Molecular biological studies of brain glutamate receptors

Abstract

Glutamate is the major excitatory neurotransmitter that depolarizes neurons and glial cells in the nervous system of vertebrates. Ionotropic glutamate receptors are integral membrane proteins that form cation-specific ion channels, and are further classified into three subclasses: the NMDA receptors, the AMPA receptors, and the KA receptors. NMDA receptors are found in almost all CNS neurons, and are thought to play a role in synaptogenesis, synaptic plasticity, and excitotoxity. The main objective of this dissertation was to better understand the mechanism of the protein kinase C (PKC) potentiation of recombinant NMDA receptors. The expression pattern of glutamate receptors in type-2 astrocytes was also determined.;First, the effect of PKC phosphorylation on the function of recombinant NMDA receptors expressed in xenopus oocytes was studied. I found that PKC potentiation of the recombinant NMDA receptor is not due to an increase in the NMDA or glycine affinity, and the voltage-dependent Mg{dollar}\sp{lcub}2+{rcub}{dollar} block of the NMDA receptors is not altered by the PKC potentiation process.;I further determined the structural features in the NR1{dollar}\sb{lcub}100{rcub}{dollar} subunit involved in PKC potentiation. I mutated serine residues in the cytoplasmic loops and the extracellular loop. Homomeric NR1{dollar}\sb{lcub}100{rcub}{dollar} and heteromeric NR1{dollar}\sb{lcub}100{rcub}{dollar}/NR2A receptors with one, two or four serine substitutions were examined for PKC potentiation, affinity for NMDA and glycine, and voltage-dependent Mg{dollar}\sp{lcub}2+{rcub}{dollar} block. Three serine residues in the predicted intracellular loops of NR1 subunit were shown to participate in PKC potentiation but have little or no effect on agonist or co-agonist affinity or on the voltage-dependent Mg{dollar}\sp{lcub}2+{rcub}{dollar} block.;The expression pattern of glutamate receptors in astrocytes cultured from different brain regions was determined by Northern blot analysis, immunocytochemistry and in situ hybridization. I found that the expression of GluR1 is highest in astrocytes from cerebellum, followed by hippocampus, neocortex and striatum, whereas the expression of GluR2 and NR1 are undetectable in these regions. Whole-cell patch-clamp recordings of these glutamate receptors demonstrated that the glutamate receptors in hippocampal astrocytes can be excited by glutamate or kainate, but not NMDA.