Anion channels modulate store-operated calcium influx in human microglia

Recent work from this laboratory has demonstrated that purinergic-mediated depolarization of human microglia inhibited a store-operated pathway for en try of Ca2+. We have used Fura-2 spectrofluorometry to investigate the effe cts on store-operated Ca2+ influx induced by replacement of NaCl with Na-gl uconate in extracellular solutions. Three separate procedures were used to activate store-operated channels. Platelet activating factor (PAF) was used to generate a sustained influx of Ca2+ in standard physiological saline so lution (PSS). The magnitude of this response was depressed by 70% after rep lacement of PSS with low Cl- PSS. A second procedure used ATP, initially ap plied in Ca2+-free PSS solution to deplete intracellular stores. The subseq uent perfusion of PSS solution containing Ca2+ resulted in a large and sust ained entry of Ca2+, which was inhibited by 75% with low Cl- PSS. The SERCA inhibitor cyclopiazonic acid (CPA) was used to directly deplete stores in zero-Ca2+ PSS. Following the introduction of PSS containing Ca2+, a maintai ned stores-operated influx of Ca2+ was evident which was inhibited by 77% i n the presence of the low Cl- PSS. Ca2+ influx was linearly reduced with ce ll depolarization in elevated K+ (7.5 to 35 mM) suggesting that changes in external Cl- were manifest as altered electrical driving force for Ca2+ ent ry. However, 50 mM external KCl effectively eliminated divalent entry which may indicate inactivation of this pathway with high magnitudes of depolari zation. Patch clamp studies showed low Cl- PSS to cause depolarizing shifts in both holding currents and reversal potentials of currents activated wit h voltage ramps. The results demonstrate that Cl- channels play an importan t role in regulating store-operated entry of Ca2+ in human microglia. (C) 2 000 Harcourt Publishers Ltd.