Drosophila Painless is a Ca2+-requiring channel activated by noxious heat
Drosophila Painless is a Ca2+-requiring channel activated by noxious heat
复制标题
DOI：
10.1523/jneurosci.2757-08.2008
复制
发表时间：
2008-10-01
影响因子：
5.3
通讯作者：
Tominaga, Makoto
中科院分区：
文献类型：
--
作者：
Sokabe, Takaaki;Tsujiuchi, Seiya;Tominaga, Makoto
热变化激活瞬时受体电位（TRP）离子通道超家族的一些成员。它们是探测环境温度的主要传感器。果蝇TRP通道无痛被认为是避免有害热的原因，因为无痛突变果蝇在热感方面表现出缺陷。然而，没有研究证明它的热响应性。我们发现，在人胚胎肾源性293 （HEK293）细胞中无痛表达是一种有害的热激活的、Ca2+可渗透的通道，其功能主要依赖于Ca2+。在Ca2+成像中，无痛介导了细胞内Ca2+ (Ca-i(2+))在加热过程中的强劲增加，并在全细胞膜片钳模式下显示出热诱发的内向电流。Ca2+通透性明显高于其他阳离子。在没有细胞外Ca2+ (Ca-o(2+))和Ca-i（2+）的情况下，热诱发电流可以忽略不计，而200 nM的Ca-i（2+）可以使无痛细胞热激活。Ca-i（2+）的存在显著加快了活化动力学。Ca-i（2+）存在时，无痛激活的温度阈值为42.6℃，而Ca-o（2+）存在时，无痛激活的温度阈值显著提高到44.1℃。温度阈值在以Ca2+依赖的方式重复加热后进一步降低。在切除膜的单通道水平上观察到无痛的Ca2+依赖性热激活。我们发现Ca2+调控位点位于Painless的n端区域。无痛表达HEK293细胞对各种热敏TRP通道激活剂（包括异硫氰酸烯丙酯）不敏感，而哺乳动物TRPA1抑制剂、钌红和樟脑可可逆地阻断无痛的热激活。我们的研究结果表明，无痛是果蝇对有毒热量的直接传感器。
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, Ca2+-permeable channel, and the function is mostly dependent on Ca2+. In Ca2+-imaging, Painless mediated a robust intracellular Ca2+ (Ca-i(2+)) 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 Ca2+ (Ca-o(2+)) and Ca-i(2+), whereas 200 nM Ca-i(2+) enabled heat activation of Painless. Activation kinetics were significantly accelerated in the presence of Ca-i(2+). The temperature threshold for Painless activation was 42.6 degrees C in the presence of Ca-i(2+), whereas the threshold was significantly increased to 44.1 degrees C when only Ca-o(2+) was present. Temperature thresholds were further reduced after repetitive heating in a Ca2+-dependent manner. Ca2+-dependent heat activation of Painless was observed at the single-channel level in excised membranes. We found that a Ca2+-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.