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（由适用提供）：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起关键作用。 CAV1.2功能的异常，包括增加的长期开放模式门控和钝性的肾上腺素反应性，与心力衰竭和肥大有关。 CAV1.2的激活增加，反过来触发Ca2+​​响应信号通路，这有助于心力衰竭和肥大的发病机理。正确靶向CAV1.2对不同的表面部位，并将其活性的激素调节对于正常的心脏生理学至关重要。 cav1.2 in 心脏与影响通道贩运，定位，周转和功能的大型超分子复合物有关。与CAV1的基础机制有关的许多流行教条。2运输和调制来自使用在异源表达系统中重组的重组通道的研究得出的。然而，使用敲门小鼠的最新结果表明，在天然心脏中没有复制关于从异源表达研究得出的几个长期存在的有关CAV1.2的调节，这强调了在实际心肌细胞中对机械研究的批判性需求。例如，成年鼠心肌细胞中Cavβ2蛋白表达的降低96％，基于异源表达研究，CAV1.2电流仅降低了约29％的降低，挑战常规智慧，即α1C运输到细胞表面绝对需要结合β。我们已经发展了 在心肌细胞的背景下表达信息丰富的α1C突变体的两种完整的新工具：（A）内部介导的蛋白质结扎可实现二氢吡啶（DHP）耐药的α1C亚基在心肌细胞中使用两个腺病毒中的二氢吡啶（DHP）抗性α1C亚基。该策略规定了生成编码整个α1C的病毒的需求，这在技术上在技术上挑战了，插入量很大。 （B转基因小鼠有条件地表达可耐力型，心脏特异性DHP耐药的α1C在成人心肌细胞中含有突变和推定调节位点的突变和截断心肌细胞中的肾上腺素能调节；（2）α1cc-terminus的作用和机制调节CAV1.2的运输和功能调节（3）阐明基础的CAV1.2 CAV1排除在t型tubulle中，具有L型Ca2+通道的相应细胞内段（α1C），这应该提供有关CA2+对心肌细胞中CA2+影响的关键新理解，这与心脏病的心脏病理学和分子式的孔隙相关性高度相关。 Ca2+通道。