ATP-evoked intracellular Ca²⁺ transients shape the ionic permeability of human microglia from epileptic temporal cortex.

ABSTRACT:

Background

Intracellular Ca²⁺ 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 Ca²⁺ concentration ([Ca²⁺]_i). Here, we aimed to functionally characterize human microglia obtained from cortices of subjects with temporal lobe epilepsy, focusing on the Ca²⁺-mediated response triggered by purinergic signaling.

Methods

Fura-2 based fluorescence microscopy was used to measure [Ca²⁺]_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 Ca²⁺-dependent whole-cell currents.

Results

In human microglia extracellular ATP evoked [Ca²⁺]_i increases depend on Ca²⁺ entry from the extracellular space and on Ca²⁺ mobilization from intracellular compartments. Extracellular ATP also induced a transient fivefold potentiation of the total transmembrane current, which was completely abolished when [Ca²⁺]_i increases were prevented by removing external Ca²⁺ and using an intracellular Ca²⁺ chelator. TRAM-34, a selective K_Ca3.1 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 Ca²⁺ transient amplitude revealed a linear correlation. Treatment of microglial cells with LPS for 48?h did not prevent the ATP-induced Ca²⁺ mobilization but completely abolished the ATP-mediated current potentiation. The absence of the Ca²⁺-evoked K⁺ current led to a less sustained ATP-evoked Ca²⁺ entry, as shown by the faster Ca²⁺ transient kinetics observed in LPS-treated microglia.

Conclusions

Our study confirms a functional role for K_Ca3.1 channels in human microglia, linking ATP-evoked Ca²⁺ transients to changes in membrane conductance, with an inflammation-dependent mechanism, and suggests that during brain inflammation the K_Ca3.1-mediated microglial response to purinergic signaling may be reduced.

SUBMITTER: Palomba NP 

PROVIDER: S-EPMC7883449 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature