TRPC4 Mediates Trigeminal Neuropathic Pain via Ca2+-ERK/P38-ATF2 Pathway in the Trigeminal Ganglion of Mice

Background

Trigeminal neuropathic pain (TNP) is a debilitating condition characterized by chronic facial pain, yet its underlying mechanisms remain incompletely understood. Transient Receptor Potential Canonical 4 (TRPC4) has been reported to promote the development of abnormal pain or pain hypersensitivity in neuropathic pain. However, the specific contribution of TRPC4 to TNP pathogenesis remains unclear.

Aim

This study aimed to investigate the role of TRPC4 in a mouse model of trigeminal neuropathic pain induced by chronic constriction of the unilateral infraorbital nerve (CION).

Methods

Adult male/female mice were subjected to either CION surgery or sham surgery. Behavioral assays were conducted to assess facial pain-like responses over a 28-day period. TRPC4 distribution in the trigeminal ganglion (TG) was evaluated using Immunofluorescence. TRPC4 inhibitor ML204 and agonist Englerin A were employed to evaluate the impact of TRPC4 on facial pain-like behaviors. A TRPC4-overexpressing HEK293 cell model was conducted via plasmid transfection. To assess the function of TRPC4, we employed cellular calcium imaging technology to investigate the effects of modulating TRPC4 function by analyzing dynamic changes in intracellular calcium ion concentrations in primary trigeminal ganglion neurons and HEK293 cells. Trpc4 shRNA was used to specifically knock down TRPC4 in the trigeminal ganglion. Western blot analysis was used to assess the activation of ERK, P38, and ATF2 signaling pathways.

Results

Mice subjected to CION exhibited persistent facial pain-like behaviors and a significant increase in TRPC4 expression in TG neurons. Trpc4 shRNA or pharmacological inhibition with ML204 attenuated CION-induced pain behaviors, while activation of TRPC4 with Englerin A induced pain-like responses in naive mice. Calcium imaging revealed that both Englerin A and TRPC4 overexpression elevated intracellular Ca²²⁺ levels in TG neurons and HEK293 cells. This Ca²²⁺ influx triggered the activation of ERK and P38, leading to enhanced ATF2 activation. Downregulation of TRPC4 in the TG reduced ERK/P38 phosphorylation and ATF2 expression and activation.

Conclusion

This study provides the first evidence that TRPC4 plays a critical role in CION-induced trigeminal neuropathic pain by promoting the activation of the downstream transcription factor ATF2 via the Ca²²⁺-ERK/P38 pathway.