| The N-methyl-D-aspartate (NMDA) subtype of excitatory, ionotropic L-glutamate neurotransmitter receptor is a cation-selective, ligand-gated ion channel composed of NRl and NR2 subunits. NMDA receptors are localised on the dendritic spines of neurons where they are tightly associated with proteins of the post-synaptic density such as the scaffolding protein, post-synaptic density 95 (PSD-95). Many studies have characterised the pharmacological properties of NMDA receptors expressed in non-neuronal cell lines which lack the proteins of the post-synaptic density. The aim of this thesis was to investigate the effect of PSD-95 on the biochemical and pharmacological properties of NMDA receptors following their co-expression in mammalian cells. Various homomeric and heteromeric combinations of NR1-1 a, NR1-2a, NR1-4b, NR2A and NR2B subunits were expressed +/- PSD-95 in human embryonic kidney 293 cells and analysed by quantitative immunoblotting, radioligand binding and immunoprecipitation. It was found that PSD-95 both enhanced the level of NR2A and NR2B subunit expression by approximately three-fold and induced a three-fold increase in the [3H]MK801 radioligand binding sites for NR1-1a/NR2A receptors. PSD-95 also increased the EC50 values for the L-glutamate and glycine enhancement of [3H]MK801 binding to NR1-1a/NR2A receptors by four-fold and six-fold respectively. PSD-95 however, had no effect on the affinity of NRl-la/NR2A receptors for L-glutamate and glycine as determined by [3H]CGP39653 and [3H]MDL105-519 displacement assays respectively suggesting that the reduced EC50 may be due to decreased channel gating. Deletion studies showed that both the biochemical and pharmacological effects of PDS-95 on expressed NMDA receptors were dependent upon the NR2 C-terminal ESDV motif, the PSD-95-binding domain. Further, mutagenesis of the two N-terminal cysteine residues of PSD-95 demonstrated that PSD-95/NMDA receptor clustering is necessary for the changes in NMDA receptor properties induced by PSD-95. These results suggest that the interaction between PSD-95 and native NMDA receptors may be important for the regulation of both receptor function and receptor number at post- synaptic sites. |
