Regulators and interactomes of CaV1.2 in health and disease

该提案概述了一个全面的5年培训计划，以发展贾里德库什纳，医学博士，成为一个 独立翻译研究员。库什纳博士是一位普通心脏病专家和内科医生， 最终目标是改善对心脏病患者的护理。为了实现这一目标，他对 了解心脏离子通道功能异常如何导致心血管疾病， 相反，心血管疾病如何导致离子通道调节的适应不良变化。为了 作为一名独立的翻译科学家和该领域的领导者，库什纳博士已经发展了自己的职业生涯， 发展计划，旨在填补他的培训具体的教育和经验的差距。这些短期 目标包括：1）接受细胞电生理学的高级培训，重点是技术 所述探针离子通道在其天然环境中起作用; 2）获得大型数据集分析的经验， 目的是解释基因和蛋白质表达的大规模变化; 3）发展以下方面的专业知识： 蛋白质组学和质谱法的使用; 4）完善他目前的技能，并发展新的技能， 心血管生理学，重点是负荷独立的措施， 收缩和舒张功能; 5）获得探测蛋白质降解途径的专业知识， 强调在健康和疾病的离子通道的破坏;和5）发展技能，他的长期关键- 学期成功，特别强调他的赠款写作技巧。哥伦比亚大学以其丰富的 支持性的研究环境，被证明是库什纳博士组织多学科研究的理想环境。 拥有专业知识的导师团队来实现这些目标。 在他提议的研究项目中，库什纳博士将使用邻近标记的结果， L-型钙离子通道，CaV1.2，以了解其在正常人中功能所必需的通道调节剂 生物学和心力衰竭（HF），一种以收缩功能和肾上腺素能储备降低为特征的病症， 患者通常经历为随着用力而增加的呼吸短促。使用转基因小鼠， CaV1.2亚基融合到工程抗坏血酸过氧化物酶APEX 2的心脏特异性表达， 探索了慢性HF小鼠心脏中变化的通道微环境。他确定 HF中CaV1.2附近超过2000种蛋白质中的71种发生显著变化。为了确定这些蛋白质 干扰CaV1.2功能，他将分析通道活动，收缩性和肾上腺素能反应性，在遗传 APEX鉴定的蛋白质的敲除。为了测量对CaV1.2表达的影响，他将使用转基因的 表达YFP标记的α1C的小鼠，该α1C在细胞外环上含有银环蛇毒素结合位点（BBS- α1C）。用荧光团结合的银环蛇毒素处理分离的心肌细胞， 流式细胞术检测CaV1.2的表达。在与APEX-1基因敲除的BBS-α1C小鼠杂交中产生HF。 通过鉴定HF中的蛋白质，他可以鉴定那些改变HF中CaV1.2表达的邻近蛋白质。

This proposal outlines a comprehensive 5-year training program to develop Jared Kushner, MD, into an independent translational investigator. Dr. Kushner is a general cardiologist and physician-scientist whose ultimate goal is to improve care for patients with heart disease. To reach this goal, he is interested in understanding how abnormal ion channel function in the heart can lead to cardiovascular disease and conversely, how cardiovascular disease can cause maladaptive changes in ion channel regulation. In order to become an independent translational scientist and leader in this field, Dr. Kushner has developed a career development plan designed to fill specific educational and experiential gaps in his training. These short-term goals include: 1) Receiving advanced training in cellular electrophysiology, with an emphasis on techniques that probe ion channel function in their native milieu; 2) Acquiring experience in the analysis of large datasets, with the goal of interpreting large-scale changes in gene and protein expression; 3) Developing expertise in proteomics and the use of mass spectrometry; 4) Refining his current skills and developing new skills in cardiovascular physiology, with emphasis on the experimental assessment of load-independent measures of systolic and diastolic function; 5) Acquiring expertise in probing pathways of protein degradation, with emphasis on the destruction of ion channels in health and disease; and 5) Developing skills critical to his long- term success, with particular emphasis on his grant-writing skills. Columbia University, with its rich and supportive research environment, proved to be an ideal setting for Dr. Kushner to organize a multi-disciplinary mentorship team with the expertise to accomplish these goals. In his proposed research project, Dr. Kushner will use results from proximity-labeling the interactome of the L-type Ca2+ channel, CaV1.2, to gain insights into regulators of the channel essential to its function in normal biology and in heart failure (HF), a condition characterized by reduced systolic function and adrenergic reserve, often experienced by patients as increased shortness of breath with exertion. Using transgenic mice with cardiac-specific expression of CaV1.2 subunits fused to the engineered ascorbate peroxidase, APEX2, he probed the changing channel microenvironment in hearts of mice that developed chronic HF. He identified significant changes in 71 of over 2000 proteins in the vicinity of CaV1.2 in HF. To determine if those proteins perturb CaV1.2 function, he will analyze channel activity, contractility, and adrenergic responsiveness in genetic knockouts of APEX-identified proteins. To measure effects on CaV1.2 expression, he will use a transgenic mouse expressing a YFP-tagged α1C containing a bungarotoxin-binding site on an extracellular loop (BBS- α1C). Treating isolated myocytes with fluorophore-conjugated bungarotoxin, he can track total and surface expression of CaV1.2 with flow-cytometry. Generating HF in BBS-α1C mice crossed with knockouts of APEX- identified proteins in HF, he can identify those neighboring proteins that alter CaV1.2 expression of HF.