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Mechanism of adrenergic CaV1.2 stimulation revealed by proximity proteomics.

ABSTRACT: Increased cardiac contractility during the fight-or-flight response is caused by ?-adrenergic augmentation of CaV1.2 voltage-gated calcium channels1-4. However, this augmentation persists in transgenic murine hearts expressing mutant CaV1.2 ?1C and ? subunits that can no longer be phosphorylated by protein kinase A-an essential downstream mediator of ?-adrenergic signalling-suggesting that non-channel factors are also required. Here we identify the mechanism by which ?-adrenergic agonists stimulate voltage-gated calcium channels. We express ?1C or ?2B subunits conjugated to ascorbate peroxidase5 in mouse hearts, and use multiplexed quantitative proteomics6,7 to track hundreds of proteins in the proximity of CaV1.2. We observe that the calcium-channel inhibitor Rad8,9, a monomeric G protein, is enriched in the CaV1.2 microenvironment but is depleted during ?-adrenergic stimulation. Phosphorylation by protein kinase A of specific serine residues on Rad decreases its affinity for ? subunits and relieves constitutive inhibition of CaV1.2, observed as an increase in channel open probability. Expression of Rad or its homologue Rem in HEK293T cells also imparts stimulation of CaV1.3 and CaV2.2 by protein kinase A, revealing an evolutionarily conserved mechanism that confers adrenergic modulation upon voltage-gated calcium channels.

SUBMITTER: Liu G

PROVIDER: S-EPMC7018383 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Mechanism of adrenergic CaV1.2 stimulation revealed by proximity proteomics.

Liu Guoxia G Papa Arianne A Katchman Alexander N AN Zakharov Sergey I SI Roybal Daniel D Hennessey Jessica A JA Kushner Jared J Yang Lin L Chen Bi-Xing BX Kushnir Alexander A Dangas Katerina K Gygi Steven P SP Pitt Geoffrey S GS Colecraft Henry M HM Ben-Johny Manu M Kalocsay Marian M Marx Steven O SO

Nature 20200122 7792

Increased cardiac contractility during the fight-or-flight response is caused by β-adrenergic augmentation of CaV1.2 voltage-gated calcium channels1-4. However, this augmentation persists in transgenic murine hearts expressing mutant CaV1.2 α1C and β subunits that can no longer be phosphorylated by protein kinase A-an essential downstream mediator of β-adrenergic signalling-suggesting that non-channel factors are also required. Here we identify the mec ...[more]

PMID: 31969708

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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.

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Mechanism of adrenergic CaV1.2 stimulation revealed by proximity proteomics.

Publications

Mechanism of adrenergic Ca<sub>V</sub>1.2 stimulation revealed by proximity proteomics.

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