Agam K, von Campenhausen M, Levy S, Ben-Ami H C, Cook B, Kirschfeld K, Minke B
Department of Physiology and the Kühne Minerva Center for Studies of Visual Transduction, The Hebrew University-Hadassah Medical School, Jerusalem, Israel.
J Neurosci. 2000 Aug 1;20(15):5748-55. doi: 10.1523/JNEUROSCI.20-15-05748.2000.
Drosophila transient receptor potential (TRP) is a prototypical member of a novel family of channel proteins underlying phosphoinositide-mediated Ca(2+) entry. Although the initial stages of this signaling cascade are well known, downstream events leading to the opening of the TRP channels are still obscure. In the present study we applied patch-clamp whole-cell recordings and measurements of Ca(2+) concentration by ion-selective microelectrodes in eyes of normal and mutant Drosophila to isolate the TRP and TRP-like (TRPL)-dependent currents. We report that anoxia rapidly and reversibly depolarizes the photoreceptors and induces Ca(2+) influx into these cells in the dark. We further show that openings of the light-sensitive channels, which mediate these effects, can be obtained by mitochondrial uncouplers or by depletion of ATP in photoreceptor cells, whereas the effects of illumination and all forms of metabolic stress were additive. Effects similar to those found in wild-type flies were also found in mutants with strong defects in rhodopsin, Gq-protein, or phospholipase C, thus indicating that the metabolic stress operates at a late stage of the phototransduction cascade. Genetic elimination of both TRP and TRPL channels prevented the effects of anoxia, mitochondrial uncouplers, and depletion of ATP, thus demonstrating that the TRP and TRPL channels are specific targets of metabolic stress. These results shed new light on the properties of the TRP and TRPL channels by showing that a constitutive ATP-dependent process is required to keep these channels closed in the dark, a requirement that would make them sensitive to metabolic stress.
果蝇瞬时受体电位（TRP）是磷酸肌醇介导的Ca(2+)内流所依赖的一类新型通道蛋白家族的典型成员。尽管这一信号级联反应的初始阶段已为人熟知，但导致TRP通道开放的下游事件仍不清楚。在本研究中，我们应用膜片钳全细胞记录技术以及离子选择性微电极对正常和突变果蝇眼睛中的Ca(2+)浓度进行测量，以分离出依赖于TRP和TRP样（TRPL）的电流。我们报告称，缺氧能迅速且可逆地使光感受器去极化，并在黑暗中诱导Ca(2+)流入这些细胞。我们进一步表明，介导这些效应的光敏感通道的开放可通过线粒体解偶联剂或光感受器细胞中ATP的耗尽来实现，而光照和所有形式的代谢应激的效应是相加的。在视紫红质、Gq蛋白或磷脂酶C存在严重缺陷的突变体中也发现了与野生型果蝇相似的效应，这表明代谢应激作用于光转导级联反应的后期。TRP和TRPL通道的基因敲除阻止了缺氧、线粒体解偶联剂和ATP耗尽的效应，从而证明TRP和TRPL通道是代谢应激的特定靶点。这些结果通过表明在黑暗中需要一个组成型的ATP依赖过程来使这些通道保持关闭，而这一需求会使它们对代谢应激敏感，从而为TRP和TRPL通道的特性提供了新的线索。
