Role of Calcineurin-Mediated Dephosphorylation in Modulation of an Inwardly Rectifying K+ Channel in Human Proximal Tubule Cells

Abstract

Activity of an inwardly rectifying K(+) channel with inward conductance of about 40 pS in cultured human renal proximal tubule epithelial cells (RPTECs) is regulated at least in part by protein phosphorylation and dephosphorylation. In this study, we examined involvement of calcineurin (CaN), a Ca(2+)/calmodulin (CaM)-dependent phosphatase, in modulating K(+) channel activity. In cell-attached mode of the patch-clamp technique, application of a CaN inhibitor, cyclosporin A (CsA, 5 microM) or FK520 (5 microM), significantly suppressed channel activity. Intracellular Ca(2+) concentration ([Ca(2+)]( i )) estimated by fura-2 imaging was elevated by these inhibitors. Since inhibition of CaN attenuates some dephosphorylation with increase in [Ca(2+)]( i ), we speculated that inhibiting CaN enhances Ca(2+)-dependent phosphorylation, which might result in channel suppression. To verify this hypothesis, we examined effects of inhibitors of PKC and Ca(2+)/CaM-dependent protein kinase-II (CaMKII) on CsA-induced channel suppression. Although the PKC inhibitor GF109203X (500 nM) did not influence the CsA-induced channel suppression, the CaMKII inhibitor KN62 (20 microM) prevented channel suppression, suggesting that the channel suppression resulted from CaMKII-dependent processes. Indeed, Western blot analysis showed that CsA increased phospho-CaMKII (Thr286), an activated CaMKII in inside-out patches, application of CaM (0.6 microM) and CaMKII (0.15 U/ml) to the bath at 10(-6) M Ca(2+) significantly suppressed channel activity, which was reactivated by subsequent application of CaN (800 U/ml). These results suggest that CaN plays an important role in supporting K(+) channel activity in RPTECs by preventing CaMKII-dependent phosphorylation.

Regulation of Renal Potassium Channels by Protein Kinases and Phosphatases

“…Moreover, Western blot analysis also revealed that CyA increased phospho-CaMKII, an active form of CaMKII (Gerges et al, 2005), as shown in Fig. 7 (Kubokawa et al, 2009). Taken together, these results strongly suggest that CaMKII-mediated phosphorylation suppresses channel activity, and CaN-mediated dephosphorylation reversibly elevates activity of the renal ATP-regulated K + channel.…”

“…That is, CyA-induced channel suppression was blocked by a CaMKII inhibitor, KN-62, as shown in Fig. 6 (Kubokawa et al, 2009). Moreover, Western blot analysis also revealed that CyA increased phospho-CaMKII, an active form of CaMKII (Gerges et al, 2005), as shown in Fig.…”

“…Taken together, these results strongly suggest that CaMKII-mediated phosphorylation suppresses channel activity, and CaN-mediated dephosphorylation reversibly elevates activity of the renal ATP-regulated K + channel. It was also demonstrated that CyA increased [Ca 2+ ] i in various cells, including renal tubule cells (Gordjani et al, 2000;Frapier et al, 2001;Bultynck et al, 2003;Kubokawa et al, 2009). Thus, the CyA-induced increase in [Ca 2+ ] i may enhance the activity of CaMKII.…”

“…Moreover, it was reported that Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) also suppressed the activity of the ATP-regulated K + channels in the basolateral membrane of the proximal tubule cells (Kubokawa et al, 2009) and the apical membrane of the principal cells in CCD (Kubokawa et al, 1995b). Indeed, the Ca 2+ -induced K + channel inhibition was not blocked by PKC inhibitor alone, but was almost completely blocked by KN-62 (Tokumitsu et al, 1990), a CaMKII inhibitor, in addition to PKC inhibitor (Kubokawa et al, 1995b(Kubokawa et al, , 2009). Thus, it is possible that both PKC and CaMKII are involved in K + channel regulation.…”

“…Thus, it is possible that both PKC and CaMKII are involved in K + channel regulation. It has also been demonstrated that cyclosporin A (CyA), an inhibitor of Ca 2+ /calmodulindependent phosphatase, calcineurin (CaN) (Hemenway & Heitman, 1999), inhibited the activity of the K + channel in both proximal tubule (Ye et al, 2006, Kubokawa et al, 2009) and principal cells of CCD (Ling & Eaton, 1993). Cyclosporin A is a a well-known immunosuppressive agent (Morris, 1981;White & Calne, 1982;Cohen, 1984), and affects various functions of the kidney other than K + channels (Epting et al, 2006;Tumlin, 1993;Grinyó & Cruzado, 2004;J.…”