Molecular and electrophysiological characterization of rat brain glutamate receptors

Abstract

Glutamate, the primary excitatory neurotransmitter in the central nervous system, acts on three main classes of ionotropic receptors, NMDA, AMPA and kainate receptors. Glutamate receptors are thought to be involved in synaptogenesis, developmental structuring and in the regulation of neuronal circuitry. The cloning and functional characterization of two new NMDA receptor splice variants is first reported here. The splice variants form functional homomeric channels that differ in agonist affinity and in sensitivity to polyamines and activators of protein kinase C (PKC). Alternative RNA splicing generates at least seven variants of the NMDA receptor which are characterized by the presence or absence of a 21-amino acid insert in the N-terminal domain, and by alternative C-terminal domains. We characterized homomeric NMDA receptor channels formed by the variant subunits in Xenopus oocytes. NMDA receptors that lack the N-terminal insert exhibited a relatively high affinity for agonists and marked potentiation by spermine. In contrast, those receptor variants with the N-terminal insert showed reduced agonist affinity and lacked spermine potentiation at saturating glycine. These findings indicate that the N-terminal insert either participates in the binding domains for agonists and polyamines or indirectly modifies their conformations. The splice variants varied in their sensitivity to activators of PKC from 3-fold to 20-fold. Structure/function studies showed that the C-terminal sequence and the presence or absence of the N-terminal insert influence sensitivity to PKC. These findings support a role for alternative splicing in generating NMDA channels with different functional properties.;Functionally diverse AMPA/kainate receptors can also be generated by assembly of non-NMDA glutamate receptor GluR1, 2, 3 and 4 subunits into homomeric and heteromeric channels. We examined GluR1, 2 and 3 gene expression in embryonic, neonatal and adult rat brain by Northern analysis. Our study shows that in each region examined, GluR1, 2 and 3 exhibit a characteristic pattern of temporal expression varying through development. Comparison of the normalized levels of GluR1, 2 and 3 mRNAs suggests a relative increase in GluR2 expression, as compared to GluR1 and 3, in specific brain regions as a function of age. Since heteromeric receptors containing GluR2 are Ca{dollar}\sp{lcub}2+{rcub}{dollar} impermeable, this result predicts a decrease in glutamate-operated Ca{dollar}\sp{lcub}2+{rcub}{dollar} permeability mediated through kainate/AMPA receptors during the first two weeks of postnatal life.