Chlorotoxin‐sensitive Ca2+‐activated Cl− channel in type R2 reactive astrocytes from adult rat brain

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Abstract
Astrocytes express four types of Cl− or anion channels, but Ca2+‐activated Cl− (ClCa) channels have not been described. We studied Cl− channels in a morphologically distinct subpopulation (∼ 5% of cells) of small (10–12 μm, 11.8 ± 0.6 pF), phase‐dark, GFAP‐positive native reactive astrocytes (NRAs) freshly isolated from injured adult rat brains. Their resting potential, −57.1 ± 4.0 mV, polarized to −72.7 ± 4.5 mV with BAPTA‐AM, an intracellular Ca2+ chelator, and depolarized to −30.7 ± 6.1 mV with thapsigargin, which mobilizes Ca2+ from intracellular stores. With nystatin‐perforated patch clamp, thapsigargin activated a current that reversed near the Cl− reversal potential, which was blocked by Cl− channel blockers, 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoate (NPPB) and Zn2+, by I− (10 mM), and by chlorotoxin (EC50 = 47 nM). With conventional whole‐cell clamp, NPPB‐ and Zn2+‐sensitive currents became larger with increasing [Ca2+]i (10, 150, 300 nM). Single‐channel recordings of inside‐out patches confirmed Ca2+ sensitivity of the channel and showed open‐state conductances of 40, 80, 130, and 180 pS, and outside‐out patches confirmed sensitivity to chlorotoxin. In primary culture, small phase‐dark NRAs developed into small GFAP‐positive bipolar cells with chlorotoxin‐sensitive ClCa channels. Imaging with biotinylated chlorotoxin confirmed the presence of label in GFAP‐positive cells from regions of brain injury, but not from uninjured brain. Chlorotoxin‐tagged cells isolated by flow cytometry and cultured up to two passages exhibit positive labeling for GFAP and vimentin, but not for prolyl 4‐hydroxylase (fibroblast), A2B5 (O2A progenitor), or OX‐42 (microglia). Expression of a novel chlorotoxin‐sensitive ClCa channel in a morphologically distinct subpopulation of NRAs distinguishes these cells as a new subtype of reactive astrocyte. GLIA 42:325–339, 2003.