Mechanism of adrenergic Ca V 1.2 stimulation revealed by proximity proteomics

Nature. 2020 Jan;577(7792):695-700. doi: 10.1038/s41586-020-1947-z. Epub 2020 Jan 22.

Abstract

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 mechanism by which β-adrenergic agonists stimulate voltage-gated calcium channels. We express α1C or β2B 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 β-adrenergic stimulation. Phosphorylation by protein kinase A of specific serine residues on Rad decreases its affinity for β 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Calcium Channels, L-Type / chemistry
  • Calcium Channels, L-Type / metabolism*
  • Calcium Channels, N-Type / metabolism
  • Cellular Microenvironment
  • Cyclic AMP / metabolism
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • Female
  • HEK293 Cells
  • Heterotrimeric GTP-Binding Proteins / metabolism
  • Humans
  • Male
  • Mice
  • Monomeric GTP-Binding Proteins / metabolism
  • Myocardium / metabolism
  • Phosphorylation
  • Protein Domains
  • Protein Subunits / chemistry
  • Protein Subunits / metabolism
  • Proteomics*
  • Receptors, Adrenergic, beta / metabolism*
  • Signal Transduction
  • ras Proteins / chemistry
  • ras Proteins / metabolism

Substances

  • CACNA1C protein, mouse
  • Cacna1b protein, mouse
  • Cacna1d protein, mouse
  • Calcium Channels, L-Type
  • Calcium Channels, N-Type
  • Protein Subunits
  • Receptors, Adrenergic, beta
  • Rem protein, mouse
  • Rrad protein, mouse
  • Cyclic AMP
  • Cyclic AMP-Dependent Protein Kinases
  • Heterotrimeric GTP-Binding Proteins
  • Monomeric GTP-Binding Proteins
  • ras Proteins