β-adrenergic control of sarcolemmal Ca V 1.2 abundance by small GTPase Rab proteins

The number and activity of Ca v 1.2 channels in the cardiomyocyte sarcolemma tunes the magnitude of Ca 2+ -induced Ca 2+ release and myocardial contraction. β-adrenergic receptor ( βAR ) activation stimulates sarcolemmal insertion of Ca V 1.2 channels. This supplements the pre-existing sarcolemmal Ca V 1.2 population, forming large ‘super-clusters’ wherein neighboring channels undergo enhanced cooperative-gating behavior, amplifying Ca 2+ influx and myocardial contractility. Here, we determine this stimulated insertion is fueled by an internal reserve of early- and recycling endosome-localized, pre-synthesized Ca V 1.2 channels. βAR -activation decreased Ca V 1.2/endosome colocalization in ventricular myocytes, as it triggered ‘emptying’ of endosomal Ca V 1.2 cargo into the sarcolemma. We examined the rapid dynamics of this stimulated insertion process with live-myocyte imaging of channel trafficking, and discovered that Ca V 1.2 are often inserted into the sarcolemma as pre-formed, multi-channel clusters. Likewise, entire clusters were removed from the sarcolemma during endocytosis, while in other cases, a more incremental process suggested removal of individual channels. The amplitude of the stimulated insertion response was doubled by co-expression of constitutively-active Rab4a, halved by co-expression of dominant-negative Rab11a, and abolished by co-expression of dominant-negative mutant Rab4a. In ventricular myocytes, βAR -stimulated recycling of Ca V 1.2 was diminished by both nocodazole and latrunculin-A, suggesting an essential role of the cytoskeleton in this process. Functionally, cytoskeletal disruptors prevented βAR-activated Ca 2+ current augmentation. Moreover, βAR -regulation of Ca V 1.2 was abolished when recycling was halted by co-application of nocodazole and latrunculin-A. These findings reveal that βAR -stimulation triggers an on-demand boost in sarcolemmal Ca V 1.2 abundance via targeted, Rab4a and Rab11a-dependent insertion of Ca V 1.2 channels is essential for βAR -regulation of cardiac Ca V 1.2. Significance Statement The L-type voltage-gated Ca 2+ channel Ca V 1.2 is essential for excitation-contraction coupling in the heart. During the fight-or-flight response, Ca V 1.2 channel activity is augmented as a result of PKA-mediated phosphorylation, downstream of β-adrenergic receptor ( βAR ) activation. We discovered that enhanced sarcolemmal abundance of Ca V 1.2 channels, driven by stimulated insertion/recycling of specific Ca V 1.2 containing endosomes, is essential for βAR -mediated regulation of these channels in the heart. These data reveal a new conceptual framework of this critical and robust pathway for on-demand tuning of cardiac EC-coupling during fight-or-flight.