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: 8759443
• 负责人: Henry M. Colecraft
• 金额: $ 62.02万
• 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 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8759443
• 关键词: Action Potentials; Address; Adenoviruses; Adrenergic Agents; Adrenergic Receptor; Adult; Arrhythmia; Binding; C-terminal; Calmodulin; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Cell surface; Cells; Cessation of life; Cleaved cell; Complex; Coupling; Data; Development; Dihydropyridines; Disease; Distal; Doxycycline; Exhibits; Gene Expression; Goals; Heart; Heart Hypertrophy; Heart failure; Hormonal; Hormones; Hypertrophy; Knock-in Mouse; L-Type Calcium Channels; Lead; Life; Ligation; Macromolecular Complexes; Mediating; Membrane; Molecular; Mus; Muscle Cells; Mutate; Mutation; N-terminal; Pathogenesis; Peptide Hydrolases; Perinatal; Phosphoric Monoester Hydrolases; Phosphotransferases; Physiological; Physiology; Play; Property; Proteins; Recombinants; Regulation; Resistance; Role; RyR2; Ryanodine Receptors; Sarcolemma; Sarcoplasmic Reticulum; Scaffolding Protein; Series; Signal Pathway; Signaling Molecule; Site; Staging; Structure; Surface; System; Techniques; Transgenic Mice; Ventricular; Virus; adrenergic; base; caveolin-3; cost effective; dihydropyridine; heart cell; hormone regulation; innovation; insight; intein; molecular pathology; 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 CaV?2 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 ??is absolutely required for ¿1C trafficking to the cell surface. We have developed two complementary novel tools to express informative ¿ 1C mutants within the context of cardiomyocytes: (a) Intein-mediated protein ligation enables robust reconstitution of dihydropyridine (DHP)-resistant ¿ 1C subunits in ventricular myocytes using two adenoviruses containing N- and C-terminal halves of ¿ 1C. This strategy circumvents the need to generate viruses encoding the entire ¿ 1C, which is technically challenging due to the large insert size. (b Transgenic mice conditionally expressing doxycycline-inducible, cardiac-specific DHP-resistant ¿ 1C 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 ??subunit binding to ¿ 1C for CaV1.2 trafficking, function and adrenergicmodulation in cardiomyocytes; (2) the role and mechanisms by which ¿ 1C 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 (¿ 1G), which is excluded from t-tubules, with the corresponding intracellular segments of the L-type Ca2+ channel (¿ 1C). 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起关键作用。CaV1.2功能异常，包括长开模式门控增加和肾上腺素能反应减弱，与心力衰竭和肥厚有关。反过来，CaV1.2激活的增加触发Ca2+响应信号通路，这有助于心力衰竭和肥厚的发病机制。适当地将CaV1.2靶向到不同的表面位置，并通过激素调节其活性，对正常的心脏生理至关重要。Cav1.2在