Most mammals, humans included, writhe in pain in response to a venomous sting from the bark scorpion. Not so the grasshopper mouse. The scorpion’s desert-dwelling cohabitant seems unperturbed by multiple stings and actually preys on the scorpions. Ashlee Rowe, formerly of the University of Texas at Austin, US, and now at Michigan State University, East Lansing, US, and her colleagues discovered how an evolutionary adaptation in the rodents’ Nav1.8 voltage-gated sodium channel neutralizes the scorpion’s bite by turning the normally painful toxin into an analgesic. The report, which appeared October 25 in Science, unmasks new possibilities for the sodium channel Nav1.8 as a target for potential pain treatments.
Sulayman Dib-Hajj, a pain researcher at Yale University, New Haven, Connecticut, US, who was not involved in the work, called it “a very elegant study and an important contribution to the field.”
Like cone snails, puffer fish, and some snakes, bark scorpions are armed with venom containing peptides that block or activate sodium, potassium, and calcium channels. The scorpion’s sting evokes intense pain by driving excitatory current through Nav1.7 sodium channels in nociceptive neurons. But Rowe and her husband Matthew Rowe had previously shown that grasshopper mice (Onychomys spp.) are resistant to the scorpion’s toxin (Rowe and Rowe, 2008).
For the current study, the Rowes worked with Harold Zakon at the University of Texas at Austin, US, to study in more detail the mice’s response to the toxin. Whereas common house mice (Mus musculus) responded to an injection of scorpion toxin in the paw with vigorous licking, a behavior indicative of pain, the desert mice groomed only briefly. Injection of formalin caused similar pain behaviors in both species of mice, suggesting that grasshopper mice were not insensitive to pain generally, but were specifically immune to the toxin’s effects.
In order to understand how the toxin might affect pain-sensing neurons, co-first author Yucheng Xiao and Theodore Cummins, both at Indiana University in Indianapolis, US, studied dissociated small sensory neurons from the dorsal root ganglia (DRG) of the grasshopper mice. They made electrophysiological recordings to measure the activity of Nav1.7 and Nav1.8, the two voltage-gated sodium channels responsible for generating and sustaining action potentials, and thus pain signaling, in nociceptive sensory neurons. In neurons from both species, Cummins told PRF, the toxin could stimulate Nav1.7-mediated excitatory current. But in the grasshopper mouse neurons, the toxin had an additional effect: It blocked Nav1.8.
Loss of the Nav1.8 current in the presence of the toxin rendered the grasshopper mouse neurons insensitive to a depolarizing stimulus that, in untreated neurons from both mouse species, elicited action potentials.
Those findings explained why the grasshopper mice were resistant to the painful effects of the toxin, and raised the possibility that the peptide might actually block pain signaling in that species. To test that idea in vivo, the researchers injected both species of mice with toxin followed by formalin. As predicted, pretreatment of the house mice with toxin sensitized the animals to subsequent formalin injection, as indicated by drastically increased licking compared to animals pretreated with saline. The grasshopper mice showed the opposite response, grooming less following toxin pretreatment compared to saline pretreatment, indicating analgesia. “The venom made the desert mice less responsive to formalin, showing that it truly works as an analgesic,” said Cummins.
“The work provides in vivo evidence of the physiological interplay between Nav1.7 and Nav1.8,” said Dib-Hajj. “It teaches us that you can block certain types of pain by blocking Nav1.8, even if current thru Nav1.7 is upregulated at the same time,” he said. That could be important in conditions where Nav1.7 overactivity plays a role, as in some inherited pain disorders (Dib-Hajj et al., 2012) and some cases of painful small-fiber neuropathy (see PRF related news story). Because of its newly appreciated role in neuropathic pain, Nav1.7 has recently become a prime target in the hunt for new pain-killing molecules. Although the new results cannot be generalized to neuropathy in humans, they do suggest that Nav1.8, too, is a viable pain target.
There may be specific advantages to targeting Nav1.8, said Cummins, thanks to its distinctive genetic differences. “Because Nav1.8 has more sequence diversity in mammals, it makes a better target than Nav1.7, which is highly conserved with other sodium channels in the brain and body,” he added. Nonspecific actions at those other sodium channels could produce dangerous side effects, and the differences in Nav1.8’s sequence provide a better chance of finding a specific inhibitor, Cummins explained.
The researchers realized that if they could figure out how evolution had tweaked the channel to bind scorpion venom, that might provide clues about how to make specific inhibitors of human channels. To that end, the team genetically engineered chimeric channels containing bits from either mouse species. Electrophysiological recordings from cells expressing the chimeric channels revealed the discrete region (domain II) of the channel that confers toxin sensitivity. By comparing sequence differences between the two species in domain II, the researchers traced the toxin interaction point to a single amino acid difference in the domain II SS2-S6 linker, a structure adjacent to the channel pore. There, an uncharged glutamine residue in the house mouse protein sequence is replaced by a negatively charged glutamic acid in the grasshopper mouse. “Negatively charged amino acids are important for binding toxins that carry a positive charge,” like that of the bark scorpion, Rowe explained.
“We know several of the specific amino acid residues that the toxin interacts with, but we do not yet know how it alters the gating mechanism,” Cummins said. The toxin appears not to change the channel’s voltage-dependent activation or inactivation. Instead, Cummins said, “It may work as a pore blocker, but we will have to work that out carefully in future experiments.”
Animal venoms have served up a plethora of toxins that have taught scientists much about ion channels. Now the bark scorpion joins the list of creatures that offer more than a painful sting. “Venom toxins can manipulate ion channels in ways that we can’t even imagine,” said Rowe. If we can understand the adaptations that animals make to withstand such toxins, then that can tell us something about how to make analgesics or other channel-targeted therapies, she said.
Gary Lewin of the Max Delbrück Center for Molecular Medicine in Berlin, Germany, who recently tracked the evolution of pain resistance in naked mole rats to changes in the Nav1.7 channel (see PRF related news story), echoed that idea. In an editorial accompanying Rowe’s paper in Science, Lewin wrote, “Drug designers could … take advantage of the millions of years of natural selection to find new approaches to tackle important drug targets like sodium channels.” For another demonstration of that concept, see the recent discovery of a specific Nav1.7 inhibitor from centipede venom (Yang et al., 2013).
Stephani Sutherland, PhD, is a neuroscientist, yogi, and freelance writer in Southern California.
Image: A southern grasshopper mouse eats the Arizona bark scorpion that it has just killed. Credit: Matthew and Ashlee Rowe, University of Texas at Austin, US.