TRPA1 is a nonselective cation channel with high Ca2+ permeability. We found that extracellular Ca2+, but not either divalent cations (Mg2+ and Ba2+) or intracellular Ca2+, is involved in heat-evoked activation of green anole lizard TRPA1 (gaTRPA1). Heat-evoked activation of chicken TRPA1 and rat snake TRPA1 did not depend solely on extracellular Ca2+. A comparison of extracellular amino acids in TRPA1 identified three negatively charged amino acid residues (glutamate and aspartate) near the outer pore vestibule that are involved in heat-evoked gaTRPA1 activation in the presence of extracellular Ca2+. These results suggest that neutralization of acidic amino acids by extracellular Ca2+ is important for heat-evoked activation of gaTRPA1, chTRPA1, and rsTRPA1. Single channel opening of gaTRPA1 was confirmed in the planner lipid bilayer system. We found that lysophosphatidic acid (LPA) is an itch mediator, but not a pain mediatorÂ by a cheek injection model of mice. Mouse dorsal root ganglion neurons directlyÂ responded to LPA depending on TRPA1 and TRPV1. LPA-induced itch-relatedÂ behaviors were decreased in TRPA1KO, TRPV1KO or TRPA1TRPV1 double KO mice.Â TRPA1 and TRPV1 channels were activated by intracellular LPA, but not byÂ extracellular LPA following LPA5 receptor activation with an activity of Ca2+-independent phospholipase A2 and phospholipase D. KK672-673 and KR977-978Â (K: lysine, R: arginine) of TRPA1 were identified as intracellular LPA interaction sites.Â Thus, targeting TRPA1, TRPV1 or PLD could be effective for cholestatic itchÂ interventions.