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. 2009 Feb;101(2):750-7.
doi: 10.1152/jn.90840.2008. Epub 2008 Nov 26.

Chloride accumulation drives volume dynamics underlying cell proliferation and migration

Christa W Habela  1 , Nola Jean Ernest, Amanda F Swindall, Harald Sontheimer
Affiliations

Chloride accumulation drives volume dynamics underlying cell proliferation and migration

Christa W Habela et al. J Neurophysiol. 2009 Feb.

Abstract

During brain development, progenitor cells migrate over long distances through narrow and tortuous extracellular spaces posing significant demands on the cell's ability to alter cell volume. This phenotype is recapitulated in primary brain tumors. We demonstrate here that volume changes occurring spontaneously in these cells are mediated by the flux of Cl- along with obligated water across the cell membrane. To do so, glioma cells accumulate Cl- to approximately 100 mM, a concentration threefold greater than predicted by the Nernst equation VSports手机版. Shunting this gradient through the sustained opening of exogenously expressed GABA-gated Cl- channels caused a 33% decrease in cell volume and impaired the ability of cells to migrate in a spatially constrained environment. Further, dividing cells condense their cytoplasm prior to mitosis, a phenomenon which is associated with the release of intracellular Cl- as indicated by a 40-mM decrease in [Cl-]i. These findings provide a new framework for considering the role of intracellular Cl- in glioma cells. Here, Cl- serves as an important osmotically active regulator of cell volume being the energetic driving force for volume changes required by immature cells in cell migration and proliferation. This mechanism that was studied in CNS malignancies may be shared with other immature cells in the brain as well. .

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FIG. 1.
FIG. 1.
Glioma cells actively accumulate chloride. A: continuous whole cell voltage-clamp recordings of a ρ1–D54 cell (top, black) and an IRES-D54 (bottom) cell held at −40 mV. Lines indicate times of GABA and picrotoxin application. Blue trace is the same ρ1–D54 cell in the presence of picrotoxin throughout recording. All traces to scale. B: example cell prior to (left) and after (right) whole cell dialysis; 40× magnification. C: example I-V curves from a gramicidin (blue) or a whole cell (black) patch recording. D: intracellular chloride concentrations calculated from the Nernst equation for cells under gramicidin or whole cell patch conditions. Unpaired t-test for gramicidin patched cells, Asterisk, P < 0.05.
FIG. 2.
FIG. 2.
Opening of chloride channels is sufficient to shrink glioma cells. A: example Coulter Counter experiment in which 10 μM GABA was added to the cells represented by closed symbols at t = 0. B: mean traces from 2-dimensional (2-D) time-lapse experiments. GABA (10 μM) was added to the culture media with or without 200 μM bumetanide at t = 0. Each trace is the mean of 3 independent experiments with 20 cells sampled from each experiment.
FIG. 3.
FIG. 3.
Cell volume is chloride dependent during growth phase but not in M phase. A: example 3-dimensional (3-D) reconstructions of a growth phase (left) and an M phase (right) cell. Images not to scale. B: mean volumes if individual control, bumetanide- or DIOA-treated growth phase (bipolar) or M phase cells. Volumes determined by 3-D microscopy. One-way ANOVA P < 0.001, Tukey- Kramer multiple comparison's test **, P < 0.01; ***, P < 0.001. P > 0.05 for comparison between all M phase cells and bumetanide vs. control bipolar cells. C: 2-D time-lapse traces representing the mean of 3 independent experiments for each condition. Cells were chronically treated for 72 h with the respective drugs prior to GABA application. The responses of a total of 51 control cells (red), 59 bumetanide-treated cells and 60 DIOA-treated cells were analyzed.
FIG. 4.
FIG. 4.
An outwardly directed chloride gradient is required for glioma cell migration. A: example fields imaged from Transwell migration inserts from control cells (top) or cells treated with 10 μM GABA prior to and during the migration assay (bottom). B: migration through a Transwell 8 μm pore is impaired in cells treated with 10 μM GABA Results are from 4 independent experiments performed in duplicate. For each assay, the number of cells that had migrated through the filter in 6 fields of view were counted and averaged. Paired t-test, *, P < 0.05.
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