Calcineurin activation by slow calcium release from intracellular stores suppresses protein kinase C regulation of L-type calcium channels in L6 cells

L-type Ca2+ channel activity was assayed in L6 cells as the rate of nifedipine-sensitive Ba2+ influx in a depolarizing medium. In the absence of extracellular Ca2+, activation of protein kinase C (PKC) with phorbol-12-myristate-13-acetate (PMA) or thymeleatoxin (TMX) inhibited Ba2+ influx by 38%. Thapsigargin (Tg), a selective inhibitor of the Ca2+-ATPase in the sarcoplasmic reticulum, evoked a rise in the cytosolic Ca2+ concentration ([Ca2+]i) in a Ca2+-free medium from 30 to ∼80nM. This [Ca2+]i increase declined slowly, giving rise to a modest elevation of [Ca2+]i that persisted for >5min. The inhibitory effects of PMA and TMX on channel activity were abolished when tested in Tg-treated cells in a Ca2+-free medium. However, when the Ca2+ ionophore, ionomycin, was applied with Tg, PMA and TMX retained their inhibitory effect on L-type Ca2+ channel activity, suggesting that a lower amplitude and prolonged release of Ca2+ stores is necessary for abrogating PKC-mediated inhibition of LCC. Cyclosporin A (5μM) and ascomycin (5μM), inhibitors of the Ca2+/calmodulin-dependent protein phosphatase, calcineurin, fully restored the inhibitory effect of PMA and TMX on channel activity. Addition of 1mM CaCl2 to the Tg-treated cells increased [Ca2+]i to ∼165nM and also restored the inhibitory effects of PMA and TMX. These results indicate that a small, relatively prolonged [Ca2+]i increase elicited by passive depletion of internal Ca2+ stores led to activation of calcineurin, giving rise to an increase in protein phosphatase activity that counteracted the inhibitory effects of PKC on channel activity. A larger increase in [Ca2+]i via store-dependent Ca2+ entry enhanced the activity of PKC sufficiently to overcome the protein phosphatase activity of calcineurin. This study is the first to demonstrate that the regulation of L-type Ca2+ channels in a myocyte model involves a balance between the differential Ca2+ sensitivities and opposing actions of PKC and calcineurin.