Requirement of extracellular Ca 2+ binding to specific amino acids for heat‐evoked activation of TRPA1

Key points We found that extracellular Ca 2+ , but not other divalent cations (Mg 2+ and Ba 2+ ) or intracellular Ca 2+ , is involved in heat‐evoked activation of green anole (ga) TRPA1. Heat‐evoked activation of chicken (ch) and rat snake (rs) TRPA1 does not depend solely on extracellular Ca 2+ . Neutralization of acidic amino acids on the outer surface of TRPA1 by extracellular Ca 2+ is important for heat‐evoked large activation of gaTRPA1, chTRPA1 and rsTRPA1.Abstract Transient receptor potential ankyrin 1 (TRPA1) is a homotetrameric non‐selective cation‐permeable channel that has six transmembrane domains and cytoplasmic N‐ and C‐termini. The N‐terminus is characterized by an unusually large number of ankyrin repeats. Although the 3‐dimensional structure of human TRPA1 has been determined, and TRPA1 channels from insects to birds are known to be activated by heat stimulus, the mechanism for temperature‐dependent TRPA1 activation is unclear. We previously reported that extracellular Ca 2+ , but not intracellular Ca 2+ , plays an important role in heat‐evoked TRPA1 activation in green anole lizards (gaTRPA1). Here we focus on extracellular Ca 2+ ‐dependent heat sensitivity of gaTRPA1 by comparing gaTRPA1 with heat‐activated TRPA1 channels from rat snake (rsTRPA1) and chicken (chTRPA1). In the absence of extracellular Ca 2+ , rsTRPA1 and chTRPA1 are activated by heat and generate small inward currents. 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 TRPA1 activation in the presence of extracellular Ca 2+ . These results suggest that neutralization of acidic amino acids by extracellular Ca 2+ is important for heat‐evoked activation of gaTRPA1, chTRPA1, and rsTRPA1, which could clarify mechanisms of heat‐evoked channel activation.