NMDA receptor (NMDAR) assemblies consist of NR1 subunits combined with at least one type of NR2 subunit (NR2A-D). NMDAR functional diversity arises largely from differences in NR2 subunit composition. For example, in cerebellar Golgi cells it is thought that NR1/NR2B and NR1/NR2D subunit-containing receptors give rise to high- (40-50 pS) and low-conductance (20-40 pS) single-channel openings, respectively (Misra et al. 2000). Further functional diversity could arise if more than one type of NR2 subunit coexists within a native receptor. We have used NR2D subunit ablated animals, to determine whether NR2B and NR2D subunits co-assemble in Golgi cells to form a distinct receptor subtype.
Parasaggital cerebellar slices were prepared from 8- to 10-day-old C57BL/6J mice (wild-type, WT), and NR2D subunit ablated (NR2D -/-) mice killed by decapitation (Ikeda et al. 1995). In outside-out patches from WT animals, application of 10 µM NMDA elicited a mixture of high- and low-conductance channels, while patches from NR2D -/- mice produced only high-conductance events. We next compared the biophysical and pharmacological properties of high-conductance (50 pS) openings in isolation. The mean single-channel conductance of these events was no different in the two strains, but the probability of opening (Popen(HIGH)) was greater in NR2D -/- (0.04 ± 0.004, mean ± S.E.M.) than in WT (0.02 ± 0.004; P < 0.05, Student’s t test). As the NR2D subunit confers a low probability of opening, when combined with NR1 (Wyllie et al. 1996) our results suggest the presence of the NR2D subunit in some high-conductance NR2B-containing assemblies in WT mice. Furthermore, the NR2B subunit-selective antagonist ifenprodil reduced Popen(HIGH) by only 31.2 ± 9.4 % (n = 7) in WT, compared with 52.4 ± 9.3 % (n = 9; P < 0.05) in NR2D -/- mice. This is consistent with the presence of an ifenprodil-insensitive NR2D subunit within NR2B subunit-containing NMDAR complexes in the WT.
In conclusion, our results indicate that deletion of the NR2D subunit results in a loss of low-conductance NMDAR channels, while the conductance of the remaining openings was unaltered. Furthermore, changes in the biophysical and pharmacological properties of the remaining 50 pS openings, suggests that a novel triheteromeric receptor subtype, containing both NR2B and NR2D subunits, was present in the WT.
This work was supported by The Wellcome Trust.