Experimental studies of N-methyl-D-aspartate receptor NR3 subunits

Abstract

This thesis focuses on the NR3A and NR3B subunits of the excitatory glutamate receptor N-methyl-D-aspartate (NMDA). Functional NMDA receptors are believed to be tetrameric complexes with combinations of NR1 and NR2 subunits, and possible addition of NR3 subunits. The NR3A subunit is highly expressed throughout the developing brain, and declines to low levels in adulthood. NR3B expression is most prominent in brain stem and spinal cord motoneurons, and in contrast to NR3A, levels are constant during development.

In paper I, we cloned and sequenced the human NR3A, which at the time had only been described in rodent. Only minor differences in homology to rat NR3A was found; one potential glycosylation and one phosphorylation site, plus a PXXP motif (SH3-binding motif). Only the short splice variant of NR3A was detected in human tissue. NR3A mRNA expression in the forebrain was most abundant in the cerebrocortical anlage, throughout the ventricular zone and cortical plate, while in the spinal cord, expression was highest in the dorsal half and the neuroepithelial layer encircling the central canal.

In paper II, we demonstrated an extensive expression of NR3A, at both mRNA and protein levels, in adult human CNS. The highest levels were found in cortical regions, mainly in pyramidal neurons of layers II/III and V, and in pons and thalamus, lower levels in claustrum and the caudate nucleus, but no detectable NR3A in cerebellum. In the spinal cord we found the largest species difference, with very low NR3A expression in human tissue contrasted to the high levels in the rat. Individual human motoneurons had remarkably high NR3A expression. Analyzing subunit assembly we found that NR3A, like NR1, was present as monomers and dimers, which suggests there are considerable intracellular pools of un-assembled NR1 and NR3A. NR3A was also found in larger complexes, such as tetramers, probably representing functional NMDA receptors. As expected for such NMDA receptors, we found that NR3A was associated with NR1, NR2A and NR2B in adult human CNS.

In paper III and IV, we identified glycine as an endogenous ligand for NR3A (paper III) and NR3B (paper IV). Subunit differences were identified by competitive binding analysis of several NR1 glycine site ligands. For both NR3A and NR3B, [3H]-glycine was displaced by the agonist D-serine and the partial agonist HA-966, but not by glutamate. Different affinity to the NR3 subunits was observed for the partial agonist D-cycloserine and the antagonist 7-chloro-kynurenate, which could not displace [3H]-glycine from NR3A (paper III), but from NR3B (paper IV). We could only partially displace [3H]-glycine bound to NR3B with the antagonist CNQX, but not L-689.560 (paper IV). The structure models built for the S1/S2 ligand-binding site of NR3A and NR3B identified potential interactions between backbone amino acids and the ligands. In paper IV, we used the NR3B model for screening of a chemical library. A selection of putative NR3B antagonist was investigated by competitive binding of [3H]-glycine. However, none of them showed detectable affinity for NR3B.

The widespread distribution of NR3A in the human CNS suggests that is has important roles in several different functions. With our pharmacological profile of NR3A and NR3B we show that these differences can exploit in developing subunit specific substrates, which could be used to elucidate the role of NR3A and NR3B in the CNS.