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.

Atopic dermatitis (AD) is the most common skin disease in children. It is characterized by relapsing inflammation, skin barrier defects, and intractable itch. However, the pathophysiology of itch in AD remains enigmatic. Here, we examine the contribution of Tmem79, a putative Membrane Associated Protein in Eicosanoid and Glutathione metabolism (MAPEG), to AD. We show that Tmem79 is expressed by both keratinocytes and sensory neurons and that loss of Tmem79 in both cell types contributes to development of the scratching phenotype observed in Tmem79 null mice. Interestingly, we find that loss of neuronal and keratinocytic Tmem79 contribute to scratching, but loss of keratinocytic Tmem79 alone is sufficient to elicit robust scratching. Tmem79 null mice accumulate dermal mast cells, which are diminished following chronic treatment with cyclooxygenase inhibitors and an EP3 receptor antagonist. In Tmem79 null mice, mast cell degranulation produces histaminergic itch in a H4R/H1R histamine receptor-dependent manner that may involve activation of TRPV1-negative afferents. Furthermore, acute antagonism of TRPA1, but not TRPV1, diminishes scratching, although compensatory mechanisms exist. Mechanistic insights from this model suggest that therapeutics targeting PGE2, H1R/H4R, or TRPA1 signaling pathways may represent useful avenues to treat Tmem79 null-associated AD itch.