Dataset Information

Cardiac CaV1.2 channels require ? subunits for ?-adrenergic-mediated modulation but not trafficking.

ABSTRACT: Ca2+ channel ?-subunit interactions with pore-forming ?-subunits are long-thought to be obligatory for channel trafficking to the cell surface and for tuning of basal biophysical properties in many tissues. Unexpectedly, we demonstrate that transgenic expression of mutant ?1C subunits lacking capacity to bind CaV? can traffic to the sarcolemma in adult cardiomyocytes in vivo and sustain normal excitation-contraction coupling. However, these ?-less Ca2+ channels cannot be stimulated by ?-adrenergic pathway agonists, and thus adrenergic augmentation of contractility is markedly impaired in isolated cardiomyocytes and in hearts. Similarly, viral-mediated expression of a ?-subunit-sequestering peptide sharply curtailed ?-adrenergic stimulation of WT Ca2+ channels, identifying an approach to specifically modulate ?-adrenergic regulation of cardiac contractility. Our data demonstrate that ? subunits are required for ?-adrenergic regulation of CaV1.2 channels and positive inotropy in the heart, but are dispensable for CaV1.2 trafficking to the adult cardiomyocyte cell surface, and for basal function and excitation-contraction coupling.

SUBMITTER: Yang L

PROVIDER: S-EPMC6355231 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Cardiac CaV1.2 channels require β subunits for β-adrenergic-mediated modulation but not trafficking.

Yang Lin L Katchman Alexander A Kushner Jared J Kushnir Alexander A Zakharov Sergey I SI Chen Bi-Xing BX Shuja Zunaira Z Subramanyam Prakash P Liu Guoxia G Papa Arianne A Roybal Daniel D Pitt Geoffrey S GS Colecraft Henry M HM Marx Steven O SO

The Journal of clinical investigation 20190107 2

Ca2+ channel β-subunit interactions with pore-forming α-subunits are long-thought to be obligatory for channel trafficking to the cell surface and for tuning of basal biophysical properties in many tissues. Unexpectedly, we demonstrate that transgenic expression of mutant α1C subunits lacking capacity to bind CaVβ can traffic to the sarcolemma in adult cardiomyocytes in vivo and sustain normal excitation-contraction coupling. However, these β-less Ca2+ channels cannot be stimulated by β-adrenerg ...[more]

PMID: 30422117

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Project description:Voltage-dependent L-type (Ca(V)1.2) Ca(2+) channels are a heteromeric complex formed from pore-forming alpha(1) and auxiliary alpha(2)delta and beta subunits. Ca(V)1.2 channels are the principal Ca(2+) influx pathway in arterial myocytes and regulate multiple physiological functions, including contraction. The macromolecular composition of arterial myocyte Ca(V)1.2 channels remains poorly understood, with no studies having examined the molecular identity or physiological functions of alpha(2)delta subunits.We investigated the functional significance of alpha(2)delta subunits in myocytes of resistance-size (100 to 200 mum diameter) cerebral arteries.alpha(2)delta-1 was the only alpha(2)delta isoform expressed in cerebral artery myocytes. Pregabalin, an alpha(2)delta-1/-2 ligand, and an alpha(2)delta-1 antibody, inhibited Ca(V)1.2 currents in isolated myocytes. Acute pregabalin application reversibly dilated pressurized arteries. Using a novel application of surface biotinylation, data indicated that >95% of Ca(V)1.2 alpha(1) and alpha(2)delta-1 subunits were present in the arterial myocyte plasma membrane. Alpha(2)delta-1 knockdown using short hairpin RNA reduced plasma membrane-localized Ca(V)1.2 alpha(1) subunits, caused a corresponding elevation in cytosolic Ca(V)1.2 alpha(1) subunits, decreased intracellular Ca(2+) concentration, inhibited pressure-induced vasoconstriction ("myogenic tone"), and attenuated pregabalin-induced vasodilation. Prolonged (24-hour) pregabalin exposure did not alter total alpha(2)delta-1 or Ca(V)1.2 alpha(1) proteins but decreased plasma membrane expression of each subunit, which reduced myogenic tone.alpha(2)delta-1 is essential for plasma membrane expression of arterial myocyte Ca(V)1.2 alpha(1) subunits. alpha(2)delta-1 targeting can block Ca(V)1.2 channels directly and inhibit surface expression of Ca(V)1.2 alpha(1) subunits, leading to vasodilation. These data identify alpha(2)delta-1 as a novel molecular target in arterial myocytes, the manipulation of which regulates contractility.

Project description:The number and activity of Cav1.2 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 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. βAR-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, βAR-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 βAR-activated Ca2+ current augmentation. Moreover, βAR-regulation of CaV1.2 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 CaV1.2 abundance via targeted Rab4a- and Rab11a-dependent insertion of channels that is essential for βAR-regulation of cardiac CaV1.2.

Dataset Information

Cardiac CaV1.2 channels require ? subunits for ?-adrenergic-mediated modulation but not trafficking.

Publications

Cardiac CaV1.2 channels require β subunits for β-adrenergic-mediated modulation but not trafficking.

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