NR2B and NR2D subunits coassemble in cerebellar Golgi cells to form a distinct NMDA receptor subtype restricted to extrasynaptic sites

Abstract

NMDA receptors (NMDARs) are thought to be tetrameric assemblies composed of NR1 and at least one type of NR2 subunit. The identity of the NR2 subunit (NR2A, -B, -C, -D) is critical in determining many of the functional properties of the receptor, such as channel conductance and deactivation time. Further diversity may arise from coassembly of more than one type of NR2 subunit, if the resulting triheteromeric assembly (NR1 plus two types of NR2) displays distinct functional properties. We have used gene-ablated mice (NR2D -/-) to examine the effects of the NR2D subunit on NMDAR channels and NMDAR EPSCs in cerebellar Golgi cells. These cells are thought to express both NR2B and NR2D subunits, a combination that occurs widely in the developing nervous system. Our experiments provide direct evidence that the low conductance NMDAR channels in Golgi cells arise from diheteromeric NR1/NR2D assemblies. To investigate whether a functionally distinct triheteromeric assembly was also expressed, we analyzed the kinetic and pharmacological properties of single-channel currents in isolated extrasynaptic patches. We found that after the loss of the NR2D subunit, the properties of the 50 pS NMDAR channels were altered. This result is consistent with the presence of a triheteromeric assembly (NR1/NR2B/NR2D) in cells from wild-type mice. However, we could find no difference in the properties of NMDAR-mediated EPSCs between wild-type and NR2D subunit ablated mice. Our experiments suggest that although both diheteromeric and triheteromeric NR2D-containing receptors are expressed in cerebellar Golgi cells, neither receptor type participates in parallel fiber to Golgi cell synaptic transmission. The presence of the NR2D subunit within an assembly may therefore result in its restriction to extrasynaptic sites.

Figure 1.

Single-channel properties of Golgi cell NMDARs in wild-type and NR2D -/- mice. A, Single-channel currents from an outside-out patch (-60 mV) excised from a Golgi cell soma in a wild-type mouse (P8). Channel activity was evoked in response to 10 μm NMDA and 10 μm glycine. A mixed population of 50, 40, and 20 pS openings was observed (mean amplitudes indicated by dashed lines) in wild-type mice. Stretches of record have been selected to illustrate examples of low-conductance openings (along with high-conductance events). Note, however, that the frequency of low-conductance openings is generally low in Golgi cells. Bottom panel shows a fitted amplitude histogram and open period_HIGH distribution obtained by time course fitting of single-channel openings. Current amplitudes were fitted with the sum of three Gaussian distributions; open periods were fitted with the sum of two exponentials (note the log scale). In both cases a maximum likelihood fitting procedure was applied to ensure best fit. B, Single-channel currents from an outside-out patch (-60 mV) from an NR2D -/- mouse (P8). Note the absence of low-conductance openings, in both the single-channel recordings and the amplitude distribution (bottom left-hand panel). Amplitudes were fitted with the sum of two Gaussian distributions (∼50 and 40 pS, corresponding to high-conductance type openings), and open periods were fitted with the sum of two exponentials.

Figure 2.

Comparison of main conductance states and open period_HIGH distributions in wild-type and NR2D -/- animals. A, Plots of the main chord conductance measurements obtained from outside-out patches in wild-type and NR2D -/- animals. In the wild-type animals, three main conductance states were present (dashed lines). Only high-conductance openings were observed in patches from NR2D -/- mice (indicated by a single dashed line; subconductances of the 50 pS channels have not been included in wild-type or NR2D -/- data). B, Slope conductance measurements of ∼50 pS channels from wild-type and NR2D -/- mice. Averaged data from patches from 16 wild-type and 7 NR2D -/- cells have been pooled, and the resulting I—V relationship has been obtained by least squares fitting to give the slope conductance. C, Comparison of all fits to the open period_HIGH data from wild-type and NR2D -/- mice. Distributions were best fit with a fast (open symbols) and a slow (filled symbols) time constant.

Figure 3.

Ifenprodil sensitivity of high-conductance NMDAR openings in wild-type and NR2D -/- mice. A, Continuous single-channel current records showing the effect of 0.1 μm ifenprodil (bottom traces) on high-conductance NMDAR openings. In both wild-type and NR2D -/- mice, P_OPEN was clearly reduced. Note that in the records from NR2D -/- NMDARs, channel openings appear longer in duration in control than in the ifenprodil. B, Comparison of the change in P_OPEN(HIGH) produced by 0.1 μm ifenprodil in wild-type and NR2D -/- NMDARs. The reduction was significantly greater in NR2D -/-. C, Concentration—inhibition relationship for the action of ifenprodil on single-channel charge transfer. Data represent 16 patches from wild-type mice (filled symbols) and 14 patches from NR2D -/- mice (open symbols). The relationship for wild-type mice was fitted with a modified Hill equation giving high- and low-affinity components with IC₅₀ of 129 nm (I_max(H) = 71.5%) and 45 μm, respectively. The relationship for NR2D -/- mice was fit with a single-component Hill equation with IC₅₀ of 37 nm. For comparison a data point showing the degree of ifenprodil block (100 nm) for NMDARs recorded in migrating cerebellar granule cells (Misra et al., 2000a) has been added to this plot (gray symbol).

Figure 4.

Properties of NMDAR mEPSCs in wild-type and NR2D -/- mice. A, Whole-cell records from a P8 Golgi cell (-30 mV) in the presence of 1 μm TTX (left-hand panel) and 1 μm TTX with AP5 (right-hand panel) to yield pure non-NMDAR-mediated mEPSCs. B, Averaged mEPSCs in TTX (left) displaying two components; the inset histogram (left) shows the peak amplitude distribution form EPSCs recorded in this cell. Averaged mEPSCs in TTX plus AP5 (right) displayed a single component with fast rise and decay. Right-hand inset shows the NMDAR-mediated component obtained by subtraction (τ = 84.9 msec, in this example). C, Comparison of NMDAR mEPSCs in wild-type (filled symbols) and NR2D -/- (open symbols) mice showing the lack of significant difference in rise-time, peak amplitude, τ, and charge transfer.

Figure 5.

Ifenprodil sensitivity of the NMDAR component of EPSCs in wild-type and NR2D -/- mice. A, Averaged EPSCs from a Golgi cell (P8, wild-type mouse) in response to a 50 Hz train of stimuli applied to the parallel fiber input. The NMDAR-mediated component was greatly reduced by 10 μm ifenprodil. The histogram compares the percentage block in wild-type and NR2D -/- mice. This was measured from charge transfer analysis and calculated relative to the maximum block achieved with 50 μm AP5.