The nicotinic α6 subunit gene determines variability in chronic pain sensitivity via cross-inhibition of P2X2/3 receptors

Abstract

Chronic pain is a highly prevalent and poorly managed human health problem. We used microarray-based expression genomics in 25 inbred mouse strains to identify dorsal root ganglion (DRG)-expressed genetic contributors to mechanical allodynia, a prominent symptom of chronic pain. We identified expression levels of Chrna6, which encodes the α6 subunit of the nicotinic acetylcholine receptor (nAChR), as highly associated with allodynia. We confirmed the importance of α6* (α6-containing) nAChRs by analyzing both gain- and loss-of-function mutants. We find that mechanical allodynia associated with neuropathic and inflammatory injuries is significantly altered in α6* mutants, and that α6* but not α4* nicotinic receptors are absolutely required for peripheral and/or spinal nicotine analgesia. Furthermore, we show that Chrna6's role in analgesia is at least partially due to direct interaction and cross-inhibition of α6* nAChRs with P2X2/3 receptors in DRG nociceptors. Finally, we establish the relevance of our results to humans by the observation of genetic association in patients suffering from chronic postsurgical and temporomandibular pain.

Fig. 1. Correlation of DRG expression of Chrna6 and mechanical allodynia after SNI in inbred mice

SNI surgery was performed on 25 inbred mouse strains, and withdrawal thresholds of the ipsilateral hind paw to von Frey fiber stimulation were measured. Symbols in A (n=139) represent mean ± SEM paw withdrawal threshold on each testing day; bars in B (n=4–6 mice/strain) represent mean ± SEM percentage of maximum possible allodynia (see Materials and Methods online). (C) The top 7 correlations (Pearson’s r; all P<0.005) between the strain means shown in B and basal DRG expression levels of ≈45,000 probesets in these same strains. (D) The correlation between allodynia and basal DRG expression (in arbitrary units) of probeset 1450426_at (Chrna6); symbols represent individual strain values. Symbol abbreviations are self-explanatory except for: Bc, BALB/cBy; B6, C57BL/6; BR, C57BR/cd; D2, DBA/2. (E) Average expression across all strains of all Chrn* genes. Bars represent basal DRG expression (in arbitrary units) ± SD.

Fig. 2. Chrna6 mRNA expression in a subset of DRG neurons

DRG neurons from adult α6*-GFP mice were stained with antibodies against GFP (green) and sensory neuron markers NF200 (A), IB4 (B) and CGRP (C) (all red). Arrows indicate neurons expressing either GFP or the sensory marker. Filled arrow heads indicate neurons co-expressing both markers. Scale bars= 100 μm.

Fig. 3. Differential mechanical allodynia after nerve injury and chronic inflammation in Chrna6 mutant mice

Increased mechanical allodynia after SNI surgery (A,B) and CFA injection (C,D) in Chrna6 KO mice, and decreased mechanical allodynia after SNI (E,F) and CFA (G,H) in Chrna6 L9’S gain-of-function mutants. In all graphs, symbols (n=5–12 mice/genotype) represent mean ± SEM paw withdrawal threshold (g) on each testing day; bars represent mean ± SEM percentage of maximum possible allodynia (see Materials and Methods). *P<0.05, **P<0.01, ***P<0.001 compared to other genotype. A replication of the KO data, using a different neuropathic assay, can be found in fig. S4.

Fig. 4. Altered anti-allodynic potency and efficacy of nicotine in Chrna6 mutant mice

Shown are dose-response relationships for the ability of systemic (i.p.; A,B), intracerebroventricular (i.c.v.; C,D), intrathecal (i.t.; E,F), and peripheral (intraplantar; i.pl.; G,H) nicotine to reverse already-developed (and maximal) mechanical allodynia produced by SNI (day 7 post-surgery; A,C,E,G) and CFA (day 3 post-injection; B,D,F,H). Symbols (n=4–8 mice/dose/genotype) represent mean ± SEM percentage of maximum possible anti-allodynia, based on the pre-SNI/CFA and post-SNI/CFA withdrawal thresholds of each mouse (see Materials and Methods). Statistical analyses are shown in table S3.

Fig. 5. Dependence of spinal and/or peripheral nicotine anti-allodynia on α6

A head-to-head comparison of supraspinal (25 μg, i.c.v.; graph A), spinal (17 μg, i.t.; graph B) and peripheral (50 μg, i.pl.; graph C) nicotine anti-allodynia against neuropathic (CCI) and inflammatory (CFA) pain in Chrna6 (α6*) and Chrna4 (α4*) WT and KO mice tested using identical parameters at the peak of allodynia (14 days post-CCI, 3 days post-CFA). Bars (n=5–6 mice/genotype/injury) represent mean ± SEM percentage of maximum possible anti-allodynia (see Materials and Methods). **P<0.01, **P<0.01, ***P<0.001 compared to analogous WT.

Fig. 6. Electrophysiological detection of crosstalk between P2X and α6* receptors in Xenopus oocytes

(A) Co-expressed P2X2/3 and α6β4 receptors; (B) co-expressed P2X2/3 and α6β4β3 receptors. Exemplar inward currents are shown, evoked with 100 μM α,βme-ATP, 100 μM ACh, or a mixture of 100 μM α,βme-ATP+ACh. (C) Co-expressed P2X2 and α6β2 receptors were tested with 100 μM ATP, 10 μM ACh, or a mixture of the two agonists. All graphs summarize experiments from n = 11–13 cells. The ‘predicted’ (Pred.) current for each cell is the arithmetic sum of the I_α,βme-ATP and I_ACh currents. The ‘Δ’ current for each cell is the predicted current minus the observed I_{α,βme-ATP+ACh} current. Error bars represent SEM. To provide measureable responses, several subunits were mutated as described in Methods.

Fig. 7. Modulation of P2X2/3 agonist-induced pain and hypersensitivity by nicotine in Chrna6 mutant mice

(A) Intrathecal administration of α,βme-ATP produces nocifensive (licking) behavior inhibited by the P2X3 antagonist, A-317491 (300 nmol, intraplantar), and systemic nicotine in WT and L9’S but not KO mice. Symbols (n=4–11 mice/dose/genotype) represent mean ± SEM samples featuring licking behavior (see Materials and Methods). *P<0.05, **P<0.01, ***P<0.001 compared to 0 dose within genotype. •P<0.05 compared to other genotypes within dose. (B) Intrathecal administration of α,βme-ATP produces mechanical allodynia reversed by A-317491 (300 nmol, intraplantar) and nicotine (0.9 mg/kg) in WT and L9’S but not KO mice. Symbols (n=4 mice/genotype or drug) represent mean ± SEM paw withdrawal threshold at each time point.

Fig. 8. Human clinical pain is affected by a promoter SNP (rs7828365) in CHRNA6

(A) Percentage of herniotomy patients reporting persistent pain 6 months after surgery, stratified by rs7828365 genotype (TT: n=8; TC: n=82; CC: n=325). (B) CPSQ composite pain scores (see Materials and Methods) of temporomandibular disorder patients stratified by rs7828365 genotype (TT: n=2; TC: n=38; CC: n=117). *P<0.05 compared to other genotypes.