Sphingolipid-gated Ca2+ release from intracellular stores of endothelial cells is mediated by a novel Ca(2+)-permeable channel.
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.
Kim
S
S
Cardiology Division, Duke University Medical Center, Durham, North Carolina 27710.
Lakhani
V
V
Costa
D J
DJ
Sharara
A I
AI
Fitz
J G
JG
Huang
L W
LW
Peters
K G
KG
Kindman
L A
LA
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000096:biosynthesis / Q000378:metabolism*; D002460:Cell Line; D004730:Endothelium, Vascular / Q000378:metabolism*; D005260:Female; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D015640:Ion Channel Gating / Q000187:drug effects; D007700:Kinetics; D009543:Nifedipine / Q000494:pharmacology; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D010455:Peptides / Q000494:pharmacology; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000494:pharmacology; D012333:RNA, Messenger / Q000008:administration & dosage / Q000378:metabolism; D013107:Sphingolipids / Q000502:physiology*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D013997:Time Factors; D014982:Xenopus laevis; D020866:omega-Conotoxin GVIA
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D010455:Peptides; D012333:RNA, Messenger; D013107:Sphingolipids; C005356:sphingosine phosphorylcholine; D010767:Phosphorylcholine; D000001:Calcimycin; D006493:Heparin; D020866:omega-Conotoxin GVIA; D009543:Nifedipine; D013110:Sphingosine; D002118:Calcium