Contribution of the tetrodotoxin-resistant voltage-gated sodium channel NaV1.9 to sensory transmission and nociceptive behavior

Abstract

The transmission of pain signals after injury or inflammation depends in part on increased excitability of primary sensory neurons. Nociceptive neurons express multiple subtypes of voltage-gated sodium channels (NaV1s), each of which possesses unique features that may influence primary afferent excitability. Here, we examined the contribution of NaV1.9 to nociceptive signaling by studying the electrophysiological and behavioral phenotypes of mice with a disruption of the SCN11A gene, which encodes NaV1.9. Our results confirm that NaV1.9 underlies the persistent tetrodotoxin-resistant current in small-diameter dorsal root ganglion neurons but suggest that this current contributes little to mechanical thermal responsiveness in the absence of injury or to mechanical hypersensitivity after nerve injury or inflammation. However, the expression of NaV1.9 contributes to the persistent thermal hypersensitivity and spontaneous pain behavior after peripheral inflammation. These results suggest that inflammatory mediators modify the function of NaV1.9 to maintain inflammation-induced hyperalgesia.

Fig. 1.

Disruption of SCN11A (Na_V1.9) in mouse ES cells and generation of Na_V1.9-/- mice. (A) Structure of WT and mutant SCN11A loci. Thick solid lines denote genomic sequence within the targeting construct. A 174-bp region (Δ) of the SCN11A coding sequence was replaced by a 6.9-kb IRES-LacZ reporter and neomycin resistance cassette (IRES-LacZ-neo). The numbers designate the exons. B and N indicate restriction sites for BamHI and NcoI, respectively. Two overlapping oligonucleotide probes used to hybridize Southern blots are indicated by p. pBS denotes Bluescript vector sequence. (B) The first reaction multiplex for each sample includes three primers (neo- and gene-specific) and simultaneously detects the endogenous (233 bp) and targeted (424 bp) alleles. The second reaction includes only gene-specific primers and detects only the endogenous allele. Reactions using either no DNA (-) or DNA obtained from F₂ mice or ES cells are shown. W, WT; H, heterozygote; K, homozygous null mutant.

Fig. 2.

Properties of the persistent sodium current in small-diameter DRG neurons. (A) TTX-resistant sodium current elicited by steps to -40, -20, 0, and +20 mV from a holding potential of -90 mV. (B) Average amplitude of the TTX-resistant sodium current as a function of test pulse voltage in WT (▪, n = 26) and Na_V1.9-/- (▪, n = 18) neurons. (C) Peak current elicited by pulses to -40 mV as a function of membrane potential during a 0.5-s prepulse. A fit of the data to the Boltzman equation yielded V_h = -44.6 mV and k = 6.6 mV. (D) Current elicited by pulses to -40 mV in control (black line) and 500 μM lidocaine (gray line).

Fig. 3.

Mechanical and thermal stimulus-induced primary afferent and behavioral response thresholds in Na_V1.9-/- mice. (A) In vitro measurement of C-fiber responses, evoked by mechanical (Left) and thermal (Right) stimulation of the skin. (B) Behavioral response thresholds of WT (▵), heterozygous (▪), and Na_V1.9-/- mice (gray circles) to mechanical stimulation (Left, n = 9–13) and radiant heating of the plantar hind paw (Right, n = 9–13).

Fig. 4.

Inflammatory hyperalgesia in Na_V1.9-/- mice. (A) Time course of spontaneous behavioral responses to intraplantar injection of formalin (n = 11, Na_V1.9+/+; n = 7, Na_V1.9+/-; n = 15, Na_V1.9-/-). (B–D) Withdrawal latencies in response to radiant heating of the paw after injection with inflammatory mediators. (B) Duration of carrageenan-induced thermal hyperalgesia in Na_V1.9-/- mice (n = 8) was shorter than in WT (n = 7) and Na_V1.9+/- mice (n = 8). Unpaired t test; **, P < 0.01. (C) CFA-induced thermal hyperalgesia was significantly less in Na_V1.9-/- (n = 9) and Na_V1.9+/- mice (n = 9), compared with WT mice (n = 10). Repeated measures ANOVA followed by Fisher's probable least-squares difference; *, P < 0.05; **, P < 0.01 (post hoc analyses only). (D) Unlike WT mice (n = 8) and Na_V1.9+/- mice (n = 8), Na_V1.9-/- mice (n = 8) failed to develop significant PGE₂-induced thermal hyperalgesia. Repeated measures ANOVA followed by Fisher's probable least-squares difference; *, P < 0.05 for Na_V1.9-/-, compared with WT mice.