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

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An Erratum to this article was published on 10 December 2009

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 Ca2+/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 μM) or FK520 (5 μM), significantly suppressed channel activity. Intracellular Ca2+ concentration ([Ca2+] i ) estimated by fura-2 imaging was elevated by these inhibitors. Since inhibition of CaN attenuates some dephosphorylation with increase in [Ca2+] i , we speculated that inhibiting CaN enhances Ca2+-dependent phosphorylation, which might result in channel suppression. To verify this hypothesis, we examined effects of inhibitors of PKC and Ca2+/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 μM) 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 μM) and CaMKII (0.15 U/ml) to the bath at 10−6 M Ca2+ 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.

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Acknowledgement

This work was supported in part by a grant from the Corporation for Private School of Japan (to M. K., 2007–2009).

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Authors and Affiliations

  1. Department of Physiology, Iwate Medical University School of Medicine, 19-1 Uchimaru, Morioka, 020-8505, Japan

    Manabu Kubokawa, Toshiyuki Kojo, You Komagiri & Kazuyoshi Nakamura

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  1. Manabu Kubokawa
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  2. Toshiyuki Kojo
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  3. You Komagiri
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  4. Kazuyoshi Nakamura
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Correspondence to Manabu Kubokawa.

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An erratum to this article can be found at http://dx.doi.org/10.1007/s00232-009-9220-2

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Kubokawa, M., Kojo, T., Komagiri, Y. et al. Role of Calcineurin-Mediated Dephosphorylation in Modulation of an Inwardly Rectifying K+ Channel in Human Proximal Tubule Cells. J Membrane Biol 231, 79–92 (2009). https://doi.org/10.1007/s00232-009-9207-z

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