Montero M, Alvarez J, Scheenen W J, Rizzuto R, Meldolesi J, Pozzan T
Department of Biochemistry and Molecular Biology and Physiology, Insituto de Biologia y Genetica Molecular, University of Valladolid and Consejo Superior de Investigaciones Cientificas, E-47005 Valladolid, Spain.
J Cell Biol. 1997 Nov 3;139(3):601-11. doi: 10.1083/jcb.139.3.601.
Two recombinant aequorin isoforms with different Ca2+ affinities, specifically targeted to the endoplasmic reticulum (ER), were used in parallel to investigate free Ca2+ homeostasis in the lumen of this organelle. Here we show that, although identically and homogeneously distributed in the ER system, as revealed by both immunocytochemical and functional evidence, the two aequorins measured apparently very different concentrations of divalent cations ([Ca2+]er or [Sr2+]er). Our data demonstrate that this contradiction is due to the heterogeneity of the [Ca2+] of the aequorin-enclosing endomembrane system. Because of the characteristics of the calibration procedure used to convert aequorin luminescence into Ca2+ concentration, the [Ca2+]er values obtained at steady state tend, in fact, to reflect not the average ER values, but those of one or more subcompartments with lower [Ca2+]. These subcompartments are not generated artefactually during the experiments, as revealed by the dynamic analysis of the ER structure in living cells carried out by means of an ER-targeted green fluorescent protein. When the problem of ER heterogeneity was taken into account (and when Sr2+ was used as a Ca2+ surrogate), the bulk of the organelle was shown to accumulate free [cation2+]er up to a steady state in the millimolar range. A theoretical model, based on the existence of multiple ER subcompartments of high and low [Ca2+], that closely mimics the experimental data obtained in HeLa cells during accumulation of either Ca2+ or Sr2+, is presented. Moreover, a few other key problems concerning the ER Ca2+ homeostasis have been addressed with the following conclusions: (a) the changes induced in the ER subcompartments by receptor generation of InsP3 vary depending on their initial [Ca2+]. In the bulk of the system there is a rapid release whereas in the small subcompartments with low [Ca2+] the cation is simultaneously accumulated; (b) stimulation of Ca2+ release by receptor-generated InsP3 is inhibited when the lumenal level is below a threshold, suggesting a regulation by [cation2+]er of the InsP3 receptor activity (such a phenomenon had already been reported, however, but only in subcellular fractions analyzed in vitro); and (c) the maintenance of a relatively constant level of cytosolic [Ca2+], observed when the cells are incubated in Ca2+-free medium, depends on the continuous release of the cation from the ER, with ensuing activation in the plasma membrane of the channels thereby regulated (capacitative influx).
两种具有不同Ca2+亲和力、特异性靶向内质网（ER）的重组水母发光蛋白异构体被同时用于研究该细胞器内腔中的游离Ca2+稳态。我们在此表明，尽管通过免疫细胞化学和功能证据显示，这两种水母发光蛋白在内质网系统中分布均匀且相同，但它们测得的二价阳离子浓度（[Ca2+]er或[Sr2+]er）却明显不同。我们的数据表明，这种矛盾是由于包裹水母发光蛋白的内膜系统中[Ca2+]的异质性所致。由于用于将水母发光蛋白发光转换为Ca2+浓度的校准程序的特点，实际上，稳态下获得的[Ca2+]er值倾向于反映的不是内质网的平均[Ca2+]值，而是一个或多个[Ca2+]较低的亚区室的[Ca2+]值。如通过内质网靶向绿色荧光蛋白对活细胞内质网结构进行动态分析所揭示的，这些亚区室并非在实验过程中人为产生的。当考虑到内质网异质性问题（并且当使用Sr2+作为Ca2+替代物时），显示该细胞器的大部分区域积累游离的[阳离子2+]er直至达到毫摩尔范围内的稳态。提出了一个基于存在高[Ca2+]和低[Ca2+]的多个内质网亚区室的理论模型，该模型紧密模拟了在HeLa细胞中积累Ca2+或Sr2+期间获得的实验数据。此外，还解决了与内质网Ca++稳态有关的其他一些关键问题，并得出以下结论：（a）由InsP3受体产生所诱导的内质网亚区室中的变化取决于它们最初的[Ca2+]。在系统的大部分区域有快速释放，而在[Ca2+]较低的小亚区室中阳离子同时被积累；（b）当内腔水平低于阈值时，受体产生的InsP3对Ca2+释放的刺激受到抑制，这表明[阳离子2+]er对内质网肌醇三磷酸受体活性有调节作用（然而，这种现象已经有报道，但仅在体外分析的亚细胞部分中）；（c）当细胞在无Ca2+培养基中孵育时观察到的胞质[Ca2+]相对恒定水平的维持，取决于阳离子从内质网的持续释放，从而导致由此调节的质膜通道的激活（容量性内流）。
