L-type calcium channel trafficking and modulation in heart

描述（由申请人提供）：心脏L型Ca 2+通道在心脏兴奋-收缩偶联、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，包括长开放模式门控增加和肾上腺素能反应性减弱，与心力衰竭和心肌肥厚相关。CaV1.2的激活增加，反过来，触发Ca 2+响应信号通路，这有助于心力衰竭和肥大的发病机制。将CaV1.2正确靶向不同的表面位点，以及激素对其活性的调节，对于正常的心脏生理学至关重要。Cav1.2 in 心脏与影响通道运输、定位、周转和功能的大的超分子复合物相关。大多数与CaV1.2运输和调节机制相关的流行教条来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，几个长期存在的“事实”CaV1.2的调节来自异源表达的研究是不复制在天然心脏，强调迫切需要在实际的心肌细胞的背景下的机制研究。例如，成年鼠心肌细胞中CaVβ2蛋白表达降低96%仅导致CaV 1.2电流降低约29%，这挑战了基于异源表达研究的传统观点，即α1C运输至细胞表面绝对需要与β结合。我们已经开发 两种互补的新工具来表达心肌细胞内的信息α1C突变体：（a）内含肽介导的蛋白连接能够使用两种含有α1C的N-和C-末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。这种策略避免了产生编码整个α1C的病毒的需要，由于插入片段大小较大，这在技术上具有挑战性。（B）在成年心肌细胞和发育的所有阶段条件性表达强力霉素诱导的、心脏特异性DHP抗性α 1 C的转基因小鼠，该α 1 C携带推定调节位点的突变和截短。我们拟在心肌细胞中研究：（1）β亚基与α1C结合在心肌细胞CaV 1.2运输、功能和肾上腺素能调节中的作用;（2）α1C C末端在心脏CaV 1.2运输和功能调节中的作用和机制;（3）阐明CaV1.2通过替换T型Ca 2+通道的胞内结构域而靶向心肌细胞中二联体的功能决定因素（α1G），其被排除在t-小管之外，与L-型Ca 2+通道（α1C）的相应细胞内片段。这三个目标，这应该提供关键的新的理解有关的Ca 2+流入心肌细胞的调节，是高度相关的了解心脏病理和心脏兴奋收缩耦合和肾上腺素能调节心脏Ca 2+通道的分子机制。

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.