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 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.
Yeromin
Andriy V
AV
Department of Physiology and Biophysics, University of California, Irvine, CA 92697-4561, USA.
Roos
Jack
J
Stauderman
Kenneth A
KA
Cahalan
Michael D
MD
D000818:Animals; D002021:Buffers; D002118:Calcium / Q000502:physiology*; D015220:Calcium Channels / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D003956:Dialysis; D004330:Drosophila; D029721:Drosophila Proteins / Q000378:metabolism / Q000502:physiology*; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D019284:Thapsigargin / Q000494:pharmacology
D002021:Buffers; D015220:Calcium Channels; D029721:Drosophila Proteins; D004533:Egtazic Acid; D019284:Thapsigargin; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium