Acute phosphatidylinositol 4,5 bisphosphate depletion destabilizes sarcolemmal expression of cardiac L-type Ca2+ channel CaV1.2.

CaV1.2 channels are critical players in cardiac excitation-contraction coupling, yet we do not understand how they are affected by an important therapeutic target of heart failure drugs and regulator of blood pressure, angiotensin II. Signaling through Gq-coupled AT1 receptors, angiotensin II triggers a decrease in PIP2, a phosphoinositide component of the plasma membrane (PM) and known regulator of many ion channels. PIP2 depletion suppresses CaV1.2 currents in heterologous expression systems but the mechanism of this regulation and whether a similar phenomenon occurs in cardiomyocytes is unknown. Previous studies have shown that CaV1.2 currents are also suppressed by angiotensin II. We hypothesized that these two observations are linked and that PIP2 stabilizes CaV1.2 expression at the PM and angiotensin II depresses cardiac excitability by stimulating PIP2 depletion and destabilization of CaV1.2 expression. We tested this hypothesis and report that CaV1.2 channels in tsA201 cells are destabilized after AT1 receptor-triggered PIP2 depletion, leading to their dynamin-dependent endocytosis. Likewise, in cardiomyocytes, angiotensin II decreased t-tubular CaV1.2 expression and cluster size by inducing their dynamic removal from the sarcolemma. These effects were abrogated by PIP2 supplementation. Functional data revealed acute angiotensin II reduced CaV1.2 currents and Ca2+ transient amplitudes thus diminishing excitation-contraction coupling. Finally, mass spectrometry results indicated whole-heart levels of PIP2 are decreased by acute angiotensin II treatment. Based on these observations, we propose a model wherein PIP2 stabilizes CaV1.2 membrane lifetimes, and angiotensin II-induced PIP2 depletion destabilizes sarcolemmal CaV1.2, triggering their removal, and the acute reduction of CaV1.2 currents and contractility.

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

The number and activity of Cav1.2 channels in the cardiomyocyte sarcolemma tunes the magnitude of Ca2+-induced Ca2+ release and myocardial contraction. ß-Adrenergic receptor (ßAR) activation stimulates sarcolemmal insertion of CaV1.2. This supplements the preexisting sarcolemmal CaV1.2 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 CaV1.2 channels. ßAR-activation decreased CaV1.2/endosome colocalization in ventricular myocytes, as it triggered "emptying" of endosomal CaV1.2 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 CaV1.2 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, ßAR-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 ßAR-activated Ca2+ current augmentation. Moreover, ßAR-regulation of CaV1.2 was abolished when recycling was halted by coapplication of nocodazole and latrunculin-A. These findings reveal that ßAR-stimulation triggers an on-demand boost in sarcolemmal CaV1.2 abundance via targeted Rab4a- and Rab11a-dependent insertion of channels that is essential for ßAR-regulation of cardiac CaV1.2.