Assembly of N-methyl-D-aspartate (NMDA) receptors.
The N-methyl-D-aspartate receptor (NMDAR) requires both NR1 and NR2 subunits to form a functional ion channel. Despite the recent advances in our understanding of the contributions of these different subunits to both the function and pharmacology of the NMDAR, the precise subunit stoichiometry of the receptor and the regions of the subunits governing subunit interactions remain unclear. Since NR2 subunits are not transported to the cell surface unless they associate with NR1 subunits, cell-surface expression of NR2A can be used to monitor the association of the different subunits in cells transfected with N- and C-terminally truncated NR1 subunits. By combining measurements of cell-surface expression of NR2A with co-immunoprecipitation experiments, and by using Blue Native gel electrophoresis to determine the oligomerization status of the subunits, we have shown that regions of the N-terminus of NR1 are critical for subunit association, whereas the truncation of the C-terminus of NR1 before the last transmembrane region has no effect on the association of the subunits. Evidence from the Blue Native gels, sucrose-gradient centrifugation and size exclusion of soluble NR1 domains suggests that NR1 subunits alone can form stable dimers. Using a cell line, which can be induced to express the NMDAR following exposure to dexamethasone, we have shown that NMDARs can be expressed at the cell surface within 5 h of the recombinant gene induction, and that there appears to be a delay between the first appearance of the subunits and their stable association.
McIlhinney
R A J
RA
MRC Anatomical Neuropharmacology Unit, Mansfield Road, Oxford OX1 3TH, UK. jeff.mcilhinney@pharm.ox.ac.uk
Philipps
E
E
Le Bourdelles
B
B
Grimwood
S
S
Wafford
K
K
Sandhu
S
S
Whiting
P
P
D000818:Animals; D006801:Humans; D007473:Ion Channels / Q000737:chemistry / Q000502:physiology; D021122:Protein Subunits; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry* / Q000378:metabolism*
D007473:Ion Channels; D021122:Protein Subunits; D016194:Receptors, N-Methyl-D-Aspartate