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Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System

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Mechanism of adrenergic Ca_V1.2 stimulation revealed by proximity proteomics

Guoxia Liu¹, Arianne Papa², Alexander N Katchman¹

¹Division of Cardiology, Department of Medicine, Columbia University, Vagelos College of Physicians and Surgeons, New York, NY, USA.

|January 24, 2020

Abstract:

Increased cardiac contractility during the fight-or-flight response is caused by β-adrenergic augmentation of Ca_V1.2 voltage-gated calcium channels^1-4. However, this augmentation persists in transgenic murine hearts expressing mutant Ca_V1.2 α_1C 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 α_1C or β_2B subunits conjugated to ascorbate peroxidase⁵ in mouse hearts, and use multiplexed quantitative proteomics^6,7 to track hundreds of proteins in the proximity of Ca_V1.2. We observe that the calcium-channel inhibitor Rad^8,9, a monomeric G protein, is enriched in the Ca_V1.2 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 Ca_V1.2, observed as an increase in channel open probability. Expression of Rad or its homologue Rem in HEK293T cells also imparts stimulation of Ca_V1.3 and Ca_V2.2 by protein kinase A, revealing an evolutionarily conserved mechanism that confers adrenergic modulation upon voltage-gated calcium channels.

Insights

Researchers discovered that the protein Rad, not just the calcium channel itself, mediates the fight-or-flight response. Protein kinase A phosphorylation of Rad relieves its inhibition of Ca_V1.2 channels, increasing cardiac contractility.

Area of Science:

Molecular Biology
Cardiovascular Physiology
Ion Channel Regulation

Background:

Beta-adrenergic stimulation enhances cardiac contractility via Ca_V1.2 calcium channels.
Previous studies suggested non-channel factors contribute to this augmentation.
Protein kinase A (PKA) is a key mediator of beta-adrenergic signaling.

Purpose of the Study:

To elucidate the precise mechanism by which beta-adrenergic agonists stimulate voltage-gated calcium channels.
To identify non-channel protein factors involved in adrenergic modulation of Ca_V1.2.

Main Methods:

Utilized transgenic mouse hearts expressing ascorbate peroxidase-conjugated Ca_V1.2 subunits.
Employed multiplexed quantitative proteomics to analyze protein proximity to Ca_V1.2.
Investigated the effect of PKA phosphorylation on the interaction between Rad and Ca_V1.2.

Main Results:

Identified the G protein Rad as enriched in the Ca_V1.2 microenvironment.
Observed Rad depletion during beta-adrenergic stimulation.
Demonstrated that PKA phosphorylation of Rad reduces its affinity for Ca_V1.2 beta subunits, increasing channel open probability and relieving inhibition.

Conclusions:

The protein Rad acts as a crucial intermediary in beta-adrenergic stimulation of Ca_V1.2 channels.
PKA-mediated phosphorylation of Rad is essential for augmenting cardiac contractility.
This mechanism of adrenergic modulation of voltage-gated calcium channels is evolutionarily conserved, also affecting Ca_V1.3 and Ca_V2.2.

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