β-Adrenergic control of sarcolemmal Ca1.2 abundance by small GTPase Rab proteins.

The number and activity of Ca1.2 channels in the cardiomyocyte sarcolemma tunes the magnitude of Ca-induced Ca release and myocardial contraction. β-Adrenergic receptor () activation stimulates sarcolemmal insertion of Ca1.2. This supplements the preexisting sarcolemmal Ca1.2 population, forming large "superclusters" wherein neighboring channels undergo enhanced cooperative-gating behavior, amplifying Ca influx and myocardial contractility. Here, we determine this stimulated insertion is fueled by an internal reserve of early and recycling endosome-localized, presynthesized Ca1.2 channels. -activation decreased Ca1.2/endosome colocalization in ventricular myocytes, as it triggered "emptying" of endosomal Ca1.2 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 Ca1.2 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, -stimulated recycling of Ca1.2 was diminished by both nocodazole and latrunculin-A, suggesting an essential role of the cytoskeleton in this process. Functionally, cytoskeletal disruptors prevented -activated Ca current augmentation. Moreover, -regulation of Ca1.2 was abolished when recycling was halted by coapplication of nocodazole and latrunculin-A. These findings reveal that -stimulation triggers an on-demand boost in sarcolemmal Ca1.2 abundance via targeted Rab4a- and Rab11a-dependent insertion of channels that is essential for -regulation of cardiac Ca1.2.