Store-operated Ca2+ Influx Causes Ca2+ Release from the Intracellular Ca2+ Channels That Is Required for T Cell Activation

Abstract

The precise control of many T cell functions relies on cytoso-

Bidirectional Coupling between Ryanodine Receptors and Ca2+ Release-activated Ca2+ (CRAC) Channel Machinery Sustains Store-operated Ca2+ Entry in Human T Lymphocytes

“…Previous studies performed on Jurkat T cells, a human lymphoblastic leukemia T cell line, suggested that RyR may be involved in regulating Ca 2ϩ signaling in T cells (18 -24). We have previously shown that in Jurkat T cells, the RyR regulate SOCE by controlling Ca 2ϩ store refilling (19). However, the conclusions drawn from studies on Jurkat T cells cannot be extended to primary human T cells because the latter express type 1 RyR (RyR1) and type 2 RyR (RyR2), whereas Jurkat T cells express type 3 RyR (RyR3) (25).…”

“…Cell Proliferation Assay-A cell division track assay was performed using the CellTrace carboxyfluorescein diacetate succinimidyl ester (CFSE) cell proliferation kit (Invitrogen) as described previously (19). Briefly, resting T cells were washed, resuspended in PBS containing 4 M CFSE at a density of 1 ϫ 10 6 cells/ml, and incubated at 37°C for 10 min.…”

“…In Mn 2ϩ quench experiments, levels of Ca 2ϩ -independent Fura-2 fluorescence F i were determined as F i ϭ F 340 ϩ ␣F 380 , where ␣ is the "isocoefficient" equal to the value of the slope of the plot of F 340 versus F 380 determined experimentally, as described previously (19,38 ] i/CPA3 , etc.) were integrated over the time in each experiment to determine the releasable store content as described previously (19,39). Values of integrated [Ca 2ϩ ] i transients induced by the CPA application subsequent to the readdition of Ca 2ϩ -containing bath solution were normalized to those induced by the CPA application prior to the readdition of Ca 2ϩ -containing bath solution.…”

“…K d was taken as 248 nM (37). In Mn 2ϩ quench experiments, levels of Ca 2ϩ -independent Fura-2 fluorescence F i were determined as F i ϭ F 340 ϩ ␣F 380 , where ␣ is the "isocoefficient" equal to the value of the slope of the plot of F 340 versus F 380 determined experimentally, as described previously (19,38 ] i/CPA3 , etc.) were integrated over the time in each experiment to determine the releasable store content as described previously (19,39).…”

“…[ ] i values were calculated from F 340 and F 380 values and Fura-2 calibration as described previously (19). K d was taken as 248 nM (37).…”