Facilitation of murine cardiac L-type Ca _v 1.2 channel is modulated by Calmodulin kinase II–dependent phosphorylation of S1512 and S1570

Abstract

Activity-dependent means of altering calcium (Ca 2+ ) influx are assumed to be of great physiological consequence, although definitive tests of this assumption have only begun to emerge. Facilitation and inactivation offer two opposing, activity-dependent means of altering Ca 2+ influx via cardiac Ca v 1.2 calcium channels. Voltage-and frequency-dependent facilitation of Ca v 1.2 has been reported to depend on Calmodulin (CaM) and/or the activity of Calmodulin kinase II (CaMKII). Several sites within the cardiac Ltype calcium channel complex have been proposed as the targets of CaMKII. Here, we generated mice with knockin mutations of α 1 1.2 S1512 and S1570 phosphorylation sites [sine facilitation (SF) mice]. Homocygote SF mice were viable and reproduced in a Mendelian ratio. Voltage-dependent facilitation in ventricular cardiomyocytes carrying the SF mutation was decreased from 1.58-to 1.18-fold. The CaMKII inhibitor KN-93 reduced facilitation to 1.28 in control cardiomyocytes. SF mutation negatively shifted the voltage-dependent inactivation and slowed recovery from inactivation, thereby making fewer channels available for activation. Telemetric ECG recordings at different heart rates showed that QT time decreased significantly more in SF than in control mice at higher rates. Our results strongly support the notion that CaMKII-dependent phosphorylation of Cav1.2 at S1512 and S1570 mediates Ca 2+ current facilitation in the murine heart. calcium channels | facilitation | heart | voltage-dependent ion channels C alcium (Ca 2+ ) current (I Ca ) through L-type channels is an important determinant of intracellular Ca 2+ transients that trigger transmitter release, secretion, and contraction (1). In the heart, the size of the intracellular Ca 2+ transient is determined by the release of Ca 2+ from intracellular stores and by the size of the L-type current (2). The availability of L-type channels to open is regulated by the membrane potential and other factors that include protein kinases, phosphatases, and Ca 2+ binding proteins (3, 4). A train of repetitive depolarizations (frequency-dependent) (5, 6) or a strong depolarizing prepulse (voltage-dependent) (7) drives the L-type calcium channels from their normal gating pattern into a mode of gating characterized by long opening and high open probability (8, 9), a process that has been termed "facilitation" and that represents one of the rare positive feedback mechanisms in signal transduction. The present consensus is that facilitation requires elevated intracellular calcium, or [Ca 2+ ], in the vicinity of the channel ("Ca In addition or alternatively to these CaM-dependent mechanisms, CDF of Ca v 1.2 has been attributed to the action of Calmodulin kinase II (CaMKII) (17)(18)(19)(20). Constitutively active CaMKII facilitated L-type current in excised patches (21). CaMKII is tethered to the C terminus in close proximity to the IQ motif, and CDF was abolished by mutating the putative interaction site (22). CaM-KII was reported by us and by others to phosphorylat...

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

“…inant role for phosphorylation of two other sites in the proximal C-terminal domain of Ca V 1.2 channels by CaMKII in voltagedependent facilitation of cardiac calcium currents in transfected cells and in murine heart in vivo (46,47). In light of those results, phosphorylation of Ca V 1.2-S1700 may be primarily involved in PKA regulation in response to activation of the sympathetic nervous system and β-adrenergic signaling.…”

Deletion of the C-terminal Phosphorylation Sites in the Cardiac β-Subunit Does Not Affect the Basic β-Adrenergic Response of the Heart and the Cav1.2 Channel

“…Recently, we reported that CDF requires phosphorylation of Ca v 1.2 at Ser 1512/1570 (27). In contrast, Colbran and co-workers (22) reported that phosphorylation of Ca v ␤ 2 by CaMKII at Thr 500 modulated CDF.…”

“…27, 30, 31. Facilitation of I Ca was measured during a triple pulse protocol with a 200-ms control pulse to 0 mV (V1) followed by a 200-ms prepulse (V pre ) to ϩ80 mV followed by 200-ms test pulse to 0 mV (V2) (27). The extent of voltage-dependent facilitation was calculated as the ratio of the peak current during V2 and V1.…”

“…Basal properties of the Ca v 1.2 current were unaffected as expected from the report that deletion of the Cacnb2 gene in the adult heart has minimal effects on I Ca (26). We crossed the ␤Stop line with the S1928A (25) and SF (S1512A and S1570A) (27) mouse lines that contain well characterized phosphorylation sites for PKA and CaMKII, respectively. Again, the basal properties of the Ca v 1.2 current were unaffected, suggesting that ␤-adrenergic regulation of the Ca v 1.2 channel may be mediated by other phosphorylation sites, e.g.…”

Mitochondrial‐Mediated Oxidative Ca 2+ /Calmodulin‐Dependent Kinase II Activation Induces Early Afterdepolarizations in Guinea Pig Cardiomyocytes: An In Silico Study

“…A growing body of evidence has demonstrated that CaMKII can also be activated by ROS 21, 22, 23, 24. Once activated, CaMKII can phosphorylate a wide range of key Ca 2+ and Na + regulatory proteins such as LCCs,25, 26, 27, 28 RyRs,29, 30, 31, 32, 33, 34, 35 phosphalamban,29, 34, 36 and Na + channels 37, 38. Importantly, Xie et al39 showed that H 2 O 2 perfusion–induced oxidative CaMKII activation leads to afterdepolarizations in isolated rabbit cardiomyocytes, likely by phosphorylation of Na + channels and LCCs.…”