Molecular identification and functional characterization of a temperature-sensitive transient receptor potential channel (TRPM8) from canine

TRPM8 belongs to the family of transient receptor potential channels and is activated by cooling and cooling agents, such as icilin and menthol. It is expressed in a subset of sensory neurons and is thought to be involved in thermosensation. Here, we report the cloning and functional characterization of canine TRPM8 (cTRPM8). cTRPM8 shares 95.1%, 94.1%, and 93.9% protein sequence identity with human, rat and mouse TRPM8, respectively. Similar to these mammalian orthologs, cTRPM8 was activated by menthol and icilin with strong outward rectification and little cation selectivity. Menthol and icilin also caused calcium-dependent desensitization. Interestingly, cTRPM8 was activated at < 17 °C, a temperature threshold lower than that reported for the other orthologs. At 22 °C, the EC50 for activation of cTRPM8 expressed in HEK293 cells by icilin and menthol was 0.06 and 4.3 μM determined by Fluorometric Imaging Plate Reader (FLIPR) and 0.4 and 85 μM by patch clamp, respectively. Mustard oil also activated cTRPM8 (FLIPR EC50 = 490 μM). Menthol activation was more potent at + 60 mV than at − 60 mV (EC50 = 53 and 124 μM, respectively, in Xenopus ooctyes). Icilin-, menthol- and mustard oil-induced intracellular Ca2+ increases were similarly blocked by N-(4-tertiarybutyl-phenyl)-4-(3-chloropyridin-2-yl) tetrahydropyrazine-1(2H)-carboxamide (BCTC) with IC50 = 2.3, 2.8 and 1.8 μM, respectively. Cooling-activated current was also inhibited by BCTC. Extracellular calcium blocked cTRPM8 in a concentration- and voltage-dependent manner (half maximal blocking [Ca2+] = 1.6 mM at − 100 mV). These results constitute the first study of cTRPM8 and support the idea that cTRPM8 functions as a transducer of cold stimuli in vivo.