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Phosphorylation and functional regulation of ClC-2 chloride channels expressed in Xenopus oocytes by M

Tetsushi Furukawa1, Takehiko Ogura, Ya-Juan Zheng

  • 1Department of Physiology, Akita University School of Medicine, Akita 010-8543, Japan.

The Journal of Physiology
|May 3, 2002
PubMed

Insights

The M-phase kinase p34(cdc2)/cyclin B phosphorylates and regulates the ClC-2 chloride channel, affecting cell volume and cell cycle progression. This interaction is counter-regulated by protein phosphatase 1.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Ion Channel Physiology

Background:

  • Cell cycle progression involves significant cellular process alterations, often mediated by ion channels.
  • Inwardly rectifying chloride channels show enhanced activity during M phase in various organisms, but the regulatory mechanisms are unclear.

Purpose of the Study:

  • To investigate the regulation of the volume-sensitive chloride channel, ClC-2, by cell cycle-specific kinases.
  • To elucidate the role of p34(cdc2)/cyclin B in modulating ClC-2 channel activity.

Main Methods:

  • In vitro and cell-free phosphorylation assays using p34(cdc2)/cyclin B and ClC-2.
  • Site-directed mutagenesis (S632A) to identify phosphorylation sites.
  • Xenopus oocyte expression system to study channel function.
  • Yeast two-hybrid system to identify interacting proteins.

Main Results:

  • p34(cdc2)/cyclin B directly phosphorylates ClC-2 at serine 632.
  • Phosphorylation by p34(cdc2)/cyclin B attenuates ClC-2 currents and reduces activation by cell swelling.
  • ClC-2 interacts with protein phosphatase 1, suggesting counter-regulation.
  • Olomoucine, a kinase inhibitor, reverses the p34(cdc2)/cyclin B-mediated attenuation.

Conclusions:

  • The M-phase kinase p34(cdc2)/cyclin B regulates ClC-2 channel activity through phosphorylation.
  • Protein phosphatase 1 may counteract p34(cdc2)/cyclin B effects on ClC-2.
  • ClC-2 channel activity is modulated by cell cycle events and cell volume, impacting cellular physiology.

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