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

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  • Ca〔2+〕-Dependent Inhibition of Inwardly Rectifying K〔+〕 Channel in Opossum Kidney Cells

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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 μM) in the presence of 10−6 M Ca2+ in cell-attached patches. The application of 10 μM 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 μM), 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 μM), 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 μM) and Ni2+ (10 μM) 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.<br>

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