A Store-operated Calcium Channel in Drosophila S2 Cells

Abstract

Using whole-cell recording in Drosophila S2 cells, we characterized a Ca²⁺-selective current that is activated by depletion of intracellular Ca²⁺ stores. Passive store depletion with a Ca²⁺-free pipette solution containing 12 mM BAPTA activated an inwardly rectifying Ca²⁺ 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 Ca²⁺ from 2 to 20 mM and was not affected by substitution of choline for Na⁺. A pipette solution containing ∼300 nM free Ca²⁺ and 10 mM EGTA prevented spontaneous activation, but Ca²⁺ current activated promptly upon application of ionomycin or thapsigargin, or during dialysis with IP₃. Isotonic substitution of 20 mM Ca²⁺ by test divalent cations revealed a selectivity sequence of Ba²⁺ > Sr²⁺ > Ca²⁺ >> Mg²⁺. Ba²⁺ and Sr²⁺ currents inactivated within seconds of exposure to zero-Ca²⁺ solution at a holding potential of 10 mV. Inactivation of Ba²⁺ and Sr²⁺ currents showed recovery during strong hyperpolarizing pulses. Noise analysis provided an estimate of unitary conductance values in 20 mM Ca²⁺ and Ba²⁺ 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 Ca²⁺ current was completely and reversibly blocked by Gd³⁺, with an IC₅₀ 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 Ca²⁺ currents. We conclude that S2 cells express store-operated Ca²⁺ channels with many of the same biophysical characteristics as CRAC channels in mammalian cells.