Calmodulin kinase is functionally targeted to the action potential plateau for regulation of L-type Ca2+ current in rabbit cardiomyocytes

摘要

L-type Ca²⁺ current (ICa−L) triggers Ca²⁺ release from the sarcoplasmic reticulum (SR) and both SR and ICa−L are potential sources of intracellular Ca²⁺ (Cai2+) for feedback regulation of ICa−L. Cai2+ bound to calmodulin (Ca²⁺–CaM) can inhibit ICa−L, while Ca²⁺–CaM can also activate Ca²⁺–CaM-dependent protein kinase II (CaMK) to increase ICa. However, it is not known whether ICa−L or the SR is the primary source of Ca²⁺ for ICa−L regulation. The L-type Ca²⁺ channel C terminus is implicated as a critical transduction element for ICa−L responses to Ca²⁺–CaM and CaMK, and the C terminus undergoes voltage-dependent steric changes, suggesting that Cai2+ control of ICa−L may also be regulated by cell membrane potential. We developed conditions to separately test the relationship of Ca²⁺–CaM and CaMK to ICa−L and SR Cai2+ release during voltage clamp conditions modelled upon time and voltage domains relevant to the cardiac action potential. Here we show that CaMK increases ICa−L after brief positive conditioning pulses, whereas Ca²⁺–CaM reduces ICa−L over a broad range of positive and negative conditioning potentials. SR Ca²⁺ release was required for both Ca²⁺–CaM and CaMK ICa−L responses after strongly positive conditioning pulses (+10 and +40 mV), while Cai2+ from ICa−L was sufficient for Ca²⁺–CaM during weaker depolarizations. These findings show that ICa−L responses to CaMK are voltage dependent and suggest a new model of L-type Ca²⁺ channel regulation where voltage-dependent changes control ICa−L responses to Ca²⁺–CaM and CaMK signalling.