Small‐conductance Cl^– channels contribute to volume regulation and phagocytosis in microglia

Abstract

The shape and volume of microglia (brain immune cells) change when they activate during brain inflammation and become migratory and phagocytic. Swollen rat microglia express a large Cl(-) current (I(Clswell)), whose biophysical properties and functional roles are poorly understood and whose molecular identity is unknown. We constructed a fingerprint of useful biophysical properties for comparison with I(Clswell) in other cell types and with cloned Cl(-) channels. The microglial I(Clswell) was rapidly activated by cell swelling but not by voltage, and showed no time-dependence during voltage-clamp steps. Like I(Clswell) in many cell types, the halide selectivity sequence was I(-) > Br(-) > Cl(-) > F(-). However, it differed in lacking inactivation, even at +100 mV with high extracellular Mg(2+), and in having a much lower single-channel conductance: 1-3 pS. Based on these fundamental differences, the microglia channel is apparently a different gene product than the more common intermediate-conductance I(Clswell). Microglia express several candidate genes, with relative mRNA expression levels of: CLIC1 > ClC3 > I(Cln) > or = ClC2 > Best2 > Best1 > or = Best3 > Best4. Using a pharmacological toolbox, we show that all drugs that reduced the microglia current (NPPB, IAA-94, flufenamic acid and DIOA) increased the resting cell volume in isotonic solution and inhibited the regulatory volume decrease that followed cell swelling in hypotonic solution. Both channel blockers tested (NPPB and flufenamic acid) dose-dependently inhibited microglia phagocytosis of E. coli bacteria. Because I(Clswell) is involved in microglia functions that involve shape and volume changes, it is potentially important for controlling their ability to migrate to damage sites and phagocytose dead cells and debris.

Reversed Na+/Ca2+Exchange Contributes to Ca2+Influx and Respiratory Burst in Microglia

“…Phagocytic microglia are rod-shaped or nearly spherical. 28 The relative order of mRNA expression for the Na + /Ca 2+ exchangers was: NCX1 > NCX3 > NCX2, where '>' denotes a significant difference from the preceding gene (p < 0.05). No significant changes were detected following a 1 h treatment with opsonized zymosan to stimulate phagocytosis and the respiratory Role of Reversed Na + /Ca 2+ Exchange in Microglia burst (see Fig.…”

“…Such a mechanism is consistent with the dramatically reduced respiratory burst when voltage-dependent (K v ) or Ca 2+ -dependent (SK) channels are blocked in microglia. 22,24,68 In addition, Cl -influx through anion channels 28,69 could help counteract the depolarization resulting from electron efflux. We recently showed that anion channels are major determinants of the membrane potential in microglia 26 and thus, should affect Ca 2+ entry, and also contribute to phagocytosis.…”

“…Transcript levels were monitored by real time quantitative RT-PCR (qRT-PCR), 27 as we recently described for microglia. 25,[28][29][30] Gene-specific primers (Table 1) were designed using the 'Primer3Output' program (http://frodo.wi.mit.edu/cgi-bin/ primer3/primer3_www.cgi). RNeasy mini kits (Qiagen, Mississauga, ON) were used to isolate RNA after degrading any contaminating DNA with DNaseI (0.1 U/ml, 15 min, 37°C; Amersham Biosciences, PQ).…”

Propofol inhibits pressure-stimulated macrophage phagocytosis via the GABAA receptor and dysregulation of p130cas phosphorylation

“…Phagocytosis in human macrophages is accompanied by changes in ion channel currents (24), and Cl Ϫ channels are important for phagocytosis in neutrophil and microglia (15,37). Propofol, also known as a GABA A receptor agonist, binds to the ␤ 2 -subunit of the GABA A receptor (4), which regulates Cl Ϫ channels (32).…”

“…Propofol binds to the ␤ 2 -subunit of the GABA A receptor (4,16), which regulates Cl Ϫ channels (32). Some investigators have reported that ion channels, such as those regulating Cl Ϫ and Ca 2ϩ fluxes, might affect phagocytosis by macrophages and microglia (15,24). The effects of propofol on macrophage phagocytic activity, therefore, may be potentially important in surgical wound healing.…”