Csutora P, Su Z, Kim H Y, Bugrim A, Cunningham K W, Nuccitelli R, Keizer J E, Hanley M R, Blalock J E, Marchase R B
Department of Cell Biology, University of Alabama, Birmingham, AL 35294-0005, USA.
Proc Natl Acad Sci U S A. 1999 Jan 5;96(1):121-6. doi: 10.1073/pnas.96.1.121.
Depletion of endoplasmic reticulum Ca2+ stores leads to the entry of extracellular Ca2+ into the cytoplasm, a process termed capacitative or store-operated Ca2+ entry. Partially purified extracts were prepared from the human Jurkat T lymphocyte cell line and yeast in which Ca2+ stores were depleted by chemical and genetic means, respectively. After microinjection into Xenopus laevis oocytes, the extracts elicited a wave of increased cytoplasmic free Ca2+ ([Ca2+]i) that spread from the point of injection across the oocyte. Extracts from cells with replete organellar Ca2+ stores were inactive. The increases depended on extracellular Ca2+, were unaffected by the inositol 1,4,5-trisphosphate (IP3) inhibitor heparin or an anti-IP3 receptor antibody and were unchanged when the endoplasmic reticulum was segregated to the hemisphere opposite the injection site by centrifugation. Confocal microscopy revealed that [Ca2+]i increases were most pronounced at the periphery of the oocyte. The patterns of [Ca2+]i increases were replicated by computer simulations based on a diffusible messenger of about 700 Da that directly activates Ca2+ influx. In addition, ICRAC, a Ca2+ release-activated Ca2+ current monitored in Jurkat cells by whole-cell patch clamp recordings, was more rapidly activated when active extracts were included in the patch pipette than by the inclusion of a Ca2+ chelator or IP3. These data support the existence in yeast and mammalian cells depleted of Ca2+ stores of a functionally conserved diffusible calcium influx factor that directly activates Ca2+ influx.
内质网Ca2+储存的耗尽会导致细胞外Ca2+进入细胞质，这一过程称为容量性或储存操纵性Ca2+内流。分别从人Jurkat T淋巴细胞系和酵母中制备了部分纯化的提取物，其中酵母的Ca2+储存分别通过化学和遗传手段耗尽。将提取物显微注射到非洲爪蟾卵母细胞中后，引发了细胞质游离Ca2+（[Ca2+]i）的增加波，该波从注射点扩散穿过卵母细胞。细胞器Ca2+储存充足的细胞提取物无活性。这些增加依赖于细胞外Ca2+，不受肌醇1,4,5-三磷酸（IP3）抑制剂肝素或抗IP3受体抗体的影响，并且当通过离心将内质网分离到与注射部位相对的半球时，增加情况不变。共聚焦显微镜显示，[Ca2+]i的增加在卵母细胞周边最为明显。基于约700 Da的可扩散信使直接激活Ca2+内流的计算机模拟复制了[Ca2+]i增加的模式。此外，通过全细胞膜片钳记录在Jurkat细胞中监测的Ca2+释放激活的Ca2+电流（ICRAC），当在膜片吸管中加入活性提取物时比加入Ca2+螯合剂或IP3时更迅速地被激活。这些数据支持在Ca2+储存耗尽的酵母和哺乳动物细胞中存在一种功能保守的可扩散钙内流因子，该因子直接激活Ca2+内流。
