Anion channels modulate store-operated calcium influx in human microglia

Abstract

Recent work from this laboratory has demonstrated that purinergic-mediated depolarization of human microglia inhibited a store-operated pathway for entry of Ca2+. We have used Fura-2 spectrofluorometry to investigate the effects on store-operated Ca2+influx induced by replacement of NaCI with Na-gluconate 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 solution (PSS). The magnitude of this response was depressed by 70% after replacement of PSS with low CI–PSS. A second procedure used ATP, initially applied in Ca2+-free PSS solution to deplete intracellular stores. The subsequent perfusion of PSS solution containing Ca2+resulted in a large and sustained 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 maintained stores-operated influx of Ca2+was evident which was inhibited by 77% in the presence of the low Cl–PSS. Ca2+influx was linearly reduced with cell depolarization in elevated K+(7.5 to 35mM) suggesting that changes in external Cl–were manifest as altered electrical driving force for Ca2+entry. However, 50mM external KCl effectively eliminated divalent entry which may indicate inactivation of this pathway with high magnitudes of depolarization. Patch clamp studies showed low Cl–PSS to cause depolarizing shifts in both holding currents and reversal potentials of currents activated with voltage ramps. The results demonstrate that Cl–channels play an important role in regulating store-operated entry of Ca2+in human microglia.