BK(Ca) channel in heart mitochondria

描述（由申请人提供）：来自心脏线粒体的大电导、Ca 2+激活的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，报告的免疫化学信号约为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很少，也没有直接证据表明其在心脏保护中的作用。由于心脏mitoBKCa与其质膜对应物BKCa（或MaxiK）具有相同的电导、Ca 2+反应性和对药理学试剂的敏感性，因此我们预计mitoBKCa与BKCa一样由四个形成孔的α亚基组装而成，单体质量约为125 kDa。1）线粒体BKCa和质膜BKCa由相同的基因编码，剪接变异为BKCa提供了优先靶向线粒体的内在信号; 2）心肌细胞质膜中BKCa的正常缺失和线粒体中BKCa的存在由线粒体BKCa骨架内的内在信号和线粒体BKCa骨架内的内在信号两者决定。线粒体BKCa直接或间接（即通过伴侣蛋白）和通过细胞特异性机制（即剪接插入物）参与心肌保护，并且）mitoBKCa通过调节线粒体钙保留能力（CRC）和渗透性转换孔（mPTP）开放而有助于心脏保护。初步数据显示：1）通过特异性抗BKCa抗体检测线粒体中约125 kDa的蛋白; 2）检测分离的心肌细胞mRNA中的所有27个组成型BKCa外显子; 3）检测含有剪接插入物DEC的BKCa同种型，（61个氨基酸的C-末端插入），但不是BKCa的组成型（无插入物的BKCa）容易靶向成年心肌细胞中的线粒体; 4）mitoBKCa亚蛋白质组作为伴侣Hsp 60被发现，Hsp 60是与线粒体输入蛋白质的折叠相关的热休克蛋白;和5）BKCa基因消融阻止了推定的BKCa通道开放剂NS 1619的心脏保护作用。总的来说，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。确定心肌线粒体BKCa的分子相关性; 2.功能验证克隆的推定的mitoBKCa的身份; 3.确定参与mitoBKCa线粒体靶向的信号机制;和4.直接讨论mitoBKCa在心脏保护中的作用。该计划的结果将打开在分子水平上研究mitoBKCa的机会，并通过以下方式推进心脏领域：解决mitoBKCa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。

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.