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Reconstitution of β-adrenergic regulation of Ca(V)1.2: Rad-dependent and Rad-independent protein kinase A mechanisms

Item Type:Preprint
Title:Reconstitution of β-adrenergic regulation of Ca(V)1.2: Rad-dependent and Rad-independent protein kinase A mechanisms
Creators Name:Katz, M. and Subramaniam, S. and Chomsky-Hecht, O. and Tsemakhovich, V. and Belkacemi, A. and Flockerzi, V. and Klussmann, E. and Hirsch, J. and Weiss, S. and Dascal, N.
Abstract:INTRODUCTION: Cardiac L-type voltage-gated Ca(V)1.2 channels are crucial in physiological regulation of cardiac excitation-contraction coupling. Adrenergic modulation of Ca(V)1.2 starts with activation of β-adrenergic receptors (AR) and culminates in protein kinase A (PKA) - induced increase of calcium influx through Ca(V)1.2 channels. To date, this cascade has never been fully reconstituted in heterologous systems; even partial reconstitution proved challenging and controversial. A recent study identified Rad, a calcium channel inhibitory protein, as an essential component of the adrenergic signaling cascade. We corroborated this finding, further characterized, and fully reconstituted, the complete β-AR Ca(V)1.2 modulation cascade in a heterologous expression system. OBJECTIVE: Our primary goal was to heterologously reconstitute the complete β-adrenergic cascade, and to investigate the role of Rad and additional molecular determinants in adrenergic regulation of cardiac Ca(V)1.2. METHODS AND RESULTS: We utilized the Xenopus oocyte heterologous expression system. We expressed Ca(V)1.2 channel subunits, without or with Rad and β1-AR or β2-AR. To activate PKA, we injected cyclic AMP (cAMP) into the oocytes, or extracellularly applied isoproterenol (Iso) to stimulate β-AR. Whole-cell Ba(2+) currents served as readout. We find and distinguish between two distinct pathways of PKA modulation of Ca(V)1.2: Rad-dependent (~80% of total) and Rad-independent. We separate the two mechanisms by showing distinct requirements for the cytosolic N- and distal C- termini of α(1C) and for the Ca(V)β subunit. Finally, for the first time, we reconstitute the complete pathway using agonist activation of either β1-AR or β2-AR. The reconstituted system reproduces the known features of β-AR regulation in cardiomyocytes, such as a >2-fold increase in Ca(V)1.2 current, a hyperpolarizing shift in activation curve, and a high constitutive activity of β2-AR. CONCLUSIONS: The adrenergic modulation of Ca(V)1.2 is composed of two distinct pathways, Rad-independent and Rad-dependent. The latter contributes most of the β-AR-induced enhancement of Ca(V)1.2 activity, crucially depends on Ca(V)β subunit, and is differently regulated by β1-AR and β2-AR. The reconstitution of the full β-AR cascade provides the means to address central unresolved issues related to roles of auxiliary proteins in the cascade, Ca(V)1.2 isoforms, and will help to develop therapies for catecholamine-induced cardiac pathologies.
Keywords:Animals, Xenopus laevis
Publisher:Cold Spring Harbor Laboratory Press
Article Number:2020.11.30.403964
Date:1 December 2020
Official Publication:https://doi.org/10.1101/2020.11.30.403964
Related to:
https://edoc.mdc-berlin.de/20286/Final version

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