A membrane-associated phosphoswitch in Rad controls adrenergic regulation of cardiac calcium channels
Arianne Papa, … , Manu Ben-Johny, Steven O. Marx
Arianne Papa, … , Manu Ben-Johny, Steven O. Marx
Published January 16, 2024
Citation Information: J Clin Invest. 2024;134(5):e176943. https://doi.org/10.1172/JCI176943.
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Research Article Cardiology

A membrane-associated phosphoswitch in Rad controls adrenergic regulation of cardiac calcium channels

Abstract

The ability to fight or flee from a threat relies on an acute adrenergic surge that augments cardiac output, which is dependent on increased cardiac contractility and heart rate. This cardiac response depends on β-adrenergic–initiated reversal of the small RGK G protein Rad–mediated inhibition of voltage-gated calcium channels (CaV) acting through the Cavβ subunit. Here, we investigate how Rad couples phosphorylation to augmented Ca2+ influx and increased cardiac contraction. We show that reversal required phosphorylation of Ser272 and Ser300 within Rad’s polybasic, hydrophobic C-terminal domain (CTD). Phosphorylation of Ser25 and Ser38 in Rad’s N-terminal domain (NTD) alone was ineffective. Phosphorylation of Ser272 and Ser300 or the addition of 4 Asp residues to the CTD reduced Rad’s association with the negatively charged, cytoplasmic plasmalemmal surface and with CaVβ, even in the absence of CaVα, measured here by FRET. Addition of a posttranslationally prenylated CAAX motif to Rad’s C-terminus, which constitutively tethers Rad to the membrane, prevented the physiological and biochemical effects of both phosphorylation and Asp substitution. Thus, dissociation of Rad from the sarcolemma, and consequently from CaVβ, is sufficient for sympathetic upregulation of Ca2+ currents.

Authors

Arianne Papa, Pedro J. del Rivero Morfin, Bi-Xing Chen, Lin Yang, Alexander N. Katchman, Sergey I. Zakharov, Guoxia Liu, Michael S. Bohnen, Vivian Zheng, Moshe Katz, Suraj Subramaniam, Joel A. Hirsch, Sharon Weiss, Nathan Dascal, Arthur Karlin, Geoffrey S. Pitt, Henry M. Colecraft, Manu Ben-Johny, Steven O. Marx

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Figure 4

Effects on Venus-Rad binding to Cerulean-β2B by the insertion of negatively charged Asp residues in the N- and C-termini of Rad.

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Effects on Venus-Rad binding to Cerulean-β2B by the insertion of negativ...
(A) Protein structure prediction with AlphaFold of human Rad (P55042) (60, 61). (B) Protein sequences of N-terminus and C-terminus of Rad, showing phosphorylated residues (highlighted yellow) and residues substituted with Asp (red font). Arrowheads mark hydrophobic residues in the basic-hydrophobic motif of Rad. (C–E) FRET efficiency (ED) between Ven-Rad mutants and Cer-β2B is plotted against the free concentration of Ven-C-2SD, C-4SD, and C-6SD Rad. The red line fits a 1:1 binding isotherm for C-2SD, C-4SD, and C-6SD Rad. The black and blue lines are the 1:1 binding isotherm for WT-Rad in the absence and presence of FSK + Cal (same as Figure 2B). (F) Graph summarizing mean Kd,EFF for the binding of β2B and WT and mutant Rad. Error bars are SEM. Black and blue dashed lines are mean values of WT Rad without and with FSK + Cal (from Figure 2). P < 0.0001 by 1-way ANOVA; ****P < 0.0001 compared with WT Rad without FSK + Cal by Dunnett’s multiple-comparison test. n = 3, 7, 6, 6, 3, and 3 from left to right. (G) Table showing changes in charge induced by either treatment with forskolin (FSK) and calyculin (Cal) or substitution of Asp residues in full-length Rad, in the N-terminal domain of Rad (NTD, residues 1–45), or in the C-terminal domain of Rad (CTD, residues 251–307), calculated using https://protcalc.sourceforge.net The change in charge on phosphorylated Ser residues by the addition of a phosphate group is –1.96 at pH 7.2, and –1.86 at pH 6.6, assuming pKa2 = 5.8 (62).