Calmodulin and ATP support activity of the Cav1.2 channel through dynamic interactions with the channel

摘要

Calmodulin (CaM) plays a critical role in regulation of Cav1.2 Ca2+ channels. CaM binds to the channel directly, maintaining channel activity and regulating it in a Ca2+-dependent manner. To explore the molecular mechanisms involved, we compared the activity of the wildtype channel (alpha 1C) and mutant derivatives, C-terminal deleted (alpha 1C Delta) and alpha 1C Delta linked to CaM (alpha 1C Delta CaM). These were co-expressed with beta 2a and alpha 2 delta subunits in HEK293 cells. In the inside-out mode, alpha 1C Delta and alpha 1C Delta showed minimal open-probabilities in a basic internal solution (run-down), whereas 1C with CaM and alpha 1C Delta CaM maintained detectable channel activity, confirming that CaM was necessary, but not sufficient, for channel activity. Previously, we reported that ATP was required to maintain channel activity of alpha 1C Delta. Unlike alpha 1C Delta, the mutant channels did not require ATP for activation in the early phase (3-5min). However, alpha 1C Delta with CaM+ATP and alpha 1C Delta CaM with ATP maintained activity, even in the late phase (after 7-9min). These results suggested that CaM and ATP interacted dynamically with the proximal C-terminal tail of the channel and, thereby, produced channel activity. In addition, okadaic acid, a protein phosphatase inhibitor, could substitute for the effects of ATP on alpha 1C but not on the mutant channels. These results supported the hypothesis that CaM and ATP maintain activity of Cav1.2 channels, further indicating that ATP has dual effects. One maintains phosphorylation of the channel and the other becomes apparent when the distal carboxyl-terminal tail is removed.