Isolation of Functional Temperature Activated Transmembrane Domain of Human TRPM8

Abstract

Transient receptor potential melastatin 8 (TRPM8) is a known cold sensor and a part of a family of thermosensitive TRP ion channels. TRPM8 functions in many physiological roles and as a potential therapeutic target for regulating pain, obesity, and withdrawal in addiction. Nevertheless, it remains unclear what areas of the channel are important for cold activation. Given that the transmembrane region is the most evolutionary conserved region, the transmembrane domain (helices S1-S6) is anticipated to be central to cold activation. Here we show the TRPM8 transmembrane domain (TRPM8-TMD) forms a tetramer in detergent DDM micelles using negative stain electron microscopy, chemical cross-linking, and NMR translational diffusion experiments. Planar lipid bilayer electrophysiology experiments indicate the TRPM8-TMD forms a functional outward rectifying ion channel in the presence of PIP2 and is sufficient for cold and menthol activation without the N-terminal and C-terminal cytoplasmic domains. Additionally, the TRPM8-TMD is activated by menthol, icilin, and testosterone at similar concentrations as required for full-length TRPM8. Taken together, these data indicate that the core functional features of TRPM8, including cold and ligand activation, are localized to the transmembrane domain.