Mechanism of differential control of NMDA receptor activity by NR2 subunits

Abstract

N-methyl-d-aspartate (NMDA) receptors (NMDARs) are a major class of excitatory neurotransmitter receptors in the central nervous system. They form glutamate-gated ion channels that are highly permeable to calcium and mediate activity-dependent synaptic plasticity. NMDAR dysfunction is implicated in multiple brain disorders, including stroke, chronic pain and schizophrenia. NMDARs exist as multiple subtypes with distinct pharmacological and biophysical properties that are largely determined by the type of NR2 subunit (NR2A to NR2D) incorporated in the heteromeric NR1/NR2 complex. A fundamental difference between NMDAR subtypes is their channel maximal open probability (P(o)), which spans a 50-fold range from about 0.5 for NR2A-containing receptors to about 0.01 for receptors containing NR2C and NR2D; NR2B-containing receptors have an intermediate value (about 0.1). These differences in P(o) confer unique charge transfer capacities and signalling properties on each receptor subtype. The molecular basis for this profound difference in activity between NMDAR subtypes is unknown. Here we show that the subunit-specific gating of NMDARs is controlled by the region formed by the NR2 amino-terminal domain (NTD), an extracellular clamshell-like domain previously shown to bind allosteric inhibitors, and the short linker connecting the NTD to the agonist-binding domain (ABD). The subtype specificity of NMDAR P(o) largely reflects differences in the spontaneous (ligand-independent) equilibrium between open-cleft and closed-cleft conformations of the NR2-NTD. This NTD-driven gating control also affects pharmacological properties by setting the sensitivity to the endogenous inhibitors zinc and protons. Our results provide a proof of concept for a drug-based bidirectional control of NMDAR activity by using molecules acting either as NR2-NTD 'closers' or 'openers' promoting receptor inhibition or potentiation, respectively.

Figure 1. The NR2 NTD+linker region controls NMDAR Po

a–c Potentiation by MTSEA of receptors incorporating NR1-A652C and the indicated NR2 subunits. NTD, N-terminal domain; ABD, agonist-binding domain. d Pooled data (mean +/− s.d.), from top to bottom: 3.2 +/− 0.3 (n=12), 30 +/− 4 (n=14), 25 +/− 6 (n=6), 25 +/− 7 (n=5), 4.0 +/− 0.3 (n=3), 32 +/− 4 (n=3), 17 +/− 2 (n=6), 6.9 +/− 0.5 (n=5), 53 +/− 7 (n=9), 270 +/− 60 (n=7), 68 +/− 12 (n=6) and 23 +/− 2 (n=5) (**p<0.001). e Po within bursts of openings for receptors incorporating NR1wt and the indicated NR2 subunit. Left: representative traces of bursts. Right (from top to bottom): 0.78 +/− 0.06 (n=3), 0.24 +/− 0.07 (n=3), 0.43 +/− 0.02 (n=3) and 0.61 +/− 0.04 (n=3) (*p<0.05, Student’s t-test). f Kinetics of inhibition by MK-801 at receptors incorporating NR1wt and NR2Dwt (τ_on = 32 s), NR2D-ΔNTD (5.7 s) or NR2D-(2A NTD+L) (1.6 s). Error bars represent s.d.

Figure 2. Locking open the NR2-NTD increases NMDAR activity

a 3D model of NR2B-NTD. b Top: chemical formula of the transferable moiety of MTSEA, MTSET and MTS-PtrEA. Middle: Recordings from NR1wt/NR2B-Y282C and control NR1wt/NR2B-Y282S receptors during MTS treatment. The potentiation upon MTS wash likely reflects the washout of a reversible pore-blocking effect of the positively charged MTS. Bottom: Schematic representations of the NTD-ABD tandem of NR2B-Y282C after MTS-modification (MTS head group in yellow). c Relative currents after application of MTSEA, MTSET and MTS-PtrEA to receptors incorporating NR1wt and the indicated NR2 subunit. See Table S1 for values.

Figure 3. The NR2 NTD+linker region controls zinc and proton sensitivities of NMDARs

a Zinc sensitivity of receptors incorporating NR1wt and (Inhib_max, IC₅₀): NR2Awt (81%, 7.5 nM [n=6]), NR2Bwt (98%, 720 nM [n = 13]), NR2Dwt (100%, 7.8 µM [n=3]), NR2B-(2A NTD) (83%, 0.20 nM [n=4]), NR2B-(2A NTD+L) (86%, 5.4 nM [n=4]) or NR2D-(2A NTD+L) (90%, 1.5 nM [n=5]). n_H in the 0.9–1.2 range. b pH_IC50 of receptors incorporating NR1wt and the indicated NR2 subunit. See Table S2 for values. (**p<0.001). c Proton sensitivity of NR1wt/NR2B-Y282C receptors before (pH_IC50 = 7.70, n_H = 1.5 [n=3]) and after (pH_IC50 = 7.34, n_H = 1.4 [n=3]) MTS-PtrEA modification. Error bars represent s.d.

Figure 4. Model for the control of NMDAR activity by the NR2 N-terminal domain

a Structural depiction of an NMDAR. The full receptor is a tetramer but only a NR1/NR2 dimer is shown. b In its ligand-free state, the NR2-NTD alternates between open- and closed-cleft conformations, the latter favoring pore closure. In the model, this equilibrium determines the subtype-specificity of NMDAR Po (ko/kc[NR2B] < ko/kc[NR2A]). The NTD is also the target of subunit-specific allosteric inhibitors such as zinc–, or ifenprodil,, which bind the NTD central cleft and promote domain closure. We hypothesize that a molecule binding in the same cleft, but preventing its closure, behaves as a positive allosteric modulator, enhancing receptor activity.