Researchers at the University of California, San Francisco (UCSF), and the University of Queensland have identified a scorpion toxin that targets the “wasabi receptor,” an ion channel protein that is found in sensory nerve cells and is responsible for what the scientists call the “sinus-clearing or eye-stinging” pain experienced when eating wasabi or chopping onions.
The studies found that the scorpion toxin, a peptide dubbed the wasabi receptor toxin, or WaTx, activates the wasabi receptor—more correctly termed TRPA1—to trigger this pain response to irritants via a previously unknown mechanism. Unusually, WaTx can enter TRPA1-containing sensory nerve cells directly through their plasma membranes, bypassing transport by channel proteins. The researchers hope that WaTx could be used to study chronic pain and inflammation, and may lead to the development of novel non-opioid pain therapies.
“It was surprising to find a toxin that can pass directly through membranes,” said John Lin King, a doctoral student in UCSF’s neuroscience graduate program and lead author of the team’s study, which is published in Cell. “This is unusual for peptide toxins. But it’s also exciting because if you understand how these peptides get across the membrane, you might be able to use them to carry things—drugs, for example—into the cell that can’t normally get across membranes.”
The researches’ investigations with the toxin are reported a paper titled “A Cell-Penetrating Scorpion Toxin Enables Mode-Specific Modulation of TRPA1 and Pain.”
TRPA1 is an ion channel that acts as a chemosensory receptor, which is directly activated by a range of environmental irritants and endogenous inflammatory agents to produce “acute and persistent pain,” the authors explained. Most chemicals that directly activate TRPA1, including plant-derived irritants and environmental toxins, are classed as chemically reactive electrophiles. “Think of TRPA1 as the body’s ‘fire alarm’ for chemical irritants in the environment,” said Lin King. “When this receptor encounters a potentially harmful compound—specifically, a class of chemicals known as ‘reactive electrophiles,’ which can cause significant damage to cells—it is activated to let you know you’re being exposed to something dangerous that you need to remove yourself from.”
Cigarette smoke, for example, is rich in reactive electrophiles that can trigger TRPA1 in the cells that line the airways, and so induce coughing and sustained airway inflammation. The receptor is also activated by chemicals in hot or spicy foods such as wasabi, onions, mustard, ginger, and garlic. These are all compounds that may have evolved to help ward off animals that might otherwise have eaten the plants. TRPA1 also functions as a “receptor-operated” channel, the team noted, which is activated by inflammatory agents. “Thus, TRPA1 is considered a promising therapeutic target for treating chronic pain, itch, and neurogenic inflammatory syndromes that are initiated or exacerbated by nociceptor activation.”
There are, however, other compounds, including menthol, and delta9-tetrahydrocannabinol—the psychoactive component in cannabis—that are natural, non-electrophilic TRPA1 agonists, and which act on the channel via a different mechanism. These are much less potent, less effective, and have less specificity for TRPA1, the authors noted.
Reasoning that there might be other, more potent non-chemically reactive TRPA1-activating compounds, they analyzed large collections of animal venoms as “evolutionarily honed chemical ‘libraries’ that contain pain-inducing defensive agents.” Their screening tests showed that venom from the Australian black rock scorpion (Urodacus manicatus) contained a strong TRPA1 activator.
Plant and environmental irritants target an intracellular site known as the allosteric nexus, and this causes the TRPA1 channel to rapidly flutter open and closed, and allow positively charged sodium and calcium ions to flow into the cell, triggering pain. Although both ions are able to enter when TRPA1 is activated by these irritants, the channel lets much more calcium than sodium into the cell, which leads to inflammation. The researchers found that WaTx also targets the allosteric nexus, but in contrast with the actions of the majority of chemical irritants, WaTx wedges itself into the allosteric nexus and keeps the TRPA1 channel open, which abolishes its preference for calcium. As a result, overall ion levels are high enough to trigger a pain response, but calcium levels remain too low to cause inflammation.
Most peptides and proteins that need to get into cells are taken up either by endocytosis, or pass through selective channels in the plasma membrane. The researchers showed that unusually, the amino acid sequence of WaTx and its resulting structure allow the peptide to directly pass through the plasma membrane, without needing to traverse through channel proteins. They claim that the findings “support a direct mechanism of peptide penetration via passive diffusion and establish that cell penetration and channel activation are distinct biophysical properties of the toxin.” And although there are a few other proteins, including the HIV Tat protein, which can penetrate into cells in the same way, WaTx contains no sequences similar to those found in Tat or in any other protein that can pass through the cell’s membrane in the same way.
The team then carried out a series of in vivo tests which showed that administering a known TRPA1 activator, mustard oil, into the paws of mice triggered pain and hypersensitivity to temperature and touch. These are hallmarks of chronic pain. The mustard oil also caused inflammation and swelling. In contrast, mice injected with WaTx responded with acute pain and hypersensitivity reactions, but without swelling. “When triggered by calcium, nerve cells can release pro-inflammatory signals that tell the immune system that something’s wrong and needs to be repaired,” Lin King said. “This ‘neurogenic inflammation’ is one of the key processes that becomes dysregulated in chronic pain. Our results suggest that you can decouple the protective acute pain response from the inflammation that establishes chronic pain. Achieving this goal, if only in principle, has been a longstanding aim in the field.”
Many animals use venom to paralyze or kill their prey, but WaTx seems to serve as a defense mechanism. While virtually every animal, including worms and humans, have a form of TRPA1, the researchers found that WaTx only activates the version found in mammals. And given that black rock scorpions don’t eat mammals, it’s likely that the toxin is mainly used to ward off mammalian predators.
“Our results provide a beautiful and striking example of convergent evolution, whereby distantly related life forms—plants and animals—have developed defensive strategies that target the same mammalian receptor through completely distinct strategies,” said David Julius, PhD, professor and chair of UCSF’s department of physiology, who is senior author of the report. The researchers hope that their findings will lead to a better understanding of acute pain, as well as provide new insights into the link between chronic pain and inflammation. The findings may even lay the groundwork for the development of new pain drugs.
The authors concluded that WaTx represents “a unique tool for future mechanistic studies of central neural pathways contributing to chronic pain.” Moreover, they noted, “… further insights into the structural basis of WaTx action may suggest strategies for targeting the allosteric nexus to diminish the inflammatory component of TRPA1-mediated pain while preserving its acute protective role in chemo-nociception.”
“The discovery of this toxin provides scientists with a new tool that can be used to probe the molecular mechanisms of pain, in particular, to selectively probe the processes that lead to pain hypersensitivity,” Lin King stated. “And for those interested in drug discovery, our findings underscore the promise of TRPA1 as a target for new classes of non-opioid analgesics to treat chronic pain.”