L-type calcium channel trafficking and modulation in heart

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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.