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Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting
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Regulation of the epithelial Na+ channel by the protein kinase CK2

Tanja Bachhuber1, Joana Almaça, Fadi Aldehni

  • 1Institut für Physiologie, Universität Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany.

The Journal of Biological Chemistry
|March 1, 2008
PubMed

Insights

Protein kinase CK2 is essential for epithelial sodium channel (ENaC) activation and membrane expression. CK2 inhibition blocks ENaC activity, suggesting a role in regulating sodium transport via the Nedd4-2 pathway.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Physiology

Background:

  • Protein kinase CK2 is a constitutively active enzyme regulating diverse cellular processes.
  • The epithelial sodium channel (ENaC) is crucial for sodium reabsorption in various epithelia.
  • CK2 phosphorylation sites are identified in the C-termini of ENaC beta- and gamma-subunits.

Purpose of the Study:

  • To investigate the role of CK2 in regulating endogenous and expressed ENaC.
  • To elucidate the impact of CK2 on ENaC activity, membrane localization, and its interaction with other regulatory pathways.

Main Methods:

  • Ussing chamber experiments on murine epithelia and cultured cells.
  • Electrophysiological recordings in Xenopus oocytes expressing wild-type and mutant ENaC.
  • Inhibition studies using selective CK2 inhibitors (TBB, heparin, poly(E:Y)).
  • Site-directed mutagenesis of CK2 phosphorylation sites and Nedd4-2 binding motifs.
  • siRNA-mediated knockdown of Nedd4-2.

Main Results:

  • Selective CK2 inhibition dose-dependently reduced amiloride-sensitive Na(+) transport in native epithelia and ENaC currents in oocytes.
  • Mutating CK2 phosphorylation sites in ENaC subunits attenuated channel activity and abolished TBB sensitivity.
  • CK2 translocation to the cell membrane was observed upon wild-type ENaC expression but not with mutated sites.
  • CK2 phosphorylation is essential for ENaC activation and contributes to its membrane expression.
  • Disruption of the Nedd4-2 binding motif rendered ENaC insensitive to CK2 inhibition, and Nedd4-2 knockdown mimicked CK2 inhibition effects.

Conclusions:

  • CK2 phosphorylation is critical for ENaC activation and influences its membrane localization.
  • CK2 appears to regulate ENaC activity, potentially by inhibiting the Nedd4-2 pathway.
  • These findings highlight CK2 as a key regulator of ENaC function in sodium transport.

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