The objective of this research is to broaden understanding of the ATP-dependent glutamate uptake system in the synaptic vesicle, particularly to investigate its regulation. There is compelling evidence that glutamate serves as a major excitatory neurotransmitter in the central nervous system. The ATP-dependent vesicular uptake is highly specific for glutamate and considered to play an important role in determining the neurotransmitter role of glutamate. Recently, we have obtained evidence that vesicular glutamate uptake is inhibited by a proteinaceous substance. I propose to (a) purify the inhibitory substance by classical and high pressure (HPLC) liquid chromatography; (b) determine physicochemical properties by SDS-polyacrylamide gel electrophoresis, gel filtration, sucrose density gradient centrifugation and amino acid analysis; (c) determine the mechanism of inhibition by examining its effects on a proton-pump ATPase, membrane potential, chloride influx, and glutamate efflux; (d) examine the role of protein phosphorylation in the inhibition by SDS-gel electrophoresis and autoradiography; (e) identify a peptide fragment which retains the inhibitory activity by HPLC; and (f) identify the protein(s) with which the inhibitor interacts, by inhibitor-conjugated affinity chromatography. This research will not only enhance understanding of the vesicular glutamate uptake system, but also may ultimately provide new insights into the investigation of those types of neuropathophysiology where abnormal glutamate transmission is implicated.
Showing the most recent 10 out of 13 publications
