# Phosphorylation-dependent regulation of calcium channels by macromolecular complexes

> **NIH NIH R01** · COLUMBIA UNIVERSITY HEALTH SCIENCES · 2022 · $729,194

## Abstract

Our overall goal is to discover details of fundamental mechanisms underlying regulation of CaV1.2 channels
that have eluded more than four decades of investigation. We propose to use novel tools and approaches to
identify novel proteins, supramolecular complexes, and signaling pathways affecting CaV1.2 channels as the
basis for targeted drug development for arrhythmias. Although it is well-established that phosphorylation by
cyclic AMP (cAMP)-PKA, but not Ca2+/calmodulin kinase II (CaMKII), is the fundamental process by which b-
adrenergic stimulation controls Ca2+ influx via CaV1.2 in the heart, the molecular targets of PKA remain
unknown. A detailed molecular understanding of CaV1.2 regulation in myocytes has been hampered by the
inability to recapitulate and then dissect in heterologous expression systems key aspects of CaV1.2 function in
myocytes. Our novel tools surmount major obstacles that have limited progress in the field, and allow us to
identify the neighboring proteome of CaV1.2 in the heart and probe molecular aspects of CaV1.2 regulation,
using biochemical and electrophysiological techniques, within the context of cardiomyocytes, but with the
power of a heterologous expression system. The failure thus far to identify any site as essential for adrenergic
modulation led us to propose an alternative hypothesis: that a combination of phosphorylation sites in a1C is
required for b-adrenergic stimulation of CaV1.2. To address this hypothesis, we generated mice with alanine-
substitutions in rabbit a1C of all conserved and non-conserved consensus PKA phosphorylation sites (“35-
mutant a1C”), and found that b-adrenergic regulation was not dependent upon any of these serine or threonine
residues. Using a similar transgenic approach, we found that b-adrenergic regulation does not require
phosphorylation of any of the 18 N-terminal, HOOK and GK domain consensus PKA phosphorylation sites in
the b2b subunit. The next step is to create transgenic mice expressing b2b subunits with all PKA consensus
sites removed (“33-mutant b2b”) and test regulation in a b2 knockout background. Thereafter, we will determine
whether phosphorylation of either a1C or b subunits is sufficient to enable b-adrenergic regulation by crossing
the 35-mutant a1C and the 33-mutant b2b mice. If adrenergic regulation is preserved, these results would shift a
four-decade paradigm: the core CaV1.2 subunits are not the required PKA targets. Other aims of the proposal
are to determine whether b-adrenergic stimulation of CaV1.2 is dependent upon a target extrinsic to CaV1.2
core subunits, and whether specifically attenuating b-adrenergic-modulation of CaV1.2 can suppress
arrhythmogenesis. The feasibility of this approach is supported by the demonstration that disrupting the b-a
interaction prevents b-adrenergic regulation of CaV1.2. The three Aims, which will provide key new
understandings concerning the regulation of Ca2+ influx in cardiomyocytes, are highly relevant towards
unders...

## Key facts

- **NIH application ID:** 10425277
- **Project number:** 5R01HL146149-04
- **Recipient organization:** COLUMBIA UNIVERSITY HEALTH SCIENCES
- **Principal Investigator:** Steven O Marx
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $729,194
- **Award type:** 5
- **Project period:** 2019-08-01 → 2023-12-14

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10425277

## Citation

> US National Institutes of Health, RePORTER application 10425277, Phosphorylation-dependent regulation of calcium channels by macromolecular complexes (5R01HL146149-04). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10425277. Licensed CC0.

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