The subterranean naked mole-rat (Heterocephalus glaber, NM-R) is a rodent that has attracted growing research interest from a biomedical and zoological perspective. NM-Rs exhibit unique physiological characteristics including eusociality, pain insensitivity, cancer resistance, an unusual immune system, a long lifespan, and the lower basal metabolic rate than expected for its body mass. In addition, NM-Rs are poikilothermic mammals. So far, the precise mechanisms of cold detection processes remain poorly understood. The transient receptor potential melastatin 8 (TRPM8) ion channel is the principal cold receptor in mammals, a better understanding of the role of TRPM8 is needed to improve our current knowledge of the thermosensory system in NM-R. We used RNAScope and immunofluorescence to better understand the expression and localization of TRPM8 in sensory and non-sensory tissues. TRPM8 is expressed in the NM-R DRGs but more TRPM8+ NF200+ neurons are found in NM-R compared to mice. Approximately 33.7% of TRPM8-positive neurons also express NF200, indicating a significant presence in myelinated sensory neurons, while 39.33% and 19.14% of these neurons co-express CGRP and TRPV1, respectively. Interestingly, TRPM8 was also observed in large-diameter neurons and was not limited to smaller neurons typically associated with cold sensing in mice. Furthermore, we identified TRPM8 expression in the near lamina I of the dorsal horn of the NM-R spinal cord, which is similar to mice. Notably, we observed abundant and specific Trpm8 mRNA expression in the neurons of grey matter within the NM-R spinal cord. A notable discovery was the identification of a unique extended N-terminal sequence in NM-R TRPM8 compared with TRPM8 sequences from more than 30 mammals. We confirmed a unique extended N-terminal variant of the TRPM8 ion channel in NM-Rs using RT-PCR and a custom Ex-trpm8 probe designed for the 415–854 bp region from the NM-R Trpm8 mRNA. To explore the functional implications of this extended N-terminal sequence, we performed calcium imaging assays using HEK293T cells expressing NM-R TRPM8. The results demonstrated that the extended sequence significantly diminishes the response of TRPM8 to cold, menthol, and icilin. When this sequence was deleted, TRPM8 function was restored to levels similar to the mouse TRPM8. Additionally, fusing this N-terminal extension to mouse TRPM8 reduced its responsiveness, suggesting that the extended sequence modulates cold sensitivity in NM-Rs. VII Further experiments using calcium imaging of cultured NM-R DRG neurons and skin-nerve preparations demonstrated that NM-Rs retain intact cold sensation, which is largely independent of TRPM8. Cold-sensitive neurons were more abundant in NM-Rs than in mice, particularly among larger neurons. Additionally, cold-sensitive fibers in NM-Rs exhibited faster conduction velocities compared to those in mice, suggesting the involvement of alternative pathways for cold detection and potential evolutionary adaptations for rapid cold sensing in subterranean environments. These findings suggest that NM-Rs may have evolved unique TRPM8 isoforms and alternative mechanisms for cold perception, which may reflect adaptations to their distinct ecological niche. These findings not only advance our understanding of cold detection in NM-Rs but also raise intriguing questions about the evolutionary divergence of TRPM8 function in this species.
