People with congenital insensitivity to pain (CIP) often come to severe bodily harm in the absence of protective painful feedback. Mutations leading to this infrequent disorder have been identified in several genes, but these don’t account for all cases. Now, researchers led by Ingo Kurth at Jena University Hospital, Germany, have identified a rare mutation in the Nav1.9 voltage-gated sodium channel in two patients with unexplained pain insensitivity. The report was published online September 15 in Nature Genetics.
Sodium channels expressed in nociceptors, most notably Nav1.7, have recently emerged as important players in pain signaling and as the culprit in several pain disorders (see Hoeijmakers et al., 2012, for review). Complete loss of Nav1.7 channels due to deletion mutations results in CIP, but mutations that make Nav1.7, or its cousin Nav1.8 (Faber et al., 2012; Han et al., 2013; Huang et al., 2013) more active have the opposite effect, leading to pain conditions including inherited erythromelalgia and small fiber neuropathy (see PRF related news story). Whereas the functions of Nav1.7 and Nav1.8 seem to be relatively straightforward in that the channels are required for neuronal firing, the ways in which Nav1.9 contributes to cell excitability are more subtle and still not entirely understood. The new findings are particularly surprising because they indicate that increased activity in Nav1.9 leads to pain insensitivity. If those findings hold up, “it would be incredibly interesting that channel excitability could cause a change [in pain sensitivity] in the opposite direction than expected,” said Jeffrey Mogil, a pain researcher at McGill University, Montreal, Canada, who was not involved in the work.
Looking for a genetic source of one girl’s insensitivity to pain, Kurth and co-authors sequenced her entire exome—the bits of DNA that encode proteins—as well as those of her healthy parents. They found a missense mutation in the daughter that was not present in the parents; so-called "de novo" mutations arise spontaneously rather than being inherited. De novo mutations are extremely rare—only zero to one is expected in any given exome—and this mutation occurred in the SCN11A gene, which encodes Nav1.9. The researchers deduced that the mutation, which resulted in an amino acid change in the sixth transmembrane segment of domain II of Nav1.9, likely contributed to the girl’s pain insensitivity. To further support this conclusion, they sequenced SCN11A exons from 58 other patients with severe sensory loss and found one patient with the same de novo mutation as the daughter, and a similar medical history. In both patients, the mutation was present in only one copy of the gene. Notably, skin biopsies revealed normal sensory innervation, indicating that the sensory neurons seemed to be structurally—if not functionally—intact.
In order to understand how the SCN11A mutation might affect pain signaling, the investigators introduced the orthologous mutation into the Scn11a locus in mice. Mice heterozygous for the mutation were somewhat less sensitive to pain than their wild-type counterparts in behavioral tests of pain threshold, but did not recapitulate the patients’ phenotype of total pain insensitivity.
Eleven of 101 mice with the mutation exhibited self-inflicted wounds—as the patients did—but Mogil pointed out that such wounds in mice don't necessarily indicate reduced pain sensation. Among all the mutant mice, heat-pain sensitivity was slightly decreased compared to controls as measured by a tail-flick latency test, but on other tests of thermal and mechanical sensitivity in the paw, mutants were similar to controls. However, the mutant animals differed in pain sensitivity under inflammatory conditions, which supports a previously established role for Nav1.9 in inflammation-related pain hypersensitivity (Lolignier et al., 2011; Priest et al., 2005). “These mice are not as sensitive as the wild-type, but certainly they are not insensitive,” Mogil said.
The researchers next asked how the mutation changed Nav1.9 function and whether that might affect neurons’ firing properties. First author Enrico Leipold made electrophysiological recordings from individual sensory neurons of the dorsal root ganglia (DRG) from wild-type mice, heterozygous knock-in mutants, and mice that contained solely mutant Scn11a. In cells expressing the mutated channel, voltage-dependent excitatory currents required less depolarization than in wild-type DRG cells, a change the authors attributed to a gain of function in the mutant Nav1.9 channels. Based on those recordings, they estimated that the voltage dependence of mutant Nav1.9 was negatively shifted by 30 millivolts (mV); that is, the channel required less depolarization from resting potential to open and pass current. They estimated a similar hyperpolarized shift in the voltage dependence of channel inactivation. Together, they surmised, these shifts in the channel’s voltage dependence might result in a persistent current through the sodium channels, which might account for the slightly depolarized resting membrane potential seen in cells with the channel mutation compared to wild-type.
How a persistent depolarizing current could render neurons insensitive to stimuli remains unclear, however. One hypothesis, put forth by the authors, is that persistent current flowing through mutant Nav1.9 channels would depolarize the cells enough to inactivate other sodium channels key to the generation of action potentials, namely, Nav1.7 and Nav1.8, which would then be chronically unavailable to trigger action potentials. The authors also suggest that calcium signaling might be disrupted, further impairing neural transmission.
Next, the team recorded from heterologous cells expressing the human version of the mutated Nav1.9 channels to compare it to the mouse mutation. Unlike the mouse DRG recordings, here the investigators assessed Nav1.9 function in isolation from other ion channels. Like the mutant mouse channel, the human mutant displayed a hyperpolarized shift in activation. However, Leipold said, “mutant mouse channels show a shift in steady-state inactivation that is not present in mutant human channels.” That shift might decrease the mutation’s effect in mice, the team speculated, which could account for the more pronounced phenotype seen in the patients. They also pointed out that age could contribute to phenotype severity, so aged mice might look different than the young animals tested here.
Some reports suggest that such a depolarizing shift in the resting membrane potential arising from increased Nav1.9 activity would make cells more, not less, likely to fire action potentials (Baker et al., 2003; Copel et al., 2009). The consequences of the mutation arise not only from how it alters the function of the channel protein—in this case, voltage dependence of gating—but also how it interacts with the specialized combination of other ion channels and signaling molecules that populate each cell type, including human pain-sensing neurons of the DRG (Rush et al., 2006).
How the insight from the current study might lead to better pain treatments, either for CIP or other conditions, remains unclear, considering the channel is also expressed in autonomic and enteric neurons, and the mutation occurs in a highly conserved region of the channel. Nevertheless, the work contributes new understanding of a sodium channel about which there is still much to learn.
Stephani Sutherland, PhD, is a neuroscientist, yogi, and freelance writer in Southern California.
Image: Nav1.9 α subunit. Domain II (DII) and domain IV (DIV) are shown. Red asterisk indicates position of amino acid change in the sixth transmembrane segment in DII, on the intracellular face of Nav1.9, a structure that also interacts with the inactivation domain (IFM motif) that mediates inactivation of the channel. Reprinted by permission from Macmillan Publishers Ltd: Nature Genetics, advance online publication, 15 Sep 2013 (doi: 10.1038/ng.2767).
