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 degrees C, a temperature threshold lower than that reported for the other orthologs. At 22 degrees C, the EC(50) for activation of cTRPM8 expressed in HEK293 cells by icilin and menthol was 0.06 and 4.3 microM determined by Fluorometric Imaging Plate Reader (FLIPR) and 0.4 and 85 microM by patch clamp, respectively. Mustard oil also activated cTRPM8 (FLIPR EC(50) = 490 microM). Menthol activation was more potent at +60 mV than at -60 mV (EC(50) = 53 and 124 microM, respectively, in Xenopus ooctyes). Icilin-, menthol- and mustard oil-induced intracellular Ca(2+) increases were similarly blocked by N-(4-tertiarybutyl-phenyl)-4-(3-chloropyridin-2-yl) tetrahydropyrazine-1(2H)-carboxamide (BCTC) with IC(50) = 2.3, 2.8 and 1.8 microM, respectively. Cooling-activated current was also inhibited by BCTC. Extracellular calcium blocked cTRPM8 in a concentration- and voltage-dependent manner (half maximal blocking [Ca(2+)] = 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.
Liu
Yi
Y
Analgesics Research Team, Drug Discovery, Johnson and Johnson Pharmaceutical Research and Development, Spring House, PA 19477-0776, USA.
Lubin
Mary Lou
ML
Reitz
Tasha L
TL
Wang
Yan
Y
Colburn
Raymond W
RW
Flores
Christopher M
CM
Qin
Ning
N
D000200:Action Potentials / Q000187:drug effects; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D003080:Cold Temperature; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D005470:Fluorometry / Q000379:methods; D005727:Ganglia, Spinal / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D008610:Menthol / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D009149:Mustard Plant; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D018408:Patch-Clamp Techniques / Q000379:methods; D010938:Plant Oils / Q000494:pharmacology; D011719:Pyrazines / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D011744:Pyrimidinones / Q000494:pharmacology; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D050053:TRPM Cation Channels / Q000235:genetics* / Q000502:physiology; D014162:Transfection; D014982:Xenopus laevis
D018076:DNA, Complementary; C475772:N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide; D010938:Plant Oils; D011719:Pyrazines; D011725:Pyridines; D011744:Pyrimidinones; D050053:TRPM Cation Channels; D008610:Menthol; C490483:icilin; D002118:Calcium; C027793:mustard oil