BK(Ca) channel in heart mitochondria

心脏线粒体中的 BK(Ca) 通道

• 批准号: 8628868
• 负责人: ENRICO STEFANI
• 金额: $ 62.2万
• 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/8628868
• 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.

描述（由申请人提供）：从心脏线粒体（Mitobkca）中的大传导，Ca2+激活的K+通道被认为在心脏保护中起作用。据报道，〜55和〜125 kDa的免疫化学信号据报道，Mitobkca分子大小尚不确定。此外，线粒体分子身份及其线粒体靶向机制仍然未知，而有关其功能特性或直接证据表明其在心脏保护中的作用的信息很少。由于心脏MitoBKCA共享电导率，Ca2+响应能力以及对药理学剂的敏感性，其质膜对应物称为BKCA（或Maxik），因此我们希望Mitobkca像BKCA一样由四个孔形成bkca组成，具有〜125 kda的单体质量的亚基。现在，我们将检验以下假设：1）Mitobkca和质膜BKCA由相同的基因编码，并且剪接变异为BKCA提供了固有信号，以实现其优先的线粒体靶向； 2）从心肌细胞等离生中正常不存在BKCA，并在线粒体中的存在均由mitobkca骨架内的固有信号（即剪接插入片段）在直接或无关（即通过伴侣）和细胞特定机制Mitobkiars Mitobkca Mitoirts contriiouns统治。保留能力（CRC）和渗透性过渡孔（MPTP）开放。初步数据显示：1）特定抗BKCA抗体在线粒体中检测约125 kDa蛋白； 2）在孤立的心肌细胞mRNA中检测所有27个本构型BKCA外显子； 3）含有剪接插入材料DEC的BKCA同工型（61个氨基酸的C末端插入仪），但不容易将BKCA的本构形式（无插入BKCA）靶向成人心肌细胞中的线粒体； 4）Mitobkca次蛋白蛋白的Mitobkca次蛋白质是与线粒体进口蛋白折叠有关的热休克蛋白； 5）BKCA基因消融阻止了推定的BKCA频道开启器NS1619的心脏保护作用。总体而言，数据支持上述假设，该假设将使用多种方法进行测试，并追求以下特定目的：1。确定心脏Mitobkca的分子相关性； 2。在功能上验证克隆的推定Mitobkca的身份； 3。确定涉及线粒体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。该计划的结果将为在分子水平上研究Mitobkca的机会开放，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，并定义其功能性能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剂提供新的靶标。