Kv1.3 Regulates The Driving Force For Calcium Entry Through P2x4 In Microglia

Potassium efflux mediated by voltage-gated potassium channels repolarizes neurons and muscle cells following an action potential. In electrically non-excitable immune cells, however, potassium efflux is responsible for the maintenance of a negative membrane potential required for entry of extracellular calcium, which affects many immune functions such as cytokine expression and release. Microglia constitute the main innate immune cells of the brain and are tasked with detecting and responding to danger-associated molecular patterns derived from local tissue damage or pathogenic insults. We and others have previously found that specific activation and differentiation modalities can induce unique repertoires of potassium channels in microglia. As such, pro‑inflammatory lipopolysaccharide (LPS)-activated microglia are characterized by high expression of the voltage-gated Kv1.3 channel. Treatment of LPS-activated cells with Kv1.3-selective inhibitors reduced release of proinflammatory markers such as IL‑1β, iNOS and COX2 expression. Here we sought to understand the mechanism of Kv1.3 inhibition on calcium entry through P2X4, one of the main ATP-gated calcium channels in microglia. Patch-clamp electrophysiology on cultured neonatal mouse microglia showed that activation of P2X4 by low concentrations of ATP induces robust inward currents and membrane depolarization. In addition to its previously described suppression of Kir2.1 and upregulation of Kv1.3, activation with LPS reduced P2X4 mRNA expression and ATP‑induced currents. Fluorescence imaging assays using the Fluo-4 calcium indicator dye confirmed that calcium entry is reduced in LPS‑stimulated cells. Pharmacological inhibition of Kv1.3 negatively affects microglial calcium responses to ATP, demonstrating its functional significance in restoring the negative membrane potential necessary for calcium entry. Since Kv1.3 inhibitors effectively reduce microglia-associated inflammatory responses in vitro and in vivo, our data explain, in part, their mode of action on regulating intracellular calcium by reducing calcium entry through P2X4 receptors.