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Multicenter Study
, 18 (4), 595-9

Genetically Determined P2X7 Receptor Pore Formation Regulates Variability in Chronic Pain Sensitivity

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Multicenter Study

Genetically Determined P2X7 Receptor Pore Formation Regulates Variability in Chronic Pain Sensitivity

Robert E Sorge et al. Nat Med.

Abstract

Chronic pain is highly variable between individuals, as is the response to analgesics. Although much of the variability in chronic pain and analgesic response is heritable, an understanding of the genetic determinants underlying this variability is rudimentary. Here we show that variation within the coding sequence of the gene encoding the P2X7 receptor (P2X7R) affects chronic pain sensitivity in both mice and humans. P2X7Rs, which are members of the family of ionotropic ATP-gated receptors, have two distinct modes of function: they can function through their intrinsic cationic channel or by forming nonselective pores that are permeable to molecules with a mass of up to 900 Da. Using genome-wide linkage analyses, we discovered an association between nerve-injury-induced pain behavior (mechanical allodynia) and the P451L mutation of the mouse P2rx7 gene, such that mice in which P2X7Rs have impaired pore formation as a result of this mutation showed less allodynia than mice with the pore-forming P2rx7 allele. Administration of a peptide corresponding to the P2X7R C-terminal domain, which blocked pore formation but not cation channel activity, selectively reduced nerve injury and inflammatory allodynia only in mice with the pore-forming P2rx7 allele. Moreover, in two independent human chronic pain cohorts, a cohort with pain after mastectomy and a cohort with osteoarthritis, we observed a genetic association between lower pain intensity and the hypofunctional His270 (rs7958311) allele of P2RX7. Our findings suggest that selectively targeting P2X7R pore formation may be a new strategy for individualizing the treatment of chronic pain.

Figures

Figure 1
Figure 1
Haplotype mapping of SNI-induced mechanical allodynia reveals a genetic association with the P451L variant of the mouse P2rx7 gene. (a) The percentage of the maximum possible allodynia in 18 inbred mouse strains (F17,82 = 2.3, P < 0.01; one-way analysis of variance (ANOVA)). Data are mean ± s.e.m. n = 8–26 mice per strain. (b) Standardized (z-score) data ordered by strain from least allodynia (left) to most allodynia (right). Strains with the Pro451 allele are shown as black bars, and strains with the Leu451 allele are shown as white bars (the NZB/BIn strain has the Leu451 allele). (c) The top five statistical associations (ranked by P value from the ANOVA model) genome wide between mechanical allodynia severity and 5,694 SNP haplotype blocks. The colored blocks represent the haplotypes of the 18 strains, listed from least allodynia (left) to most allodynia (right). The most common haplotype is shown in red, the second most common haplotype is shown in blue, and the third most common haplotype is shown in green. The genetic effect score represents the proportion of the observed interstrain phenotypic difference explained by the genetic variation within that haplotype block. The chromosomal (Chr.) position of the start of the haplotype block is given in Mb, as are the number of SNPs within the block. (d–g) Comparisons of all mice, grouped by P451L genotype (that is, carrying the Pro451 allele or the Leu451 allele). All data are mean ± s.e.m. Baseline withdrawal thresholds (d), ipsilateral mechanical allodynia (e), contralateral allodynia (negative values represent hypoalgesia) (f) and ipsilateral allodynia (measured as the average percent change from baseline on postoperative days 5 and 7) (g) of an independently tested cohort of mice. Baseline was measured in noninjured mice, and allodynia was measured after SNI in each strain. n = 8–26 mice per strain. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the opposite genotype, Student's t test.
Figure 2
Figure 2
BzATP causes calcein dye loss from the representative Pro451-carrying A/J mouse strain but not the Leu451-carrying B10.D2 mouse strain. (a-f) Peritoneal macrophages from A/J or B10.D2 mice were loaded with the dye calcein AM. (a) Photomicrographs of differential interference contrast or calcein fluorescence in A/J and B10.D2 macrophages. Scale bars, 25 μm. (b-e) Representative traces of calcein fluorescence, as measured by a photomultiplier detector. Cells were stimulated with the P2X7R agonist BzATP. Co2+ did not quench the calcein fluorescence in B10.D2 cells until after the addition of ionomycin (Iono). (d,e) Calcein fluorescence of A/J macrophages pretreated with the P2X7R antagonist BBG (d) or the pannexin 1 inhibitor Panx (e) before BzATP stimulation. (f) Calcein dye loss for control A/J macrophages, A/J macrophages with no pretreatment and A/J macrophages pretreated with BBG, carbenoxolone (CBX), Panx or the scrambled inactive version of Panx, scr Panx, before BzATP stimulation, as well as contro B10.D2 macrophages and B10.D2 macrophages stimulated with BzATP with no pretreatment. Data are mean ± s.e.m. (g) Western blot of P2X7R protein expression in whole-brain homogenates. (h) BzATP stimulation evoked a rise in intracellular [Ca2+] in A/J and B10.D2 macrophages loaded with the fluorescent Ca2+ indicator dye fura-2 AM. 340/380, the ratio of fluorescence at 340 nm to that at 380 nm. (i) The peak rise in intracellular [Ca2+] evoked by BzATP in cells loaded with fura-2 AM. ΔF/F represents the peak BzATP-evoked calcium response expressed relative to baseline. Data are mean ± s.e.m. n = 5-7 mice for each experimental group. ***P < 0.001 compared to vehicle treated; ANOVA followed by Tukey's post-hoc test.
Figure 3
Figure 3
Treatment with the TAT-P451 peptide prevents and reverses pore formation induced by BzATP and reverses the mechanical allodynia caused by peripheral nerve injury (SNI) or inflammation (CFA) in A/J mice. (a,b) Representative traces of calcein fluorescence of A/J macrophages treated with TAT-P451 or TAT-451L before BzATP stimulation. (c) Calcein dye loss in A/J macrophages treated as in a and b. Data are mean ± s.e.m. ***P < 0.001 compared to the control group; ANOVA followed by Tukey's post-hoc test. (d) YO-PRO uptake in cells treated with TAT-P451, TAT-451L or saline after 10 min BzATP stimulation. Data are mean ± s.e.m. ***P < 0.001 compared to saline; ANOVA followed by t test. (e) BzATP-evoked rise in intracellular [Ca2+] in fura-2 AM–loaded A/J macrophages pretreated with TAT-P451 or TAT-451L. (f) Peak rise in intracellular [Ca2+] evoked by BzATP in A/J and B10.D2 macrophages loaded with fura-2 AM and pretreated with TAT-P451 or TAT-451L. Data are mean ± s.e.m. For all in vitro experiments, each experimental group represents n = 5–7 mice. (g,h) Reversal of mechanical allodynia induced by nerve injury (SNI) (g) or inflammation (CFA) (h) by intravenous administration of TAT-P451 (but not TAT-451L) in A/J but not B10.D2 mice. Data are mean ± s.e.m. Anti-allodynia indicates reversal of allodynia. n = 6–13 mice per strain per peptide treatment group. *P < 0.05, ***P < 0.001 compared to all other groups; ANOVA followed by t test.
Figure 4
Figure 4
Genetic association of the human P2RX7 gene with chronic pain. (a) Significance of the association (expressed as –log P) between 23 SNPs in and near the human P2RX7 gene and the amount of chronic pain in two cohorts, the PMP and osteoarthritis (OA) cohorts. A third set of P values (purple) is shown for the combination of the first two P values, assessed by the optimally weighted z-test. The SNPs genotyped in both cohorts are ordered by genomic position. The horizontal line indicates the P < 0.05 significance threshold corrected for the number of effectively independent multiple comparisons after accounting for the linkage disequilibrium (LD) between the SNPs. (b) The LD between each pair of SNPs in terms of D′ values: red indicates strong LD (D′ = 1), with lighter shades of pink indicating decreasing LD. Blue and white indicate D ′ = 1 and D′ < 1, respectively, with low confidence (by log10 odds score) in the value of D′. No haplotype blocks (regions of widespread high LD) were observed among the SNPs. (c) The positions of the 23 genotyped SNPs in a 90.3-kb region containing the P2RX7 gene. The intron and exon structure of the 52.7-kb gene itself is shown in the inset. SNPs with significant associations are shown in red.

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