Three Is Not a Crowd: A Trio of Molecular Sensors Protects Organisms Against Acute Heat
For decades, researchers have looked for the molecular sensors tuned to potentially harmful heat. Now, a team led by Joris Vriens and Thomas Voets, University of Leuven, Belgium, has pieced together what is likely the key set of ion channels, all from the transient receptor potential (TRP) family, that serve this role.
Using genetically engineered mice lacking TRPV1 and TRPM3, the researchers found that nociceptors from these animals could still detect acute heat. Yet when they applied a TRPA1 blocker to the neurons, this sensitivity disappeared. What’s more, in contrast to the double knockout mice, those without TRPV1, TRPM3, and TRPA1 exhibited few to no pain-related behaviors.
The results support the long-held idea of a fault-tolerant mechanism for avoiding extreme heat, with the trio of TRP channels collectively guaranteeing that organisms can compensate even if one or two of the channels somehow become disabled.
“The data are very strong,” said Michael Caterina, Johns Hopkins University, Baltimore, US, who did not take part in the research. “The new information here is not the concept of a fault-tolerant mechanism but the precise identification of what will probably be the three most important channels involved in painful heat detection.”
“This study upholds roles for TRP channels as molecular determinants of thermosensation, and perhaps highlights the idea that sensitivity to noxious temperatures involves direct activation of TRP channels by heat (TRPV1 and TRPM3) as well as indirect activation by products of tissue injury (TRPA1),” wrote David Julius, University of California, San Francisco, US, in an email to PRF. Julius was also not involved in the work.
The findings were published online March 14 in Nature, along with a News and Views by Rose Hill and Diana Bautista, University of California, Berkeley, US.
Residual sensitivity
In 2000, Caterina, Julius, and their colleagues confirmed a then-controversial hypothesis—that TRPV1 serves as the common receptor for both intense heat and the chemical capsaicin, the pungent component of chili peppers. Building upon earlier work, the group found that TRPV1 knockout mice responded less strongly to both stimuli than their wild-type littermates (Caterina et al., 2000).
Still, they responded. And at the cellular level, so did C-fiber nociceptors. As for how, the researchers proposed that additional molecules must also have been contributing. “These might be other members of the TRP family of ion channels or completely unrelated proteins,” they wrote in the pages of Science.
In the years thereafter, TRPV2, TRPV3, and TRPV4 emerged as obvious candidates, but one by one, each was ruled out. In 2011, however, Vriens and Voets discovered that TRPM3 was expressed in a large swath of nociceptors, including those carrying TRPV1. And by genetically removing TRPM3 in mice, they could blunt, though not completely eliminate, the animals’ reflexes to noxious heat (Vriens et al., 2011; see related PRF news story).
“It turned out TRPM3 was another heat sensor,” said Vriens.
To test if there was yet a third player, first author Ine Vandewauw and her colleagues created knockout mice missing TRPV1 and TRPM3. Then, using calcium imaging, they measured how sensory neurons isolated from the trigeminal ganglion of these animals responded to a temperature of 45 degrees Celsius (113 degrees Fahrenheit). Remarkably, about 40 percent of the cells still became active, suggesting there was indeed another heat sensor. The double knockout mice also showed heat avoidance responses in the tail-immersion and hot-plate assays, albeit less so than their genetically intact counterparts.
But what accounted for this residual sensitivity? Two clues came from experiments using drugs known to target TRPA1. For most of the residually active neurons, mustard oil—a TRPA1 agonist—excited the cells. Conversely, a TRPA1 antagonist quelled their remaining activity. And so, “we came up with the idea to build a triple knockout” that would lack all three TRP channels, said Vriens.
Trigeminal neurons from these triple knockouts were silent when exposed to the high temperatures. A cocktail of antagonists against all three channels had a similar effect on cells from wild-type mice. And, for the two behavioral assays, almost all of the triple knockout mice reached the cut-off times, with some sustaining tissue damage three days after a tail-immersion test set at 57 degrees Celsius. However, the animals’ ability to sense strong pressure and low temperatures were left intact, arguing that the observed deficits were specific to noxious heat detection.
Redundant parts
Overall, the new work shows what researchers could previously only infer about the molecular sensors of noxious heat. “If you knock out two of the channels, as long as you have one left over, you’ll still have acute heat sensing,” said Vriens. In other words, no single channel is absolutely essential.
But that doesn’t necessarily mean they are equally important under normal circumstances. “We do think that TRPV1 might be the most important one,” said Voets. In support of this idea, double knockout mice lacking TRPV1 and either of the other two channels seemed to be the most impaired, compared to those without TRPM3 and TRPA1.
“If one looks at the pairwise analysis of double mutants in this study, any of those containing a TRPV1 deficiency show the most robust defect in heat sensation. Thus, it seems that TRPV1 is the critical component that’s likely being activated directly by temperature and helping to set the initial threshold,” said Julius. “The others are combining with that to give the full behavioral effect.”
“What’s the most surprising component of all of this is that TRPA1 ended up being responsible for one of three somewhat redundant mechanisms, because intrinsic heat sensitivity of the channel is something that has not been demonstrated in mammals under physiological conditions,” said Caterina.
Unlike other TRP channels, TRPA1’s role in thermal nociception depends on the production of reactive oxygen species (ROS) after tissue damage (see PRF related news story). Consistent with this, the current paper reported that one ROS, hydrogen peroxide (H202), could heighten sensory neurons’ responses to heat in the double (TRPV1/TRPM3) knockout mice but not the triple knockouts also missing TRPA1.
More questions than answers
Although identifying the TRP trio ties up many loose ends, it also raises what might prove to be even tougher questions to answer. For one, at this point, “it’s difficult to parse out what the fractional or temporal effect is of each of these channels,” said Julius. TRPV1 may be the most important of the three, but it’s not clear exactly how much of the work it’s doing, and when, relative to the other two.
Then, there are behaviors that seem to rely on other thermosensors. Indeed, in a two-temperature choice test, the triple knockout mice avoided an area heated to 45 degrees Celsius less so than double knockout mice did. But both groups spent more time in an area held at an innocuous temperature of 30 degrees.
“This finding suggests that the sensing of painful heat might not have a substantial role in determining a preference for pleasurable temperatures over painful ones,” wrote Hill and Bautista in the accompanying News and Views. Are the channels underlying this preference also part of a fault-tolerant mechanism for guiding animals toward innocuous warmth?
Lastly, does the same redundancy drive chronic pain? “It seems that all three of the channels play a role in pathological conditions, but their roles are a little bit different,” said Vriens. He and Voets are now trying to study this aspect in various models of persistent pain. “At the translational level, we are developing antagonists against one of the three TRP channels as a novel class of analgesic drugs, which we hope to be able to test in humans in the coming years.”
Matthew Soleiman is a science writer residing in Nashville, Tennessee. Follow him on Twitter @MatthewSoleiman.
Image credit: yehorlisnyi/123RF Stock Photo
Article Contributors:
