Ryanodine Receptors Control Cytosolic Calcium Elevation Following Activation of Store-Operated Calcium Entry in Activated but not Resting Human T Lymphocytes

Abstract

Previously we have shown that in Jurkat T lymphocytes, the ryanodine (RyR) receptors are activated by store-operated Ca2+ entry (SOCE) and that inhibition of RyR significantly reduced elevation in intracellular Ca2+ concentration ([Ca2+]i) following SOCE. Because Jurkat T cells differ from normal human T cells, we explored contribution of RyR into Ca2+ signaling in two functional human T lymphocyte subsets: resting and activated. Resting T cells were isolated from the peripheral blood of healthy humans and activated in vitro using anti-CD3 and anti-CD28 antibodies. Assessing the [Ca2+]i dynamics in activated T cells using fura-2, a Ca2+ indicator, revealed that RyR blockers ryanodine (Ry) and dantrolene (Da) significantly reduced Ca2+ elevation upon SOCE activation, while increasing Ca2+ content within the store, which is consistent with our previous findings in Jurkat T cells. In contrast, in resting T cells neither Ry nor Da affected [Ca2+]i elevation upon SOCE activation at physiological concentration (2 mM) of extracellular Ca2+. However, the inhibitory effects of RyR blockers were observed in resting T cells in the presence of the elevated extracellular Ca2+concentration (10 mM). Using Mn2+ quench of fura 2 fluorescence approach we further explored whether inhibition of [Ca2+]i elevation in the presence of RyR blockers could be attributed to termination of SOCE due to the Ca2+ accumulation within the store. We found that rates of Mn2+ quench were identical in the presence and absence of RyR blockers, indicating that within a given timeframe enhanced Ca2+ accumulation within the store did not affect SOCE. We conclude that in activated human T cells Ry-sensitive store serves as an intermediate compartment for SOCE and that RyR controls [Ca2+]i dynamics by regulating [Ca2+]i release from the store.