Ca2+-dependent inhibition of inwardly rectifying K+ channel in opossum kidney cells

The effect of intracellular Ca2+ on the activity of the inwardly rectifying ATP-regulated K+ channel with an inward conductance of about 90 pS was exa mined by using the patch-clamp technique in opossum kidney proximal tubule (OKP) cells. The activity of the inwardly rectifying K+ channel rapidly dec lined with an application of ionomycin (1 muM) in the presence of 10(-6) M Ca2+ in cell-attached patches. The application of 10 muM phorbor-12-myrista te-acetate (PMA) with 10(-6) M Ca2+ reduced the K+ channel activity. Althou gh the channel activity was not influenced by an increase of bath Ca2+ from 10(-7.5) to 10(-6) M, the activity was inhibited by protein kinase C (PKC, 1 U/ml) with 10(-6) M Ca2+ in inside-out patches. The inhibitory effect of Ca2+ with ionomycin on the channel activity was diminished by the pretreat ment with a specific PKC inhibitor, GF 109203X (5 muM), in cell-attached pa tches. By contrast, the application of Ca2+/calmodulin kinase II (CaMK II, 300 pM) dramatically increased this channel activity in inside-out patches. In cell-attached patches, the addition of both GF 109203X and cyclospolin A (5 muM), a potent inhibitor of protein phosphatase 2B (calcineurin), inst ead stimulated the K+ channel activity with ionomycin and 10(-6) M Ca2+. Th e addition of protein phosphatase 2B (calcineurin) (2 U/ml) to the bath wit h calmodulin (1 muM) and Ni2+ (10 muM) to stimulate calcineurin inhibited t he channel activity in inside-out patches. Furthermore, the inhibitory effe ct of PKC or calcineurin on this channel activity was abolished by a remova l of Ca2+ from bath solution. These results suggest that Ca2+-dependent inh ibitory effect on the inwardly rectifying K+ channel in OKP cells was mainl y mediated by Ca2+-PKC-mediated phosphorylation, and that the Ca2+ -calmodu lin-dependent phosphorylation process may be counterbalanced by the Ca2+ -c almodulin-dependent dephosphorylation process.