Researchers identify gene mutation capable of regulating pain

(A) Venn diagram identifying 5 missense TRPV1 variants that intersect when examining human TRPV1 missense variants from gnomAD and avian genetic divergence from the human TRPV1 sequence. (B) Location of these 5 missense variants within the rat TRPV1 structure (PDB ID: 3J5P). Alignment of the mammalian and avian TRP region (I696–S711 within the red dashed box; non-conserved amino acids 708 and 710 are shown in white). (C) 3D structure of WT TRPV1, K710N and T708I based on the closed-state rat TRPV1 molecular model (PDB ID: 3J5P). Polar contacts are indicated by red or blue dashed lines. (D–G) Calcium influx in response to 1 μM capsaicin (Cap) with (D) WT TRPV1, (E) K710N, (F) T708I, or (G) K710N/T708I TRPV1 mutants shown as Fura-2AM 340 ratio: 380 nm. (H) AUC (total amount of calcium influx) and (I) percentage of maximal change in Fura-2AM ratio were calculated for TRPV1 mutants relative to WT TRPV1. n = 8 cells/group from 3 independent experiments. Data are expressed as mean ± SEM. Significance was determined by 1-way ANOVA followed by Tukey’s post hoc test. Credit: Journal of Clinical Investigation (2022). DOI: 10.1172/JCI163735

Pain afflicts at least 1.5 billion people worldwide, and despite the availability of various pain relievers, not all forms of pain are treatable. In addition, pain medications can have side effects such as dependence and tolerance, especially in the case of morphine and other opioids.

In search of new painkillers, researchers at the Butantan Institute’s Specialized Pain and Signaling Laboratory (LEDS) in São Paulo, Brazil, studied TRPV1, a sensory neuron receptor that picks up noxious stimuli, including the heat and burning sensation conveyed by chili peppers, and discovered a possible pain dysesthesia mutation in the gene encoding this protein. They report their findings in an article published in Journal of Clinical Investigation.

The study was conducted in collaboration with Stanford University and Emory University in the United States and University Hospital Münster in Germany. The researchers analyzed a number of mutations in humans and also benefited from existing knowledge about birds, which unlike mammals have a TRPV1 receptor that is naturally resistant to noxious insults and even spicy food, but can sense pain.

“There are more than 1,000 TRPV1 mutations in humans, and there is nothing new about trying to disable the receptor in order to relieve pain, but these efforts have not been successful until now,” said Vanessa Olzon Zambelli, a researcher at LEDS and co-author of the study. first author of the article.

“First, many drugs that result from this process interfere with body temperature regulation. Second, TRPV1 is an important channel for heat signaling, and completely altering its activity cancels normal pain, blocking the burning sensation, the which has a protective function.”

The researchers began by exploring a genome database to compare the genetic sequences of avian and human TRPV1. Using a computational approach, they identified five bird mutations believed to be associated with pain resistance.

Cryogenic electron microscopy (which does not require large sample sizes or crystallization and is therefore suitable for imaging structures at near-atomic resolution) showed that the five bird mutations were located at K710, an amino acid residue thought to control gating (opening and closing) of the TRPV1 channel.

Researchers identify gene mutation capable of regulating pain

Graphical summary. Credit: Journal of Clinical Investigation (2022). DOI: 10.1172/JCI163735

Mutations can also exist in humans, but they are very rare, so the researchers decided to find out what would happen if they were “transplanted” into mammals. When they tested these variants in genetically engineered cells, they found that the function of the channel was indeed altered. They then used the CRISPR/Cas9 gene-editing technique to create mice with the K710N mutation, which they had previously discovered reduces the receptor’s response to capsaicin in cells. Capsaicin is the active substance in pepper.

The researchers did not observe allegorical behavior (indicating pain avoidance) in mice with the K710N mutation that were injected with capsaicin and given peppery chicken chow, in contrast to the behavior of normal mice, which lifted their paws to avoid touching the capsaicin. probably because even skin contact caused pain.

Mice with the K710N mutation also showed less hypersensitivity to nerve injury, while their response to noxious heat remained intact. Furthermore, blocking the K710 region in normal mice reduced acute behavioral responses to noxious stimuli and returned nerve injury-induced pain hypersensitivity to baseline levels.

In addition to modulating pain, TRPV1 also plays an important role in protection against other stimuli. For example, recent evidence suggests that it serves in non-neuronal cells as an intracellular molecular sensor that protects against glucose-induced cellular stress or tissue ischemia. Additional tests performed as part of this study involving cardiomyocytes (heart muscle cells) exposed to hydrogen peroxide, high glucose, and a model of cerebral ischemia confirmed the protective effect even with the mutation.

Translational analysis

The second part of the study consisted of an attempt to pharmacologically reduce the function of the receptor. To this end, the researchers developed a peptide, V1-cal, which selectively acted on the K710 region. V1-cal-treated and capsaicin-treated mice showed less pain sensitivity and reduced neuropeptide release leading to neurogenic inflammation and edema without temperature change. Finally, chronic pain was also significantly improved.

“We now want to add value to this study by validating the results under optimal laboratory conditions [required by regulatory agencies]identify small molecules other than the peptide that can be more easily synthesized, perform preclinical tests and, if those are successful, start a clinical trial,” Zambelli said.

More information:
Shufang He et al, A human TRPV1 genetic variant within the channel gating domain modulates pain sensitivity in rodents, Journal of Clinical Investigation (2022). DOI: 10.1172/JCI163735

Reference: Researchers identify gene mutation capable of regulating pain (2023, March 4) Retrieved March 4, 2023, from

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