Researchers identify gene mutation that can regulate pain

(A) Venn diagram identifying 5 missense TRPV1 variants that overlap when examining the human TRPV1 missense variants from the 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 mammalian and avian TRP domains (I696–S711 within the red dotted 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 molecular model of rat TRPV1 (PDB ID: 3J5P). Pole contacts are marked in red or by 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 mutations shown as fura- 2AM ratio of 340:380 nanometers. (H) The AUC (total amount of calcium influx) and (I) the percentage of maximal change in fura-2AM ratio for TRPV1 mutants relative to WT TRPV1 were calculated. 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

At least 1.5 billion people suffer from pain worldwide, and despite the availability of various pain-relieving drugs, not all forms of pain are treatable. In addition, painkillers can have side effects such as dependence and tolerability, especially with morphine and other opioids.

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

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

“There are more than 1,000 TRPV1 mutations in humans, and turning off the receptor to reduce pain is nothing new, but these attempts have so far been unsuccessful,” said Vanessa Olzon Zambelli, a researcher at LEDS and co-first author of the article .

“First, many drugs resulting from this process interfere with body temperature regulation. Second, TRPV1 is an important heat signaling channel, and completely altering its activity abolishes physiological pain and disrupts the burning heat sensation that has a protective function. ”

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

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

Researchers identify gene mutation that can regulate pain

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

The mutations can also be present 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 actually altered. Next, they used the CRISPR/Cas9 gene editing technique to create mice with the K710N mutation, which they had previously found reduced the receptor’s response to capsaicin in cells. Capsaicin is the active ingredient in pepper.

The researchers observed no nociceptive behavior (suggesting pain avoidance) in mice with the K710N mutation injected with capsaicin and given peppery chicken chow, in contrast to the behavior of normal mice, which raised their paws to avoid touching the capsaicin , presumably because even skin contact caused pain.

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

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

Translational Analysis

The second part of the study consisted of trying to pharmacologically reduce the function of the receptor. To this end, researchers designed a peptide, V1-cal, that acted selectively on the K710 region. Mice treated with V1-cal and treated with capsaicin showed less nociceptive behavior and reduced release of neuropeptides, leading to neurogenic inflammation and edema without changing temperature. Finally, the chronic pain also improved significantly.

“We now want to upgrade this study by validating the results under best-practice laboratory conditions [required by regulatory agencies]to identify other small molecules besides the peptide that can be more easily synthesized, to conduct preclinical studies and, if successful, to start a clinical study,” said Zambelli.

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

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

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