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Comparative Study
. 2004 Feb;123(2):167-82.
doi: 10.1085/jgp.200308982.

A store-operated calcium channel in Drosophila S2 cells

Andriy V Yeromin  1 , Jack Roos, Kenneth A Stauderman, Michael D Cahalan
Affiliations
Comparative Study

A store-operated calcium channel in Drosophila S2 cells

Andriy V Yeromin et al. J Gen Physiol. 2004 Feb.

Abstract

Using whole-cell recording in Drosophila S2 cells, we characterized a Ca(2+)-selective current that is activated by depletion of intracellular Ca2+ stores. Passive store depletion with a Ca(2+)-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 approximately 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 approximately 50 nM, and was also blocked by 20 microM SKF 96365 and by 20 microM 2-APB. At concentrations between 5 and 14 microM, 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.

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Figures

F<sc>igure</sc> 1.
Figure 1.
Thapsigargin-dependent Ca2+ entry in S2 cells. Fluo-4 fluorescence changes were monitored using a FLIPR384. After 20 s of recording, the 384-well pipette-tip head was lowered into the solution creating an offset artifact in the recording. This offset artifact is unrelated to a cellular response and is dependent on the fluid volume in each well at the start of the experiment and the extent of tip penetration into the solution. 10 s after lowering the pipette-tip head, either thapsigargin (TG, 1 μM final, circles) or DMSO (triangles) was injected (arrow). CaCl2 was then added to achieve a final concentration of 1.8 mM. Traces were zeroed at time 0, and each data point represents the mean (±SEM) of 192 replicates.
F<sc>igure</sc> 2.
Figure 2.
Development of inwardly rectifying Ca2+ current during dialysis with BAPTA. Pipette solution 9 (passive store depletion). (A) I-V relations during voltage ramps from −110 to +110 mV in external solutions containing 2 or 20 mM Ca2+. (B) Time course of Ca2+ currents measured at −110 mV at varying times after break-in to achieve whole-cell recording. Open and solid bars above represent exposure to 2 (□) or 20 (▪) mM Ca2+ (solutions 1 and 3). Arrows a and b indicate the time corresponding to I-V curves in A. (C) Leak-subtracted I-V relations of Ca2+ current in Na+ or choline (Ch) external solutions (1 and 2). Leak traces were recorded in the presence of Na+ or choline before current development. (D) Time course of Ca2+ current after break-in. Solid and open bars represent alternating exposure to Na+ (□) and choline (Ch; ▪). Arrows a and b indicate the time corresponding to I-V curves in C.
F<sc>igure</sc> 3.
Figure 3.
Activation of Ca2+ current by store depletion. Pipette solution 11 (Ca2+ buffered to 310 nM). (A) Control currents with no activation of Ca2+ current (note current scale). (B) IP3-activated Ca2+ currents (10 μM IP3 added to pipette), during exposure to 2 (□) or 20 (▪) mM Ca2+. Note complex changes in current during exposure to varying external Ca2+. (C) Thapsigargin (TG, 1 μM) applied externally at indicated time (gray bars), during exposure to 2 (□) or 20 (▪) mM Ca2+. (D) Ionomycin (10 μM) applied externally at indicated times (gray bars), during exposure to 2 (□) or 20 (▪) mM Ca2+.
F<sc>igure</sc> 4.
Figure 4.
Selectivity among divalent cations. Pipette solution 9. Bars indicate exposure to varying divalent cations at indicated concentrations. Currents at −110 mV are shown during exposure to 2 (□) or 20 (▪) mM Ca2+, or to a test divalent cation (gray square). Labeled I-V curves were acquired at indicated times on corresponding development time courses. (A) Ion selectivity; Ca2+ >> Mg2+. (B) I-V curves comparing 2 and 20 mM Ca2+ with 20 mM Mg2+. (C) Ba2+ current. Note partial inactivation of current after changing to Ba2+. (D) Ba2+ I-V relation; note “hook” and steeper inward rectification with Ba2+. (E) Sr2+ current. Note partial inactivation of current after changing to Sr2+. (F) Sr2+ I-V relation. Note steeper inward rectification with Sr2+.
F<sc>igure</sc> 5.
Figure 5.
Ca2+, Sr2+, and Ba2+ currents during pulses. Pipette solution 9. (A) Comparison of normalized currents at −110 mV with 20 mM Ca2+, Sr2+, or Ba2+. Note time-dependent increase in current with Sr2+ and Ba2+. (B) Currents in response to voltage pulses ranging from −110 to +110 in 10-mV increments from the holding potential of 10 mV. Currents were recorded during exposure to 20 mM Ca2+ (left) or 20 mM Ba2+ (right). (C) Corresponding I-V curves (not leak subtracted) at beginning (squares) and end (triangles) of pulses. Note increased inward rectification at end of pulse with Ba2+.
F<sc>igure</sc> 6.
Figure 6.
Noise analysis of divalent currents. Pipette solution 9. Currents at varying times, indicated to the right of each trace, in exemplary experiments with 20 mM Ca2+ (A) and 20 mM Ba2+ (C). Data were recorded at 5 kHz sampling and post-filtered at 1 kHz. Currents are shown during the last 180 ms of a 660 ms pulse to −110 mV. Ba2+ currents were corrected for ∼3% residual activation by subtraction of a linear function. This procedure did not significantly affect the value of single-channel current. (B, D) Variance analysis of Ca2+ and Ba2+ currents. Background-subtracted variances (σ2 − σ0 2) plotted as a function of the mean current during the development of Ca2+ current (B) or Ba2+ current (D). Background variance was between 0.5 and 0.6 pA2 in five experiments. The labeled points correspond to traces in A and C. Data are fitted by a linear function (n = 58, correlation coefficient 0.81 for Ca2+; and n = 189, correlation coefficient 0.95 for Ba2+). The slopes indicate a single channel current i of 9.7 ± 0.4 fA for Ca2+ and 80.8 ± 0.8 fA for Ba2+.
F<sc>igure</sc> 7.
Figure 7.
Store-operated current carried by Na+ in divalent-free solution. Pipette solution 9. (A) Time course of currents after development of Ca2+ current with 2 mM external Ca2+ (□), and during subsequent exposure to divalent-free Na+-containing solution (▪; solution 7). (B) Corresponding Ca2+ and Na+ I-V curves. (C) Cs+ does not carry measurable monovalent current. After development of Ca2+ current (□), transient inward currents upon divalent withdrawal were only seen with Na+ (▪; solution 7) but not Cs+ (gray square, solution 8). (D) Corresponding I-V curves.
F<sc>igure</sc> 8.
Figure 8.
Pharmacological sensitivity to Gd3+, SKF 96365, and 2-APB. Pipette solution 9. (A) Gd3+ block. After exposure to 2 Ca2+ (□), Gd3+ added to 20 mM Ca2+-containing external solution (▪) at progressively higher concentrations (gray bars) caused reversible block of Ca2+ current. (B) Dose-response curve for Gd3+ fitted to: I/I0 = IC50/(IC50 + [Gd3+]), with IC50 value of 46 nM; combined data for 12 cells. (C) Effect of SKF 96365 (20 μM) on Ca2+ current. (D) Effect of 2-APB at indicated concentrations on Ca2+ current.
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