Van Coppenolle Fabien, Vanden Abeele Fabien, Slomianny Christian, Flourakis Matthieu, Hesketh John, Dewailly Etienne, Prevarskaya Natalia
Laboratoire de Physiologie Cellulaire, INSERM EMI 0228, Université de Lille 1, Bâtiment SN3, 59655 Villeneuve d'Ascq CEDEX, France.
J Cell Sci. 2004 Aug 15;117(Pt 18):4135-42. doi: 10.1242/jcs.01274. Epub 2004 Jul 27.
Under resting conditions, the endoplasmic reticulum (ER) intraluminal free calcium concentration (Ca(2+)) reflects a balance between active uptake by Ca(2+)-ATPases and passive efflux via 'leak channels'. Despite their physiological importance and ubiquitous leak pathway mechanism, very little is known about the molecular nature of these channels. As it has been suggested that the open translocon pore complex of the ER is permeable to ions and neutral molecules, we hypothesized that the ribosome-bound translocon would be permeable to calcium after treatment with puromycin, a translation inhibitor that specifically releases polypeptide chains. At this time, the translocon channel is left open. We measured the fluctuations in cytoplasmic and luminal calcium concentrations using fluorescent dyes (fura-2 and magfura-2, respectively). The calcium release induced by thapsigargin (a Ca(2+)-ATPase inhibitor) was lower after puromycin treatment. Puromycin also reduced the Ca(2+) level when perfused into the medium, but was ineffective after anisomycin pre-treatment (an inhibitor of the peptidyl transferase). Puromycin had a similar effect in the presence of heparin and ryanodine. This puromycin-evoked Ca(2+) decrease was specific to the translocon. We conclude that the translocon complex is a major calcium leak channel. This work reveals a new role for the translocon which is involved in the control of the Ca(2+) and could therefore supervise many physiological processes, including gene expression and apoptosis.
在静息状态下，内质网（ER）腔内游离钙浓度（Ca(2+)）反映了Ca(2+)-ATP酶的主动摄取与通过“渗漏通道”的被动外流之间的平衡。尽管这些通道在生理上具有重要意义且渗漏途径机制普遍存在，但对其分子性质却知之甚少。由于有人提出内质网开放的易位子孔复合体对离子和中性分子具有通透性，我们推测在用嘌呤霉素（一种特异性释放多肽链的翻译抑制剂）处理后，与核糖体结合的易位子对钙具有通透性。此时，易位子通道保持开放。我们分别使用荧光染料（fura-2和magfura-2）测量细胞质和内质网腔内钙浓度的波动。在用嘌呤霉素处理后，毒胡萝卜素（一种Ca(2+)-ATP酶抑制剂）诱导的钙释放减少。当将嘌呤霉素灌注到培养基中时，它也会降低Ca(2+)水平，但在茴香霉素（一种肽基转移酶抑制剂）预处理后则无效。在存在肝素和ryanodine的情况下，嘌呤霉素也有类似作用。这种由嘌呤霉素引起的Ca(2+)降低是易位子特有的。我们得出结论，易位子复合体是主要的钙渗漏通道。这项工作揭示了易位子的一个新作用，即参与对Ca(2+)的调控，因此可能监督包括基因表达和细胞凋亡在内的许多生理过程。
