Zinc alleviates pain through high-affinity binding to the NMDA receptor NR2A subunit

Abstract

Zinc is abundant in the central nervous system and regulates pain, but the underlying mechanisms are unknown. In vitro studies have shown that extracellular zinc modulates a plethora of signaling membrane proteins, including NMDA receptors containing the NR2A subunit, which display exquisite zinc sensitivity. We created NR2A-H128S knock-in mice to investigate whether Zn2+-NR2A interaction influences pain control. In these mice, high-affinity (nanomolar) zinc inhibition of NMDA currents was lost in the hippocampus and spinal cord. Knock-in mice showed hypersensitivity to radiant heat and capsaicin, and developed enhanced allodynia in inflammatory and neuropathic pain models. Furthermore, zinc-induced analgesia was completely abolished under both acute and chronic pain conditions. Our data establish that zinc is an endogenous modulator of excitatory neurotransmission in vivo and identify a new mechanism in pain processing that relies on NR2A NMDA receptors. The study also potentially provides a molecular basis for the pain-relieving effects of dietary zinc supplementation.

Figure 1

Targeting the NMDA receptor NR2A subunit gene in mice. (a) The NR2A-H128S mutant allele was created by homologous recombination. The scheme shows the WT NR2A allele, the targeting vector, the targeted allele and the H128S knock-in allele. The CAT codon encoding histidine 128 (H128) in exon 1 was replaced by the TCC codon encoding serine (S), and a loxP-flanked (‘floxed’) neo cassette was introduced 3′ from exon 1 to select for embryonic stem cells harboring the knock-in allele. The final mutant allele was obtained after excision of the neo cassette by a Cre recombinase treatment of embryonic stem cells. White box, exon 1; star in white box shows the H128S mutation; dark line, intronic sequences; Ba, BamH I; Bs, Bsu361; Sp, SpeI (restriction sites); triangles, loxP sites; neo box, neomycin-resistance cassette; gray bars, probes for Southern blot analysis; lines above gene indicate expected labeled DNA fragments in Southern blot analysis. (b) Southern blot analysis of WT and targeted alleles in the selected embryonic stem cell clone. Genomic DNA was digested with Bsu361 and BamHI, and hybridized with 5′and 3′ external probes, respectively or the neo probe. Expected bands at each size as indicated in a are obtained. (c) Genomic DNA sequence analysis using tail biopsies from WT (left panel) and knock-in homozygous mutant (right panel) mice, showing the replacement of the CAT codon by the mutated TCC codon. (d) Genotyping of the NR2A-H128S knock-in line. PCR analysis using mutation-specific primers (strategy on left) reveals WT (280 bp) and mutant (315 bp) alleles (right). White arrow, forward primer for WT DNA, black arrow, forward primer for mutant DNA, gray arrows, reverse primer. Analysis of genomic DNA from WT NR2A, homozygous NR2A-H128S and heterozygous NR2A WT/NR2A-H128S mice is shown. The 35-bp differences between mutant and WT alleles results from the remaining loxP site in the mutant allele.

Figure 2

High-affinity zinc inhibition of NMDA currents is lost in NR2A-H128S mice. (a) Sensitivity to subunit-specific modulators of brain NMDARs from WT and knock-in NR2A-H128S (KI) mice transplanted into Xenopus oocytes. Inhibition by 20 nM zinc was abolished in KI mice (upper traces; 2.3 ± 1.3%, n = 6 versus 30.3 ± 4.2% n = 10 for WT; mean ± s.d., *P < 0.001, Student’s t-test), whereas inhibition by the NR2B-selective antagonist ifenprodil was unchanged (lower traces; 25.2 ± 3.2%, n = 6 versus 24.8 ± 2.4% n = 13 for WT; P = 0.7). NMDA currents were induced by co-application of 300 μM NMDA, 100 μM glycine and 10 μM strychnine (N + G + S). (b) Hippocampal Schaeffer collateral to CA1 NMDA EPSCs from KI mice were insensitive to 300 nM zinc, contrasting with the marked inhibition seen in WT mice (peak current ratio: 0.48 ± 0.03 n = 4 versus 0.93 ± 0.06 n = 5 for KI; *P < 0.001, Student’s t-test). Same for NMDA EPSCs recorded in the dorsal horn of the spinal cord (0.59 ± 0.05, n = 6 for WT versus 1.03 ± 0.07, n = 6 for KI; mean ± s.d., *P < 0.001). (c) Unaltered protein expression levels in KI mice compared with those of WT mice in forebrain (top) and spinal cord (bottom). Lower band, α-tubulin control (α-tub). Full-length blots are presented in Supplementary Figure 1. For each protein, quantification was performed on two or three different WT-KI couples; no significant changes in expression was detected (P > 0.05, one-sample Student’s t-test; see Supplementary Fig. 2).

Figure 3

NR2A-H128S mice show enhanced basal pain sensitivity in response to radiant heat and capsaicin. (a–j) Tail immersion (TI), hot plate (HP), tail flick (TF), Hargreaves (HT), von Frey filaments (VF), tail pressure (TP) and chemical tests with capsaicin (CAP) and TIP39 (TIP) were used to evaluate basal pain sensitivity in response to thermal (a–d, g), mechanical (e,f) and chemical (h–j) noxious stimuli (see Online Methods). No genotype effect was observed in TI withdrawal at three temperatures (a), in the HP response (b), in the mechanical responses (e,f) or in the TIP response (i). By contrast, significant hypersensitivity of NR2A-H128 mice was detected in responses to radiant heat stimuli (c,d) and capsaicin (h,j). Furthermore, TF test with three different heat rates showed thermal hypersensitivity at 0.9 and 2.2 °C s⁻¹ (g). Data are expressed as means ± s.e.m. of eight mice per group. *P < 0.05 and **P < 0.001, NR2A-H128S mutants versus controls, Student’s t-test.

Figure 4

NR2A-H128S mice show increased mechanical allodynia under chronic pain. Thermal and mechanical sensitivity of mutant knock-in (KI) mice and their WT controls was examined under CFA inflammatory or SNL neuropathic pain. Raw values (left) and percentage pain (right: (value_{contralateral} − value_ipsilateral)/value_{contralateral}) are shown. Dotted line, baseline value. (a,b) Thermal sensitivity (Hargreaves test). Areas under the curve (AUC) of percentage pain showed no genotype difference in intensity and duration of thermal hyperalgesia under either CFA (a) or SNL (b) conditions. (c,d) Mechanical sensitivity (von Frey test). Upon CFA injection (c), as well as after partial sciatic nerve ligation (d), mechanical allodynia developed with higher intensity and duration in mutant mice (AUC of percentage pain: CFA, 316 ± 15 for WT mice versus 510 ± 7.3 for KI mice; SNL, 565 ± 12 for WT mice versus 778 ± 14 for KI mice). Data are expressed as means ± s.e.m. of eight mice per group. *P < 0.05, **P < 0.01 and ***P < 0.001, NR2A-H128S mutants versus controls for individual time points, Student’s t-test.

Figure 5

Zinc analgesia is abolished in NR2A-H128S mice. Zinc analgesia was examined in mutant knock-in (KI) mice and control WT mice after intrathecal (i.t.; 0.2 nM, left panels) or subcutaneous (s.c.; 0.1–1 mg per kilogram body weight, right panels) ZnCl₂ administration. Baseline thresholds (BL) were measured in naive mice before the drug injection or induction of chronic pain. (a,b) Acute thermal pain (TF; raw values (left panels) and percentage of maximum possible effect (%MPE, right panels; see Online Methods)) shows zinc analgesia in WT but not KI mice. Data are expressed as means ± s.e.m. of eight mice per group. *P < 0.05, **P < 0.01 and ***P < 0.001, zinc-treated group versus saline-treated group for individual time points, Student’s t-test. (c,d) CFA inflammatory pain. Both i.t. or s.c. zinc administrations inhibited CFA-induced thermal hyperalgesia (HT, left panels) and mechanical allodynia (VF, right panels). These antihyperalgesic and antiallodynic effects were absent in mutant mice. (e,f) SNL neuropathic pain. As for inflammatory pain, both i.t. or s.c. zinc inhibited thermal hyperalgesia (HT, left panels) and mechanical allodynia (VF, right panels) induced by sciatic nerve ligation. These antihyperalgesic and antiallodynic effects were absent in KI mice. For both CFA and SNL experimental series, data are expressed as means ± s.e.m. of eight mice per group. ***P < 0.001 for zinc-treated versus saline-treated groups, ^§§§P < 0.001 for mutants versus controls, two-way repeated measures analysis of variance (ANOVA) followed by Bonferroni–Dunn test.