High intracellular chloride delays the activation of the volume-sensitive chloride conductance in mouse L-fibroblasts

Abstract

1. The relationship between cell volume and volume-sensitive Cl- conductance during hyposmotic cell swelling of patched cells and the effects of intracellular chloride on the conductance have been studied in mouse L-fibroblasts. To this end, swelling-activated current and cell volume were measured simultaneously in cells dialysed with low-Cl- (16 mM) or high-Cl- (130 mM) solutions using the whole-cell patch-clamp technique and videomicroscopy. 2. The increase in cell volume of patched cells and the volume-sensitive conductance saturated during a 4-5 min exposure to mildly hyposmotic solutions (15-20 % less than isosmotic). The swelling of patched cells varied considerably and was greater than the swelling of intact cells. No correlation between the maximal values of the volume-sensitive conductance and the maximal volumes of swollen cells was evident for cells dialysed with the low-Cl- solutions. 3. The amplitude of the volume-sensitive conductance decreased with a reduction in either extracellular or intracellular Cl- concentration; the size of the maximal conductance was not modulated by intracellular Cl- ions. 4. The activation of the volume-sensitive conductance was slower in high-Cl- cells than in low-Cl- cells whether it was induced by hypotonic cell swelling or by cell inflation; in low-Cl- cells the conductance saturated before the cell volume had reached its maximal value. 5. It is concluded that in patched cells an increase in cell volume triggers activation of the volume-sensitive Cl- conductance but does not determine its amplitude and that the rate of activation of the conductance is affected by the intracellular Cl- concentration.

Figure 1. Hyposmotic swelling-induced current in L-fibroblasts

A, time course of the membrane conductance change in cells dialysed with the high-Cl⁻ (140 mM CsCl) solution in the absence of hyposmotic challenge (□, n= 5) and during application of the hyposmotic (240 mosmol kg⁻¹) solution (▪, n= 11). B, mean I-V characteristics for the hyposmotically swollen cells (n= 11) measured at the break-in (curve 1) and at 4.5 min (curve 2). C, comparison of hyposmotic swelling of intact and patched cells. ○, data for intact cells (the right Y-axis scale), n= 16. The regression lines were drawn with a slope of 18.5 % min⁻¹ for the 0.5-1.25 min interval and 2.9 % min⁻¹ for the 1.25-4.25 min interval. •, data for patched cells (the left Y-axis scale), n= 10. The cells were dialysed with a low-Cl⁻, gluconate-substituted pipette solution. D, correlation between the maximal swollen volumes and the swelling-induced conductances in cells dialysed with low-Cl⁻ solutions (n= 26) and swollen by a 15 % decrease in the extracellular osmolality (to 255 mosmol kg⁻¹). The slope of the regression line is 1.27 ± 1.90 (P= 0.510, F= 0.448). Dotted lines show the 95 % confidence intervals. Holding potential was -37 mV.

Figure 2. Chloride dependence of the swelling-induced current

A, mean (n= 7) I-V characteristics of the swelling-induced current (curve 2) in cells dialysed with the high-Cl⁻ (130 mM KCl) pipette solution. The extracellular hyposmotic solution (240 mosmol kg⁻¹) contained 124 mM Cl⁻. Holding potential was 3 mV. Curve 1 in this (and the next) panel shows the pre-swelling I-V characteristics. The thin line shows a 2nd order polynomial fit to the I-V curve for the low-Cl⁻ cells (from panel B) for comparison. B, mean (n= 6) I-V characteristics of the swelling-induced current (curve 2) in cells dialysed with the low-Cl⁻ (16 mM Cl⁻, aspartate-substituted) solution. Holding potential was -37 mV. C, comparison of the maximal values of the swelling-induced conductance at three different transmembrane Cl⁻ gradients (see the text). Number of cells in each group is shown above the corresponding bars.

Figure 3. Kinetics of hyposmotic cell swelling and the swelling-induced conductance in patched cells at two intracellular Cl⁻ concentrations

A, comparison of maximal volumes of swollen patched cells from the same coverslip dialysed with either the low-Cl⁻ (gluconate-substituted pipette solution,) or high-Cl⁻ solution (▪) during application of the 255 mosmol kg⁻¹ (-15 %) hyposmotic solution. The mean volumes of swollen cells in each group are shown by the horizontal interrupted lines. B, comparison of maximal swelling-induced conductances in the same cells. The interrupted lines show the mean conductances in each cell group. C, time course of volume changes in the low-Cl⁻ (•, n= 4) and the high-Cl⁻ (○, n= 3) cells. Short horizontal lines (here and in panel D) cross the fitted curves at their mid-points. D, time course of the swelling-induced conductance in the low-Cl⁻ (▪) and the high-Cl⁻ (□) cells. Here and in the following figures, the pre-swelling conductance value has been subtracted from all conductance values.

Figure 4. Comparison of the time courses of cell swelling and the activation of the volume-sensitive conductance in cells dialysed with different solutions

Each panel compares the time courses of cell swelling (V) and activation of the volume-sensitive Cl⁻ conductance (G) within one cell group. A, high-Cl⁻ cells (n= 7). B, low-Cl⁻ cells with aspartate as a Cl⁻ substitute (n= 18). C, low-Cl⁻ cells with gluconate as a Cl⁻ substitute (n= 7 for volume and n= 13 for conductance data). D, low-Cl⁻ cells with glutamate as a Cl⁻ substitute (n= 6 for volume and n= 8 for conductance data).

Figure 5. Kinetics of inflation-induced Cl⁻ conductance at two intracellular Cl⁻ concentrations

Time courses of the inflation-induced Cl⁻ conductance in individual low-Cl⁻ (A) or high-Cl⁻ (B) cells. Each cell was inflated immediately after the first measurement of the conductance (time zero in each plot). The lines are the single-phase exponential association curves fitted to the conductance values in individual cells within the first 3 min. The individual half-activation times are 0.20 (○), 0.39 (□), 0.65 (▵), 0.38 (•), 0.40 (▪) and 0.33 min (▴) for the low-Cl⁻ cells and 1.13 (○), 0.93 (□), 1.10 (▵), 1.87 (•), 1.01 (▪) and 0.59 min (▴) for the high-Cl⁻ cells.