Rare, inherited pain disorders remain largely mysterious, but researchers continue to identify genetic mutations in voltage-gated sodium channels (Nav) that may give rise to them. Now, Enrico Leipold, Ingo Kurth, and colleagues at Jena University Hospital, Germany, have identified a mutation in SCN11A, the gene encoding Nav1.9, in a family afflicted with an early-onset, cold-aggravated episodic pain disorder. The gain-of-function mutation made Nav1.9 channels hyperexcitable, even at low temperature. The report, published December 8 in Nature Communications, also provides new data about a gain-of-function SCN11A mutation linked to pain insensitivity that was previously identified by the current authors.
“They have done a nice, comprehensive analysis of the properties of this newly identified mutation,” said Ted Cummins, a neuroscientist at Indiana University, Indianapolis, US, who was not involved in the work. “Certainly the finding helps hammer home the point that Nav1.9 is important in nociception, and it seems to play an especially important role in cold sensation.”
The multidisciplinary team of researchers, located at Stanford University, California, US; Aachen University Hospital, Germany; and University of Cologne, Germany, studied a six-year-old girl who suffered from episodes of pain brought on by cold ambient temperature, as did her father, aunt, cousin, and grandmother, who exhibited similar symptoms. Often occurring at the end of the day, painful episodes lasted about 20-30 minutes and began at about one year of age. The girl’s father described his pain as fiery hot, usually beginning in the joints and spreading to the extremities. In the affected adults, skin biopsies revealed peripheral neuropathy in small-fiber sensory nerve endings. Whole-exome sequencing of the girl and her cousin revealed a rare shared mutation in a highly conserved region of SCN11A important for sensing voltage. The mutation was also found in other affected but not unaffected family members, and it has never been reported in healthy individuals. By those criteria, Kurth told PRF, the researchers concluded that the SCN11A mutation, which results in a substitution of an alanine for a valine at position 1184 of the protein (p.V1184A), likely contributed to the pain disorder.
Increased channel availability
To determine the mutation’s effect on ion channel behavior, first author Enrico Leipold and colleagues performed electrophysiological experiments using heterologous cells expressing either wild-type or p.V1184A human Nav1.9. Cells expressing p.V1184A channels passed more excitatory current than wild-type channels. Analysis of the channels’ biophysical properties revealed that the mutant channels opened slightly faster than wild-type channels, and closed more slowly. Most importantly, the voltage dependence of channel opening was shifted, such that mutant channels required less depolarization in order to open. “We interpret these data to mean that the mutation increases functional availability of the channel. There are a certain number of channels in the cell membrane, and at a given voltage, only a certain percentage is available to open—this percentage is larger for the mutants,” Leipold told PRF.
The mutation had no effect on channels’ fast inactivation—the process by which channels close and become refractory, or unable to open again, for a period of time. Cummins said “They didn’t look at slow inactivation,”—a kinetically separate process that has been implicated in cold sensitivity. To be fair, Cummins added, “Nav1.9 channels are extremely hard to work with, and they did an excellent job with the biophysical analysis—those are really difficult experiments.”
Because some patients’ pain was brought on by exposure to cold—such as going barefoot on a cold floor or being out in chilly weather—Leipold recorded from neurons in a bath at 30 degrees Celsius, close to normal skin temperature, and at 20 degrees. All ion channels have some inherent temperature dependence, Leipold said; they generally open faster at higher temperatures and have slower activation at lower temperatures—which is reflected in the excitability of neurons. As expected, mouse dorsal root ganglion (DRG) neurons transfected with wild-type human Nav1.9 fired fewer action potentials at 20 degrees compared to 30 degrees. Cells expressing mutant channels also fired fewer action potentials at the lower temperature, but the reduction was not as great as in the wild-type. “The hyperexcitability introduced by the mutation is relatively larger at lower temperatures,” Leipold told PRF. “It seems the mutation makes cells resistant to cooling,” which normally slows firing, he said, and which could be linked to the patients’ cold sensitivity.
It remains unclear how cold temperature triggers pain in patients with the p.V1184A mutation, but it likely involves activation of an unidentified molecular cold sensor. “It may be that Nav1.9 serves simply as an amplifier of signals generated by cold-transducers,” Kurth said. That idea sprang from a study by French researchers published in Cell Reports in May (Lolignier et al., 2015; see also associated PRF comment). In that study, the authors created transgenic mice lacking Nav1.9. Although the number of sensory neurons responding to cold temperature was similar between wild-type and knockout mice, cells lacking Nav1.9 fired far fewer action potentials in response to noxious cold. In the oxaliplatin model of chemotherapy-induced cold hypersensitivity, pain behaviors were attenuated in mice lacking Nav1.9 compared to wild-type. Together, the results point to a crucial role for Nav1.9 in transducing the sensation of noxious cold in conjunction with molecular cold sensors. Some cold-sensing molecules have been identified, such as the transient receptor potential channel M8 (TRPM8), but researchers believe that novel cold sensors remain to be discovered.
The interactions between temperature and pain are complex and incompletely understood. While some patients anecdotally complain that neuropathic pain conditions worsen in the cold, other pain conditions are improved by cooling. For example, another rare pain disorder, primary erythromelalgia, arises from mutations in the SCN9A gene (which encodes the sodium channel Nav1.7) and is brought on by elevated temperature and alleviated by cool temperature. Researchers in Taiwan found that elevated temperature exacerbated the mutant channels’ hyperactivity (Wu et al., 2013).
Two mutations, two very different phenotypes
Previous research has identified a number of gain-of-function mutations in SCN11A (as well as SCN9A, and SCN10A—the gene encoding Nav1.8) in patients with unexplained painful small fiber neuropathy (SFN), a more common pain condition (see PRF related news). Conversely, some cases of rare congenital insensitivity to pain (CIP) arise from a mutation that silences Nav1.7. Those findings are consistent with the idea that increased neuronal excitability in pain-sensing neurons causes or exacerbates pain. In contrast, Kurth and Leipold previously identified a gain-of-function mutation, p.L811P, in Nav1.9 in several people with severe pain insensitivity—a result that has been difficult to explain (see PRF related news).
In an attempt to determine how a mutation that makes cells hyperexcitable could also quiet them, Leipold and colleagues recorded long trains of action potentials from cells expressing wild-type, p.V1184A, or p.L811P mutant channels. A 30-second injection of current produced sustained firing of stable action potentials in cells with wild-type or p.V1184A channels, but action potentials diminished in amplitude and integrity in cells with p.L811P channels. Nevertheless, said Cummins, “they’re still firing.” The authors suggest that chronic dysfunction or developmental effects introduced by the gain-of-function mutation might over time lead to changes in calcium signaling or gene expression that could result in loss of nociceptor signaling.
“When we are looking at disease mutations and trying to determine how they cause a phenotype, we always have to ask, What are we missing?” said Cummins. In this case, when two mutations produce such wildly different phenotypes, he said, “there’s still something there we haven’t figured out yet.”
Stephani Sutherland, PhD, is a neuroscientist, yogi, and freelance writer in Southern California.
Image credit: Leipold et al., 2015