Phorbol myristate acetate-dependent interaction of protein kinase Cα and the neuronal glutamate transporter EAAC1

Abstract

Sodium-dependent transporters clear extracellular glutamate in the mammalian CNS. Activation of protein kinase C (PKC) rapidly increases the activity of the neuronal glutamate transporter EAAC1 (excitatory amino acid carrier-1). This effect is associated with redistribution of EAAC1 to the cell membrane and appears to be dependent on a particular PKC subtype, PKCα. In the present study, we sought to determine whether this specificity for regulation of EAAC1 is associated with the formation of EAAC1-PKCα complexes. In C6 glioma cells, activation of PKC with phorbol 12-myristate 13-acetate (PMA) induced formation of EAAC1-PKCα complexes but did not induce formation of complexes with PKCα, a PKC not thought to regulate EAAC1. Formation of these complexes was blocked by inhibitors of PKC. Confocal microscopy revealed that PMA caused EAAC1 and PKCα to colocalize in clusters at or near the cell surface. The EAAC1-PKCα complexes were also observed in rat brain synaptosomes, demonstrating that this interaction is not restricted to C6 cells. These data demonstrate that EAAC1 and PKCα interact in a PKC-dependent manner that is associated with EAAC1 redistribution. Although PKC activation has been implicated in the regulation of many different neurotransmitter transporters, this study provides the first example of an interaction between a neurotransmitter transporter and PKC. PKCα also forms complexes with GluR2 (glutamate receptor subunit 2) and causes a reduction in the levels of GluR2-containing AMPA receptors at the plasma membrane. Together, these data suggest that PKCα may simultaneously trigger the redistribution of EAAC1 and glutamate receptors.