BK(Ca) channel in heart mitochondria

描述(由申请人提供)：来自心肌线粒体的大电导、钙激活的钾通道(MitoBKCa)被认为在心脏保护中发挥作用。MitoBKCa分子大小不确定，已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尚不清楚，关于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。由于心肌mitoBKCa与质膜类似物BKCa(或Maxik)具有相同的电导、钙敏感性和对药物的敏感性，我们预计mitoBKCa类似于BKCa由四个孔形成的亚单位组装而成，单体质量约为125 kDa。我们现在将检验假设：1)mitoBKCa和质膜BKCa由相同的基因编码，剪接变异为BKCa提供了具有选择性线粒体靶向的内在信号；2)心肌细胞质膜上正常缺乏BKCa而线粒体的存在直接或间接(即通过伴侣)受mitoBKCa骨架内的内在信号(即剪接插入)和细胞特有机制的控制，并且mitoBKCa通过调节线粒体钙保持能力和通透性转换孔的开放来促进心肌保护。初步数据表明：1)特异性抗BKCa抗体在线粒体中检测到~125 kDa的蛋白质；2)在分离的心肌细胞mRNAs中检测到所有27个组成的BKCa外显子；3)含有剪接插入DEC(61个氨基酸的末端插入片段)的BKCa亚型而不是BKCa的组成形式(无插入的BKCa)很容易针对成年心肌细胞的线粒体；4)mitoBKCa亚蛋白质组发现作为伴侣的Hsp60，一种与线粒体进口蛋白的折叠相关的热休克蛋白；5)BKCa基因的被激活阻止了可能的BKCa通道开放剂NS1619的心脏保护作用。总体而言，这些数据支持上述假设，这些假设将通过多种方法进行检验，并追求以下具体目标：1.确定心肌mitoBKCa的分子相关性；2.从功能上验证克隆的mitoBKCa的身份；3.确定mitoBKCa线粒体靶向的信号机制；以及4.直接研究mitoBKCa在心脏保护中的作用。该计划的结果将为在分子水平上研究mitoBKca提供机会，并通过以下方式推动心脏领域的发展：解决mitoBKca的特性，提供有关其靶向机制的信息，并确定其功能特性和在心脏保护中的作用。进一步了解线粒体BKCa心脏保护作用的潜在分子机制(S)将为转化为治疗学提供新的靶点。

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.