A Store-operated Calcium Channel in Drosophila S2 Cells

Using whole-cell recording in Drosophila S2 cells, we characterized a Ca(2+)-selective current that is activated by depletion of intracellular Ca(2+) stores. Passive store depletion with a Ca(2+)-free pipette solution containing 12 mM BAPTA activated an inwardly rectifying Ca(2+) current with a reversal potential >60 mV. Inward currents developed with a delay and reached a maximum of 20–50 pA at −110 mV. This current doubled in amplitude upon increasing external Ca(2+) from 2 to 20 mM and was not affected by substitution of choline for Na(+). A pipette solution containing ∼300 nM free Ca(2+) and 10 mM EGTA prevented spontaneous activation, but Ca(2+) current activated promptly upon application of ionomycin or thapsigargin, or during dialysis with IP(3). Isotonic substitution of 20 mM Ca(2+) by test divalent cations revealed a selectivity sequence of Ba(2+) > Sr(2+) > Ca(2+) >> Mg(2+). Ba(2+) and Sr(2+) currents inactivated within seconds of exposure to zero-Ca(2+) solution at a holding potential of 10 mV. Inactivation of Ba(2+) and Sr(2+) currents showed recovery during strong hyperpolarizing pulses. Noise analysis provided an estimate of unitary conductance values in 20 mM Ca(2+) and Ba(2+) of 36 and 420 fS, respectively. Upon removal of all external divalent ions, a transient monovalent current exhibited strong selectivity for Na(+) over Cs(+). The Ca(2+) current was completely and reversibly blocked by Gd(3+), with an IC(50) value of ∼50 nM, and was also blocked by 20 μM SKF 96365 and by 20 μM 2-APB. At concentrations between 5 and 14 μM, application of 2-APB increased the magnitude of Ca(2+) currents. We conclude that S2 cells express store-operated Ca(2+) channels with many of the same biophysical characteristics as CRAC channels in mammalian cells.