L-type calcium channel trafficking and modulation in heart

描述(申请人提供)：心肌L型钙通道在心肌兴奋-收缩偶联、动作电位时程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迟钝，与心力衰竭和肥厚有关。CaV1.2激活增加，进而触发钙反应信号通路，参与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，并对其活动进行激素调节，对于正常的心脏生理至关重要。Cav1.2英寸 心脏与影响通道运输、定位、周转和功能的大的超分子复合体有关。关于CaV1.2运输和调节机制的大部分流行教条都来自于使用在异源表达系统中重组的重组通道的研究。然而，最近使用敲打小鼠的结果表明，来自异源表达研究的几个长期存在的关于CaV1.2调控的“事实”在自然心脏中没有重复，强调了在实际心肌细胞的背景下进行机制研究的迫切需要。例如，成年小鼠心肌细胞Cavβ2蛋白表达降低96%，仅导致CaV1.2电流减少约29%，挑战了基于异源表达研究的传统观点，即与β结合是α1C转运到细胞表面所必需的。我们已经开发出 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)内含素介导的蛋白连接使抗二氢吡啶的α1C亚基能够通过两种腺病毒在心室肌细胞中强劲地重组，所述两种腺病毒分别含有α1C的N-端和C-端。这一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)转基因小鼠有条件地表达多西环素诱导的、心脏特异的抗dhp的α1C，这些转基因小鼠在成年心肌细胞和发育的所有阶段都存在假定的调控位点的突变和截断。我们建议在心肌细胞中确定：(1)β亚单位与α1C结合在心肌细胞中对CaV1.2的运输、功能和肾上腺素调节的作用；(2)α1C末端调节心肌中CaV1.2的运输和功能调节的作用和机制；(3)通过用L型钙通道(α1C)的相应细胞内片段取代T管中排除的T型钙通道(α1G)的胞内结构域，阐明CaV1.2功能靶向心肌细胞二联体的决定因素。这三个目标对于了解心肌细胞钙离子内流的调控机制、心肌兴奋收缩偶联和肾上腺素能调节心肌钙通道的分子机制具有重要意义。

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.