The voltage-gated sodium channel Na(v)1.9 is an effector of peripheral inflammatory pain hypersensitivity

Abstract

We used a mouse with deletion of exons 4, 5, and 6 of the SCN11A (sodium channel, voltage-gated, type XI, alpha) gene that encodes the voltage-gated sodium channel Na(v)1.9 to assess its contribution to pain. Na(v)1.9 is present in nociceptor sensory neurons that express TRPV1, bradykinin B2, and purinergic P2X3 receptors. In Na(v)1.9-/- mice, the non-inactivating persistent tetrodotoxin-resistant sodium TTXr-Per current is absent, whereas TTXr-Slow is unchanged. TTXs currents are unaffected by the mutation of Na(v)1.9. Pain hypersensitivity elicited by intraplantar administration of prostaglandin E2, bradykinin, interleukin-1beta, capsaicin, and P2X3 and P2Y receptor agonists, but not NGF, is either reduced or absent in Na(v)1.9-/- mice, whereas basal thermal and mechanical pain sensitivity is unchanged. Thermal, but not mechanical, hypersensitivity produced by peripheral inflammation (intraplanatar complete Freund's adjuvant) is substantially diminished in the null allele mutant mice, whereas hypersensitivity in two neuropathic pain models is unchanged in the Na(v)1.9-/- mice. Na(v)1.9 is, we conclude, an effector of the hypersensitivity produced by multiple inflammatory mediators on nociceptor peripheral terminals and therefore plays a key role in mediating peripheral sensitization.

Figure 1.

A, Targeting construct for producing Na_v1.9^−/− mice by deleting exons 4–6 of SCN11a. The region of Na_v1.9^−/− mRNA encoded by exon 5 is absent in the knock-out mice (primer1). B, Top, Triple-labeled immunohistochemistry for lacZ (green), NF200 (red), and peripherin (blue) in a DRG section from a Na_v1.9^−/− mouse showing lacZ expression in small neurons. Scale bar, 50 μm. Bottom, In situ hybridization for Na_v1.9mRNA in WT mice showing expression similar to lacZ in −/− mice. Scale bar, 100 μm.

Figure 2.

Basal behavioral responses. Responsiveness against mechanical (von Frey threshold), heat (hotplate latency time at 50, 52, and 55°C) and cold (cold plate latency time at 0 °C) stimulations are identical in WT, Na_v1.9^+/−, and Na_v1.9^−/− naive mice (n = 15).

Figure 3.

Sodium currents in DRG neurons. A, In WT mice, 45% of small (<25 μm) DRG neurons have a TTXr current that can be separated into TTXr-Per and TTXr-Slow components by voltage steps from −90 mV holding potential to −30 mV and from −90 to 10 mV, respectively. In the Nav1.9^−/− mice, no small DRG neurons have a TTXr-Per current. B, The voltage-dependent activation in WT mice of TTXr in those small DRG neurons with both TTXr-Slow and TTXr-Per is more hyperpolarized than those cells with just TTXr-Slow. In KO mice, the activation curve reflects a composite of TTXr in small cells that have lost TTXr-Per and those with only TTXr-Slow. Voltage dependence of inactivation of TTXr is similar in WT mice and Nav1.9^−/− mice.

Figure 4.

Detection of Na_v1.9 expression in the DRG neurons. DRG neurons double labeled for Na_v1.9 (red) and B₂ bradykinin, TRPV1 capsaicin, or P2X₃ ATP receptors (green). Na_v1.9 colocalized well with any of these receptors. Scale bars, 50 μm.

Figure 5.

Peripheral pain hypersensitivity in Na_v1.9 mice. A–C, In WT mice, intraplantar bradykinin (BK) injection produced immediate licking and flinching (A) and a later decrease in mechanical threshold (B) and hotplate response latency (C). In Na_v1.9^−/− mice, BK elicited a reduced licking time and no mechanical or thermal hypersensitivity. D, E, Intraplantar capsaicin produced in WT mice immediate licking (D) and delayed reduction in mechanical threshold (E). Na_v1.9^−/− mice had reduced licking time and a higher mechanical threshold after injection than WT. *p < 0.05, **p < 0.01 vs preinjection baseline. ^#p < 0.05, ^##p < 0.01 versus WT. n = 7. Cap, Capsaicin.

Figure 6.

Pain hypersensitivity after purinergic receptor agonist injection. The P2X receptor agonist αβ-met-ATP and the P2Y receptor agonist UTP both produced transient thermal hyperalgesia in WT but not Na_v1.9^−/− mice. *p < 0.05, **p < 0.01 versus preinjection baseline. n = 7.

Figure 7.

Pain hypersensitivity in response to PGE₂. A, B, Intraplantar PGE₂ induced transient mechanical (A) and thermal (B) pain hypersensitivity in WT but not in Na_v1.9^−/− mice. C, D, Intrathecal PGE₂ produced identical mechanical (C) and thermal (D) hyperalgesia in WT, Na_v1.9^+/−, and Na_v1.9^−/− mice. *p < 0.05, **p < 0.01 versus preinjection baseline. n = 7.

Figure 8.

Pain hypersensitivity in response to IL-1β and NGF. A, B, Intraplantar IL-1β induced mechanical (A) and thermal (B) hyperalgesia in WT mice that was significantly reduced in −/− mice. C, D, Intraplantar NGF induced similar mechanical (C) and thermal (D) pain hypersensitivity in WT and Na_v1.9^−/− mice. *p < 0.05, **p < 0.01 versus preinjection baseline. n = 7.

Figure 9.

Peripheral inflammation. A, In Na_v1.9^−/− mice, inflammatory heat pain hypersensitivity was diminished. CFA injection produced a significant reduction in hotplate latency for 7 d in WT and Na_v1.9^+/− mice. However, thermal hyperalgesia was essentially absent in the Na_v1.9^+/− mice, with the response latency at 50, 52, and 55°C significantly longer in Na_v1.9^−/− mice. *p < 0.05, **p < 0.01 versus preinjection baseline. ^#p < 0.05, ^##p < 0.01 versus WT. n = 8. C, The relative numbers of Na_v1.9-positive neuron profiles increased in the DRG after inflammation. Scale bar, 100 μm.

Figure 10.

Mechanical sensitivity after peripheral inflammation. Mechanical threshold against von Frey stimulation reduced after peripheral inflammation similarly in WT, Na_v1.9^+/−, and Na_v1.9^−/− mice. *p < 0.05, **p < 0.01 versus preinjection baseline. n = 8.