A membrane-associated phosphoswitch in Rad controls adrenergic regulation of cardiac calcium channels
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
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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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 8

Schematics of role of membrane affinity and interaction of Rad and CaVβ in β-adrenergic regulation of CaV1.2.

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Schematics of role of membrane affinity and interaction of Rad and CaVβ ...
(A) CaVβ is close to the membrane because it is bound to CaVα, a membrane-embedded channel protein. In the basal state, the binding of Rad and CaVβ (and consequent channel inhibition) is promoted by the association of positively charged Rad with the negatively charged plasma membrane. Since the intrinsic affinity of Rad for β is low, the binding of Rad to β depends on the compensating, high local concentration of Rad at the membrane. Rad binding to β leads to inhibition of Ca2+ channels. (B) Adrenergic stimulation causes PKA activation and phosphorylation of the C-terminus of Rad, which decreases the net positive charge of the C-terminus of Rad. This change in charge disrupts Rad’s attachment to the membrane (arrow 1), thereby diluting it and favoring its dissociation from CaVβ (arrow 2). Rad-less Ca2+ channels have increased open probability compared with Rad-bound channels. (C) Rad is constitutively attached to the membrane via appending the CAAX motif to its C-terminus. Despite the change in charge induced by PKA phosphorylation of Rad, the CAAX motif prevents Rad’s detachment from the membrane, and thus the binding of Rad to CaVβ is preserved and the Ca2+ channel remains inhibited.