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Updated: Apr 22, 2026

Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting
Published on: December 9, 2022
Regulation of ClC-2 activity by SPAK and OSR1
Jamshed Warsi1, Zohreh Hosseinzadeh, Bernat Elvira
1Department of Physiology I, University of Tübingen, 72076 Tübingen, Germany.
Background/Aims:
SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1) are powerful regulators of diverse transport processes. Both kinases are activated by cell shrinkage and participate in stimulation of regulatory cell volume increase (RVI). Execution of RVI involves inhibition of Cl- channels. The present study explored whether SPAK and/or OSR1 regulate the activity of the Cl- channel ClC-2.
Methods:
To this end, ClC-2 was expressed in Xenopus laevis oocytes with or without additional expression of wild type SPAK, constitutively active SPAK(T233E), WNK1 insensitive inactive SPAK(T233A), catalytically inactive SPAK(D212A), wild type OSR1, constitutively active OSR1(T185E), WNK1 insensitive inactive OSR1(T185A), and catalytically inactive OSR1(D164A). Cl- channel activity was determined by dual electrode voltage clamp.
Results:
Expression of ClC-2 was followed by the appearance of a conductance (GCl), which was significantly decreased following coexpression of wild-type SPAK, SPAK(T233E), wild type OSR1 or OSR1(T185E), but not by coexpression of SPAK(T233A), SPAK(D212A), OSR1(T185A), or OSR1(D164A). Inhibition of ClC-2 insertion by brefeldin A (5 μM) resulted in a decline of GCl which was similar in the absence and presence of SPAK or OSR1, suggesting that SPAK and OSR1 did not accelerate the retrieval of ClC-2 protein from the cell membrane.
Conclusion:
SPAK and OSR1 are powerful negative regulators of the cell volume regulatory Cl- channel ClC-2.
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