TRPM8, a member of the transient receptor potential (TRP) channel superfamily, is expressed in thermosensitive neurons, in which it functions as a cold and menthol sensor. TRPM8 and most other temperature-sensitive TRP channels (thermoTRPs) are voltage gated; temperature and ligands regulate channel opening by shifting the voltage dependence of activation. The mechanisms and structures underlying gating of thermoTRPs are currently poorly understood. Here we show that charge-neutralizing mutations in transmembrane segment 4 (S4) and the S4-S5 linker of human TRPM8 reduce the channel's gating charge, which indicates that this region is part of the voltage sensor. Mutagenesis-induced changes in voltage sensitivity translated into altered thermal sensitivity, thereby establishing the strict coupling between voltage and temperature sensing. Specific mutations in this region also affected menthol affinity, which indicates a direct interaction between menthol and the TRPM8 voltage sensor. Based on these findings, we present a Monod-Wyman-Changeux–type model explaining the combined effects of voltage, temperature and menthol on TRPM8 gating.
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We thank G. Droogmans, F. Mahieu, J. Vriens, R. Vennekens and J. Prenen for helpful suggestions and criticisms, and A. Patapoutian and M. Bandell for the Y745H and L1009R mutants. This work was supported by grants from the Human Frontiers Science Program (RGP32/2004), the Belgian Federal Government (IUAP P5/05), the Research Foundation–Flanders (G.0136.00, G.0172.03 and G.0565.07) and the Research Council of the KU Leuven (GOA2004/07 and EF/95/010).
The authors declare no competing financial interests.
Examples of the method used to determine Popen. (PDF 131 kb)
Fits of two published models describing the temperature dependence of TRPM8 to experimental data. (PDF 119 kb)
[3H]menthol displacement experiments on nontransfected HEK 293 cells and on cells expressing TRPM2, WT TRPM8 and TRPM8 mutants. (PDF 83 kb)
Plasma membrane expression and current densities for different TRPM8-TRPM5 chimeras. (PDF 249 kb)
Additional predictions of the MWC model. (PDF 99 kb)
Parameters that describe the expression level, voltage dependence, temperature dependence and menthol sensitivity of WT and mutant TRPM8. (PDF 218 kb)
Procedure for determination of activation curves, comparison of two models describing the temperature dependence of TRPM8, and mathematical description of the MWC and HH models. (PDF 264 kb)
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Voets, T., Owsianik, G., Janssens, A. et al. TRPM8 voltage sensor mutants reveal a mechanism for integrating thermal and chemical stimuli. Nat Chem Biol 3, 174–182 (2007). https://doi.org/10.1038/nchembio862
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