Drosophila painless is a Ca²⁺-requiring channel activated by noxious heat

Thermal changes activate some members of the transient receptor potential (TRP) ion channel super family. They are primary sensors for detecting environmental temperatures. The Drosophila TRP channel Painless is believed responsible for avoidance of noxious heat because painless mutant flies display defects in heat sensing. However, no studies have proven its heat responsiveness. We show that Painless expressed in human embryonic kidney-derived 293 (HEK293) cells is a noxious heat-activated, Ca ²⁺-permeable channel, and the function is mostly dependent on Ca ²⁺. In Ca²⁺-imaging, Painless mediated a robust intracellular Ca²⁺ (Ca²⁺_i) increase during heating, and it showed heat-evoked inward currents in whole-cell patch-clamp mode. Ca²⁺ permeability was much higher than that of other cations. Heat-evoked currents were negligible in the absence of extracellular Ca ²⁺ (Ca²⁺_o) and Ca²⁺_i, whereas 200 nM Ca²⁺_i enabled heat activation of Painless. Activation kinetics were significantly accelerated in the presence of Ca ²⁺_i. The temperature threshold for Painless activation was 42.6°C in the presence of Ca²⁺_i, whereas the threshold was significantly increased to 44.1°C when only Ca ²⁺_o was present. Temperature thresholds were further reduced after repetitive heating in a Ca²⁺-dependent manner. Ca ²⁺-dependent heat activation of Painless was observed at the single-channel level in excised membranes. We found that a Ca ²⁺-regulatory site is located in the N-terminal region of Painless. Painless-expressing HEK293 cells were insensitive to various thermosensitive TRP channel activators including allyl isothiocyanate, whereas mammalian TRPA1 inhibitors, ruthenium red, and camphor, reversibly blocked heat activation of Painless. Our results demonstrate that Painless is a direct sensor for noxious heat in Drosophila.