UCSF

Transient Receptor Potential (TRP) cation channels function as cellular sensors in uni- and multicellular eukaryotes. Despite intensive study, the mechanisms of TRP channel activation by chemical or physical stimuli remain poorly understood. Here we present strategies to unravel these biophysical mechanisms based on genetic and evolutionary approaches. First, to identify amino acid residues crucial for TRP channel gating, we developed an unbiased, high-throughput genetic screen in yeast that uncovered rare, constitutively active mutants of the capsaicin receptor, TRPV1. We show that mutations within the pore helix domain dramatically increase basal channel activity and responsiveness to chemical and thermal stimuli. Mutation of corresponding residues within two related TRPV channels leads to comparable effects on their activation properties. Our data suggest that conformational changes in the outer pore region are critical for determining the balance between open and closed states, providing evidence for a general role for this domain in TRP channel activation. We also report the cloning and characterization of an ortholog of a cold and menthol receptor, TRPM8, from the cold-blooded African clawed frog Xenopus laevis. This TRPM8 ortholog responds to menthol but displays a striking shift in its temperature-response profile toward lower temperatures compared to the rat and chicken receptors, with a positive correlation between TRPM8 response threshold and species core body temperature. Our results suggest that species-specific differences in thermosensitivity can be attributed at least in part to intrinsic biophysical properties of temperature-gated channels in peripheral sensory neurons.