Direct Protein Kinase C-dependent Phosphorylation Regulates the Cell Surface Stability and Activity of the Potassium Chloride Cotransporter KCC2

John A. Payne,Jeffery R. Williams,Joshua A. Walker,Stephen J. Moss,Henry H.C. Lee,Richard J. Goodier

doi:10.1074/jbc.m705053200

Abstract

The potassium chloride cotransporter KCC2 plays a major role in the maintenance of transmembrane chloride potential in mature neurons; thus KCC2 activity is critical for hyperpolarizing membrane currents generated upon the activation of gamma-aminobutyric acid type A and glycine (Gly) receptors that underlie fast synaptic inhibition in the adult central nervous system. However, to date an understanding of the cellular mechanism that neurons use to modulate the functional expression of KCC2 remains rudimentary. Using Escherichia coli expression coupled with in vitro kinase assays, we first established that protein kinase C (PKC) can directly phosphorylate serine 940 (Ser(940)) within the C-terminal cytoplasmic domain of KCC2. We further demonstrated that Ser(940) is the major site for PKC-dependent phosphorylation for full-length KCC2 molecules when expressed in HEK-293 cells. Phosphorylation of Ser(940) increased the cell surface stability of KCC2 in this system by decreasing its rate of internalization from the plasma membrane. Coincident phosphorylation of Ser(940) increased the rate of ion transport by KCC2. It was further evident that phosphorylation of endogenous KCC2 in cultured hippocampal neurons is regulated by PKC-dependent activity. Moreover, in keeping with our recombinant studies, enhancing PKC-dependent phosphorylation increased the targeting of KCC2 to the neuronal cell surface. Our studies thus suggest that PKC-dependent phosphorylation of KCC2 may play a central role in modulating both the functional expression of this critical transporter in the brain and the strength of synaptic inhibition.

Highlights

The activity of KCC2 results in low intracellular ClϪ concentrations that are responsible for hyperpolarizing responses of GABAA and Gly receptors in fast synaptic inhibition within the central nervous system [4, 8]
Deficits in KCC2 activity are of importance in epilepsy and other central nervous system pathologies [26]; comprehending the cellular mechanisms neurons use to regulate the activity of this protein is of significance
We have begun to examine the molecular sites of phosphorylation for individual protein kinases within KCC2 and the role that these residues play in regulating its functional expression

Summary

Introduction

Together these biochemical and imaging experiments indicate that phosphorylation of KCC2 on Ser940 increases transporter cell surface expression levels. Together this series of experiments revealed that activation of KCC2 by PKC was dependent upon Ser940, the major site of phosphorylation for this kinase within this protein. This revealed that activation of PKC with PDBu produced a highly significant increase (p Ͻ 0.01) in the proportion of KCC2 expressed on the neuronal cell surface (CS) to 295.6 Ϯ 9.8% of control, an effect that was blocked by a specific PKC inhibitor (Fig. 6D).

Results

Conclusion