Calmodulin reverses rundown of L-type Ca²⁺ channels in guinea pig ventricular myocytes

Abstract

Calmodulin (CaM) is implicated in regulation of Ca(2+) channels as a Ca(2+) sensor. The effect of CaM on rundown of L-type Ca(2+) channels in inside-out patch form was investigated in guinea pig ventricular myocytes. Ca(2+) channel activity disappeared within 1-3 min and did not reappear when the patch was excised and exposed to an artificial intracellular solution. However, application of CaM (0.03, 0.3, 3 microM) + 3 mM ATP to the intracellular solution within 1 min after patch excision resulted in dose-dependent activation of channel activity. Channel activity averaged 11.2%, 94.7%, and 292.9%, respectively, of that in cell-attached mode. Channel activity in inside-out patch mode was induced by CaM + ATP at nanomolar Ca(2+) concentrations ([Ca(2+)]); however, increase to micromolar [Ca(2+)] rapidly inactivated the channel activity induced, revealing that the effect of CaM on the channel was Ca(2+) dependent. At the 2nd, 4th, 6th, 8th, and 10th minutes after patch excision, CaM (0.75 microM) + ATP induced Ca(2+) channel activity to 150%, 100%, 96.9%, 29.3%, and 16.6%, respectively, revealing a time-dependent action of CaM on the channel. CaM added with adenosine 5'-(beta,gamma-imido)triphosphate (AMP-PNP) also induced channel activity, although with much lower potency and shorter duration. Protein kinase inhibitors KN-62, CaM-dependent protein kinase (CaMK)II 281-309, autocamtide-related CaMKII inhibitor peptide, and K252a (each 1-10 microM) did not block the effect of CaM, indicating that the effect of CaM on the Ca(2+) channel was phosphorylation independent. Neither CaM nor ATP alone induced Ca(2+) channel activity, showing a cooperative effect of CaM and ATP on the Ca(2+) channel. These results suggest that CaM is a crucial regulatory factor of Ca(2+) channel basal activity.

The Distinct Roles of Calmodulin and Calmodulin Kinase II in the Reversal of Run-Down of L-Type Ca2+ Channels in Guinea-Pig Ventricular Myocytes

“…We previously found that run-down of L-type Ca 2+ -channel activities is reversed by CaM + ATP (14) and that CaMKII-mediated phosphorylation is also involved in the maintenance of basal activity of the channels (15). In the present study, we have confirmed that playing distinct roles, both CaM and CaMKII are required in the reversal of run-down of Ca 2+ -channel activities.…”

“…3A (middle lane, open circle), 5 min after run-down, different concentrations of CaM were applied. However, even with concentrations of 7 μM, 10 times of 0.7 μM, the concentration that we previously used (14), no obvious recovery of the channel activity was observed. The averaged relative channel activities from 5 to 10 min after the formation of inside-out patches are 28.2 ± 11.1% (n = 4), 23.5 ± 3.2% (n = 4), and 42.5 ± 11.2% (n = 6) for CaM at 0.7, 2.1, and 7 μM, respectively, indicating that the time-dependent attenuation of CaM's effects in the reversal of run-down of Ca 2+ channels is not due to the insufficient dose of CaM.…”

“…-channel activities after run-down (14) and that CaMKII inhibitors block cytoplasmic factorinduced recovery of the channel activity after run-down in the inside-out patches (15), indicating that both CaM and CaMKII are involved in maintenance of the basal activity of Ca 2+ channels. However, the detailed mechanism has not yet been determined.…”

Cyclosporin and Timothy syndrome increase mode 2 gating of CaV1.2 calcium channels through aberrant phosphorylation of S6 helices

“…''Mode 2'' gating was discovered as a pharmacological effect of the dihydropyridine Bay K8644 (4), but subsequent work has implicated reversible protein phosphorylation of the channel by calcium͞calmodulin-dependent protein kinase type II (CaMKII) in the regulation of mode 2 gating in cells that were not treated with dihydropyridines (5). Although the molecular mechanism of this effect and its physiological consequences are only beginning to be investigated (6)(7)(8)(9), it is likely that unregulated mode 2 gating would be cytotoxic because the individual channel openings in mode 2 are Ͼ10 times longer on average than the openings in mode 1.…”

Dynamic Alterations in the CaV1.2/CaM/CaMKII Signaling Pathway in the Left Ventricular Myocardium of Ischemic Rat Hearts

“…Our previous study has shown that the run-down of Ca V 1.2 activity is reversed by CaM and ATP (Xu et al, 2004). The direct binding of CaM with the IQ motif of the Ca 2 + -channel a1-subunit has been reported to be involved in the molecular mechanism by which it exerts its Ca 2 + -dependent effects, namely, CDI and CDF.…”