title: Ca(2+)-dependent inhibition of inwardly rectifying K(+) channel in opossum kidney cells.
abstract: The effect of intracellular Ca(2+) 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 microM) in the presence of 10(-6) M Ca(2+) in cell-attached patches. The application of 10 microM phorbor-12-myristate-acetate (PMA) with 10(-6) M Ca(2+) reduced the K(+) channel activity. Although the channel activity was not influenced by an increase of bath Ca(2+) from 10(-7.5) to 10(-6) M, the activity was inhibited by protein kinase C (PKC, 1 U/ml) with 10(-6) M Ca(2+) in inside-out patches. The inhibitory effect of Ca(2+) with ionomycin on the channel activity was diminished by the pretreatment with a specific PKC inhibitor, GF 109203X (5 microM), in cell-attached patches. By contrast, the application of Ca(2+)/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 microM), a potent inhibitor of protein phosphatase 2B (calcineurin), instead stimulated the K(+) channel activity with ionomycin and 10(-6) M Ca(2+). The addition of protein phosphatase 2B (calcineurin) (2 U/ml) to the bath with calmodulin (1 microM) and Ni(2+) (10 microM) 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 Ca(2+) from bath solution. These results suggest that Ca(2+)-dependent inhibitory effect on the inwardly rectifying K(+) channel in OKP cells was mainly mediated by Ca(2+)-PKC-mediated phosphorylation, and that the Ca(2+)-calmodulin-dependent phosphorylation process may be counterbalanced by the Ca(2+)-calmodulin-dependent dephosphorylation process.
authors.affiliation: Department of Physiology II, Osaka Medical College, Takatsuki, 569-8686 Japan. ymori@art.osaka-med.ac.jp
authors.forename: Y
authors.initials: Y
authors.lastname: Mori
authors.forename: A
authors.initials: A
authors.lastname: Kawasaki
authors.forename: A
authors.initials: A
authors.lastname: Takamaki
authors.forename: I
authors.initials: I
authors.lastname: Kitano
authors.forename: R
authors.initials: R
authors.lastname: Yoshida
authors.forename: M
authors.initials: M
authors.lastname: Kubokawa
authors.forename: T
authors.initials: T
authors.lastname: Kubota
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D019703:Calcineurin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002147:Calmodulin / Q000494:pharmacology; D018929:Cell Culture Techniques; D004594:Electrophysiology; D007687:Kidney Tubules, Proximal / Q000502:physiology*; D009893:Opossums / Q000502:physiology*; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D015221:Potassium Channels / Q000502:physiology*; D011493:Protein Kinase C / Q000378:metabolism*
chemical_list: D002147:Calmodulin; D015221:Potassium Channels; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019703:Calcineurin; D002118:Calcium