title: Requirement of extracellular Ca2+binding to specific amino acids for heat-evoked activation of TRPA1.
abstract: We found that extracellular Ca2+, but not other divalent cations (Mg2+and Ba2+) or intracellular Ca2+, 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 Ca2+. Neutralization of acidic amino acids on the outer surface of TRPA1 by extracellular Ca2+is important for heat-evoked large activation of gaTRPA1, chTRPA1 and rsTRPA1.
  
  
  
  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 Ca2+, but not intracellular Ca2+, plays an important role in heat-evoked TRPA1 activation in green anole lizards (gaTRPA1). Here we focus on extracellular Ca2+-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 Ca2+, 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 Ca2+. These results suggest that neutralization of acidic amino acids by extracellular Ca2+is important for heat-evoked activation of gaTRPA1, chTRPA1, and rsTRPA1, which could clarify mechanisms of heat-evoked channel activation.
authors.affiliation: Division of Cell Signaling, Okazaki Institute for Integrative Bioscience (National Institute for Physiological Sciences), National Institutes of Natural Sciences, Okazaki, 444-8787, Japan.|Department of Physiological Sciences, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, 444-8585, Japan.
authors.forename: Erkin
authors.initials: E
authors.lastname: Kurganov
authors.affiliation: Division of Cell Signaling, Okazaki Institute for Integrative Bioscience (National Institute for Physiological Sciences), National Institutes of Natural Sciences, Okazaki, 444-8787, Japan.|Department of Physiological Sciences, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, 444-8585, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Saito
authors.affiliation: Division of Cell Signaling, Okazaki Institute for Integrative Bioscience (National Institute for Physiological Sciences), National Institutes of Natural Sciences, Okazaki, 444-8787, Japan.
authors.forename: Claire
authors.initials: C
authors.lastname: Tanaka Saito
authors.affiliation: Division of Cell Signaling, Okazaki Institute for Integrative Bioscience (National Institute for Physiological Sciences), National Institutes of Natural Sciences, Okazaki, 444-8787, Japan.|Department of Physiological Sciences, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, 444-8585, Japan.|Institute for Environmental and Gender-Specific Medicine, Juntendo University, Urayasu, 279-0021, Japan.
authors.forename: Makoto
authors.identifier: 0000-0003-3111-3772
authors.initials: M
authors.lastname: Tominaga
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D002645:Chickens; D004305:Dose-Response Relationship, Drug; D045604:Extracellular Fluid / Q000187:drug effects / Q000378:metabolism*; D057809:HEK293 Cells; D006358:Hot Temperature; D006801:Humans; D008116:Lizards; D009154:Mutation / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D012911:Snakes; D000074025:TRPA1 Cation Channel; D050051:Transient Receptor Potential Channels / Q000235:genetics / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D015220:Calcium Channels; D009419:Nerve Tissue Proteins; D000074025:TRPA1 Cation Channel; C473350:TRPA1 protein, human; D050051:Transient Receptor Potential Channels; D002118:Calcium
keywords: TRPA1; calcium; heat; mutation