Dynamics of [Ca2+] in the endoplasmic reticulum and cytoplasm of intact HeLa cells - A comparative study
Dynamics of [Ca2+] in the endoplasmic reticulum and cytoplasm of intact HeLa cells - A comparative study
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DOI：
10.1074/jbc.272.44.27694
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发表时间：
1997-10-31
影响因子：
4.8
通讯作者：
Alvarez, J
中科院分区：
文献类型：
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作者：
Barrero, MJ;Montero, M;Alvarez, J
我们使用腔肠素 n 重构的内质网靶向、低 Ca2+ 亲和力水母发光蛋白，在 22 摄氏度和 37 摄氏度下测量了完整 HeLa 细胞内质网 ([Ca2+](er)) 中的 [Ca2+]。 22 摄氏度时水母发光蛋白消耗速度要慢得多，这使得可以对 [Ca2+](er) 的动力学进行更长时间的研究。稳态 [Ca2+](er) (500-600 μM) 不受温度影响，尽管泵送速率和渗漏速率在 22°C 下均降低。比较了添加浓度增加的激动剂和/或 Ca2+-ATPase 抑制剂后或在无 Ca2+ 培养基中孵育后 [Ca2+](er) 和细胞质 [Ca2+] ([Ca2+](c)) 的行为。我们发现激动剂会引起 [Ca2+](er) 快速但相对较小的下降，这足以导致 [Ca2+](c) 急剧增加。 Ca2+ 释放的终止是通过 [Ca2+](c) 对肌醇 1,4,5-三磷酸受体的反馈抑制来控制的，这种机制似乎旨在释放产生所需 [Ca2+](c) 信号所需的最少量 Ca2+。我们还表明，当 [Ca2+](er) 降低到低于约 150 μM 的阈值时，即使没有 Ca2+ 泵送或 [Ca2+](c) 增加，Ca2+ 释放也会逐渐受到抑制。该效应与 [Ca2+](er) 对肌醇 1,4,5-三磷酸门控通道的调节一致。
We have measured the [Ca2+] in the endoplasmic reticulum ([Ca2+](er)) of intact HeLa cells at both 22 degrees C and 37 degrees C using endoplasmic reticulum-targeted, low Ca2+ affinity aequorin reconstituted with coelenterazine n. Aequorin consumption was much slower at 22 degrees C, and this allowed performing a much longer study of the dynamics of [Ca2+](er). The steady-state [Ca2+](er) (500-600 mu M) was not modified by the temperature, although both the rates of pumping and leak were decreased at 22 degrees C, The behavior of both [Ca2+](er) and cytoplasmic [Ca2+] ([Ca2+](c)) after the addition of increasing concentrations of agonists and/or Ca2+-ATPase inhibitors, or following incubation in Ca2+-free medium were compared. We show that agonists induce a fast but relatively small decrease in [Ca2+](er), which is enough to produce a sharp increase in [Ca2+](c). Termination of Ca2+ release is controlled by feedback inhibition of the inositol 1,4,5-trisphosphate receptors by [Ca2+](c), a mechanism that appears to be designed to release the minimum amount of Ca2+ necessary to produced the required [Ca2+](c) signal. We also show that Ca2+ release is inhibited progressively when [Ca2+](er) decreases below a threshold of about 150 mu M, even in the absence of Ca2+ pumping or [Ca2+](c) increase. This effect is consistent with a regulation of the inositol 1,4,5-trisphosphate-gated channels by [Ca2+](er).