Characterisation of TRPM8 as a pharmacophore receptor
Introduction
The only known temperature sensors in the nerve endings of mammals belong to the transient receptor potential (TRP) superfamily of cation channels. Until now, seven temperature-sensitive TRP channels have been described. Four members of the vanilloid subfamily are activated by increases of temperature above 25 °C (TRPV4 [1], [2]), 31 °C (TRPV3 [3], [4], [5]), 43 °C (TRPV1 [6]), 52 °C (TRPV2 [7]). TRPM4 and TRPM5, two members of the melastatin subfamily, are activated by temperatures in the range from 15 to 35 °C [8]. TRPM8 is stimulated by temperatures below 28 °C [9], [10] whereas TRPA1 (ANKTM1) may be sensitive to cold (<18 °C [11], [12], [13], [14]).
The trp-p8 or TRPM8 cDNA was initially identified and cloned by screening for mRNAs up-regulated in prostate cancer [15]. It is also expressed in a number of non prostatic primary tumors of breast, colon, lung and skin origin whereas transcripts encoding TRPM8 were hardly detected or not detected in the corresponding normal human tissues ([15]; reviewed in [16], [17]). Using a different approach to identify and to isolate cDNA which encode proteins sensitive to cooling compounds like menthol and icilin, McKemy et al. [9] and Peier et al. [10] cloned the TRPM8 cDNA using RNA from trigeminal neurons. They also showed that TRPM8 is a Ca2+ permeable cation channel.
In this study, we describe the action of prototype cooling agents of various chemical structures on TRPM8 channel activity. The data presented suggest that all compounds used act as TRPM8 agonists with the carboxamide WS-12 exerting the highest potency of all known agonists.
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Cell culture, transfection and stable cell lines
Human embryonic kidney (HEK-293) cells were from the American Type Culture Collection (1573-CRL, ATCC, Manassas, USA) and cultured as previously described [18]. HEK cells were transiently transfected with 3 μg DNA in 9 μl of the PolyFect® reagents (Qiagen, Hilden, Germany) for the experiments shown in Fig. 5. The bicistronic expression plasmid pdiCaT-Lb contained the entire protein-coding regions of the human TRPM8 (GeneBank™, accession number AY090109, [15]) followed by an internal ribosomal
Menthol and icilin induced Ca2+ signals in TRPM8 expressing HEK cells
TRPM8 is a Ca2+ permeable channel [9], [10]. Thus, it is possible to monitor its activity in Ca2+ imaging experiments upon application of cooling compounds such as menthol and icilin [26]. The extracellular application of menthol leads to a dose-dependent increase of the fura-2 fluorescence ratio indicating an elevation of the free cytosolic Ca2+ concentration (Fig. 3A). Increasing the menthol concentration accelerated the initial Ca2+ upstroke. The EC50 was 10.4 μM (Fig. 3B) which is similar to
Discussion
TRPM8 is a cation channel which is activated by low temperature and cooling compounds [9], [27]. Naturally occurring monoterpens such as menthol and the synthetic super-cooling agent icilin [25] are commonly used to activate and study TRPM8 channel activity. Here, chemicals with diverse structures are shown to act as TRPM8 agonists. The carboxamides (WS-12, CPS-369, CPS-113), the carboxylic acid ester (WS-30) and the phosphine oxide (WS-148) all activated TRPM8 channels with EC50 in nM to low
Note added in proof
After submission of this paper, similar observations were reported [39].
Acknowledgements
We thank Prof. Edward T. Wei from the University of California at Berkeley for the generous gift of all compounds tested in this study. The help of Dr. Stefan Philipp in the generation of the stable HEK-TRPM8 cell lines by cell sorting is gratefully acknowledged. We also like to thank Mrs. Heidi Löhr, Birgit Spohrer and Christine Jung for excellent technical assistance. This work was supported by the Wilhelm Sander-Stiftung, the Fonds der Chemischen Industrie and the Forschungsausschuss der
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