T cell receptor-mediated Ca2+ signaling: Release and influx are independent events linked to different Ca2+ entry pathways in the plasma membrane

In this study, we showed that cross-linking CD3 molecules on the T cell surface resulted in Ca²⁺ release from the intracellular stores followed by a sustained Ca²⁺ influx. Inhibition of release with TMB-8 did not block the influx. However, inhibition of phospholipase C activity suppressed both Ca²⁺ release and influx. Once activated, the influx pathway remained open in the absence of further hydrolysis of PIP2. Thapsigargin, a microsomal Ca²⁺ -ATPase inhibitor, stimulated Ca²⁺ entry into the cells by a mechanism other than emptying Ca²⁺ stores. In addition, Ca²⁺ entry into the Ca²⁺ -depleted cells was stimulated by low basal level of cytosolic Ca²⁺, not by the emptying of intracellular Ca²⁺ stores. Both the Ca²⁺ release and influx were dependent on high and low concentrations of extracellular Ca²⁺. At low concentrations, Mn²⁺ entered the cell through the Ca²⁺ influx pathway and quenched the sustained phase of fluorescence; whereas, at higher Mn²⁺ concentration both the transient and the sustained phases of fluorescence were quenched. Moreover, Ca²⁺ release was inhibited by low concentrations of Ni²⁺, La³⁺, and EGTA, while Ca²⁺ influx was inhibited by high concentrations. Thus, in T cells Ca²⁺ influx occurs independently of IP3-dependent Ca²⁺ release. However, some other PIP2 hydrolysis-dependent event was involved in prolonged activation of Ca²⁺ influx. Extracellular Ca²⁺ influenced Ca²⁺ release and influx through the action of two plasma membrane Ca²⁺ entry pathways with different pharmacological and biochemical properties.