The beta3 subunit of the Na+,K+-ATPase mediates variable nociceptive sensitivity in the formalin test

Abstract

It is widely appreciated that there is significant inter-individual variability in pain sensitivity, yet only a handful of contributing genetic variants have been identified. Computational genetic mapping and quantitative trait locus analysis suggested that variation within the gene coding for the beta3 subunit of the Na+,K+-ATPase pump (Atp1b3) contributes to inter-strain differences in the early phase formalin pain behavior. Significant strain differences in Atp1b3 gene expression, beta3 protein expression, and biophysical properties of the Na+,K+ pump in dorsal root ganglia neurons from resistant (A/J) and sensitive (C57BL/6J) mouse strains supported the genetic prediction. Furthermore, in vivo siRNA knockdown of the beta3 subunit produced strain-specific changes in the early phase pain response, completely rescuing the strain difference. These findings indicate that the beta3 subunit of the Na+,K+-ATPase is a novel determinant of nociceptive sensitivity and further supports the notion that pain variability genes can have very selective effects on individual pain modalities.

Figure 1

Haplotype mapping implicates Atp1b3 as a candidate gene underlying variable sensitivity to formalin pain. A, Sensitivity of 16 inbred mouse strains in the early phase of the formalin test (5%, 20 μl). Bars represent mean ± SEM percentage of samples featuring licking/biting behavior from 0−5 min post-injection (n=10−38/strain). B, Standardized scores (z-scores) of data in panel a, arranged from least sensitive (left) to most sensitive (right). C, Top 10 genome-wide correlations (arranged by p-value) between 5,694 SNP haplotype blocks and formalin test z-scores. Colored blocks represent the haplotype of the 16 strains, in the left-to-right order shown in graph B (most common haplotype in red, 2^nd most common in blue, 3^rd most common in green, least common in yellow). The effect score represents the proportion of the observed inter-strain phenotypic difference that could be explained by genetic variation within that haplotype block.

Figure 2

Expression of β subunits in dorsal root ganglion tissue. qPCR analysis of A, Atp1b1, B, Atp1b2, and C, Atp1b3 mRNA expression in lumbar DRG tissue. Bars represent the mean ± SEM ratios for n=3 technical replicates performed on pooled DRG tissue from n=5−6 mice/strain. D, Representative photomicrographs of DRG sections from A/J (left) and C57BL/6J (middle) mice stained with an antibody against β₃ protein. Scale bar = 30 μm. E, Quantification of β₃-like immunoreactivity in DRG neurons from A/J and C57BL/6J mice. Bars represent the mean ± SEM pixel intensity; numbers represent the number of individual neuronal soma in DRG sections from n=4−5 mice/strain. *p<0.05 by Student's t-test.

Figure 3

DRG neurons from A/J mice have greater Na⁺,K⁺ pump currents and are ouabain insensitive compared to C57BL/6J mice. A, Measurement of the resting membrane potential and B, the stimulus threshold required to elicit an action potential in dissociated DRG neurons from A/J and C57BL/6J mice (mean ± SEM ; n=14−34 neurons/strain). **p<0.01 by Student's t-test. C, Representative traces of the Na⁺,K⁺ pump current, defined as the ouabain (1 mM)-sensitive fraction of the holding current, in dissociated DRG neurons from A/J and C57BL/6J mice. D, Quantification of Na⁺,K⁺ pump current density in A/J and C57BL/6J DRG neurons (mean ± SEM ; n=15−16 neurons/strain). ***p<0.001 by Student's t-test. Measurement of the ability of neurons to maintain repetitive firing using a 10Hz stimulation train over 300 s. The maximal action potential amplitude of A/J and C57BL/6J DRG neurons was recorded at in the presence of no drug (E) and 10 μM ouabain (F,G). There was a significant difference between strains (F_1,400 = 149.1, p<0.001) in panel E and between C57BL/6J drug treatment groups (F_1,400 = 56.38, p<0.001) in panel G as determined by ANOVA.

Figure 4

Rescue of the strain difference in early phase formalin pain behavior by intrathecal siRNA against Atp1b3. A, Wide field and B, confocal microscopy of 20-μm sections of lumbar DRG tissue from mice treated with three daily i.t injections of i-Fect + 0.5 μg/day of Cy3-labeled scrambled siRNA (red). Nuclei were counterstained with DAPI (blue). Scale bars = 100 μm (wide field) and 20 μμm (confocal).C, qPCR analysis of Atp1b3 mRNA expression in lumbar DRG tissue from C57BL/6J and A/J mice treated with i-Fect transfection reagent alone (control) or i-Fect plus one of two siRNAs targeting Atp1b3 (siRNA-1 and siRNA-2). Bars represent the percent of Atp1b3 mRNA expression as compared to the respective control groups ± SEM on pooled DRG tissue from n=6 mice/strain. *p<0.05 by Bonferroni post hoc test compared to same-strain control group. D, Early phase (0−5 min) and E, late phase (5−60 min) formalin-induced licking/biting in A/J and C57BL/6J mice treated with i-Fect transfection reagent alone (i-Fect), i-Fect plus a scrambled siRNA (scrm siRNA), or i-Fect plus one of two unique siRNAs targeting Atp1b3 (siRNA-1 and siRNA-2). Bars represent mean ± SEM total time spent licking (in panel D) or the percentage of sampled intervals showing licking behavior (in panel E) (n=8−13 mice/strain/treatment). *p<0.05 by Bonferroni post hoc test compared to same-strain saline group. F, Quantification of Na⁺,K⁺ pump current density in DRG neurons from A/J mice previous treated with i-Fect plus a scrambled siRNA (scrm siRNA) or i-Fect plus siRNA-2 for three days prior to measurement (mean ± SEM ; n=7−8 neurons/group). **p<0.01 by Student's t-test.