Direct protein kinase C-dependent phosphorylation regulates the cell surface stability and activity of the potassium chloride cotransporter KCC2.
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.
Lee
Henry H C
HH
Department of Neuroscience, School of Medicine, University of Pennsylvania, Pennsylvania 19104, USA.
Walker
Joshua A
JA
Williams
Jeffery R
JR
Goodier
Richard J
RJ
Payne
John A
JA
Moss
Stephen J
SJ
D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D004705:Endocytosis; D004926:Escherichia coli / Q000378:metabolism; D006624:Hippocampus / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D009474:Neurons / Q000378:metabolism; D010766:Phosphorylation; D011189:Potassium Chloride / Q000737:chemistry*; D011493:Protein Kinase C / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011963:Receptors, GABA-A / Q000737:chemistry; D027981:Symporters / Q000737:chemistry*; D000096922:K Cl- Cotransporters
D011189:Potassium Chloride; D011493:Protein Kinase C; D011963:Receptors, GABA-A; D027981:Symporters; D000096922:K Cl- Cotransporters