The α2δ-1-NMDA Receptor Complex Is Critically Involved in Neuropathic Pain Development and Gabapentin Therapeutic Actions

Abstract

α2δ-1, commonly known as a voltage-activated Ca²⁺ channel subunit, is a binding site of gabapentinoids used to treat neuropathic pain and epilepsy. However, it is unclear how α2δ-1 contributes to neuropathic pain and gabapentinoid actions. Here, we show that Cacna2d1 overexpression potentiates presynaptic and postsynaptic NMDAR activity of spinal dorsal horn neurons to cause pain hypersensitivity. Conversely, Cacna2d1 knockdown or ablation normalizes synaptic NMDAR activity increased by nerve injury. α2δ-1 forms a heteromeric complex with NMDARs in rodent and human spinal cords. The α2δ-1-NMDAR interaction predominantly occurs through the C terminus of α2δ-1 and promotes surface trafficking and synaptic targeting of NMDARs. Gabapentin or an α2δ-1 C terminus-interfering peptide normalizes NMDAR synaptic targeting and activity increased by nerve injury. Thus, α2δ-1 is an NMDAR-interacting protein that increases NMDAR synaptic delivery in neuropathic pain. Gabapentinoids reduce neuropathic pain by inhibiting forward trafficking of α2δ-1-NMDAR complexes.

Keywords: chronic pain; dorsal root ganglion; glutamate; pregabalin; presynaptic; synaptic plasticity; synaptic trafficking; synaptic transmission; thrombospondin; voltage-gated calcium channels.

Figure 1. α2δ-1 Overexpression Induces Pain Hypersensitivity and Increases Pre- and Postsynaptic NMDAR Activity of Spinal Dorsal Horn Neurons

(A) Time course of changes in the tactile and pressure withdrawal thresholds and heat withdrawal latency after a single intrathecal injection of the Cacna2d1 vector or control vector (n = 7 rats in each group). Data are expressed as means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 (versus respective baseline), one-way ANOVA followed by Dunnett’s post hoc test. (B and C) Effects of a single intrathecal injection of 5 µg AP5 (B) or intraperitoneal injection of 10 mg/kg memantine (C) on the tactile and pressure withdrawal thresholds in rats treated with the Cacna2d1 vector or control vector (n = 8 rats in each group). Data are expressed as means ± SEM. *p < 0.05; **p < 0.01 (versus baseline before drug injection, time 0), one-way ANOVA followed by Dunnett’s post hoc test. (D) Original traces and mean changes of NMDAR currents elicited by puff application of 100 µM NMDA to spinal dorsal horn neurons in rats 5 weeks after treatment with the Cacna2d1 vector or control vector (n = 12 neurons in each group). Data are expressed as means ± SEM. *p < 0.05 (versus control vector-treated rats), two-tailed Student’s t test. (E and F) Representative traces and cumulative plots (E) and mean changes (F) of miniature excitatory postsynaptic currents (mEPSCs) of spinal dorsal horn neurons before (baseline), with (AP5), and after (washout) bath application of 50 µM AP5. Slice recordings were performed using rat spinal cords 5 weeks after treatment with the control vector (n = 10 neurons) or Cacna2d1 vector (n = 11 neurons). Data are expressed as means ± SEM. *p < 0.05 (versus baseline). ^#p < 0.05, compared with the baseline value in the control vector-treated group, one-way ANOVA followed by Tukey’s post hoc test.

Figure 2. α2δ-1 Contributes to Increased Pre- and Postsynaptic NMDAR Activity of Spinal Dorsal Horn Neurons after Nerve Injury

(A) Representative recording traces and mean changes of NMDAR currents elicited by puff application of 100 µM NMDA to spinal dorsal horn neurons of sham control rats (n = 11 neurons) and SNL rats injected with control siRNA (n = 12 neurons) or Cacna2d1-specific siRNA (n = 12 neurons). Data are expressed as means ± SEM. *p < 0.05 (versus sham group), one-way ANOVA followed by Tukey’s post hoc test. (B) Original traces, cumulative plots, and mean changes in the baseline frequency and amplitude of mEPSCs and the AP5 effect in spinal dorsal horn neurons recorded from sham control rats (n = 14 neurons) and SNL rats receiving control siRNA (n = 13 neurons) or Cacna2d1-specific siRNA (n = 12 neurons) before (baseline), with (AP5), and after (washout) bath application of 50 µM AP5. Data are expressed as means ± SEM. *p < 0.05 (versus baseline); ^#p < 0.05 (versus baseline in sham group), one-way ANOVA followed by Tukey’s post hoc test. (C) Original current traces and mean changes in NMDAR currents elicited by puff application of 100 µM NMDA to spinal dorsal horn neurons in wild-type (WT; n = 12 neurons in each group) and Cacna2d1 KO (n = 11 neurons in each group) mice 3 weeks after spared nerve injury (SNI) or sham surgery. Data are expressed as means ± SEM. *p < 0.05 (versus WT sham group), one-way ANOVA followed by Tukey’s post hoc test. (D) Representative traces and mean changes in baseline values and the AP5 effect on the frequency and amplitude of mEPSCs of spinal dorsal horn neurons in wild-type (WT; n = 11 neurons in each group) and Cacna2d1 KO (n = 12 neurons in each group) mice subjected to spared nerve injury (SNI) or sham surgery. Data are expressed as means ± SEM. *p < 0.05 (versus baseline); #p < 0.05 (versus baseline in the WT sham group), one-way ANOVA followed by Tukey’s post hoc test. (E and F) Representative traces (E) and mean changes (F) in baseline values and the AP5 effect on the amplitude of EPSCs of spinal dorsal horn neurons monosynaptically evoked by dorsal root stimulation in wild-type (WT) and Cacna2d1 KO mice subjected to spared nerve injury (SNI; n = 11 neurons in each group) or sham surgery (n = 12 neurons in each group). Data are expressed as means ± SEM. *p < 0.05 (versus baseline); ^#p < 0.05 (versus baseline in the WT sham group); one-way ANOVA followed by Tukey’s post hoc test.

Figure 3. α2δ-1 Physically Interacts with NMDARs In Vivo and In Vitro

(A and B) Reciprocal coimmunoprecipitation analysis shows the protein-protein interaction between α2δ-1 and NMDAR subunits in the membrane extracts of dorsal spinal cord tissues of rats from the sham control and SNL groups 3 weeks after surgery. (A) Proteins were immunoprecipitated first with a rabbit anti-α2δ-1 or anti-IgG antibody. Western immunoblotting (IB) was performed by using mouse anti-GluN1, anti-GluN2A, or anti-GluN2B antibodies. (B) Proteins were immunoprecipitated initially with a mouse anti-GluN1 or anti-IgG antibody. IB was performed by using a rabbit anti-α2δ-1, anti-α2δ-2, or anti-α2δ-3 antibody. IgG and input (tissue lysates only, without immunoprecipitation) were used as negative and positive controls, respectively. Similar data were obtained from 4 independent experiments. (C and D) Reciprocal coimmunoprecipitation analysis shows the α2δ-1 and GluN1 interaction in the membrane extracts of two human lumbar spinal cord tissue samples (labeled as S1 and S2). (C) Proteins were immunoprecipitated first with a rabbit anti–α2δ-1 or anti-IgG antibody. Western immunoblotting (IB) was performed by using a mouse anti-GluN1 antibody. (D) Proteins were immunoprecipitated initially with a mouse anti-GluN1 or anti-IgG antibody. IB was performed by using a rabbit anti–α2δ-1 antibody. Similar data were obtained from spinal cord tissues of each of the 4 human donors. (E and F) Coimmunoprecipitation analysis shows that α2δ-1 heterodimerized with NMDAR subunits in membrane extracts of HEK293 cells. HEK293 cells were cotransfected as indicated on the left side of the gel images. (E) GluN1/GluN2A/YFP-α2δ-1 (top images) or GluN1/GluN2B/YFP-α2δ-1 (bottom images). (F) FLAG-GluN1/ GluN2A (left) or FLAG-GluN1/GluN2B (right) with α2δ-1, α2δ-2, or α2δ-3. IRES-α2δ-1 (no-tag) was used as a negative control for YFP-α2δ-1 in (E), and untagged GluN1 (no-tag) was used as a negative control for FLAG-GluN1 in (F). Proteins were immunoprecipitated first with anti-GFP (E) or anti-FLAG antibody (F) using membrane fractions of HEK293 cells. IB was performed by using the antibodies indicated on the right side of the gel images. Data were from 5 independent experiments. (G and H) α2δ-1 interacts with NMDARs predominantly through its C-terminal domain. Coimmunoprecipitation analysis of FLAG-GluN1/GluN2A (G) and FLAG-GluN1/GluN2B (H) interactions with various YFP-tagged α2δ-1 constructs, indicated above the gel images, coexpressed in HEK293 cells. Coimmunoprecipitation and IB were performed using the antibodies indicated on the right side of the gel images. Data were from 5 independent experiments. (I) Schematic representation of α2δ-1 constructs and chimeras used for coexpression with NMDAR subunits in HEK293 cells. S, signal peptide; NT, putative N terminus; VWA, von Willebrand factor type-A domain; δ, δ peptide; CT, C-terminal domain. Pink boxes represent the domains of α2δ-2, and blue boxes represent the domains of α2δ-3. All α2δ-1 constructs and chimeras used for coimmunoprecipitation experiments were tagged with yellow fluorescent protein (YFP).

Figure 4. Gabapentin Restores NMDAR Activity Increased by Nerve Injury or α2δ-1 Coexpression In Vivo and In Vitro

(A) Original traces and mean effects of gabapentin (GBP; 100 µM for 30 min) on currents elicited by puff application of 100 µM NMDA or AMPA to spinal dorsal horn neurons in rats that had undergone sham surgery (n = 12 neurons in the vehicle group; n = 13 neurons in the gabapentin group) or SNL (n = 11 neurons in the vehicle group; n = 12 neurons in gabapentin group) 3 weeks after surgery. Data are expressed as means ± SEM. *p < 0.05 (versus sham rats treated with vehicle), one-way ANOVA followed by Tukey’s post hoc test. (B and C) Representative traces and cumulative probabilities (B) and mean changes (C) of baseline values and the AP5 effect on the frequency and amplitude of mEPSCs of spinal dorsal neurons recorded from rats subjected to sham surgery (n = 10 neurons) or SNL (n = 12 neurons in the vehicle group; n = 11 neurons in the gabapentin group). Data are expressed as means ± SEM. *p < 0.05 (versus respective baseline); #p < 0.05, compared with the baseline in sham group, one-way ANOVA followed by Tukey’s post hoc test. (D) Original traces and mean changes show the effect of α2δ-1 coexpression and gabapentin treatment (100 µM for 30 min) on whole-cell NMDAR currents in HEK293 cells expressing GluN1/GluN2A (n = 10 cells in each group) or GluN1/GluN2B (n = 12 cells in each group). Current responses were elicited by application of 300 µM NMDA plus 10 µM glycine. Data are expressed as means ± SEM. *p < 0.05 (versus respective vehicle control); #p < 0.05, compared with the current reconstituted with GluN1/GluN2A or GluN1/GluN2B alone, one-way ANOVA followed by Tukey’s post hoc test. (E) Effect of α2δ-1 coexpression and gabapentin treatment on the conductance-voltage relationship of NMDAR channels in HEK293 cells expressing GluN1/GluN2A (n = 12 cells for GluN1/GluN2A with α2δ-1; n = 10 cells for GluN1/GluN2A without α2δ-1; n = 13 cells for GluN1/GluN2A with α2δ-1 and gabapentin) or GluN1/GluN2B (n = 10 cells in each group) in the presence of Mg²⁺. In cells expressing GluN1/GluN2A, coexpression of α2δ-1 increased NMDAR conductance at −40 and −60 mV in the presence of 2 mM Mg²⁺, and the increase was reversed by treatment with gabapentin (100 µM for 30 min). Currents were normalized by values obtained at +40 mV. Data are expressed as means ± SEM. *p < 0.05 (versus cells expressing GluN1/GluN2A alone at the same voltage), two-way ANOVA followed by Tukey’s post hoc test.

Figure 5. Gabapentin Diminishes Membrane Surface Expression of α2δ-1-Bound NMDARs

(A) Luminescence resonance energy transfer (LRET) between terbium-labeled GluN2A (GluN2A*), GluN1, and YFP-α2δ-1. Black curve: without gabapentin; red curve: with gabapentin. Left: LRET lifetime signals show that gabapentin resulted in loss of the LRET signal between terbium labeled-GluN2A* and YFP-α2δ-1 on themembrane. Middle: gabapentin had no effect on the LRET signal when YFP-α2δ-1 was replaced with its R217A mutant. Right: donor-only curves of terbium-labeled GluN2A* and unlabeled α2δ-1. Data were from 4 or 5 independent experiments (Table S1). (B) LRET between terbium-labeled GluN1 (GluN1*), GluN2A, and YFP-α2δ-1. Black curve: without gabapentin; red curve: with gabapentin. Left: LRET lifetime signals show that gabapentin diminished the interaction between terbium labeled-GluN1* and YFP-α2δ-1 on the membrane. Middle: gabapentin had no effect on the LRET signal when YFP-α2δ-1 was replaced with its R217A mutant. Right: donor-only curves of terbium-labeled GluN1* and unlabeled α2δ-1. Data were from 4 or 5 independent experiments (Table S1). (C) Coimmunoprecipitation and immunoblotting (IB) analysis shows that gabapentin (GBP; 100 µM for 30 min) diminished the expression of α2δ-1-bound NMDARs in the membrane extract of HEK293 cells. Left: the GluN1/GluN2A heterodimer was cotransfected with YFP-α2δ-1, a YFP-α2δ-1 mutant (R217A, also called R241A), or IRES-α2δ-1 (no tag) in HEK293 cells. Right: the GluN1/GluN2B heterodimer was cotransfected with YFP-α2δ-1, a YFP-α2δ-1 mutant (R217A, also called R241A), or IRES-α2δ-1 (no tag) in HEK293 cells. Data were from four independent experiments. (D and E) Membrane surface protein analysis (D) and mean levels (E) show that gabapentin treatment reversed the α2δ-1 coexpression-induced increase in NMDAR surface expression. Immunoblotting was performed using antibodies against GluN1, GluN2A, GluN2B, and α2δ-1 for the membrane surface proteins isolated with biotinylation. HEK293 cells were cotransfected and treated with gabapentin or vehicle as indicated above the gel images. Na⁺/K⁺-ATPase, a known membrane protein marker, was used as an internal control. Data are expressed as means ± SEM (n = 5 independent experiments). *p < 0.05 (versus GluN1/GluN2A or GluN1/GluN2B alone), one-way ANOVA followed by Tukey’s post hoc test; ^#p < 0.05 (versus GluN1/GluN2A/α2δ-1 or GluN1/GluN2B/α2δ-1 without gabapentin), two-tailed Student’s t test. See also Table S1.

Figure 6. Uncoupling α2δ-1-NMDAR Interaction via the C Terminus of α2δ-1 Reduces Neuropathic Pain

(A and B) Original gel images and quantification data show the effect of α2δ-1Tat peptide on the α2δ-1-GluN1 interaction. (A) HEK293 cells were cotransfected with GluN1, GluN2A, and α2δ-1 or FLAG-α2δ-1. (B) HEK293 cells were cotransfected with α2δ-1, GluN2A, and GluN1 or FLAG-GluN1. Forty-eight hours after transfection, the transfected cells were incubated with 0.01, 0.1, or 1 µM α2δ-1Tat peptide for 30 min. The cell membranes were then isolated and used for coimmunoprecipitation using anti-FLAG antibody. Western blotting was conducted using (A) an anti-GluN1 antibody or (B) an anti-α2δ-1 antibody (n = 6 replicates). Data are expressed as means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 (versus control group), one-way ANOVA followed by Dunnett’s post hoc test. (C and D) Effects of a single intrathecal injection (C) or intraperitoneal injection (D) of α2δ-1Tat peptide on the tactile, pressure, and heat withdrawal thresholds in sham and SNL rats (n = 8 rats in each group). Data are expressed as means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 (versus baseline before drug injection), one-way ANOVA followed by Dunnett’s post hoc test.

Figure 7. α2δ-1-Bound NMDARs Are Critically Involved In Nerve-Injury-Induced Synaptic Targeting and Activity of NMDARs

(A and B) Representative gel images (A) and quantification data (B) show the protein levels of α2δ-1 and NMDAR subunits in spinal cord synaptosomes of sham and SNL rats (n = 6 samples from 6 rats in each group). The spinal cord slices were incubated with control vehicle (Cont), 100 µM gabapentin (GBP), 1 µM α2δ-1Tat peptide (P+), or 1 µM scrambled control peptide (P−) for 30 min. PSD-95, a known postsynaptic protein, was used as an internal control. Data are expressed as means ± SEM. *p < 0.05 (versus vehicle control), one-way ANOVA followed by Dunnett’s post hoc test. (C) Original traces and mean effects of the α2δ-1Tat peptide or scrambled control peptide (1 µM for 30 min) on currents elicited by puff application of 100 µM NMDA to spinal dorsal horn neurons in SNL (n = 12 neurons in each group) rats 3 weeks after surgery. Data are expressed as means ± SEM. *p < 0.05 (versus sham rats), one-way ANOVA followed by Tukey’s post hoc test. (D and E) Representative traces and cumulative probabilities (D), and mean changes (E) of baseline values and the AP5 effect on the frequency and amplitude of mEPSCs of spinal dorsal neurons recorded from SNL rats (n = 12 neurons in the control peptide group; n = 11 neurons in the α2δ-1Tat peptide group). Data are expressed as means ± SEM. *p < 0.05 (versus respective baseline value); #p < 0.05 (versus baseline in the control peptide group), one-way ANOVA followed by Tukey’s post hoc test.