Document Detail


Role of calcineurin-mediated dephosphorylation in modulation of an inwardly rectifying K+ channel in human proximal tubule cells.
MedLine Citation:
PMID:  19865787     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Manabu Kubokawa; Toshiyuki Kojo; You Komagiri; Kazuyoshi Nakamura
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2009-10-29
Journal Detail:
Title:  The Journal of membrane biology     Volume:  231     ISSN:  1432-1424     ISO Abbreviation:  J. Membr. Biol.     Publication Date:  2009 Oct 
Date Detail:
Created Date:  2010-01-21     Completed Date:  2010-03-26     Revised Date:  2010-05-20    
Medline Journal Info:
Nlm Unique ID:  0211301     Medline TA:  J Membr Biol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  79-92     Citation Subset:  IM    
Affiliation:
Department of Physiology, Iwate Medical University School of Medicine, 19-1 Uchimaru, Morioka, 020-8505, Japan. mkubokaw@iwate-med.ac.jp
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MeSH Terms
Descriptor/Qualifier:
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / analogs & derivatives,  pharmacology
Blotting, Western
Calcineurin / metabolism*
Calcium / metabolism
Calcium-Calmodulin-Dependent Protein Kinase Type 2 / antagonists & inhibitors,  genetics,  physiology
Cell Line
Cyclosporine / pharmacology
Humans
Indoles / pharmacology
Kidney Tubules, Proximal / cytology*
Maleimides / pharmacology
Patch-Clamp Techniques
Phosphorylation
Potassium Channels, Inwardly Rectifying / drug effects,  metabolism*
Protein Kinase C / antagonists & inhibitors
Chemical
Reg. No./Substance:
0/Indoles; 0/Maleimides; 0/Potassium Channels, Inwardly Rectifying; 127191-97-3/KN 62; 133052-90-1/bisindolylmaleimide I; 59865-13-3/Cyclosporine; 7440-70-2/Calcium; 84477-87-2/1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; EC 2.7.11.13/Protein Kinase C; EC 2.7.11.17/Calcium-Calmodulin-Dependent Protein Kinase Type 2; EC 3.1.3.16/Calcineurin
Comments/Corrections
Erratum In:
J Membr Biol. 2010 Feb;233(1-3):143

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