High-throughput investigation of temperature- and ligand-activation of the TRPM8 channel.

Abstract

The TRPM8 channel functions as the main cold sensor in sensory nerves, where decreases in temperature cause opening of the cation-conducting channel pore. Activation of TRPM8 in neurons is modulated by voltage, lipids, G-proteins, protein kinases, cytosolic calcium and natural compounds such as menthol. These mechanisms tune neural excitability and are likely important for how sensory stimuli are detected, integrated, and perceived as cold, warmth, pain, or analgesia. The molecular mechanisms of activation and regulation of the TRPM8 channel by chemical and physical stimuli are largely unknown, including the mechanism of cold-activation. Here we set out to systematically identify which residues in the transmembrane domain of the TRPM8 channel are determinant for its sensitivity to menthol or cold. We generated a library containing all possible single-residue substitutions and deletions in the transmembrane domain of the channel. This library was benchmarked using patch-clamp electrophysiology and we have developed a high-throughput fluorescence-based assay of ion channel activity. Finally, we will use Sort-seq methods to link genotypes to phenotypes for each of the variants in our libraries. Through this method, it will be possible to link individual residue changes to functional outcomes in TRPM8.