Reconstitution of β-adrenergic regulation of Ca_V1.2: Rad-dependent and Rad-independent protein kinase A mechanisms

AbstractIntroductionCardiac L-type voltage-gated CaV1.2 channels are crucial in physiological regulation of cardiac excitation-contraction coupling. Adrenergic modulation of CaV1.2 starts with activation of β-adrenergic receptors (AR) and culminates in protein kinase A (PKA) - induced increase of calcium influx through CaV1.2 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 CaV1.2 modulation cascade in a heterologous expression system.ObjectiveOur 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 CaV1.2.Methods and ResultsWe utilized theXenopusoocyte heterologous expression system. We expressed CaV1.2 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 Ba2+currents served as readout. We find and distinguish between two distinct pathways of PKA modulation of CaV1.2: 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 α1Cand for the CaVβ 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 CaV1.2 current, a hyperpolarizing shift in activation curve, and a high constitutive activity of β2-AR.ConclusionsThe adrenergic modulation of CaV1.2 is composed of two distinct pathways, Rad-independent and Rad-dependent. The latter contributes most of the β-AR-induced enhancement of CaV1.2 activity, crucially depends on CaVβ 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, CaV1.2 isoforms, and will help to develop therapies for catecholamine-induced cardiac pathologies.