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. 2000 Apr;115(4):405-16.
doi: 10.1085/jgp.115.4.405.

Membrane cholesterol content modulates activation of volume-regulated anion current in bovine endothelial cells

I Levitan  1 , A E Christian, T N Tulenko, G H Rothblat
Affiliations

Membrane cholesterol content modulates activation of volume-regulated anion current in bovine endothelial cells

I Levitan et al. J Gen Physiol. 2000 Apr.

Abstract

Activation of volume-regulated anion current (VRAC) plays a key role in the maintenance of cellular volume homeostasis. The mechanisms, however, that regulate VRAC activity are not fully understood. We have examined whether VRAC activation is modulated by the cholesterol content of the membrane bilayer. The cholesterol content of bovine aortic endothelial cells was increased by two independent methods: (a) exposure to a methyl-beta-cyclodextrin saturated with cholesterol, or (b) exposure to cholesterol-enriched lipid dispersions. Enrichment of bovine aortic endothelial cells with cholesterol resulted in a suppression of VRAC activation in response to a mild osmotic gradient, but not to a strong osmotic gradient. Depletion of membrane cholesterol by exposing the cells to methyl-beta-cyclodextrin not complexed with cholesterol resulted in an enhancement of VRAC activation when the cells were challenged with a mild osmotic gradient. VRAC activity in cells challenged with a strong osmotic gradient were unaffected by depletion of membrane cholesterol. These observations show that changes in membrane cholesterol content shift VRAC sensitivity to osmotic gradients. Changes in VRAC activation were not accompanied by changes in anion permeability ratios, indicating that channel selectivity was not affected by the changes in membrane cholesterol. This suggests that membrane cholesterol content affects the equilibrium between the closed and open states of VRAC channel rather than the basic pore properties of the channel. We hypothesize that changes in membrane cholesterol modulate VRAC activity by affecting the membrane deformation energy associated with channel opening.

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Figures

Figure 1
Figure 1
Modulation of free cholesterol content in BAEC by MβCD. (A) Cells were exposed to 5 mM MβCD: cholesterol (8:1 mol:mol) in DMEM, pH 7.2, with no serum. (B) Cells were exposed to 5 mM MβCD that was not complexed with cholesterol. Control cells were treated with serum-free medium. The bars show means ± SD (n = 3). Free cholesterol content in cells exposed to 30 min of saturated MβCD; cholesterol was significantly different from that in control cells (P < 0.05). Longer exposures in this experiment do not show statistical significance because of the high variation between the samples. Therefore, this experiment (120-min exposure) was repeated two more times (n = 3 in each experiment). In both experiments, the difference was statistically significant at the level of P < 0.01. The difference between the cholesterol content in cells exposed to free MβCD for 30, 60, or 120 min was significantly different from that in control for all exposure times (P < 0.01).
Figure 3
Figure 3
VRAC activation in cells depleted of cholesterol. The experimental protocol is as described for Fig. 2. (A and C) Current traces from individual cells challenged with a mild (A) or strong (C) osmotic gradient. (B and D) Average time courses of VRAC current densities in response to a mild (B) or strong (D) osmotic gradient. Note that average VRAC time courses in control cells in this experiment are different from those in the previous experiment for both osmotic gradients. This difference reflects the variability between the experimental batches of cells that results from uncontrollable fluctuations in the conditions of cell culture. To minimize this difference, VRAC activity in cells depleted of cholesterol is compared with VRAC activity in control cells recorded on the same day, as described in detail in results. Similar to the previous experiment, plateau VRAC current densities in cells depleted of cholesterol were significantly different from those in control cells when the cells were challenged with a mild osmotic gradient (P < 0.01; B), but were not different in cells challenged with a strong osmotic gradient (D).
Figure 2
Figure 2
VRAC activation in cells enriched with cholesterol. (A and C) Families of current traces recorded from individual cells that were challenged with a mild osmotic gradient (extracellular/intracellular osmotic ratio of 0.85; A) or with a strong osmotic gradient (extracellular/intracellular osmotic ratio of 0.70; C). Currents were elicited by linear voltage ramps from a holding potential of −60 to +60 mV, and recorded 50, 200, 350, 500, and 650 s after the establishment of the whole cell configuration. (B and D) Average time courses of VRAC current densities that developed in cells exposed to saturated MβCD:cholesterol solutions (120 min) and in control cells in response to a mild osmotic gradient (B) or to a strong osmotic gradient (D). Current densities were calculated by normalizing the maximal current amplitudes of each individual ramp by the cell capacitance. Since cell capacitance does not change significantly during the experiment, the currents were normalized to the initial cell capacitance measured immediately after the establishment of the whole-cell configuration. Plateau VRAC current densities (calculated by averaging the current densities between 350 and 600 s after the establishment of the whole cell configuration) in cells enriched with cholesterol were significantly smaller from those in control cells when the cells were challenged with a mild osmotic gradient (P < 0.01; B). There was no significant difference between the plateau VRAC current densities in cholesterol-enriched and control cells when the cells were challenged with a strong osmotic gradient (D).
Figure 5
Figure 5
VRAC activation in BAEC is suppressed by cholesterol-enriched lipid dispersions. (A) Families of current traces recorded from cells exposed to cholesterol-free liposomes (0:1 FC:PL molar ratio), to liposomes with 1:2 FC:PL ratio and to cholesterol-rich lipid dispersions with 2:1 FC:PL ratio. The experimental protocol was similar to that described in Fig. 2. (B) Average time courses of VRAC development for cells exposed to 0:1; 2:1, and 1:2 FC:PL lipid dispersions. Average plateau current densities in cells exposed to 2:1 FC:PL lipid dispersions were significantly smaller than those in cells exposed to 0:1 FC:PL (P < 0.05). There is no difference between cells exposed to 0:1 or 1:2 FC:PL.
Figure 4
Figure 4
The dependence of mean peak current density on membrane cholesterol content. The points represent mean peak VRAC current densities plotted as a function of average levels of free cholesterol in cells exposed to various experimental conditions. All cells were challenged with a mild osmotic gradient. The conditions are specified near each point. The data is described by linear fit with a correlation coefficient of 0.98. (The fit was calculated using Igor WaveMetrics software.) Peak current densities in cells enriched with cholesterol and cells depleted from cholesterol were significantly different from those in control cells (P < 0.05).
Figure 6
Figure 6
Modulation of membrane cholesterol has no effect on VRAC anion permeability ratios. Permeability ratios for glutamate−/Cl− (▪) and aspartate−/Cl− (□) were calculated from the values of measured reversal potentials. Reversal potentials were measured in at least five cells for each experimental group. Data points represent means ± SEM. There was no significant difference between VRAC permeability ratios in cells enriched with or depleted from cholesterol.
Figure 8
Figure 8
Cell capacitance of BAEC is not altered by modulation of membrane cholesterol. (A) Cell capacitance was measured immediately after the establishment of the whole cell configuration. The bars represent the average cell capacitance of cells exposed to MβCD:cholesterol (n = 19), cells exposed to empty MβCD (n = 16) for at least 60 min, and control cells (n = 18). There is no significant difference in cell capacitance between these experimental groups. (B) Cell capacitance does not change during cell swelling. Cells were challenged by a transmembrane osmotic gradient and capacitance measured every 10 s for the duration of the experiment. The values of cell capacitance at each time point were normalized to the initial capacitance measured at time 0.
Figure 7
Figure 7
Voltage-dependent inactivation of VRAC is not altered by the modulation of membrane cholesterol. (A) Families of traces recorded in response to a two-pulse voltage protocol with a 500-ms conditioning pulse from −60 to +140 mV, followed immediately with a short 10-ms test pulse to +100 mV. (B) Inactivation ratios were calculated as Itest,V/I control test, where Itest,V is the current amplitude of a test pulse after a conditioning pulse to voltage V, and Icontrol test is the current amplitude of a test pulse applied from a holding potential of −60 mV. The points represent means ± SEM (n = 7 for each experimental group). There was no significant difference in the voltage dependence of the inactivation between cells enriched with or depleted from cholesterol.
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