HEK-293 cells possess a carbachol- and thapsigargin-sensitive intracellular Ca2+ store that is responsive to stop-flow medium changes and insensitiveto caffeine and ryanodine

Because HEK-293 cells are widely used for the functional expression of chan nels, exchangers and transporters involved in Ca2+ homoeostasis, the proper ties of intracellular Ca2+ stores and the methods used for measuring intrac ellular Ca2+ release in HEK-293 cells were evaluated. Ca2+ imaging was used to show caffeine-, carbachol- and thapsigargin-induced Ca2+ release in HEK -293 cells transfected with ryanodine receptor (RyR) cDNA, but only carbach ol- and thapsigargin-induced Ca2+ release in untransfected HEK-293 cells. I ntracellular Ca2+ 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-f low procedure. Stop-flow medium-change-induced Ca2+ release in HEK-293 cell s was independent of caffeine and ryanodine, demonstrating that it did not occur through RyR channels. Consistent with these observations was the obse rvation that the level of expression of endogenous RyR proteins was below t he limits of detection by Western blotting or [H-3]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 recom binant RyR molecules. These results are inconsistent with those of Querfurt h et al. [Querfurth, Haughey, Greenway, Yacono, Golan and Geiger (1998) Bio chem. J. 334, 79-86], who reported higher levels of endogenous RyR expressi on in untransfected HEK-293 cells.