The goal of this proposal is to study the role of phosphorylation in the modulation of glutamate receptor channel function. Glutamate is the principle excitatory neurotransmitter in the central nervous system, and it participates in most physiological brain functions, as well as in many neurological diseases. Protein phosphorylation of glutamate receptors may play a major role in mechanisms underlying synaptic plasticity. Four cDNA clones for glutamate receptors (GluR) recently isolated from rat brain show ion channel activity in response to kainate, AMPA, quisqualate, and glutamate.
The specific aims of this proposal are to express these cDNA clones in human embryonic kidney 293 cells, characterize the receptors' electrophysiological properties using whole cell and excised outside-out patch clamp techniques, and study their regulation by protein phosphorylation. Protein kinases will be selectively modulated in these cells with activators and inhibitors, and the physiological consequences analyzed at both the whole cell current and single channel level. Specific cloned protein kinases will be coexpressed with GluR receptors in order to examine effects of protein kinases not endogenously expressed in these cells. In addition, site-specific mutagenesis will be employed to alter potential phosphorylation sites on the cloned receptors, and the functional effects of phosphorylation of wild-type and mutant receptors will then be compared electrophysiologically. The level of receptor phosphorylation in each of the above experiments will be determined by prelabeling cells with 32-P-orthophosphate and immunoprecipitating the GluR receptors. Preliminary studies support the feasibility of this approach. Successful expression of one of these clones (GluR1) in the kidney cells has been confirmed by immunoprecipitation and Western blots, with antibodies to GluR1 that have been prepared against synthetic oligopeptide sequences. In addition, kainate-induced current has been recorded in the transfected cells, by using the whole cell patch clamp technique. The proposed research will provide a better understanding of basic mechanisms for regulation of glutamate receptors. Glutamate receptors may play a major role in neuronal cell death occurring after hypoxic-ischemic injury and in neurodegenerative disorders such as Amyotrophic Lateral Sclerosis (ALS), Huntington's Disease and Alzheimer's Disease. Development of drugs targeted to glutamate receptor regulation rather than to blockade of the receptor itself may be effective in preventing such neuronal damage, with fewer side effects.
