14-3-3 regulation of cardiac L-type calcium channels and EC-coupling

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Project Summary: The voltage-gated L-type calcium channel (CaV1.2) is essential for cardiac excitation- contraction (EC)-coupling and dysregulation of the channel is implicated in many forms of heart disease. 14-3-3 is a ubiquitous protein that interacts with numerous cellular proteins to affect multiple physiological processes including cell growth, apoptosis, and ion channel trafficking. It preferentially binds phospho-serine/threonine residues on target proteins to regulate their trafficking, cooperativity, phosphorylation state, and/or activity. In HEK293 cells, 14-3-3 enhances trafficking of another voltage gated Ca2+ channel (CaV2.2) and has been shown to indirectly alter CaV1.2 trafficking via interactions with CaVβ subunits, however direct evidence and information about the extent and phosphorylation-dependence of this regulation is still needed. In addition, there have been no investigations into the role of 14-3-3 in CaV1.2 channel trafficking/regulation in cardiomyocytes. We address these gaps in knowledge in the current application. Since 14-3-3 has been reported to facilitate cooperative gating of the voltage-dependent cardiac Na+ channel, NaV1.5, we will also investigate the role of 14-3-3 in cooperative interactions of CaV1.2. This gating modality of CaV1.2 occurs when allosteric interactions form between C-terminal tails of adjacent channels in a cluster such that the opening of one channel can be communicated to other attached channels to enhance their open probability and amplify whole-cell Ca2+ influx. Our group has previously shown that PKA-mediated phosphorylation of CaV1.2 channels triggers enhanced trafficking of these channels into the sarcolemma of ventricular myocytes, producing larger channel clusters that facilitate enhanced cooperative gating behavior and augmented whole-cell Ca2+ currents. This helps tune cardiac EC-coupling to meet the enhanced demand during fight-or-flight. However, the molecular details of this enhanced trafficking are unclear. Here we propose that 14-3-3 plays a role in this response. We have identified several putative binding sites for 14-3-3 on the C-tail of CaV1.2 and other critical regulatory sites, including consensus PKA phosphorylation sites. This project aims to test the hypothesis that 14-3-3 regulates CaV1.2 trafficking, resulting in enhanced channel clustering on the sarcolemma that facilitates cooperative interactions and amplifies Ca2+ influx. We further propose that these interactions are strengthened by channel phosphorylation providing a means to tune CaV1.2 channel activity and EC-coupling to meet demand. In this two-year predoctoral project, we will rigorously test this hypothesis in three Specific Aims. Aim 1 tests the hypothesis that 14-3-3 interacts with CaV1.2 in a phosphorylation-dependent manner. Aim 2 tests the hypothesis that CaV1.2 channel trafficking, sarcolemmal clustering, and cooperative interactions are enhanced by 14-3-3. Aim 3 focuses on the functional effects of this regulation on cardiac EC-coupling. Alterations in CaV1.2 channel trafficking and regulation are associated with numerous cardiomyopathies given its central role in cardiac EC- coupling; thus our goals are relevant to the mission of the NHLBI.