β-Adrenergic control of sarcolemmal CaV1.2 abundance by small GTPase Rab proteins

The number and activity of Ca(v)1.2 channels in the cardiomyocyte sarcolemma tunes the magnitude of Ca2+-induced Ca2+ release and myocardial contraction. beta-Adrenergic receptor (beta AR) activation stimulates sarcolemmal insertion of Ca(V)1.2. This supplements the preexisting sarcolemmal Ca(V)1.2 population, forming large superclusters wherein neighboring channels undergo enhanced cooperativegating 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(V)1.2 channels. beta AR-activation decreased Ca(V)1.2/endosome colocalization in ventricular myocytes, as it triggered emptying of endosomal Ca(V)1.2 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(V)1.2 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, beta AR-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 beta AR-activated Ca2+ current augmentation. Moreover, beta AR-regulation of Ca(V)1.2 was abolished when recyclingwas halted by coapplication of nocodazole and latrunculin-A. These findings reveal that beta AR-stimulation triggers an on-demand boost in sarcolemmal Ca(V)1.2 abundance via targeted Rab4a- and Rab11a-dependent insertion of channels that is essential for beta AR-regulation of cardiac Ca(V)1.2.

Automated summary

The number and activity of Ca(v)1.2 channels in cardiomyocytes regulates the magnitude of Ca2+-induced Ca2+ release and myocardial contraction. Stimulation of beta-Adrenergic receptors leads to insertion of Ca(V)1.2 channels into the sarcolemma, enhancing cooperative gating behavior and increasing Ca2+ influx and contractility. This process is fueled by an internal reserve of pre-synthesized channels localized in endosomes, regulated by Rab proteins.