Adrenergic Ca _V 1.2 Activation via Rad Phosphorylation Converges at α _1C I-II Loop

Abstract

Rationale: Changing activity of cardiac Ca V 1.2 channels under basal conditions, during sympathetic activation, and in heart failure is a major determinant of cardiac physiology and pathophysiology. Although cardiac CaV1.2 channels are prominently up-regulated via activation of protein kinase A, essential molecular details remained stubbornly enigmatic. Objective: The primary goal of this study was to determine how various factors converging at the Ca V 1.2 I-II loop interact to regulate channel activity under basal conditions, during β-adrenergic stimulation, and in heart failure. Methods and Results: We generated transgenic mice with expression of Ca V 1.2 α 1C subunits with: 1) mutations ablating interaction between α 1C and β subunits; 2) flexibility-inducing polyglycine substitutions in the I-II loop (GGG-α 1C ); or 3) introduction of the alternatively spliced 25-amino acid exon 9* mimicking a splice variant of α 1C up-regulated in the hypertrophied heart. Introducing three glycine residues that disrupt a rigid IS6-AID helix markedly reduced basal open probability despite intact binding of Ca V β to α 1C I-II loop, and eliminated β-adrenergic agonist stimulation of Ca V 1.2 current. In contrast, introduction of the exon 9* splice variant in α 1C I-II loop, which is increased in ventricles of patients with end-stage heart failure, increased basal open probability but did not attenuate stimulatory response to β-adrenergic agonists when reconstituted heterologously with β 2B and Rad or transgenically expressed in cardiomyocytes. Conclusions: Ca 2+ channel activity is dynamically modulated under basal conditions, during β-adrenergic stimulation, and in heart failure by mechanisms converging at the α 1C I-II loop. Ca V β binding to α 1C stabilizes an increased channel open probability gating mode by a mechanism that requires an intact rigid linker between the β subunit binding site in the I-II loop and the channel pore. Release of Rad-mediated inhibition of Ca 2+ channel activity by β-adrenergic agonists/PKA also requires this rigid linker and β binding to α 1C .

PDE1 inhibition modulates Cav1.2 channel to stimulate cardiomyocyte contraction

“…The lack of myofilament or SR modifications by PDE1i led us to consider the opening of Ca v 1.2 – the key first step in EC coupling. As recently revealed by the Marx laboratory 11, 12 , PKA phosphorylates the binding protein Rad to dis-inhibit the channel and increase conductance. In larger mammalian cells such as those from dog, rabbit, and guinea pig, the relative role of Ca v 1.2 versus SR Ca 2+ is 1:2 and near 1:1 in failing hearts 32, 33 .…”

“…This suggests that while proximate to the channel, the two PDEs reside in different nanodomains relative to G s -coupled GPCRs. Beta-adrenergic stimulated Ca v 1.2 current requires Rad phosphorylation which otherwise constitutively suppresses current via a complex with the channel’s β and α 1C subunits 11, 12 . Proximity protein analysis has identified PDE4 isoforms, PDE3A, and PDE1C as being near Ca v 1.2.…”

“…The intracellular mechanisms whereby PDE1i augments contractility remain unknown. PKA activation by β-AR stimulation leads to phosphorylation of the regulatory protein Rad to increase Ca v 1.2 (L-type calcium channel) conductance 11, 12 . Concomitant PKA phosphorylation of phospholamban (PLN) disinhibits the sarcoplasmic reticulum (SR) Ca 2+ ATPase (SERCA) to increase SR calcium uptake and calcium-induced calcium release 13, 14 .…”

Mechanisms and Regulation of Cardiac CaV1.2 Trafficking

“…Until very recently, the prevailing thought was that Ca V β subunit interactions with α 1c were indispensable for channel trafficking to the cardiomyocyte surface membrane where the channels fulfill a duality of functions at the t-tubules in triggering Ca 2+ induced Ca 2+ release from RyR2, and contributing to loading of the SR [ 34 ], and play a further role at the caveolae in activation of calcineurin-NFAT signaling pathways [ 35 ]. The role of the Ca V β-subunit in membrane targeting of the channels has been extensively reviewed elsewhere [ 36 , 37 ] and so we mention it only briefly here to highlight some more recent advances [ 38 , 39 ]. In heterologous expression systems and in neurons, interactions between β-subunits and α 1 are fundamentally required for channel trafficking to the plasma membrane [ 7 , 19 , 24 , 40 , 41 , 42 , 43 , 44 ].…”

GRKs and Epac1 Interaction in Cardiac Remodeling and Heart Failure

“…The latter triggers a massive release of Ca 2+ (through the opening of RyR2) from the sarcoplasmic reticulum (SR) to induce myofilament contraction. This process is known as Ca 2+ -induced Ca 2+ release (CICR) [ 2 , 22 , 23 ]. Then, the PKA-dependent phosphorylation of PLB relieves its tonic inhibition of SR Ca 2+ ATPase activity, thereby promoting Ca 2+ uptake into the SR, and relaxation.…”