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Mechanism of adrenergic CaV1.2 stimulation revealed by proximity proteomics

Abstract

Increased cardiac contractility during the fight-or-flight response is caused by β-adrenergic augmentation of CaV1.2 voltage-gated calcium channels1,2,3,4. 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.

Fig. 1: Phosphorylation of α1C and β subunits by PKA is not required for β-adrenergic regulation of CaV1.2.
Fig. 2: Changes in the CaV1.2 subdomain proteome upon β-adrenergic agonist activation of PKA signalling in the heart.
Fig. 3: Phosphorylation of Rad is required for cAMP–PKA-mediated activation of CaV1.2.
Fig. 4: RGK GTPases confer adrenergic regulation to CaV1.2, CaV1.3 and CaV2.2 channels via binding to β.

Data and material availability

All transgenic mice are available from S.O.M. under a material agreement with Columbia University. All data are available in the main text, the Extended Data or the Supplementary Information. Proteomics raw data and search results were deposited in the PRIDE archive and can be accessed via the ProteomeXchange under accession numbers PXD014499, PXD014500 and PXD014501. The FRET software is accessible on github at https://github.com/manubenjohny/FACS_FRET. Source data for Figs. 14 and Extended Data Figs. 1, 2, 6, 8 are provided with the paper.

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Acknowledgements

We thank A. Karlin for helpful discussions and editing the manuscript; B. Soda for creating the cell-model illustration; and G. A. Bradshaw for technical assistance. This publication was supported by the National Institutes of Health (NIH; grants R01 HL113136, R01 HL121253 and R01 HL146149) and by the National Center for Advancing Translational Sciences (grant UL1TR001873). These studies used the resources of the Herbert Irving Comprehensive Cancer Center Flow Cytometry Shared Resources, funded in part through Center Grant P30CA013696. Images were collected (and analysed) in the Confocal and Specialized Microscopy Shared Resource of the Herbert Irving Comprehensive Cancer Center at Columbia University, supported by NIH grant P30 CA013696 (National Cancer Institute). A.P. was supported by NIH grant T32 HL120826 and National Science Foundation (NSF) Division of Graduate Education (DGE) grant 1644869. D.R. was supported by grants T32 HL120826 and F31 HL142178. J.K. was supported by grant T32 HL007343 and the Glorney–Raisbeck Fellowship from the New York Academy of Medicine, and J.A.H. was supported by grant T32 HL007854. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

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Contributions

The following authors designed research and analysed data: G.L., A.P., A.N.K., S.I.Z., D.R., J.A.H., J.K., L.Y., B.-X.C., A.K, S.P.G., G.S.P., H.M.C., M.B.-J., M.K. and S.O.M. The following authors performed research and analysed data: G.L., A.P., A.N.K., S.I.Z., D.R., J.A.H., J.K., L.Y., B.-X.C., A.K, K.D., G.S.P., H.M.C., M.B.J., M.K. and S.O.M. The following authors wrote the paper with input from all authors: S.O.M., M.K., H.M.C., G.S.P. and M.B.J. All authors provided feedback and agreed on the final manuscript.

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Correspondence to Marian Kalocsay or Steven O. Marx.

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Peer review information Nature thanks Donald Bers, Alice Ting and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Putative PKA phosphorylation sites in α1C and β2B subunits.

a, Left column, the 35 putative PKA phosphorylation sites in rabbit α1C. Centre, the 51 residues in red are either predicted phosphorylation sites or within the immediate region of the predicted phosphorylation sites. Right, all 51 residues were replaced with alanine in 35-mutant α1C transgenic mice. b, Combined bar and column scatter plot of Boltzmann function parameters, V50. Data are mean ± s.e.m. **P < 0.01; ***P < 0.001; ****P < 0.0001 by paired two-tailed t-test. pWT α, n = 19; 35-α mutant, n = 14; 28-β mutant, n = 16; 35-α mutant × 28-β mutant, n = 24. Specific P values can be found in the associated Source Data (see Supplementary Information). c, Graph showing isoproterenol- and forskolin-induced increases in nisoldipine-resistant current, stratified by total basal current density before nisoldipine treatment. d, Left, the 28 putative PKA phosphorylation sites in the N-terminal (NT), Hook, GK and C-terminal (CT) domains of β2B. Centre, the 37 residues in red are either predicted phosphorylation sites or within the immediate vicinity of predicted phosphorylation sites, and were mutated to alanine in the 28-mutant GFP-tagged β2B transgenic mice (right). e, Fluorescence imaging of isolated cardiomyocytes expressing the GFP-tagged 28-β mutant. Representative of images from more than five biologically independent mice. f, Anti-β-subunit immunoblot of cleared lysates from doxycycline-fed 35-mutant α1C transgenic (TG) mice or 35-mutant α1C × GFP-tagged 28-mutant β2B expressing mice hearts. Representative of immunoblots obtained from at least three biologically independent mice. g, Anti-Flag antibody (upper) and anti-β antibody (lower) immunoblots of anti-Flag antibody immunoprecipitations from cleared lysates of hearts from pWT, 35-α and three mice expressing 35-α × GFP-tagged-28-β. Representative images from two independent experiments. For source gel data, see Supplementary Fig. 1. Source data

Extended Data Fig. 2 Trafficking and function of APEX2-conjugated CaV1.2 subunits in heart.

a, Exemplar current–voltage relationship of Ca2+ currents from cardiomyocytes of α1C–APEX2 mice, acquired in the absence (black trace) and presence (red trace) of nisoldipine. Insets show series of whole-cell CaV1.2 currents recorded from a series of voltage steps between −40 mV and +60 mV from a holding potential of −50 mV. Scale bars, horizontal 100 ms, vertical 10 pA/pF. Representative of five experiments. b, Time course of changes in sarcomere length after superfusion of nisoldipine-containing solution. Representative of seven experiments. c, Percentage sarcomere shortening in the presence of nisoldipine. Data are mean ± s.e.m. ****P < 0.0001 by unpaired two-tailed t-test. n = 12 and 7 cardiomyocytes from left to right. NTG, nontransgenic. d, Immunofluorescence of cardiomyocytes isolated from mice expressing α1C–APEX2 and β2B–APEX2, exposed to biotin-phenol and H2O2 or no H2O2. Staining is with anti-V5 and Alexa594-conjugated secondary antibodies and streptavidin-conjugated Alexa488, and nuclear labelling is with DAPI. Scale bar, 5 μm. Representative of 13 and 8 cardiomyocytes from 2 and 3 mice respectively. e, Streptavidin–HRP blot of lysates from isolated ventricular cardiomyocytes, representative of five similar experiments. f, Exemplar whole-cell CaV1.2 currents recorded from cardiomyocytes of α1C–APEX2 transgenic mice. Black trace, 300 nM nisoldipine; blue trace, 200 nM isoproterenol plus nisoldipine. Representative of nine cells from two biologically independent mice. g, As in f, except from β2B–APEX2 mice. Black trace, control; blue trace, 200 nM isoproterenol. Representative of seven experiments from two biologically independent mice. h, i, Anti-phospho-phospholamban immunoblot of proteins from cardiomyocytes isolated from α1C–APEX2 and β2B–APEX2 mice. Cardiomyocytes were exposed to either vehicle or 1 μM isoproterenol after incubation with biotin–phenol. Blots are representative of three independent experiments from at least five biologically independent mice for each genotype. j, As in h and i, except that cardiomyocytes were isolated from non-transgenic mice without incubation with biotin–phenol. Blot is representative of three independent experiments from three biologically independent mice. k, As  h and i, except that whole heart was exposed to 1 μM isoproterenol for 5 min after infusion of biotin-phenol. This blot is representative of at least five biologically independent mice for no isoproterenol and at least five biologically independent mice for isoproterenol. For source gel data, see Supplementary Fig. 1. Source data

Extended Data Fig. 3 Analysis of proteins quantified by mass spectrometry in cardiomyocytes isolated from α1C–APEX2 and β2B–APEX2 mice.

a, Proteins with a ratio of more than 2 (measured by normalized TMT signal/noise) in the indicated experimental conditions, compared with a no-labelling control (no H2O2), were sorted by spectral counts. The 150 proteins with the highest peptide counts are displayed in this colour-coded table. α1C–APEX2 and β2B–APEX2 data were collected in biological duplicate experiments. Supplementary Table 1 shows all 3,883 proteins quantified by multiplexed SPS MS3 TMT mass spectrometry. b, Prefuse force-directed map of proteins from a. Peptide counts were used as weight. Proteins mapping to the GO term ‘Z disc’ are in green, to ‘membrane’ in yellow, and to both are in purple. α1C–APEX2 and β2B–APEX2 are in blue. c, GO term (cellular localization) enrichment for proteins in a. See Supplementary Table 2 for the full table.

Extended Data Fig. 4

Two-way hierarchical clustering of scaled data from Fig. 2. a, Dendrogram showing two-way hierarchical clustering of scaled TMT s/n data for streptavidin-purified proteins from α1C–APEX2 cardiomyocytes after stimulation with vehicle or isoproterenol. Shown are scaled relative TMT protein quantification data for 1,951 proteins from biological quintuplicate α1C–APEX2 mice. Clustering used Ward’s minimum-variance method. b, Dendrogram showing two-way hierarchical clustering of scaled relative quantification data for 1,936 proteins from biological triplicate β2B–APEX2 experiments. Heterogeneity between cardiomyocyte preparations from different mice is apparent. c, Dendrogram showing two-way hierarchical clustering of scaled relative quantification data for 2,610 proteins from whole-organ α1C–APEX without or with perfusion of isoproterenol. Prominent heterogeneity in relative protein quantification between hearts is apparent. The position of Rad is indicated by a red line. In this experiment, the individual hearts were not paired. d, Dendrogram showing two-way hierarchical clustering of scaled TMT s/n data from non-transgenic mouse cardiomyocytes stimulated with isoproterenol or with vehicle. Scaled data for 4,622 quantified proteins from a biological quadruplicate experiment are displayed. Pairing of samples is apparent.

Extended Data Fig. 5 Isoproterenol-induced change in Rad detected by mass spectrometry.

a, Right, the MS2 spectrum (top) and TMT quantification parameters (bottom) for a Rad peptide changed upon treatment of murine hearts with isoproterenol. Shown is the MS2 spectrum that was used to identify the Rad peptide IFGGIEDGPEAEAAGHTYDR. Left, m/z ratios for b and y ions identified in the spectrum and their deviation from theoretical m/z ratios. We measured the precursor mass as 778.71 Da with a charge of +3. Peptide modifications were +229.16 Da for TMT on the peptide N terminus and lysine residues, +57.02 Da for cysteine alkylation and +15.99 for methionine oxidation. Shown are ion injection times, isolation specificity, sum of signal-to-noise (SN) over all TMT channels, TMT raw intensities, adjusted intensities and final SN intensities used for relative quantification, as well as synchronous precursor selection (SPS) ion m/z ratios (isolated in the ion trap with Notch filtering; ‘Notch mz’ denotes the ion m/z ratio of individual isolated SPS ions prior to HCD fragmentation and MS3). b, Table showing gene names of proteins with P < 0.05 for the three approaches: cardiomyocytes isolated from α1C–APEX and β2B–APEX mice, and α1C–APEX hearts. Genes in yellow are common to all groups, but note that for Mast2, the fold change is not consistent. Data are mean fold changes for five pairs of biologically independent pairs of α1C–APEX2 cardiomyocyte samples, three pairs of biologically independent pairs of β2B–APEX cardiomyocyte samples, and ten α1C–APEX2 hearts, five without isoproterenol and five with isoproterenol. Non-adjusted unpaired two-tailed t-test. c, Venn diagram showing the data from b. Rrad, Rad; Prkaca, PKA catalytic subunit; Acss1, acyl-CoA synthetase short-chain family member 1. Rad is the only protein that is consistently changed amongst the three approaches.

Extended Data Fig. 6 Rad is required for forskolin-induced activation of heterologously expressed CaV1.2 channels.

a, Exemplar whole-cell CaV1.2 currents elicited from step depolarizations recorded from HEK293T cells expressing Rad. Voltage command steps were applied every 10 s before (black traces) and during (blue traces) forskolin treatment. Representative of at least ten cells. b, Methodology used for generating G/V curves. i, Upper, a 200-ms voltage ramp from −60 mV to +60 mV was applied every 10 s. Lower, current traces, each an average of three traces before (black) and three traces after (blue) forskolin treatment. ii, Conversion of time scale to applied voltage. iii, Conversion to GV relationship. Fold change was calculated at Gmax. c, Graph showing forskolin-induced fold change in current, stratified by basal current density. d, Exemplar traces of Ba2+ currents in the absence and presence of Rad elicited by voltage ramp every 10 s. Black traces, before forskolin treatment; blue traces after treatment; no Rad, 7 cells; Rad, 16 cells. e, Boltzmann function parameter V50. Data are mean ± s.e.m. **P < 0.01 by paired two-tailed t-test. n = 7 and 16, from left to right. f, g, Ratio of Ba2+ current after forskolin treatment to Ba2+ current before treatment for cells transfected without and with Rad. Representative of analyses for three cells for each condition. hl, Distribution of sweep-by-sweep average Po (single-trial Po) for different conditions. h, In the absence of Rad, sweeps with no openings or blank sweeps are rare (10%); most sweeps exhibit either intermediate or high levels of openings. i, The fraction of blank sweeps is increased with expression of Rad. j, If the PKA catalytic domain is also coexpressed with Rad, the fraction of blank sweeps is reduced and there is a resurgence of the high-activity mode. k, Pale blue lines show conditional Po–voltage relationships obtained for sweeps exhibiting high activity in the absence of Rad and PKA. The dark blue line is the Boltzmann fit. l, As in k, but with Rad and PKA expression. Source data

Extended Data Fig. 7 PKA phosphorylation sites in mouse Rad.

a, Serine/threonine residues (in purple) that are mutated to alanine in the 14-SA mutant. b, Mass-spectrometry identification of phosphorylated residues on Rad enriched with an anti-GFP nanobody matrix, from HEK cells expressing GFP–Rad and treated with forskolin. The number of spectral counts is plotted against the position of the phosphorylated amino acids in Rad. We detected 534 aggregated phosphopeptides in two independent experiments. c, Database entry for phosphorylation sites identified previously in Rad (https://phosphomouse.hms.harvard.edu/site_view.php?ref=IPI00133102). The highest level of Rad phosphorylation was detected in the heart (left panel). The lower right panel shows peptides detected with phosphorylated serine residues on positions 25, 38 and 300 (in bold red; mapped to the Rad expression constructs used here; blue highlighting indicates sequence covered by peptides). d, Serine residues mutated to alanine in the 4-SA mutant (arrows).

Extended Data Fig. 8 Binding of Rad and β2B is required for regulation of forskolin-induced stimulation of voltage-gated Ca2+ channels.

a, The Rad protein sequence shown here indicates the residues Arg208 and Leu235 that were substituted with alanine (yellow). b, The β2B protein sequence indicates the residues Asp244, Asp320 and Asp322 that were substituted with alanine (yellow), resulting in attenuation of Rad binding to the β subunit, as described previously28,29. c, Ba2+ currents from CaV1.2 channels, elicited by voltage ramp every 10 s from −60 mV to +60 mV over 200 ms, before (black) and after (blue) treatment with forskolin. Representative of 20 (top) and 15 (bottom) cells. d, Boltzmann function parameter V50. Data are mean ± s.e.m. ***P < 0.001 by paired two-tailed t-test. The data for wild-type Rad are the same as in Fig. 3h. Specific P values can be found in the associated Source Data. n = 16, 19 and 13, from left to right. e, Fold change in Gmax in Cav1.3 channels. Data are mean ± s.e.m. P < 0.0001 by one-way ANOVA; ****P < 0.0001 by Dunnett’s test. The data for wild-type Rad and wild-type β2B are the same as in Fig. 4e. n = 7, 7 and 9 cells, from left to right. f, Fold change in Gmax in Cav2.2 channels. Data are mean ± s.e.m. P < 0.001 by one-way ANOVA; ***P < 0.001 by Dunnett’s test. Data for wild-type Rad and wild-type β2B are as in Fig. 4h. n = 11, 7 and 8 cells, from left to right. Source data

Extended Data Fig. 9 Phosphorylation-dependent dissociation of Rad, β3 and β4 subunits.

a, b, FRET two-hybrid binding isotherms were determined for Cer-tagged β3 (a) and β4 (b) subunits, and N-terminal Ven-tagged wild-type (left) or 4-SA mutant (right) Rad. FRET efficiency (ED) is plotted against the free concentration Ven–WT or Ven–4SA-mutant Rad. The solid line fits a 1/1 binding isotherm. Coexpression of the PKA catalytic subunit weakened binding in cells expressing wild-type Rad, but not in cells expressing 4-SA mutant Rad. c, Bar graph summarizing mean Kd,EFF for β2B, β3 and β4, and wild-type and 4-SA mutant Rad, expressed without and with the PKA catalytic subunit. Data are mean ± 95% confidence intervals; error bars show 95% confidence intervals for the pooled nonlinear fits based on the Jacobians computed. The sample size for each condition is 1,580–10,364 cells, acquired via two independent transfections and then pooled. The distribution of data in this graph is reflected in Fig. 4b, c, and in a, b.

Extended Data Fig. 10 ClustalW alignment of Rad sequences and RGK GTPases.

a, Conservation of phosphorylation sites from mouse Rad (Ser25, Ser38, Ser272 and Ser300) in other species. Blue highlights basic amino acids (arginine, lysine and histidine), and red highlights serine and threonine. b, C-terminal phosphorylation sites are conserved in other species. The equivalent of the Ser25 phosphorylation site is conserved in Rem1, and the equivalent of Ser38 is probably conserved in Gem. Phosphorylation sites in mouse Rad (Ser25, Ser38, Ser272 and Ser300) are indicated with arrows. Blue highlights basic amino acids (arginine, lysine and histidine), and red highlights serine and threonine.

Supplementary information

Supplementary Figure 1

Raw, uncropped data for gels.

Reporting Summary

Supplementary Table 1

Mass spectrometry data acquired from isolated cardiomyocytes of α1C-APEX2 and β2B-APEX2 mice. The full table with 3883 proteins quantified by multiplexed SPS MS3 TMT mass spectrometry. Comparison of no biotin-label vs. biotin-label. n=2 α1C mice and n=2 β2B mice.

Supplementary Table 2

Gene Ontology analysis. Proteins with a ratio of >2 (measured by normalized TMT signal/noise) in the experimental conditions compared to a no-labeling control (no H2O2) were sorted by spectral counts. Peptide counts were used as weight. Table was generated for the 150 proteins with highest peptide counts as output of the BINGO tool (Maere, S., Heymans, K. & Kuiper, M. Bioinformatics 21, 3448-3449, doi:10.1093/bioinformatics/bti551 (2005)) to determine statistically overrepresented Gene Ontology (GO) categories. Table shows GO term (cellular localization) enrichment for proteins in Extended Data Fig. 3a.

Supplementary Table 3

Mass spectrometry data acquired from isolated cardiomyocytes of α1C-APEX2 mice. Data were used to create volcano plot of fold change for relative protein quantification by TMT mass spectrometry of α1C-APEX2 samples (Fig. 2e). Data shown are means for 5 pairs of biologically independent samples. Non-adjusted unpaired two-tailed t-test between isoproterenol vs. no isoproterenol.

Supplementary Table 4

Mass spectrometry data acquired from isolated cardiomyocytes of β2B-APEX2 mice. Data were used to create volcano plot of fold change for relative protein quantification by TMT mass spectrometry of β2B-APEX2 samples (Fig. 2f). Data shown are means for 3 pairs of biologically independent samples. Non-adjusted unpaired two-tailed t-test between isoproterenol vs. no isoproterenol.

Supplementary Table 5

Mass spectrometry data acquired from hearts of α1C-APEX2 mice. Data were used to create volcano plot of fold change for relative protein quantification by TMT mass spectrometry of α1C-APEX2 samples (Fig. 2h). Data shown are means for 10 hearts, 5 without isoproterenol and 5 with isoproterenol. Non-adjusted unpaired two-tailed t-test between isoproterenol vs. no isoproterenol.

Supplementary Table 6

Mass spectrometry data acquired from hearts of non-transgenic mice. Data were used to create volcano plot of fold change for relative protein quantification by TMT mass spectrometry (Fig. 2j). Data shown are means for 4 pairs of biologically independent samples. Non-adjusted unpaired two-tailed t-test between isoproterenol vs. no isoproterenol.

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Liu, G., Papa, A., Katchman, A.N. et al. Mechanism of adrenergic CaV1.2 stimulation revealed by proximity proteomics. Nature 577, 695–700 (2020). https://doi.org/10.1038/s41586-020-1947-z

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