Kinetics of empty store-activated Ca²⁺ influx in HeLa cells

The intracellular Ca²⁺ indicator Indo-1 was used to monitor changes in cytosolic [Ca²⁺] ([Ca²⁺](i)) in single HeLa cells upon readmission of external Ca²⁺ after a short incubation in Ca²⁺-free solution. HeLa cells were responsive to histamine but not to caffeine, and their histamine- sensitive store was totally depleted by a 60-min exposure to 2 μM thapsigargin. The resting [Ca²⁺](i) in thapsigargin-treated cells was higher than in control cells and low amplitude [Ca²⁺](i) oscillations were observed in about 20% of the cells. Readmission of external Ca²⁺ after a brief withdrawal of extracellular Ca²⁺ resulted in a transient [Ca²⁺](i) rise, which then decayed to the same elevated [Ca²⁺](i) measured before the Ca²⁺ withdrawal period. The [Ca²⁺](i) rise was associated with an increased rate of Mn²⁺ entry, measured as the rate of quenching of intracellular Fura-2. The same procedure did not affect the [Ca²⁺](i) in control cells not pretreated with thapsigargin. The amplitude of this [Ca²⁺](i) transient in thapsigargin pretreated cells depended on the duration of prior incubation in Ca²⁺-free medium. The [Ca²⁺](i) rise induced by elevating the extracellular [Ca²⁺] from 1.5 to 10 mM was more pronounced if the [Ca²⁺](i) during the initial incubation in 1.5 mM Ca²⁺ was first lowered by depolarizing the cells. We conclude that an empty store stimulates a Ca²⁺ entry pathway consisting of two components: a continuously elevated basal leak and a second component that is transient due to the high [Ca²⁺](i)-induced inhibition of the Ca²⁺ entry pathway. This inhibition and the subsequent recovery from it as the [Ca²⁺](i) is brought to resting levels could cause the oscillatory Ca²⁺ entry that we recorded in a fraction of the thapsigargin-treated cells.