Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 6, 8967

Endogenous Opioids Contribute to Insensitivity to Pain in Humans and Mice Lacking Sodium Channel Nav1.7

Affiliations

Endogenous Opioids Contribute to Insensitivity to Pain in Humans and Mice Lacking Sodium Channel Nav1.7

Michael S Minett et al. Nat Commun.

Abstract

Loss-of-function mutations in the SCN9A gene encoding voltage-gated sodium channel Nav1.7 cause congenital insensitivity to pain in humans and mice. Surprisingly, many potent selective antagonists of Nav1.7 are weak analgesics. We investigated whether Nav1.7, as well as contributing to electrical signalling, may have additional functions. Here we report that Nav1.7 deletion has profound effects on gene expression, leading to an upregulation of enkephalin precursor Penk mRNA and met-enkephalin protein in sensory neurons. In contrast, Nav1.8-null mutant sensory neurons show no upregulated Penk mRNA expression. Application of the opioid antagonist naloxone potentiates noxious peripheral input into the spinal cord and dramatically reduces analgesia in both female and male Nav1.7-null mutant mice, as well as in a human Nav1.7-null mutant. These data suggest that Nav1.7 channel blockers alone may not replicate the analgesic phenotype of null mutant humans and mice, but may be potentiated with exogenous opioids.

Figures

Figure 1
Figure 1. Deletion of Nav1.7 leads to altered gene expression in DRG neurons.
(a) The pie chart shows the number of significantly differentially expressed (DE) genes (analysis of variance P-value<0.01) between Nav1.7 KO mice (n=3) and wild-type littermates (n=3), annotated with the top enriched Gene Ontology (GO) terms regarding the Biological Process (BP). For the enrichments analysis, we used methods based both on gene counts, namely the classical Fisher test, and gene ranks and scores, namely the Kolmogorov–Smirnov-type tests. (b) Example list of the fold change of differentially expressed genes in sensory neurons from Nav1.7 KO mice. Complete data are available at GEO and ArrayExpress, accession code GSE61373.
Figure 2
Figure 2. Scn9a deletion increases Penk mRNA expression in DRG neurons.
Reverse transcriptase–PCR (RT–PCR) analysis of Penk and Ceacam10 mRNA expression in DRG relative to Gapdh mRNA levels in wild-type littermates and Nav1.7 KO mice. (a) Increased expression of Penk mRNA was observed in both female Nav1.7 KOs (light pink column, n=3) compared with female littermates (dark pink column, n=3), as well as male Nav1.7 KOs (light blue column, n=3) compared with male littermates (dark blue column, n=3). No difference was observed between wild-type females and wild-type males. (b) Penk mRNA expression was not altered in Nav1.8 KOs compared with littermates (female Nav1.8 KOs: light pink column, n=3; female littermates: dark pink column, n=3; male Nav1.8 KOs: light blue column, n=3; male littermates: dark blue column, n=3). (c) Ceacam10 mRNA was downregulated in both female Nav1.7 KOs (light pink column, n=3) compared with female littermates (dark pink column, n=3) and male Nav1.7 KOs (light blue column, n=3) compared with male littermates (dark blue column, n=3). (d) Downregulation of Ceacam10 mRNA was also observed in Nav1.8 KOs compared with littermates (female Nav1.8 KOs: light pink column, n=3; female littermates: dark pink column, n=3; male Nav1.8 KOs: light blue column, n=3; male littermates: dark blue column, n=3). All results are shown as a fold change from wild-type littermate. (e) L5–L6 spinal cord sections were double-labelled with met-enkephalin (Green) and IB-4 (Red). Right panel insert shows higher magnification of lamina I and II within the dorsal horn. (f) Quantification of immunostaining signal shows less Met-enkephalin immunoreactivity in littermate dorsal horns (n=3) compared with the Nav1.7 KO dorsal horn (n=3). Data are shown as mean±s.e.m. All RT–PCR data are expressed as mean±s.e.m. with significance indicated by *P<0.05, **P<0.01 and ***P<0.001 (one-way analysis of variance with Bonferroni post test (a,b)). Immunoreactivity signal is expressed as mean±s.e.m. with significance indicated by *P<0.05 (Student's t-test).
Figure 3
Figure 3. Intracellular sodium concentration regulates Penk expression.
Time-course reverse transcriptase–PCR (RT–PCR) experiments were performed to quantify expression of Penk and Ceacam10 mRNA in cultured DRG neurons in response to intracellular changes in cation concentration (n=6 per group). (a) Monensin-mediated intracellular sodium rise decreased Penk mRNA expression in DRG neurons. (b) Under the same conditions, Ceacam10 mRNA level was upregulated. (c) Penk expression was not altered by intracellular calcium rise induced by ionomycin, (d) whereas Ceacam10 mRNA was significantly upregulated after 1 h of incubation with the calcium ionophore. (e) Exposure (6 h) to the voltage-gated sodium channel blocker TTX induces a dose-dependent upregulation of Penk expression. (f) At the highest TTX concentration, 500 nM, Ceacam10 expression level was not altered. All RT–PCR data are expressed as mean±s.e.m. with significance indicated by *P<0.05, **P<0.01 and ***P<0.001 (one-way analysis of variance with Dunnett's post test or Student's t-test).
Figure 4
Figure 4. Mechanical and thermal sensory deficits in Nav1.7-null mice are reversed with the opioid antagonist naloxone.
Hargreaves ((a,c) n=6 female and n=6 male per genotype) and Randall-Selitto ((b,d) n=3 female and n=3 male per genotype) pain behavioural tests show higher pain thresholds of Nav1.7 KO mice compared with littermates. Systemic naloxone reduces thermal and pressure pain thresholds of male and female Nav1.7 KO mice but has no effect on littermates. Thermally evoked (e) and mechanically evoked (f) firing of deep dorsal horn WDR neurons is lower in Nav1.7 KO mice (n=17 neurons) compared with littermate controls (n=24 neurons). Systemic naloxone sensitizes evoked neuronal firing to both low and high threshold thermal stimulation, as well as high threshold and dynamic (brush) mechanical stimulation, with no effect on control littermates. (g) Ceacam10 KO and heterozygous KO mice show clear thermal analgesia in the Hargreaves test and this is unaffected by naloxone administration (n=6 per genotype). Data are expressed as mean±s.e.m. *P<0.05, **P<0.01 and ***P<0.001 significance levels indicate differences between KOs and littermates using a Student's t-test and $P<0.05, $$P<0.01 and $$$ P<0.001 indicate differences following naloxone administration using a paired t-test.
Figure 5
Figure 5. Naloxone dramatically reverses the analgesia associated with Nav1.7-null mutations in a CIP individual.
Perception of phasic pain. The probability of detecting 9-ms radiant heat pulses was assessed in a Nav1.7-null patient (a) and in three age-matched healthy controls (b). Neodymium:yttrium-aluminum-perovskite (Nd:YAP) laser pulses (El.En, Italy) selectively stimulate intra-epidermal free-nerve endings, thus providing a pure nociceptive input without touch. The human Nav1.7 null did not detect any stimuli in baseline and saline conditions. The probability of detecting the stimulus dramatically increased to 80% of stimuli detected during the infusion of 12 mg naloxone, almost reaching the detection levels of matched healthy controls. (c,d) Tonic pain was elicited by 25 s laser stimuli, whereas the participants rated online the intensity of the heat sensation on a visual analogue scale (0=no sensation, 100=worst pain imaginable) throughout the laser application. Again, naloxone strongly enhanced tonic pain sensations in the Nav1.7-null patient (c) throughout the time course of the stimulation without effect in the control subjects (d).

Similar articles

See all similar articles

Cited by 47 articles

See all "Cited by" articles

References

    1. Breivik H., Collett B., Ventafridda V., Cohen R. & Gallacher D. Survey of chronic pain in Europe: prevalence, impact on daily life, and treatment. Eur. J. Pain 10, 287–333 (2006). - PubMed
    1. Eijkelkamp N. et al. . Neurological perspectives on voltage-gated sodium channels. Brain 135, 2585–2612 (2012). - PMC - PubMed
    1. Waxman S. G. et al. . Sodium channel genes in pain-related disorders: phenotype-genotype associations and recommendations for clinical use. Lancet Neurol. 13, 1152–1160 (2014). - PubMed
    1. Zhang Q. et al. . Na+ current properties in islet alpha- and beta-cells reflect cell-specific Scn3a and Scn9a expression. J. Physiol. 592, 4677–4696 (2014). - PMC - PubMed
    1. Weiss J. et al. . Loss-of-function mutations in sodium channel Nav1.7 cause anosmia. Nature 472, 186–190 (2011). - PMC - PubMed

Publication types

Associated data

Feedback