Evidence that protein kinase Cα interacts with and regulates the glial glutamate transporter GLT-1

Many of the sodium-dependent neurotransmitter transporters are rapidly (within minutes) regulated by protein kinase C (PKC), with changes in activity being correlated with changes in transporter trafficking to or from the plasma membrane. Our recent studies suggest that one of the classical subtypes of PKC, PKC alpha, may selectively mediate redistribution of the neuronal glutamate transporter, excitatory amino acid carrier (EAAC)1, and show that PKC alpha can be co-immunoprecipitated with EAAC1. When the glial glutamate transporter GLT-1a is transfected into C6 glioma cells, this transporter is internalized in response to activation of PKC, but the PKC subtype involved in this regulation is unknown. In the present study, expression of the phorbol ester-activated subtypes of PKC was examined in C6 glioma transfected with GLT-1. Of the classical subtypes, only PKC alpha was detected, and of the non-classical subtypes, PKC delta and PKC epsilon were detected. In this system, phorbol ester-dependent internalization of GLT-1 was blocked by a general inhibitor of PKCs (bisindolylmaleimide II) and by concentrations of Go6976 that selectively block classical PKCs, but not by an inhibitor of PKC delta (rottlerin). PKC alpha immunoreactivity was found in GLT-1 immunoprecipitates obtained from transfected C6 cells and from crude rat brain synaptosomes, a milieu that better mimics in vivo conditions. The amount of PKC alpha in both types of immunoprecipitate was modestly increased by phorbol ester, and this increase was blocked by a PKC antagonist. These studies suggest that PKC alpha may be required for the regulated redistribution of GLT-1.