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Updated: May 31, 2026
Pull-down of Calmodulin-binding Proteins
Published on: January 23, 2012
Interaction between Calcineurin and Ca/Calmodulin Kinase-II in Modulating Cellular Functions
Manabu Kubokawa1, Kazuyoshi Nakamura, You Komagiri
1Department of Physiology, Iwate Medical University School of Medicine, 2-1-1 Nishitokuda, Yahaba, Iwate 028-3694, Japan.
Abstract:
Roles of calcineurin (CaN), a Ca(2+)/calmodulin- (CaM-) dependent protein phosphatase, and Ca(2+)/CaM-dependent protein kinase-II (CaMKII) in modulating K(+) channel activity and the intracellular Ca(2+) concentration ([Ca(2+)](i)) have been investigated in renal tubule epithelial cells. The channel current through the cell membrane was recorded with the patch-clamp technique, and [Ca(2+)](i) was monitored using fura-2 imaging. We found that a CaN-inhibitor, cyclosporin A (CyA), lowered the K(+) channel activity and elevated [Ca(2+)](i), suggesting that CyA closes K(+) channels and opens Ca(2+)-release channels of the cytosolic Ca(2+)-store. Moreover, both of these responses were blocked by KN-62, an inhibitor of CaMKII. It is suggested that the CyA-mediated response results from the activation of CaMKII. Indeed, Western blot analysis revealed that CyA increased phospho-CaMKII, an active form of CaMKII. These findings suggest that CaN-dependent dephosphorylation inhibits CaMKII-mediated phosphorylation, and the inhibition of CaN increases phospho-CaMKII, which results in the stimulation of CaMKII-dependent cellular actions.
Insights
Calcineurin inhibition by cyclosporin A reduces K+ channel activity and increases intracellular calcium by activating CaMKII in kidney cells. This highlights a signaling pathway involving calcineurin, CaMKII, and calcium regulation.
Area of Science:
- Cellular physiology
- Molecular signaling
- Renal tubule function
Background:
- Calcineurin (CaN) and Ca(2+)/calmodulin-dependent protein kinase-II (CaMKII) are key regulators of cellular processes.
- Their roles in modulating renal tubule epithelial cell K+ channel activity and intracellular calcium ([Ca(2+)](i)) require further elucidation.
Purpose of the Study:
- To investigate the specific roles of CaN and CaMKII in regulating K+ channel activity and [Ca(2+)](i) in renal tubule epithelial cells.
- To determine the signaling pathway through which CaN inhibition affects these cellular parameters.
Main Methods:
- Patch-clamp technique to record K+ channel currents across the cell membrane.
- Fura-2 imaging to monitor intracellular calcium concentration ([Ca(2+)](i)).
- Western blot analysis to detect the active form of CaMKII (phospho-CaMKII).
Main Results:
- Cyclosporin A (CyA), a CaN inhibitor, decreased K+ channel activity and increased [Ca(2+)](i).
- These effects were blocked by KN-62, a CaMKII inhibitor, indicating CaMKII involvement.
- CyA treatment led to increased levels of phospho-CaMKII, suggesting CaMKII activation.
Conclusions:
- CaN inhibition by CyA leads to CaMKII activation, resulting in decreased K+ channel activity and increased intracellular calcium.
- This suggests a regulatory mechanism where CaN-dependent dephosphorylation normally inhibits CaMKII activity.
- Inhibition of CaN releases this inhibition, promoting CaMKII-mediated cellular actions in renal tubule epithelial cells.
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