GLUTAMATE-RECEPTOR MODULATION BY PROTEIN-PHOSPHORYLATION

Glutamate-gated ion channels mediate most excitatory synaptic transmis sion in the mammalian central nervous system and play major roles in s ynaptic plasticity, neuronal development, and in some neuropathologica l conditions. Recent studies have suggested that protein phosphorylati on of neuronal glutamate receptors by cyclic AMP-dependent protein kin ase (PKA) and protein kinase C (PKC) may regulate their function and p lay a role in some forms of synaptic plasticity. To test whether these protein kinase effects are due to direct phosphorylation of the recep tors and to further examine the sites and mechanisms by which the rece ptors are modulated, we transiently expressed recombinant glutamate re ceptors in HEK-293 cells and studied their biochemical and biophysical properties. Our results indicate that the kainate-preferring receptor GluR6 is phosphorylated by PKA, primarily on a single serine in the p roposed major intracellular loop. Moreover, using the whole cell patch clamp recording technique, we have shown that phosphorylation at this site increases the amplitude of the GluR6-mediated glutamate current without significantly altering its dose-response, current-voltage rela tion or desensitization kinetics. In other experiments, we have demons trated that the NMDA receptor subunit NR1 is phosphorylated by PKC on several distinct sites, and most of these sites are located within a s ingle alternatively spliced exon in the C-terminal domain, These findi ngs suggest that RNA splicing can regulate NMDA receptor phosphorylati on and that, contrary tb the previously proposed membrane topology mod el, the NR1 C-terminus is intracellular. Furthermore, in HEK-293 cells co-transfected with NR2A and NR1 subunits containing the C-terminal e xon with the PKC phosphorylation sites, our preliminary studies indica te that the NMDA-evoked current is potentiated by intracellular PKC. W e are currently examining PKC effects on the NMDA-evoked current respo nses of mutant NR1 receptors that lack the C-terminal phosphorylation sites. These studies provide evidence that glutamate receptors are dir ectly phosphorylated and functionally modulated by protein kinases. Mo reover, by identifying phosphorylation sites within the receptor prote ins, our results provide information about the structure and membrane topology of these receptors.