14-3-3 regulates cardiac CaV1.2 clustering, expression, and trafficking

The voltage-gated L-type Ca2+ channel (CaV1.2) is essential for cardiac excitation-contraction coupling. CaV1.2 channel phosphorylation downstream of several physiological signaling cascades, regulates its activity, recycling, and trafficking to tune excitation-contraction coupling and contractility. A ubiquitously expressed protein called 14-3-3 has been previously proposed to affect CaV1.2 trafficking however the molecular details of this regulation are lacking. 14-3-3 preferentially binds phospho-serine/threonine residues on target proteins to affect many cellular processes and is known to regulate the trafficking and activity of other cardiac ion channels including NaV1.5 and hERG. Altered expression and function of 14-3-3 has additionally been implicated in cardiac pathologies including hypertrophy. Accordingly, we tested the hypothesis that 14-3-3 regulates cardiac CaV1.2 trafficking and activity in a phosphorylation-dependent manner, resulting in enhanced channel clustering that facilitates cooperative interactions and amplifies Ca2+ influx. Proximity ligation assays in cardiomyocytes revealed enhanced proximity between CaV1.2 and 14-3-3 upon stimulation with the β-adrenergic receptor agonist isoproterenol. Single molecule localization microscopy confirmed that a population of 14-3-3 colocalized with CaV1.2 and the level of colocalization increased with isoproterenol. Notably, only the 14-3-3-associated CaV1.2 population displayed increased cluster size with isoproterenol. In transfected tsA-201 cells, confocal imaging revealed reduced CaV1.2 surface expression when 14-3-3 was inhibited, and increased expression when 14-3-3 was overexpressed. Similarly, isoproterenol-stimulated augmentation of sarcolemmal CaV1.2 expression in cardiomyocytes was abrogated by pharmacological inhibition of 14-3-3. Functionally, voltage clamp experiments revealed whole-cell Ca2+ currents are reduced with 14-3-3 inhibition and increased when 14-3-3 was overexpressed. These data support a model where 14-3-3 interacts with CaV1.2 in a phosphorylation-dependent manner to promote its enhanced trafficking/recycling, clustering, and activity during β-adrenergic stimulation.