Narayanan Bisni, Islam Mohammad N, Bartelt Diana, Ochs Raymond S
Department of Pharmaceutical Sciences, St. John's University, Jamaica, New York 11439, USA.
J Biol Chem. 2003 Nov 7;278(45):44188-96. doi: 10.1074/jbc.M306538200. Epub 2003 Aug 28.
We examined capacitative calcium entry (CCE) in Jurkat and in L6 skeletal muscle cells. We found that extracellular Ca2+ can enter the endoplasmic reticulum (ER) of both cell types even in the presence of thapsigargin, which blocks entry into the ER from the cytosol through the CaATPase. Moreover, extracellular Ca2+ entry into the ER was evident even when intracellular flow of Ca2+ was in the direction of ER to cytosol due to the presence of caffeine. ER Ca2+ content was assessed by two separate means. First, we used the Mag-Fura fluorescent dye, which is sensitive only to the relatively high concentrations of Ca2+ found in the ER. Second, we transiently expressed an ER-targeted derivative of aequorin, which reports Ca2+ by luminescence. In both cases, the Ca2+ concentration in the ER increased in response to extracellular Ca2+ after the ER had been previously depleted despite blockade by thapsigargin. We found two differences between the Jurkat and L6 cells. L6, but not Jurkat cells, inhibited Ca2+ uptake at very high Ca2+ concentrations. Second, ryanodine receptor blockers inhibited the appearance of cytosolic Ca2+ during CCE if added before Ca2+ in both cases, but the L6 cells were much more sensitive to ryanodine. Both of these can be explained by the known difference in ryanodine receptors between these cell types. These findings imply that the origin of cytosolic Ca2+ during CCE is the ER. Furthermore, kinetic data demonstrated that Ca2+ filled the ER before the cytosol during CCE. Our results suggest a plasma membrane Ca2+ channel and an ER Ca2+ channel joined in tandem, allowing Ca2+ to flow directly from the extracellular space to the ER. This explains CCE; any decrease in ER [Ca2+] relative to extracellular [Ca2+] would provide the gradient for refilling the ER through a mass-action mechanism.
我们研究了Jurkat细胞和L6骨骼肌细胞中的容量性钙内流（CCE）。我们发现，即使存在毒胡萝卜素（thapsigargin），细胞外Ca2+仍可进入这两种细胞类型的内质网（ER），毒胡萝卜素会阻断Ca2+通过CaATPase从细胞质进入内质网。此外，由于咖啡因的存在，即使细胞内Ca2+流动方向是从内质网到细胞质，细胞外Ca2+进入内质网的现象依然明显。内质网Ca2+含量通过两种独立方法进行评估。首先，我们使用了Mag - Fura荧光染料，它仅对内质网中相对高浓度的Ca2+敏感。其次，我们瞬时表达了一种靶向内质网的水母发光蛋白衍生物，它通过发光来报告Ca2+。在这两种情况下，尽管有毒胡萝卜素的阻断作用，但在内质网先前耗尽后，内质网中的Ca2+浓度会因细胞外Ca2+而增加。我们发现Jurkat细胞和L6细胞之间存在两个差异。在非常高的Ca2+浓度下，L6细胞而非Jurkat细胞会抑制Ca2+摄取。其次，在两种情况下，如果在添加Ca2+之前加入ryanodine受体阻滞剂，它们会抑制CCE期间细胞质中Ca2+的出现，但L6细胞对ryanodine更为敏感。这两者都可以用这些细胞类型之间已知的ryanodine受体差异来解释。这些发现意味着CCE期间细胞质中Ca2+的来源是内质网。此外，动力学数据表明，在CCE期间Ca2+先填充内质网，然后才进入细胞质。我们的结果表明，质膜Ca2+通道和内质网Ca2+通道串联连接，使Ca2+能够直接从细胞外空间流入内质网。这解释了CCE；相对于细胞外[Ca2+]，内质网[Ca2+]的任何降低都会通过质量作用机制为内质网重新填充提供梯度。
