Chloride Influx Provokes Lamellipodium Formation in Microglial Cells

Abstract

Lamellipodium extension and retraction is the driving force for cell migration. Although several studies document that activation of chloride channels are essential in cell migration, little is known about their contribution in lamellipodium formation. To address this question, we characterized chloride channels and transporters by whole cell recording and RT-PCR, respectively, as well as quantified lamellipodium formation in murine primary microglial cells as well as the microglial cell-line, BV-2, using time-lapse microscopy. The repertoire of chloride conducting pathways in BV-2 cells included, swelling-activated chloride channels as well as the KCl cotransporters, KCC1, KCC2, KCC3, and KCC4. Swelling-activated chloride channels were either activated by a hypoosmotic solution or by a high KCl saline, which promotes K⁺ and Cl^- influx instead of efflux by KCCs. Conductance through swelling-activated chloride channels was completely blocked by flufenamic acid (200μM), SITS (1mM) and DIOA (10μM). By exposing primary microglial cells or BV-2 cells to a high KCl saline, we observed a local swelling, which developed into a prominent lamellipodium. Blockade of chloride influx by flufenamic acid (200μM) or DIOA (10μM) as well as incubation of cells in a chloride-free high K⁺ saline suppressed formation of a lamellipodium. We assume that local swellings, established by an increase in chloride influx, are a general principle in formation of lamellipodia in eukaryotic cells.

Swelling activated Cl- channels in microglia

“…2 In murine microglia, membrane stretch slowly induced a Cl -current, and cell morphology was altered by several drugs, including SITS and DIDS. 16,31 The underlying mechanism likely differs from I Clswell in rat microglia, which is not blocked by SITS or DIDS at normal membrane potentials. 1,2 It is important to note that SITS and DIDS are potent inhibitors of Cl -/HCO 3 -exchangers, which confounds their use in cell function studies.…”

Physiology of Microglia

“…Interestingly, SITS was less potent in respect to the inhibition of phagocytosis, whereas DIOA had no effect (304). Furthermore, the inhibition of swelling-activated Cl Ϫ channels in primary cultured microglia by flufenamic acid (200 M) or DIOA (10 M) or the incubation of the cells in Cl Ϫ -free high K ϩ solution suppressed the formation of a lamellipodia (1040). The volume-sensitive Cl Ϫ channel blockers NPPB, tamoxifen, and DIDS prevented the rapid process outgrowth of microglial processes initiated in response to brain tissue damage.…”

“…Chloride channels and transporters were characterized by whole cell recording and RT-PCR (1040). The repertoire of chloride-conducting pathways in murine primary microglial cells includes the K-Cl cotransporters, KCC1, KCC2, KCC3, and KCC4, as well as swelling-activated chloride channels.…”

CFTR Involvement in Cell Migration and Epithelial Restitution

“…As previously mentioned localized osmotic swelling can also contribute to the force that powers lamellipodia protrusion [59]. Solute uptake serves as a driving force for fluid uptake into the cell, often involving electroneutral transporters that couple cation uptake with Cl -transport (e.g.…”