1. Archive
2. Vol.51, No.3

Ca²⁺-Dependent Inhibition of Inwardly Rectifying K⁺ Channel in Opossum Kidney Cells

Abstract: 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 examined by using the patch-clamp technique in opossum kidney proximal tubule (OKP) cells. The activity of the inwardly rectifying K⁺ channel rapidly declined with an application of ionomycin (1 M) in the presence of 10⁻⁶ M Ca²⁺ in cell-attached patches. The application of 10 M phorbor-12-myristate-acetate (PMA) with 10⁻⁶ M Ca²⁺ reduced the K⁺ channel activity. Although 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 pretreatment with a specific PKC inhibitor, GF 109203X (5 M), in cell-attached patches. 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 M), a potent inhibitor of protein phosphatase 2B (calcineurin), instead stimulated the K⁺ channel activity with ionomycin and 10⁻⁶ M Ca²⁺. The addition of protein phosphatase 2B (calcineurin) (2 U/ml) to the bath with calmodulin (1 M) and Ni²⁺ (10 M) to stimulate calcineurin inhibited the channel activity in inside-out patches. Furthermore, the inhibitory effect of PKC or calcineurin on this channel activity was abolished by a removal of Ca²⁺ from bath solution. These results suggest that Ca²⁺-dependent inhibitory effect on the inwardly rectifying K⁺ channel in OKP cells was mainly mediated by Ca²⁺-PKC-mediated phosphorylation, and that the Ca²⁺-calmodulin-dependent phosphorylation process may be counterbalanced by the Ca²⁺-calmodulin-dependent dephosphorylation process.

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