ responsiveness, and sensitivity to pharmacological agents with its plasma membrane counterpart known as BKCa (or MaxiK), we expect that mitoBKCa is assembled like BKCa by four pore-forming a subunits with a monomeric mass of ~125 kDa. We will now test the hypotheses that: 1) mitoBKCa and plasma membrane BKCa are encoded by the same gene and splice variation provides BKCa with intrinsic signals for its preferential mitochondrial targeting; 2) the normal absence of BKCa from the cardiomyocyte plasmalemma and presence in mitochondria is ruled by both an intrinsic signal(s) within mitoBKCa backbone (i.e. splice insert) either directly or indirectly (i.e. via a chaperone), and by cell-specific mechanisms, and ) mitoBKCa contributes to cardioprotection by regulating mitochondrial calcium retention capacity (CRC) and permeability transition pore (mPTP) opening. Preliminary Data shows: 1) the detection of a ~125 kDa protein in mitochondria by specific anti-BKCa antibodies; 2) the detection of all 27 constitutive BKCa exons in isolated cardiomyocyte mRNAs; 3) that BKCa isoform containing splice insert DEC (C-terminal insert of 61 amino acids) but not the constitutive form of BKCa (insertless BKCa) is readily targeted to mitochondria in adult cardiomyocytes; 4) that mitoBKCa subproteome uncovered as a partner Hsp60, a heat shock protein relevant for folding of mitochondrial imported proteins; and 5) that BKCa gene ablation prevents the cardioprotective action of putative BKCa channel opener NS1619. Overall the data support the above hypotheses, which will be tested using multiple approaches and pursuing the following Specific Aims to: 1. Identify the molecular correlate of cardiac mitoBKCa; 2. Functionally validate the identity of cloned putative mitoBKCa; 3. Determine signal mechanisms involved in mitoBKCa mitochondrial targeting; and 4. Directly address the role of mitoBKCa in cardioprotection. The outcomes of this program will open the opportunity to study mitoBKCa at the molecular level and advance the cardiac field by: solving mitoBKCa identity, providing information on its targeting mechanisms, and defining its functional properties and role in cardioprotection. Further understanding of the underlying molecular mechanism(s) of mitoBKCa cardioprotective effects will provide new targets for translation into therapeutics. PUBLIC HEALTH RELEVANCE: One of the mechanisms involved in protecting the heart from lack of oxygen like that occurring during heart infarct is thought to be the opening of a mitochondria potassium channel named mitoBKCa. Here, we propose to unveil mitoBKCa molecular identity, the mechanisms that target it to mitochondria and directly demonstrate its role in protecting the heart from injury by oxygen deprivation. The results of this investigation wll allow the advancement of cardioprotective medicine and provide new molecular targets for therapeutics.

描述（由申请人提供）：来自心脏线粒体的大电导 Ca2+ 激活 K+ 通道 (mitoBKCa) 被认为在心脏保护中发挥作用。 MitoBKCa 分子大小不确定，报道的免疫化学信号为 ~55 和 ~125 kDa。此外，mitoBKCa 的分子特性及其线粒体靶向机制仍然未知，而关于其功能特性的信息或其在心脏保护中作用的直接证据也很少。由于心脏 mitoBKCa 与其质膜对应物 BKCa（或 MaxiK）具有相同的电导性、Ca2+ 反应性和对药物的敏感性，因此我们预计 mitoBKCa 与 BKCa 一样由四个成孔亚基组装而成，单体质量约为 125 kDa。我们现在将测试以下假设：1) mitoBKCa 和质膜 BKCa 由相同基因编码，剪接变异为 BKCa 提供优先线粒体靶向的内在信号； 2) 心肌细胞质膜中 BKCa 的正常缺失和线粒体中的存在是由 mitoBKCa 主链（即剪接插入）直接或间接（即通过分子伴侣）内的内在信号和细胞特异性机制决定的，并且 ) mitoBKCa 通过调节线粒体钙来促进心脏保护 保留能力（CRC）和渗透性过渡孔（mPTP）开口。初步数据显示：1) 通过特异性抗 BKCa 抗体检测到线粒体中 ~125 kDa 的蛋白质； 2)分离的心肌细胞mRNA中全部27个组成型BKCa外显子的检测； 3) 含有剪接插入片段 DEC（61 个氨基酸的 C 端插入片段）但不含有 BKCa 组成型（无插入片段的 BKCa）的 BKCa 亚型很容易靶向成年心肌细胞中的线粒体； 4) mitoBKCa 亚蛋白质组被发现作为伙伴 Hsp60，一种与线粒体输入蛋白折叠相关的热休克蛋白； 5) BKCa 基因消融阻止了假定的 BKCa 通道开放剂 NS1619 的心脏保护作用。总体而言，数据支持上述假设，将使用多种方法进行测试，并追求以下具体目标： 1. 确定心脏 mitoBKCa 的分子相关性； 2. 功能验证克隆的假定 mitoBKCa 的身份； 3.确定mitoBKCa线粒体靶向涉及的信号机制； 4.直接阐述mitoBKCa在心脏保护中的作用。该计划的成果将为在分子水平上研究 mitoBKCa 提供机会，并通过以下方式推进心脏领域的发展：解决 mitoBKCa 的身份，提供有关其靶向机制的信息，并定义其功能特性和在心脏保护中的作用。进一步了解 mitoBKCa 心脏保护作用的潜在分子机制将为转化为治疗提供新的靶点。 公众健康相关性：保护心脏免受缺氧（如心肌梗塞期间发生的缺氧）影响的机制之一被认为是名为 mitoBKCa 的线粒体钾通道的开放。在这里，我们建议揭示 mitoBKCa 的分子特性、将其靶向线粒体的机制，并直接证明其在保护心脏免受缺氧损伤中的作用。这项研究的结果将促进心脏保护医学的进步，并为治疗提供新的分子靶点。