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

Nat Neurosci. 2006 Apr;9(4):493-500. doi: 10.1038/nn1665. Epub 2006 Mar 5.


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 approximately 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • CHO Cells
  • Cold Temperature*
  • Cricetinae
  • Gene Library
  • Menthol / chemistry
  • Menthol / metabolism*
  • Mice
  • Molecular Sequence Data
  • Molecular Structure
  • Mutagenesis
  • Patch-Clamp Techniques
  • Protein Structure, Tertiary
  • Pyrimidinones / metabolism
  • TRPM Cation Channels / chemistry
  • TRPM Cation Channels / genetics
  • TRPM Cation Channels / metabolism*


  • Pyrimidinones
  • TRPM Cation Channels
  • TRPM8 protein, mouse
  • Menthol
  • icilin