Mechanism of adrenergic CaV1.2 stimulation revealed by proximity proteomics

Abstract

Increased cardiac contractility during fight-or-flight response is caused by β-adrenergic augmentation of Ca V 1.2 channels 1-4. In transgenic murine hearts expressing fully PKA phosphorylation-site-deficient mutant Ca V 1.2 α 1C and β subunits, this regulation persists, implying involvement of extra-channel factors. Here, we identify the mechanism by which β-adrenergic agonists stimulate voltage-gated Ca 2+ channels. We expressed α 1C or β 2B subunits conjugated to ascorbate-peroxidase 5 in mouse hearts and used multiplexed, quantitative proteomics 6,7 to track hundreds of proteins in proximity of Ca V 1.2. We observed that the Ca 2+ channel inhibitor Rad 8,9 , a monomeric G-protein, is enriched in the Ca V 1.2 micro-environment but is depleted during β-adrenergic stimulation. PKA-catalyzed phosphorylation of specific Ser residues on Rad decreases its affinity for auxiliary β-Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

The quest to identify the mechanism underlying adrenergic regulation of cardiac Ca2+ channels

“…Furthermore, Hofmann's group recalculated Catterall's data and showed that in both groups' knock-in mice, the β-adrenergic stimulation for wild-type and mutant channels were equivalent [58]. This confirmed our initial findings [54], which we have further substantiated with additional transgenic mice [59,60].…”

“…Finally, we crossed 35-mutant α 1C with 28-mutant β 2B transgenic mice. These mutant channels also displayed a normal isoproterenol-or forskolininduced increase in peak Ca 2+ current [59]. These results indicate that β-adrenergic stimulation of Ca V 1.2 does not involve direct phosphorylation of α 1C or β 2 subunits.…”

“…To test this, we mutated all 51 conserved and non-conserved serine and threonine residues within the 35 intracellular PKA consensus phosphorylation sites of rabbit α 1C to alanine ("35-mutant α 1C "). In cardiomyocytes, the nisoldipine-insensitive 35-mutant Ca 2+ currents were both up-regulated and activated at more negative potentials in response to isoproterenol or forskolin [59].…”

“…As channel regulation only reliably manifests in native tissues, we extended this method to native tissues and whole organs of transgenic mice. We generated transgenic mice with doxycycline-inducible, cardiomyocyte-specific expression of DHP-resistant-α 1C or β 2B proteins with ascorbate-peroxidase (APEX2) and a V5 epitope conjugated to the N-termini, enabling biotin-labeling of proteins within~20 nm of the Ca 2+ channels [59]. After exposure to isoproterenol, the enrichment of Rad, a member of the Rad and Gem/Kir Ras-related GTP-binding protein (RGK) family of GTPbinding proteins known for their capacity to inhibit all high-voltage-activated Ca 2+ channels [64,65], was decreased from the neighborhood of Ca V 1.2.…”

Designer genetically encoded voltage‐dependent calcium channel inhibitors inspired by RGK GTPases

“…This inhibition is relieved by protein kinase A phosphorylation of Rad, and is the long sought‐after mechanism by which β‐adrenergic agonists increase cardiac Ca V 1.2 to enhance inotropy during the fight‐or‐flight response (Liu et al . 2020).…”