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

Michael Bandell^1, 2, Adrienne E Dubin³, Matt J Petrus², Anthony Orth², Jayanti Mathur², Sun Wook Hwang^1, 4 & Ardem Patapoutian^1, 2

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

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

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

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

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.

	Top