# Phosphorylation-dependent regulation of calcium channels by macromolecular complexes

> **NIH NIH R01** · COLUMBIA UNIVERSITY HEALTH SCIENCES · 2024 · $705,980

## Abstract

Project Summary
Acute β-adrenergic activation of protein kinase A (PKA) during the “fight or flight” response increases Ca2+
influx through CaV1.2 Ca2+ channels in cardiomyocytes to augment contractility. Yet in heart failure, triggering
the same signaling systems to compensate for decreased cardiac output—but chronically rather than acutely—
is cardiotoxic, exacerbating the failing heart and promoting life-threatening arrhythmias. By elucidating the
mechanisms by which β-adrenergic signaling modulates heart function we seek to ultimately develop targeted
and specific therapies for arrhythmias, heart failure, and other cardiovascular disorders. In studies completed
in years 1-4 of this application, we identified the mechanism of adrenergic regulation of cardiac Ca2+ channels.
We showed that the small RGK G-protein, Rad, a known inhibitor of high-voltage activated Ca2+ channels is
the key PKA target in the CaV1.2 complex. Rad simultaneously interacts with the inner leaflet of the
sarcolemma and CaVβ, thus holding CaV1.2 in a low open probability gating mode. PKA phosphorylation of two
Rad C-terminal Ser residues releases Rad from the membrane, reduces affinity for CaVβ, and relieves CaV1.2
inhibition. Using phosphosite knock-in mice with Ala-substitutions of the four PKA-phosphorylated residues in
Rad (4SA-Rad mice), we showed that phosphorylation of Rad is essential for β-adrenergic augmentation of
Ca2+ influx. Even with intact PKA signaling to other proteins modulating Ca2+ handling, preventing adrenergic
activation of Ca2+ channels in 4SA-Rad mice has profound physiological effects: reduced heart rate with
increased pauses, reduced basal contractility, near-complete attenuation of β-adrenergic contractile response,
and diminished exercise capacity. Conversely, expression in mice of mutant Ca2+ channel β-subunits that
cannot bind Rad (2DA-β2B knock-in mice, 3DA-β2B transgenic mice) is sufficient to enhance basal Ca2+ influx
and contractility to the augmented levels seen in WT mice with adrenergic activation. With these new
fundamental insights, we reason that we can utilize Rad-mediated modulation of Ca2+ influx as innovative
therapies for heart failure and arrhythmias, and for age-related loss of adrenergic reserve, while avoiding the
side-effects inherent to other CaV1.2 agonist/antagonist strategies. We propose three Specific Aims to
elucidate the effects of attenuating or enhancing Ca2+ influx in the heart via Rad-dependent mechanisms:
Aim 1: To determine whether blocking the augmentation of Ca2+ influx by catecholamines attenuates
arrhythmogenesis. Aim 2: To determine whether preventing the catecholamine augmentation of Ca2+ influx
attenuates stress-induced cardiomyopathy. Aim 3: To determine how aging alters adrenergic regulation of Ca2+
handling and cardiac contractility. Feasibility is supported by all mice lines being readily available and expertise
in all methods being established by the long-standing collaborations of the investigativ...

## Key facts

- **NIH application ID:** 10826630
- **Project number:** 2R01HL146149-05A1
- **Recipient organization:** COLUMBIA UNIVERSITY HEALTH SCIENCES
- **Principal Investigator:** Steven O Marx
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $705,980
- **Award type:** 2
- **Project period:** 2019-08-01 → 2027-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10826630

## Citation

> US National Institutes of Health, RePORTER application 10826630, Phosphorylation-dependent regulation of calcium channels by macromolecular complexes (2R01HL146149-05A1). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10826630. Licensed CC0.

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