Kim S, Lakhani V, Costa D J, Sharara A I, Fitz J G, Huang L W, Peters K G, Kindman L A
Cardiology Division, Duke University Medical Center, Durham, North Carolina 27710.
J Biol Chem. 1995 Mar 10;270(10):5266-9. doi: 10.1074/jbc.270.10.5266.
Sphingolipid-gated Ca2+ signaling is mediated through Ca(2+)-permeable channels. In this report, we characterize the properties of the channel in a human endothelial cell line (EA.hy926). Ca2+ release from intracellular stores is not antagonized by nifedipine, omega conotoxin G-VIa, or heparin. To further characterize the molecular properties of the channel, we developed a novel assay to directly measure efflux of Ca2+ from intracellular stores of permeabilized Xenopus oocytes. Following size fractionation by sucrose gradient, poly(A)+ RNA from EA.hy926 cells is microinjected into oocytes of Xenopus laevis. We find that the mRNA encoding Ca2+ release activity is approximately 1.5-2.0 kilobases in length. The sphingolipid-gated Ca(2+)-permeable channel is thus likely to be a novel Ca(2+)-permeable channel distinct from other characterized intracellular Ca2+ channels such as the ryanodyne receptor and the inositol 1,4,5-trisphosphate receptor. The method described here provides a new approach to further characterizing this channel and other intracellular Ca2+ channels.
鞘脂门控的Ca2+信号传导是通过Ca(2+)通透通道介导的。在本报告中，我们描述了一种人类内皮细胞系（EA.hy926）中该通道的特性。硝苯地平、ω-芋螺毒素G-VIa或肝素均不能拮抗细胞内钙库的Ca2+释放。为了进一步描述该通道的分子特性，我们开发了一种新的检测方法，以直接测量经通透处理的非洲爪蟾卵母细胞内钙库的Ca2+外流。通过蔗糖梯度进行大小分级分离后，将来自EA.hy926细胞的聚腺苷酸加尾RNA（poly(A)+ RNA）显微注射到非洲爪蟾的卵母细胞中。我们发现，编码Ca2+释放活性的mRNA长度约为1.5 - 2.0千碱基。因此，鞘脂门控的Ca(2+)通透通道可能是一种新型的Ca(2+)通透通道，不同于其他已鉴定的细胞内Ca2+通道，如兰尼碱受体和肌醇1,4,5-三磷酸受体。本文所述方法为进一步描述该通道及其他细胞内Ca2+通道提供了一种新途径。
