Chlorotoxin-sensitive Ca2+-activated Cl- channel in type R2 reactive astrocytes from adult rat brain.
Astrocytes express four types of Cl(-) or anion channels, but Ca(2+)-activated Cl(-) (Cl(Ca)) channels have not been described. We studied Cl(-) channels in a morphologically distinct subpopulation ( approximately 5% of cells) of small (10-12 micro m, 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 Ca(2+) chelator, and depolarized to -30.7 +/- 6.1 mV with thapsigargin, which mobilizes Ca(2+) 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 Zn(2+), by I(-) (10 mM), and by chlorotoxin (EC(50) = 47 nM). With conventional whole-cell clamp, NPPB- and Zn(2+)-sensitive currents became larger with increasing [Ca(2+)](i) (10, 150, 300 nM). Single-channel recordings of inside-out patches confirmed Ca(2+) 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 Cl(Ca) 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 Cl(Ca) channel in a morphologically distinct subpopulation of NRAs distinguishes these cells as a new subtype of reactive astrocyte.
Department of Neurosurgery, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Stanislava
S
Dalton
Volodymyr
V
Gerzanich
Mingkui
M
Chen
Yafeng
Y
Dong
Yaroslav
Y
Shuba
J Marc
JM
Simard
D000818:Animals; D001253:Astrocytes / Q000145:classification / Q000187:drug effects / Q000378:metabolism*; D015415:Biomarkers; D001921:Brain / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D018118:Chloride Channels / Q000187:drug effects / Q000378:metabolism*; D005434:Flow Cytometry; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D005911:Gliosis / Q000378:metabolism / Q000503:physiopathology; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051381:Rats; D012604:Scorpion Venoms / Q000494:pharmacology*; D014746:Vimentin / Q000378:metabolism
D015415:Biomarkers; D018118:Chloride Channels; D005904:Glial Fibrillary Acidic Protein; D012604:Scorpion Venoms; D014746:Vimentin; C091539:Chlorotoxin; D002118:Calcium