PKA and phosphatases attached to the CaV1.2 channel regulate channel activity in cell-free patches

Abstract

Calmodulin (CaM) + ATP can reprime voltage-gated L-type Ca2+ channels (CaV1.2) in inside-out patches for activation, but this effect decreases time dependently. This suggests that the CaV1.2 channel activity is regulated by additional cytoplasmic factors. To test this hypothesis, we examined the role of cAMP-dependent protein kinase A (PKA) and protein phosphatases in the regulation of CaV1.2 channel activity in the inside-out mode in guinea pig ventricular myocytes. CaV1.2 channel activity quickly disappeared after the patch was excised from the cell and recovered to only 9% of that in the cell-attached mode on application of CaM + ATP at 10 min after the inside out. However, immediate exposure of the excised patch to the catalytic subunit of PKA + ATP or the nonspecific phosphatase inhibitor okadaic acid significantly increased the CaV1.2 channel activity recovery by CaM + ATP (114 and 96%, respectively) at 10 min. Interestingly, incubation of the excised patches with cAMP + ATP also increased CaM/ATP-induced CaV1.2 channel activity recovery (108%), and this effect was blocked by the nonspecific protein kinase inhibitor K252a. The channel activity in the inside-out mode was not maintained by either catalytic subunit of PKA or cAMP + ATP in the absence of CaM, but was stably maintained in the presence of CaM for more than 40 min. These results suggest that PKA and phosphatase(s) attached on or near the CaV1.2 channel regulate the basal channel activity, presumably through modulation of the dynamic CaM interaction with the channel.