Using patch-clamp technique, we studied the role of the Ca 2+/calmodulin kinase II (CaMK II)-mediated phosphorylation process on the K+ channel with an inward conductance of 90 pS in opossum kidney proximal tubule cells (OKPCs). The intracellular Ca2+ concentration ([Ca]i) was measured by use of the fluorescent dye fura 2. The following results were obtained: (i) In cell-attached patches, the channel activity was inhibited by a decrease in [Ca]i induced by perfusion with low Ca2+ (10-8 M), La3+ (100 μM), or EGTA/AM (100 μM) contained in the bath solution. The application of KN-62 (10 μM) or KN-93 (5 μM), inhibitors of CaMK II, also inhibited the channel activity. (ii) The membrane potential measured with nystatin-perforated patches was significantly decreased by the fall in [Ca]i induced by the perfusion with EGTA- or La3+-containing solution. Also, the application of KN-62 (10 μM) or KN-93 (5 μM) to the bath significantly decreased the membrane potential. (iii) In inside-out patches, the channel activity was significantly stimulated by the application of CaMK II (300 pM) at 10-7 M Ca2+ in the bath. Furthermore, the application of KN-62 (10 μM) to the bath significantly decreased the channel activity. Our findings show that the constitutive activity of inwardly rectifying K + channel at physiological [Ca]i is mediated by the Ca2+/CaMK II pathway in OKPCs.
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