A human TRPV1 genetic variant within the channel gating domain regulates pain sensitivity in rodents
Shufang He, … , Ye Zhang, Eric R. Gross
Shufang He, … , Ye Zhang, Eric R. Gross
Published December 6, 2022
Citation Information: J Clin Invest. 2023;133(3):e163735. https://doi.org/10.1172/JCI163735.
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Research Article Neuroscience Vascular biology

A human TRPV1 genetic variant within the channel gating domain regulates pain sensitivity in rodents

Abstract

Pain signals are relayed to the brain via a nociceptive system, and in rare cases, this nociceptive system contains genetic variants that can limit the pain response. Here, we questioned whether a human transient receptor potential vanilloid 1 (TRPV1) missense variant causes a resistance to noxious stimuli and, further, whether we could target this region with a cell-permeable peptide as a pain therapeutic. Initially using a computational approach, we identified a human K710N TRPV1 missense variant in an otherwise highly conserved region of mammalian TRPV1. After generating a TRPV1K710N-knockin mouse using CRISPR/Cas9, we discovered that the K710N variant reduced capsaicin-induced calcium influx in dorsal root ganglion neurons. The TRPV1K710N rodents also had less acute behavioral responses to noxious chemical stimuli and less hypersensitivity to nerve injury, while their response to noxious heat remained intact. Furthermore, blocking this K710 region in WT rodents using a cell-penetrating peptide limited acute behavioral responses to noxious stimuli and returned pain hypersensitivity induced by nerve injury to baseline levels. These findings identify K710 TRPV1 as a discrete site that is crucial for the control of nociception and provide insights into how to leverage rare genetic variants in humans to uncover fresh strategies for developing pain therapeutics.

Authors

Shufang He, Vanessa O. Zambelli, Pritam Sinharoy, Laura Brabenec, Yang Bian, Freeborn Rwere, Rafaela C.R. Hell, Beatriz Stein Neto, Barbara Hung, Xuan Yu, Meng Zhao, Zhaofei Luo, Chao Wu, Lijun Xu, Katrin J. Svensson, Stacy L. McAllister, Creed M. Stary, Nana-Maria Wagner, Ye Zhang, Eric R. Gross

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Figure 2

Construction of CRISPR/Cas9-edited TRPV1K710N-knockin mice followed by thermal and behavioral testing.

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Construction of CRISPR/Cas9-edited TRPV1K710N-knockin mice followed by t...
(A) Schematic diagram for the generation of CRISPR/Cas-9 edited TRPV1K710N-knockin mice. In the design of gRNA, the ssODNs lead to the K710N mutation (red) and 2 silent mutations (green) in the PAM (purple). (B) Representative DNA-Seq for WT TRPV1 and TRPV1K710N heterozygotes (double peak) or TRPV1K710N homozygotes (single peak). Red arrows indicate the designed mutation; green arrows indicate the silent mutations. (C) Tail withdrawal latency (seconds) in response to hot water at 48°C and 55°C (n = 9/group). (D) Body temperature detected with a rectal temperature probe in WT TRPV1 and homozygous TRPV1K710N mice (n = 10/group). (E) Representative image showing the difference in response to stepping on capsaicin-laced food for WT TRPV1 and TRPV1K710N mice. (F) Paw withdrawal behavior of WT TRPV1 and TRPV1K710N mice exposed to capsaicin-laced bird food (Sizzle N’ Heat Bird food) or regular bird food (Porch N’ Patio Bird food) (n = 10/group). (G) Experimental protocol for nociceptive behavior by intraplantar capsaicin injection into WT TRPV1 and TRPV1K710N mice. (H) Nociceptive behavior (paw licking/flicking) (n = 10/group). Data are expressed as the mean ± SEM. Significance was determined by unpaired t test (C and D) and 2-way ANOVA followed by Tukey’s post hoc test (F and H).