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

Abstract

The number and activity of Cav1.2 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 CaV1.2 channels. This supplements the pre-existing sarcolemmal CaV1.2 population, forming large super-clusters 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, pre-synthesized CaV1.2 channels. {beta}AR-activation decreased CaV1.2/endosome colocalization in ventricular myocytes, as it triggered emptying of endosomal CaV1.2 cargo into the sarcolemma. We examined the rapid dynamics of this stimulated insertion process with live-myocyte imaging of channel trafficking, and discovered that CaV1.2 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, {beta}AR-stimulated recycling of CaV1.2 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 CaV1.2 was abolished when recycling was halted by co-application of nocodazole and latrunculin-A. These findings reveal that {beta}AR-stimulation triggers an on-demand boost in sarcolemmal CaV1.2 abundance via targeted, Rab4a and Rab11a-dependent insertion of CaV1.2 channels is essential for {beta}AR-regulation of cardiac CaV1.2. Significance StatementThe L-type voltage-gated Ca2+ channel CaV1.2 is essential for excitation-contraction coupling in the heart. During the fight-or-flight response, CaV1.2 channel activity is augmented as a result of PKA-mediated phosphorylation, downstream of {beta}-adrenergic receptor ({beta}AR) activation. We discovered that enhanced sarcolemmal abundance of CaV1.2 channels, driven by stimulated insertion/recycling of specific CaV1.2 containing endosomes, is essential for {beta}AR-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.