Three voltage-gated sodium channels—Nav1.7, 1.8, and 1.9—all participate in tuning the excitability of pain-sensing nociceptors. Genetic changes that alter the function of two of the channels, 1.7 and 1.8, and boost nociceptor activity have been found in patients with painful small-fiber neuropathy (SFN), a common condition with often mysterious origins. Now, an international consortium of researchers has rounded out the Nav triad by identifying gain-of-function mutations in Nav1.9 in some cases of painful SFN. The new findings reveal a previously unsuspected genetic cause for painful peripheral neuropathy and further validate Nav1.9 as a potential target for new drugs aimed at quieting pain neurons. The study, a collaborative effort between US and European researchers, was published April 27 in Brain.
In a commentary accompanying the paper, David Bennett of the University of Oxford, UK, who was not involved in the current study, wrote that the researchers “present the first evidence for a causative role of missense mutations in Nav1.9 in painful neuropathy.” As a result, he wrote, “Mutations in Nav1.7, 1.8, and 1.9 have now all been associated with painful neuropathy as a consequence of dorsal root ganglion [DRG] cell hyperexcitability and as such are targets for the development of novel analgesics.”
For the study, neurologists Ingemar Merkies and Catharina Faber at the University Medical Centre, Maastricht, the Netherlands, and Giuseppe Lauria at IRCCS Foundation, "Carlo Besta" Neurological Institute, Milan, Italy, carefully characterized a cohort of 393 patients with pure or predominantly small-fiber neuropathy. They previously identified mutations in SCN9A and SCN10A, encoding Nav1.7 and Nav1.8, respectively, in some of the cohort (see PRF related news story on Faber et al., 2012; also Faber et al., 2012). The team now reports eight variants in SCN11A—the gene that encodes Nav1.9—in 12 of the remaining 345 subjects. The variants were all heterozygous, and seven caused amino acid sequence changes in the Nav1.9 protein. None of the eight variants were found in 694 chromosomes from healthy control subjects.
Stephen Waxman, Sulayman Dib-Hajj, and their team at Yale University School of Medicine, New Haven, and the Veterans Affairs Medical Center, West Haven, both in Connecticut, US, followed up on the genetic work with what Bennett described as “a detailed and elegant functional analysis” of two of the missense mutations. Each mutation was found in two patients; one resulted in a leucine-to-proline change at position 1158 (L1158P), and the other caused an isoleucine-to-threonine swap at position 381 (I381T).
First authors Jianying Huang, Chongyang Han, Mark Estacion, and Dymtro Vasylyev—whom Dib-Hajj called “four highly specialized and talented electrophysiologists”—carried out the analysis. Using patch-clamp recording techniques on isolated mouse dorsal root ganglia (DRG) neurons made to express only the human channels, the researchers determined that both mutations altered the channels’ gating properties. Importantly, DRG neurons expressing mutant channels alongside the rodent channel were significantly more prone to fire both spontaneous and evoked action potentials than those expressing wild-type human channels.
The neuronal excitability experiments could be misleading due to high expression levels of the mutant channels and/or the presence of the rodent channel. To address that problem, the authors used a sophisticated dynamic clamp technique, where they "injected” currents into mouse DRG neurons that lacked their own Nav1.9 to mimic physiological levels of wild-type human channels or a 50/50 combination of wild-type and mutant channels. The results confirmed that currents generated by the I381T mutant channels were capable of causing changes in cell excitability similar to those measured in the expression experiments, suggesting that the effects were “not an aberrant effect of overexpression of a mutant protein,” Waxman told PRF.
Recently, gain-of-function mutations in Nav1.9 were found in two families with a rare inherited episodic pain condition (see PRF related news story on Zhang et al., 2013), and the new work links Nav1.9 mutations to a more common condition, painful SFN. However, a different gain-of-function mutation has also been found in two people with insensitivity to pain (see PRF related news story on Leipold et al., 2013). In that study, the authors postulated that a constant, slight depolarization of the membrane potential induced by the Nav1.9 mutant would inactivate sodium channels and render cells unexcitable. Based on their electrophysiological studies, Waxman said his group found no evidence that a depolarized resting potential in DRG neurons reduced their excitability, leaving open the question of how the mutation described by Leipold et al. might lead to pain insensitivity.
SFN pain symptoms vary from patient to patient, but usually include burning pain and tingling that originates in the hands or feet, along with other unpleasant sensations. About half of SFN cases can be traced to nerve damage due to diabetes, cancer, toxins, or other insults. But for the rest of patients, SFN has unexplained origins. Could the newly identified Nav1.9 mutations cause SFN in some? Waxman told PRF, “The mutations unequivocally cause DRG neuron hyperexcitability, which is a major contributor to pain.”
SFN also features damage to small-diameter nerve endings, detectable with a skin biopsy. Waxman suggests that in addition to pain, overactivity of Nav1.9 or other mutant sodium channels could contribute to this unexplained neurodegeneration and that quieting the channels might curtail the damage. Bennett, too, suggested in his editorial that drugs aimed at quieting Nav1.9, besides acting as analgesics, “may have the added advantage of also being disease modifying.”
Together with last year's findings in rare disorders, the finding of Nav1.9 mutations in SFN breathes new life into that channel as a potential therapeutic target. Pharmaceutical companies are actively pursuing Nav1.7 and Nav1.8 as analgesic drug targets, but industry has thus far shied away from Nav1.9 because of the lack of a connection to human pain and also because of technical difficulties associated with studying the channel. Dib-Hajj told PRF, “Until now, people did not really know what to make of this channel.” In light of the new findings, though, Dib-Hajj said, “Now they can’t ignore it.”
Of course, efforts to develop a Nav1.9 blocker would be aimed not only at the few patients with mutations in the channel, but also at a much broader population suffering from SFN or even neuropathic pain more generally. “It is clear to us that Nav1.9 behaves as a threshold channel, that it is important to setting the resting membrane potential of neurons, and it controls excitability … in the case of the wild-type channel, too, not just mutants. So if you can attenuate Nav1.9 [activity], chances are you will ameliorate pain,” said Dib-Hajj. “That is based on biology, not wishful thinking.” Still, when it comes to a Nav1.9-specific blocker, Dib-Hajj said, “We predict that patients would benefit, but the proof will be in the pudding.”
Stephani Sutherland, PhD, is a neuroscientist, yogi, and freelance writer in Southern California.
Image: Adapted from Huang et al., 2014, by permission of Oxford University Press.