Abstract
The L-type Ca (2+) channel (Ca (V) 1.2) is essential for cardiac excitation-contraction coupling. To contribute to the inward Ca (2+) flux that drives Ca (2+) -induced-Ca (2+) -release, Ca (V) 1.2 channels must be expressed on the sarcolemma; thus the regulatory mechanisms that tune Ca (V) 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 (2+) channel trafficking in non-myocytes, however whether 14-3-3 has similar effects on Ca (V) 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 (V) 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 (V) 1.2 in a phosphorylation-dependent manner and regulates cardiac Ca (V) 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 (V) 1.2. The degree of 14-3-3/Ca (V) 1.2 colocalization increased upon stimulation of β -adrenergic receptors with isoproterenol. Notably, only the 14-3-3-associated Ca (V) 1.2 population displayed increased cluster size with isoproterenol, revealing a role for 14-3-3 as a nucleation factor that directs Ca (V) 1.2 super-clustering. 14-3-3 overexpression increased basal Ca (V) 1.2 cluster size and Ca (2+) currents in ventricular myocytes, with maintained channel responsivity to isoproterenol. In contrast, isoproterenol-stimulated augmentation of sarcolemmal Ca (V) 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 (V) 1.2 in a phosphorylation-dependent manner to promote enhanced trafficking/recycling, clustering, and activity during β -adrenergic stimulation. SIGNIFICANCE STATEMENT: The L-type Ca (2+) channel, Ca (V) 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 β -adrenergic signaling conditions. Proteins that modulate the trafficking and/or activity of Ca (V) 1.2 are interesting both from a physiological and pathological perspective, since alterations in Ca (V) 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 (2+) channels, but how it may interact with Ca (V) 1.2 in the heart has never been studied. Examining factors that affect Ca (V) 1.2 at rest and during β -adrenergic stimulation is crucial for our ability to understand and treat disease and aging conditions where these pathways are altered.