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High-throughput random mutagenesis screen reveals TRPM8 residues specifically required for activation by menthol

Nature Neuroscience volume 9, pages 493–500 (2006)Cite this article

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Abstract

Menthol is a cooling compound derived from mint leaves and is extensively used as a flavoring chemical. Menthol activates transient receptor potential melastatin 8 (TRPM8), an ion channel also activated by cold, voltage and phosphatidylinositol-4,5-bisphosphate (PIP2). Here we investigated the mechanism by which menthol activates mouse TRPM8. Using a new high-throughput approach, we screened a random mutant library consisting of ∼14,000 individual TRPM8 mutants for clones that are affected in their response to menthol while retaining channel function. We identified determinants of menthol sensitivity in two regions: putative transmembrane segment 2 (S2) and the C-terminal TRP domain. Analysis of these mutants indicated that activation by menthol involves a gating mechanism distinct and separable from gating by cold, voltage or PIP2. Notably, TRP domain mutations mainly attenuated menthol efficacy, suggesting that this domain influences events downstream of initial binding. In contrast, S2 mutations strongly shifted the concentration dependence of menthol activation, raising the possibility that S2 influences menthol binding.

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Figure 1: Screening of a random mutant library identifies menthol-insensitive TRPM8 mutants.
Figure 2: Selective loss of menthol sensitivity by mutations in the S2 and TRP domains of TRPM8.
Figure 3: Structural requirements for menthol sensitivity.
Figure 4: Mutation of a tripeptide in the TRP domain alters ligand selectivity.
Figure 5: Icilin sensitivity is lost in Y745H and L1009R.

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Change history

  • 19 March 2006

    Corrected affiliation for an author

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Acknowledgements

We thank N. Gray, L. Miraglia, J. Zhang, M. Medina, A. Saghatelian, B. Cravatt, T. Jegla, V. Lee and S. Peters for valuable contributions and input. This work was supported by NINDS grant NS046303. M.B. is supported by a postdoctoral fellowship from the American Heart Association. A.P. is Damon Runyon Fellow and a member of the H. Dorris Neurological Research Institute.

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Authors and Affiliations

  1. Department of Cell Biology, The Scripps Research Institute, La Jolla, 92037, California, USA

    Michael Bandell, Sun Wook Hwang & Ardem Patapoutian

  2. Genomics Institute of the Novartis Research Foundation, San Diego, 92121, California, USA

    Michael Bandell, Matt J Petrus, Anthony Orth, Jayanti Mathur & Ardem Patapoutian

  3. Department of Molecular Biology, The Scripps Research Institute, La Jolla, 92037, California, USA

    Adrienne E Dubin

  4. Korea University Graduate School of Medicine, Seoul, 136-705, Korea

    Sun Wook Hwang

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Correspondence to Ardem Patapoutian.

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Supplementary information

Supplementary Fig. 1

Examples of currents generated by cold and menthol in wild type, Y745H and L1009R. (PDF 683 kb)

Supplementary Table 1

Additional mutants for which a >3 fold decrease in menthol/cold response ratio was observed in the secondary screen (see text). (PDF 13 kb)

Supplementary Methods (PDF 72 kb)

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Bandell, M., Dubin, A., Petrus, M. et al. High-throughput random mutagenesis screen reveals TRPM8 residues specifically required for activation by menthol. Nat Neurosci 9, 493–500 (2006). https://doi.org/10.1038/nn1665

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