β-Adrenergic regulation requires direct anchoring of PKA to cardiac Ca _V 1.2 channels via a leucine zipper interaction with A kinase-anchoring protein 15

Abstract

.2 channels and PKA in the transverse tubules of isolated ventricular myocytes. Site-directed mutagenesis studies reveal that AKAP15 directly interacts with the distal C terminus of the cardiac Ca V1.2 channel via a leucine zipper-like motif. Disruption of PKA anchoring to Ca V1.2 channels via AKAP15 using competing peptides markedly inhibits the ␤-adrenergic regulation of CaV1.2 channels via the PKA pathway in ventricular myocytes. These results identify a conserved leucine zipper motif in the C terminus of the Ca V1 family of Ca 2؉ channels that directly anchors an AKAP15-PKA signaling complex to ensure rapid and efficient regulation of L-type Ca 2؉ currents in response to ␤-adrenergic stimulation and local increases in cAMP.V oltage-gated L-type calcium (Ca 2ϩ ) channels play a pivotal role in the regulation of a wide range of cellular processes, including membrane excitability, Ca 2ϩ homeostasis, protein phosphorylation, and gene regulation. In cardiac myocytes, Ca 2ϩ influx through Ca V 1.2 channels contributes to the plateau phase of the cardiac action potential and is responsible for initiating excitation-contraction coupling (1-3). Voltage-gated L-type Ca 2ϩ channels are multisubunit complexes composed of a poreforming ␣ 1 subunit and auxiliary ␤ and ␣ 2 ␦ subunits (4). In cardiac muscle, a distinct ␣ 1 subunit (␣ 1 1.2a) (5, 6), an ␣ 2 ␦ subunit (7), and several isoforms of ␤ subunits [␤ 1b and ␤ 2a-d (8-11)] have been identified and implicated to form the Ca V 1.2 channel. As for the skeletal muscle Ca V 1.1 channel (12, 13), two size forms of the ␣ 1 1.2a subunit of Ca V 1.2 channels, Ϸ240 and 210 kDa, are present in cardiac muscle and differ by truncation at the C terminus (14). Whereas the majority of Ca 2ϩ channel ␣ 1 subunits isolated from cardiac muscle are truncated (11,14,15), the cleaved distal C terminus remains associated with the truncated ␣ 1 subunit of Ca V 1.2 after proteolytic processing, and peptides derived from it can regulate channel activity (16,17). Ca V 1.2 channels can be modulated by a variety of receptormediated processes, including stimulation through activation of the ␤-adrenergic receptor͞cAMP signaling pathway (4, 18-21). Activation of ␤-adrenergic receptors increases cardiac L-type Ca 2ϩ currents through cAMP-dependent protein kinase A (PKA)-mediated phosphorylation of the Ca V 1.2 channel protein and͞or associated proteins (22, 23). As for skeletal muscle Ca v 1.1 channels (24, 25), both the ␣ 1 and ␤ subunits of Ca V 1.2 channels are substrates for phosphorylation by PKA (14,(26)(27)(28). PKA phosphorylates only the full-length form of the ␣ 1 subunit, on a single site containing serine 1928 in the C-terminal domain (14,26). In contrast, the C-terminal truncated ␣ 1 subunit is not a substrate for phosphorylation by PKA in vitro (14,26). Ca V 1.2 channels consisting of only ␣ 1 subunits can be regulated by PKA, implicating phosphorylation of serine 1928 in channel regulation (29, 30). Mutation of serine 1928 to alanine prevents PKAdependent phosphorylation and the low l...

Myoscape controls cardiac calcium cycling and contractility via regulation of L-type calcium channel surface expression

“…In this context, the overexpression of Myoscape resulted in enhanced global calcium transients with higher calcium amplitudes and LTCC currents. These results were additive to the effects of isoproterenol, implying that Myoscape acts independently of the adrenergic control of LTCC activity via PKA-dependent phosphorylation91326. Conversely, Myoscape ablation reduced LTCC currents irrespective of beta-adrenergic costimulation, which is associated with reduced global calcium transients and impaired contractile performance of cardiomyocytes.…”

“…Specifically, it contains various phosphorylation residues that allow fast regulatory responses13, as well as the IQ motif, which is a specific interaction domain for calmodulin15. Calmodulin represents the major calcium-dependent modulator of LTCC function, promoting either calcium-dependent inactivation (CDI) or calcium-dependent facilitation (CDF)161718.…”

Sites of proteolytic processing and noncovalent association of the distal C-terminal domain of CaV1.1 channels in skeletal muscle

“…AKAP15 directly targets PKA to the distal C terminus of cardiac and skeletal muscle Ca V 1 channels through a modified leucine zipper interaction (9,10). Mutation of this motif prevents PKA anchoring, and disruption of this interaction with dominantnegative peptides prevents PKA-dependent potentiation of Ca 2ϩ channel activity in skeletal and cardiac myocytes (9,10).…”

“…AKAP15 directly targets PKA to the distal C terminus of cardiac and skeletal muscle Ca V 1 channels through a modified leucine zipper interaction (9,10). Mutation of this motif prevents PKA anchoring, and disruption of this interaction with dominantnegative peptides prevents PKA-dependent potentiation of Ca 2ϩ channel activity in skeletal and cardiac myocytes (9,10). Thus, anchoring of PKA to Ca V 1.1 channels via AKAP15 favors rapid phosphorylation and modulation of these channels as part of the overall regulation of contractile force in response to motor nerve stimulation (11,12).…”

β-Adrenergic–regulated phosphorylation of the skeletal muscle Ca V 1.1 channel in the fight-or-flight response

“…The C terminus is a substrate for PKA, and the sites of most rapid phosphorylation of purified Ca V 1.1 and Ca V 1.2 in vitro are located in the distal C-terminal domain (19,25,26). Despite proteolytic processing, the distal C terminus (dCT) beyond the point of proteolytic truncation is required for interaction with A Kinase Anchoring Protein 15 (AKAP15) (27)(28)(29), which in turn is required for β-adrenergic regulation of Ca V 1.1 and Ca V 1.2 channels in intact skeletal and cardiac myocytes (28)(29)(30)(31). Coexpression of the dCT as a separate protein by transfection in nonmuscle cells leads to binding to the proximal C-terminal domain of truncated Ca V 1.1 and Ca V 1.2 channels (23,24) and to dramatic autoinhibition of Ca V 1.2 channel activity (24).…”