Trafficking and dynamics of Cav1.2 in the heart

Excitation-contraction coupling in ventricular cardiomyocytes critically depends on an intimate association between surface voltage-gated Ca2+ channels (CaV1.2) and intracellular ryanodine receptors (RyR2) that gate Ca2+ release from the sarcoplasmic reticulum (SR). The determinants and mechanisms underlying CaV1.2 targeting to these dyads and trafficking dynamics in ventricular myocytes are poorly understood but critical for insights into the molecular bases for cardiac pathophysiology, and potentially, the development of new therapies. Studies aimed at understanding mechanisms of CaV1.2 trafficking in heart cells are challenging due to the large size of the pore-forming a1C subunit and difficulties in maintaining adult ventricular myocytes in culture. Here, we have developed a novel transgenic mouse model featuring cardiac-specific doxycycline-inducible expression of a yellow fluorescent protein-tagged, dihydropyridine-insensitive a1C with a bungarotoxin-binding site (BBS) inserted in the third extracellular loop of domain II. We will use this transgenic mouse approach combined with super resolution imaging, electrophysiology, time lapse confocal microscopy, flow cytometry, and fluorescence recovery after photobleaching to investigate mechanisms and dynamics of CaV1.2 trafficking in adult cardiomyocytes. Furthermore, we hypothesize that the capacity to localize to dyadic junctions in heart is unique to CaV1.2 and conferred by specific determinants encoded within intracellular elements of a1C. We propose to generate a new transgenic mouse model featuring cardiac-specific, doxycycline-inducible expression of BBS- and YFP-tagged CaV2.2 a1B subunit. We will compare and contrast the dyadic targeting and trafficking dynamics of CaV2.2 versus CaV1.2. If the trafficking patterns of these two channels in cardiomyocytes differ, as we expect, then these experiments would establish a firm basis for future studies using chimeric channels to identify specific determinants on CaV1.2 that enable dyadic targeting. (AHA Program: Predoctoral Fellowship)