Understanding the evolution of cold sensing and menthol activation in TRPM8.

Abstract

Transient receptor potential melastatin 8 (TRPM8) is a polymodally gated ion channel activated by a variety of stimuli including menthol, cold, and pH. These chemical and physical stimuli ultimately regulate the ion conductance pore. While the menthol binding site and ion conduction pathway gate have been identified, the physics that underlies the mechanism of activation among different stimuli remains elusive. Particularly, the temperature modulation, how TRPM8 converts small changes in temperature into a conformational change, ultimately resulting in signal transduction is not well understood. Here to shed light into polymodal regulation of TRPM8, we perform a comparative analysis of conformational dynamics and functional characterizations of ancestral and extant human TRPM8. We use ancestral sequence reconstruction, a computational molecular evolution tool, to explore that ability to disentangle TRPM8 activation modes, which presumably emerged distinctly over evolutionary time. Based on the extant sequences of TRPM8, ancestral reconstruction was used to determine the DNA sequences of the last common primate, mammalian, and vertebrate ancestors. Resurrection and experimental analysis of these ancestral TRPM8 proteins show them to be functional in heterologous mammalian cells. Whole-cell patch-clamp electrophysiology and calcium mobilization assays confirm our hypothesis that the cold and menthol responses differ in terms of emergence and sensitivity between the ancestral constructs suggesting a new method to dissect polymodal protein behavior. Furthermore, comparative conformational analysis of ancestral and extant TRMP8 through the Dynamic Flexibility and Dynamic Coupling analysis also further support this hypothesis and provide dynamical finger prints for menthol and cold sensation.