L-type calcium channel trafficking and modulation in heart

心脏中 L 型钙通道的运输和调节

• 批准号: 9266817
• 负责人: Henry M. Colecraft
• 金额: $ 57.85万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2018-07-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9266817
• 关键词: Action Potentials; Address; Adenoviruses; Adrenergic Agents; Adult; Arrhythmia; Binding; C-terminal; Calmodulin; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Cell surface; Cells; Cleaved cell; Complex; Coupling; Data; Development; Dihydropyridines; Disease; Distal; Doxycycline; Exhibits; Gene Expression; Goals; Heart; Heart Hypertrophy; Heart failure; Hormonal; Hypertrophy; Knock-in Mouse; L-Type Calcium Channels; Lead; Life; Ligation; Macromolecular Complexes; Mediating; Membrane; Molecular; Mus; Muscle Cells; Mutate; Mutation; N-terminal; Pathogenesis; Pathologic; Pathology; Peptide Hydrolases; Perinatal mortality demographics; Phosphoric Monoester Hydrolases; Phosphotransferases; Physiological; Physiology; Play; Property; Proteins; Recombinants; Regulation; Resistance; Role; Ryanodine Receptors; Sarcolemma; Sarcoplasmic Reticulum; Scaffolding Protein; Series; Signal Pathway; Signaling Molecule; Site; Structure; Surface; System; Techniques; Transgenic Mice; Ventricular; Virus; base; beta-2 Adrenergic Receptors; caveolin-3; cost effective; heart cell; hormone regulation; innovation; insight; intein; mouse model; mutant; novel; protein expression; public health relevance; reconstitution; tool; trafficking; voltage

DESCRIPTION (provided by applicant): The cardiac L-type Ca2+ channel plays a key role in cardiac excitation-contraction coupling, action potential duration, and gene expression. Abnormalities in CaV1.2 function, including increased long-opening-mode gating and blunted adrenergic responsiveness, are associated with heart failure and hypertrophy. The increased activation of CaV1.2, in turn, triggers Ca2+-responsive signaling pathways, which contribute to the pathogenesis of heart failure and hypertrophy. Proper targeting of CaV1.2 to distinct surface sites, and hormonal regulation of their activity, is vital for normal cardiac physiology. Cav1.2 in heart is associated with large supramolecular complexes that impact on channel trafficking, localization, turnover, and function. Much of the prevailing dogma relating to mechanisms underlying CaV1.2 trafficking and modulation is derived from studies using recombinant channels reconstituted in heterologous expression systems. However, recent results using knock-in mice indicate that several long-standing "facts" about CaV1.2 regulation derived from heterologous expression studies are not replicated in native heart, emphasizing the critical need for mechanistic studies in the context of actual cardiomyocytes. For instance, a 96% reduction of CaVβ2 protein expression in adult murine cardiomyocytes caused only a ~29% reduction in CaV1.2 currents, challenging conventional wisdom, based on heterologous expression studies, that binding to β is absolutely required for α1C trafficking to the cell surface. We have developed two complementary novel tools to express informative α1C mutants within the context of cardiomyocytes: (a) Intein-mediated protein ligation enables robust reconstitution of dihydropyridine (DHP)-resistant α1C subunits in ventricular myocytes using two adenoviruses containing N- and C-terminal halves of α1C. This strategy circumvents the need to generate viruses encoding the entire α1C, which is technically challenging due to the large insert size. (b Transgenic mice conditionally expressing doxycycline-inducible, cardiac-specific DHP-resistant α1C harboring mutations and truncations of putative regulatory sites in adult cardiomyocytes and at all stages of development. We propose to determine in cardiomyocytes: (1) the role of β subunit binding to α1C for CaV1.2 trafficking, function and adrenergicmodulation in cardiomyocytes; (2) the role and mechanisms by which α1C C-terminus regulates CaV1.2 trafficking and functional modulation in heart; (3) elucidate determinants underlying CaV1.2 functional targeting to dyads in cardiomyocytes by replacing the intracellular domains of the T-type Ca2+ channel (α1G), which is excluded from t-tubules, with the corresponding intracellular segments of the L-type Ca2+ channel (α1C). The three Aims, which should provide key new understandings concerning the regulation of Ca2+ influx in cardiomyocytes, are highly relevant towards understanding cardiac pathologies and the molecular mechanisms responsible for cardiac excitation-contraction coupling and adrenergic modulation of the cardiac Ca2+ channel.

描述（申请人提供）：心脏L型Ca2+通道在心脏兴奋-收缩耦合、动作电位持续时间和基因表达中发挥关键作用。 CaV1.2 功能异常，包括长开放模式门控增加和肾上腺素能反应性减弱，与心力衰竭和肥厚相关。 CaV1.2 激活的增加反过来会触发 Ca2+ 响应信号通路，从而导致心力衰竭和肥厚的发病机制。 CaV1.2 正确靶向不同的表面位点及其活性的激素调节对于正常的心脏生理学至关重要。 Cav1.2英寸 心脏与影响通道运输、定位、周转和功能的大型超分子复合物相关。许多与 CaV1.2 运输和调节机制相关的流行教条源自使用在异源表达系统中重建的重组通道的研究。然而，最近使用敲入小鼠的结果表明，来自异源表达研究的有关 CaV1.2 调节的几个长期存在的“事实”并未在天然心脏中复制，这强调了在实际心肌细胞背景下进行机制研究的迫切需要。例如，成年小鼠心肌细胞中 CaVβ2 蛋白表达减少 96%，仅导致 CaV1.2 电流减少约 29%，这挑战了基于异源表达研究的传统观点，即与 β 的结合对于 α1C 运输到细胞表面是绝对必需的。我们开发了 两种互补的新工具在心肌细胞中表达信息丰富的α1C突变体：（a）内含肽介导的蛋白质连接能够使用两种含有α1C N端和C端一半的腺病毒在心室肌细胞中稳健地重建二氢吡啶（DHP）抗性α1C亚基。该策略避免了生成编码整个 α1C 的病毒的需要，由于插入片段较大，这在技术上具有挑战性。 （b 转基因小鼠条件性表达多西环素诱导型、心脏特异性 DHP 抗性 α1C，其在成年心肌细胞和发育的所有阶段中含有假定调节位点的突变和截短。我们建议确定心肌细胞中：（1）β 亚基与 α1C 结合对于 CaV1.2 运输、功能和发育的作用。 心肌细胞中的肾上腺素能调节； (2) α1C C末端调节心脏CaV1.2运输和功能调节的作用和机制； (3) 通过替换 T 型 Ca2+ 通道 (α1G) 的胞内结构域，阐明 CaV1.2 功能靶向心肌细胞二元体的决定因素，即 与 L 型 Ca2+ 通道 (α1C) 的相应细胞内片段一起被排除在 t 管之外。这三个目标应该提供有关心肌细胞 Ca2+ 内流调节的关键新理解，与理解心脏病理学以及负责心脏兴奋-收缩耦合和心脏肾上腺素能调节的分子机制高度相关。 Ca2+通道。