β-Adrenergic control of sarcolemmal Ca_V1.2 abundance by small GTPase Rab proteins

The number and activity of Ca_v1.2 channels in the cardiomyocyte sarcolemma tunes the magnitude of Ca²⁺-induced Ca²⁺ release and myocardial contraction. β-Adrenergic receptor (βAR) activation stimulates sarcolemmal insertion of Ca_V1.2. This supplements the preexisting sarcolemmal Ca_V1.2 population, forming large "superclusters" wherein neighboring channels undergo enhanced cooperative-gating behavior, amplifying Ca²⁺ influx and myocardial contractility. Here, we determine this stimulated insertion is fueled by an internal reserve of early and recycling endosome-localized, presynthesized Ca_V1.2 channels. βAR-activation decreased Ca_V1.2/endosome colocalization in ventricular myocytes, as it triggered "emptying" of endosomal Ca_V1.2 cargo into the t-tubule sarcolemma. We examined the rapid dynamics of this stimulated insertion process with live-myocyte imaging of channel trafficking, and discovered that Ca_V1.2 are often inserted into the sarcolemma as preformed, multichannel clusters. Similarly, 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 coexpression of constitutively active Rab4a, halved by coexpression of dominant-negative Rab11a, and abolished by coexpression of dominant-negative mutant Rab4a. In ventricular myocytes, βAR-stimulated recycling of Ca_V1.2 was diminished by both nocodazole and latrunculin-A, suggesting an essential role of the cytoskeleton in this process. Functionally, cytoskeletal disruptors prevented βAR-activated Ca²⁺ current augmentation. Moreover, βAR-regulation of Ca_V1.2 was abolished when recycling was halted by coapplication of nocodazole and latrunculin-A. These findings reveal that βAR-stimulation triggers an on-demand boost in sarcolemmal Ca_V1.2 abundance via targeted Rab4a- and Rab11a-dependent insertion of channels that is essential for βAR-regulation of cardiac Ca_V1.2.