Abstract
BACKGROUND: Intracellular Ca(2+) modulates several microglial activities, such as proliferation, migration, phagocytosis, and inflammatory mediator secretion. Extracellular ATP, the levels of which significantly change during epileptic seizures, activates specific receptors leading to an increase of intracellular free Ca(2+) concentration ([Ca(2+)](i)). Here, we aimed to functionally characterize human microglia obtained from cortices of subjects with temporal lobe epilepsy, focusing on the Ca(2+)-mediated response triggered by purinergic signaling. METHODS: Fura-2 based fluorescence microscopy was used to measure [Ca(2+)](i) in primary cultures of human microglial cells obtained from surgical specimens. The perforated patch-clamp technique, which preserves the cytoplasmic milieu, was used to measure ATP-evoked Ca(2+)-dependent whole-cell currents. RESULTS: In human microglia extracellular ATP evoked [Ca(2+)](i) increases depend on Ca(2+) entry from the extracellular space and on Ca(2+) mobilization from intracellular compartments. Extracellular ATP also induced a transient fivefold potentiation of the total transmembrane current, which was completely abolished when [Ca(2+)](i) increases were prevented by removing external Ca(2+) and using an intracellular Ca(2+) chelator. TRAM-34, a selective K(Ca)3.1 blocker, significantly reduced the ATP-induced current potentiation but did not abolish it. The removal of external Cl(-) in the presence of TRAM-34 further lowered the ATP-evoked effect. A direct comparison between the ATP-evoked mean current potentiation and mean Ca(2+) transient amplitude revealed a linear correlation. Treatment of microglial cells with LPS for 48 h did not prevent the ATP-induced Ca(2+) mobilization but completely abolished the ATP-mediated current potentiation. The absence of the Ca(2+)-evoked K(+) current led to a less sustained ATP-evoked Ca(2+) entry, as shown by the faster Ca(2+) transient kinetics observed in LPS-treated microglia. CONCLUSIONS: Our study confirms a functional role for K(Ca)3.1 channels in human microglia, linking ATP-evoked Ca(2+) transients to changes in membrane conductance, with an inflammation-dependent mechanism, and suggests that during brain inflammation the K(Ca)3.1-mediated microglial response to purinergic signaling may be reduced.