Swelling activated Cl- channels in microglia: Biophysics, pharmacology and role in glutamate release

Microglia have a swelling-activated Cl⁻ current (which we call I_Clswell), and while some of its biophysical properties and functional roles have been elucidated, its molecular identity is unknown. To relate this current to cell functions and determine whether it is regulated by mechanisms other than cell swelling, it is important to establish both biophysical and pharmacological fingerprints. Here, we used rat microglia and a cell line derived from them (MLS-9) to study biophysical, regulatory and pharmacological properties of I_Clswell. The whole-cell current was activated in response to a hypo-osmotic bath solution, but not by voltage, and was time-independent during long voltage steps. The halide selectivity sequence was I⁻>Br⁻>Cl⁻ (Eisenman sequence I) and importantly, the excitatory amino acid, glutamate was permeant. Current activation required internal ATP, and was not affected by the guanine nucleotides, GTPγS or GDPβS, or physiological levels of internal Mg²⁺. The same current was activated by a low intracellular ionic strength solution without an osmotic gradient. I_Clswell was reversibly inhibited by known Cl⁻ channel blockers (NPP B, flufenamic acid, glibenclamide, DCPIB), and by the glutamate release inhibitor, riluzole. Cell swelling evoked glutamate release from primary microglia and MLS-9 cells, and this was inhibited by the blockers (above), and by IAA-94, but not by tamoxifen or the Na⁺/K⁺/Cl⁻ symport inhibitor, bumetanide. Together, these results confirm the similarity of I_Clswell in the two cell types, and point to a role for this channel in inflammation-mediated glutamate release in the CNS.Key words: rat microglia, MLS-9 cells, swelling-activated anion channels, VRAC, Cl⁻ channel biophysics, Cl⁻ channel pharmacology, ionic-strength, ATP-dependence, glutamate release