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，Ca2+激活的K+通道（mitoBKCa）被认为在心脏保护中起作用。MitoBKCa分子大小是不确定的，有报道的免疫化学信号在~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，而其功能特性的信息或其在心脏保护中的作用的直接证据也很少。由于心脏mitoBKCa与质膜上的BKCa（或MaxiK）具有相同的导电性、Ca2+响应性和对药物的敏感性，我们预计mitoBKCa像BKCa一样通过四个孔形成一个单体质量约为125 kDa的亚基来组装。我们现在将验证以下假设：1)mitoBKCa和质膜BKCa由相同的基因编码，剪接变异为BKCa提供了其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的存在是由mitoBKCa主干内的内在信号（即剪接插入）直接或间接（即通过伴侣）以及细胞特异性机制决定的，并且mitoBKCa通过调节线粒体钙保留能力（CRC）和通透性过渡孔（mPTP）开放来促进心脏保护。初步数据表明：1)通过特异性抗bkca抗体检测线粒体中~125 kDa蛋白；2)分离心肌细胞mrna中27个组成BKCa外显子的检测；3)含有剪接插入DEC（61个氨基酸的c端插入物）但不含BKCa组成形式（无插入的BKCa）的BKCa异构体很容易靶向成人心肌细胞的线粒体；4)发现mitoBKCa亚蛋白质组作为合作伙伴Hsp60，这是一种与线粒体输入蛋白折叠相关的热休克蛋白；5) BKCa基因消融可阻止假定的BKCa通道开启剂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。鉴定心肌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线粒体靶向的信号机制；和4。直接解决mitoBKCa在心脏保护中的作用。该项目的结果将为在分子水平上研究mitoBKCa提供机会，并通过解决mitoBKCa的身份，提供其靶向机制的信息，并确定其功能特性和在心脏保护中的作用来推进心脏领域。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药物提供新的靶点。

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.