14-3-3 promotes sarcolemmal expression of cardiac Ca 1.2 and nucleates isoproterenol-triggered channel super-clustering.

UNLABELLED

The L-type Ca channel (Ca 1.2) is essential for cardiac excitation-contraction coupling. To contribute to the inward Ca flux that drives Ca -induced-Ca -release, Ca 1.2 channels must be expressed on the sarcolemma; thus the regulatory mechanisms that tune Ca 1.2 expression to meet contractile demand are an emerging area of research. A ubiquitously expressed protein called 14-3-3 has been proposed to affect Ca channel trafficking in non-myocytes, however whether 14-3-3 has similar effects on Ca 1.2 in cardiomyocytes is unknown. 14-3-3 preferentially binds phospho-serine/threonine residues to affect many cellular processes and is known to regulate cardiac ion channels including Na 1.5 and hERG. Altered 14-3-3 expression and function have been implicated in cardiac pathologies including hypertrophy. Accordingly, we tested the hypothesis that 14-3-3 interacts with Ca 1.2 in a phosphorylation-dependent manner and regulates cardiac Ca 1.2 trafficking and recycling. Confocal imaging, proximity ligation assays, super-resolution imaging, and co-immunoprecipitation revealed a population of 14-3-3 colocalized and closely associated with Ca 1.2. The degree of 14-3-3/Ca 1.2 colocalization increased upon stimulation of -adrenergic receptors with isoproterenol. Notably, only the 14-3-3-associated Ca 1.2 population displayed increased cluster size with isoproterenol, revealing a role for 14-3-3 as a nucleation factor that directs Ca 1.2 super-clustering. 14-3-3 overexpression increased basal Ca 1.2 cluster size and Ca currents in ventricular myocytes, with maintained channel responsivity to isoproterenol. In contrast, isoproterenol-stimulated augmentation of sarcolemmal Ca 1.2 expression and currents in ventricular myocytes were abrogated by 14-3-3 inhibition. These data support a model where 14-3-3 interacts with Ca 1.2 in a phosphorylation-dependent manner to promote enhanced trafficking/recycling, clustering, and activity during -adrenergic stimulation.

SIGNIFICANCE STATEMENT

The L-type Ca channel, Ca 1.2, plays an essential role in excitation-contraction coupling in the heart and in part regulates the overall strength of contraction during basal and fight- or-flight -adrenergic signaling conditions. Proteins that modulate the trafficking and/or activity of Ca 1.2 are interesting both from a physiological and pathological perspective, since alterations in Ca 1.2 can impact action potential duration and cause arrythmias. A small protein called 14-3-3 regulates other ion channels in the heart and other Ca channels, but how it may interact with Ca 1.2 in the heart has never been studied. Examining factors that affect Ca 1.2 at rest and during -adrenergic stimulation is crucial for our ability to understand and treat disease and aging conditions where these pathways are altered.