HEK-293 cells possess a carbachol- and thapsigargin-sensitive intracellular Ca2+ store that is responsive to stop-flow medium changes and insensitive to caffeine and ryanodine.
Because HEK-293 cells are widely used for the functional expression of channels, exchangers and transporters involved in Ca(2+) homoeostasis, the properties of intracellular Ca(2+) stores and the methods used for measuring intracellular Ca(2+) release in HEK-293 cells were evaluated. Ca(2+) imaging was used to show caffeine-, carbachol- and thapsigargin-induced Ca(2+) release in HEK-293 cells transfected with ryanodine receptor (RyR) cDNA, but only carbachol- and thapsigargin-induced Ca(2+) release in untransfected HEK-293 cells. Intracellular Ca(2+) release in untransfected HEK-293 cells was also observed if medium changes were performed by aspirating and replacing fresh medium (stop-flow), but not if medium changes were performed by a continuous over-flow procedure. Stop-flow medium-change-induced Ca(2+) release in HEK-293 cells was independent of caffeine and ryanodine, demonstrating that it did not occur through RyR channels. Consistent with these observations was the observation that the level of expression of endogenous RyR proteins was below the limits of detection by Western blotting or [(3)H]ryanodine binding. Thus the level of endogenous expression of RyR is so low in HEK-293 cells as to provide a negligible background in relation to functional analysis of recombinant RyR molecules. These results are inconsistent with those of Querfurth et al. [Querfurth, Haughey, Greenway, Yacono, Golan and Geiger (1998) Biochem. J. 334, 79-86], who reported higher levels of endogenous RyR expression in untransfected HEK-293 cells.
Tong
J
J
Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada M5G 1L6.
Du
G G
GG
Chen
S R
SR
MacLennan
D H
DH
D002110:Caffeine / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology*; D002460:Cell Line; D006801:Humans; D012433:Ryanodine / Q000494:pharmacology*; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics; D013050:Spectrometry, Fluorescence; D019284:Thapsigargin / Q000494:pharmacology*; D014162:Transfection
D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D002110:Caffeine; D019284:Thapsigargin; D002217:Carbachol; D002118:Calcium