Sphingolipid-gated Ca²⁺ release from intracellular stores of endothelial cells is mediated by a novel Ca²⁺-permeable channel

Sphingolipid-gated Ca²⁺ signaling is mediated through Ca²⁺-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, ω 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 ∼1.5-2.0 kilobases in length. The sphingolipid-gated Ca²⁺-permeable channel is thus likely to be a novel Ca²⁺-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.