Voltage-gated proton currents in microglia of distinct morphology and functional state

doi:10.1016/S0306-4522(98)00710-6

Neuroscience

Volume 91, Issue 4, July 1999, Pages 1415-1424

https://doi.org/10.1016/S0306-4522(98)00710-6 Get rights and content

Abstract

Whole-cell patch-clamp measurements were performed to investigate voltage-gated proton currents (I_PR) in cultured murine microglia of distinct morphology and functional state. We studied I_PR in ameboid microglia of untreated cultures, in ameboid microglia which had been activated by lipopolysaccharide, and in ramified microglia which had been exposed to astrocyte-conditioned medium. Proton currents of these three microglia populations did not differ regarding their activation threshold or the voltage dependence of steady-state activation. Moreover, pharmacological properties of I_PR were similar: proton currents were sensitive to extracellularly applied Zn²⁺ or La³⁺, and could be abolished by each of those at a concentration of 100 μM. In the presence of extracellular Na⁺, I_PR was decreased to a similar small extent due to activity of the Na⁺/H⁺ exchanger in all microglial populations. In contrast, proton currents of microglia differed between the three cell populations with respect to their current density and their time-course of activation: in comparison with untreated microglia, the current density of I_PR was reduced by about 50% in microglia after their treatment with either lipopolysaccharide or astrocyte-conditioned medium. Moreover, I_PR activated significantly more slowly in cells exposed to lipopolysaccharide or astrocyte-conditioned medium than in untreated cells.

It can be concluded that the distinct H⁺ current characteristics of the three microglial populations do not correlate with the functional state of the cells.

Section snippets

Cell culture

Microglia were obtained from brain cell cultures of newborn NMRI mice, supplied by Hamann–Winkelmann (Borchen, Germany). Mixed brain cell cultures were prepared as described previously.¹² In detail, brain cortices had been enzymatically dissociated (15 min at 37°C with 0.25% trypsin, type XI, Sigma, Germany) and a single-cell suspension was achieved by repeated triturations. Cells were seeded into tissue culture flasks at a density of 2–4×10⁶/5 ml in Dulbecco's modified Eagle's medium (DMEM)

H⁺ currents in untreated ameboid microglia, in lipopolysaccharide-stimulated ameboid microglia and in astrocyte-conditioned medium-treated ramified microglia

Voltage-gated proton currents (I_PR) were elicited by depolarizing voltage pulses from the holding potential of −60 mV in 20 mV increments to potentials of up to +80 mV. Voltage commands were applied for 2000 ms duration every 40 s. All measurements were performed at pH_i=6.0 and pH_o=7.5. As shown in Fig. 1A, the threshold of activation of H⁺ currents in untreated microglia was −40 mV. Amplitude of outward H⁺ currents increased at more depolarizing voltage commands. Proton currents were normalized to

Discussion

Voltage-gated proton currents were detected in microglia of distinct morphology and functional state. In response to depolarizing voltage pulses, slowly activating outward H⁺ currents were observed (i) in unstimulated ameboid microglia, (ii) in activated ameboid microglia stimulated with LPS, and (iii) in resting ramified microglia treated with ACM. H⁺ currents of all three microglia populations showed rather similar properties with respect to their activation threshold, steady-state activation

Conclusions

In contrast to microglial Na⁺ and K⁺ channels,¹⁰ voltage-gated H⁺ channels were always expressed in microglia independent of their functional state. Voltage dependence of steady-state activation and pharmacological properties of H⁺ currents appear to be identical in microglia cultured under various conditions. Changes in microglial H⁺ currents, namely a decrease in current density and an increase in activation time-constant, occurred in a similar manner in microglia either after their

Acknowledgements

The authors wish to thank Sieglinde Latta for the excellent preparation of cell cultures and Astrid Düerkop for technical assistance. We are very grateful to Dr Thomas E. DeCoursey for many helpful discussions and comments on the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft grant SFB 507/C3 (to C.E. and U.H.) and a fellowship of the Alexander von Humboldt-Foundation (to C.E.).

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Voltage-gated proton currents in microglia of distinct morphology and functional state

Abstract

Section snippets

Cell culture

H+ currents in untreated ameboid microglia, in lipopolysaccharide-stimulated ameboid microglia and in astrocyte-conditioned medium-treated ramified microglia

Discussion

Conclusions

Acknowledgements

Biophys. J.

Neurosci. Lett.

Neuroscience

Int. Rev. Cytol.

Biochim. biophys. Acta

Neurosci. Lett.

Trends Neurosci.

Neuroscience

Neurosci. Lett.

Expl Cell. Res.

Anat. Anz.

Expl Neurol.

Effect of bacterial wall lipopolysaccharide (LPS) on morphology, motility, and cytoskeletal organization of microglia in cultures

J. Neurosci. Res.

pHi regulation in alveolar macrophages: relative roles of Na+-H+ antiport and H+-ATPase

Am. J. Physiol.

The voltage-activated hydrogen ion conductance in rat alveolar epithelial cells is determined by the pH gradient

J. gen. Physiol.

Voltage-activated hydrogen ion currents

J. Membrane Biol.

Na+-H+ antiport detected through hydrogen ion currents in rat alveolar epithelial cells and human neutrophils

J. gen. Physiol.

Voltage-activated proton currents in human THP-1 monocytes

J. Membrane Biol.

Ion channel expression in PMA-differentiated human THP-1 macrophages

J. Membrane Biol.

H⁺ currents in untreated ameboid microglia, in lipopolysaccharide-stimulated ameboid microglia and in astrocyte-conditioned medium-treated ramified microglia

pH_i regulation in alveolar macrophages: relative roles of Na⁺-H⁺ antiport and H⁺-ATPase

Na⁺-H⁺ antiport detected through hydrogen ion currents in rat alveolar epithelial cells and human neutrophils