Correspondence to: Irena Levitan, 1010 Vagelos Research Labs., Institute for Medicine and Engineering, 3340 Smith Walk, University of Pennsylvania, Philadelphia, PA 19104-6306. Fax:215-573-7227 E-mail:ilevitan{at}mail.med.upenn.edu.
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Abstract |
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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-ß-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-ß-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.
Key Words: anion channels, volume regulation, cholesterol
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INTRODUCTION |
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A variety of physiological and pathophysiological factors increase the intracellular osmolarity and challenge cellular volume homeostasis (
Transitions between the closed and open states of ion channels are thought to result from conformational changes of the channel proteins (
If membrane deformation energy constitutes a significant contribution to the energetic cost of VRAC activation, then changes in the mechanical properties of the membrane bilayer that affect membrane deformation energy would be expected to alter VRAC activity. Membrane deformation energy can been approximated by a phenomenological spring model in which bilayer mechanical properties are characterized by a bilayer stiffness coefficient A, defined as A = G0def/µ2, where µ is the sum of the deformation depths of the monolayers of the membrane (
In the present study, we show that enriching endothelial cells with cholesterol using two independent methods suppresses VRAC activation, while depletion of membrane cholesterol enhances activation of the current. These observations support our hypothesis that membrane deformation energy is involved in the regulation of VRAC activity. Further studies, however, are needed to discriminate between the roles of membrane deformation energy and of specific sterolprotein interactions in the regulation of VRAC activity.
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METHODS |
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Tissue Culture
Bovine endothelial cells were grown in DMEM culture medium (Cell Grow; Fisher Scientific/Mediatech) supplemented with 10% fetal bovine serum (GIBCO BRL). Cell cultures were maintained in a humidified incubator at 37°C with 5% CO2. The cells were fed and split every 34 d. We used cells between passages 10 and 30. There was no difference in VRAC activation between the earlier and later passages.
Cholesterol Enrichment
Two methods have been used to enrich the cells with cholesterol: exposure of cells to methyl-ß-cyclodextrin (MßCD) solution saturated with cholesterol and exposure of cells to cholesterol-enriched lipid dispersions.
MßCD-cholesterol solution was prepared as described previously (
Cholesterol-rich liposomes (dispersions) were prepared as described previously (
Cholesterol Measurement
Lipid was extracted from the washed cell monolayer using isopropanol as previously described (
Electrophysiological Recording
Solutions.
External recording solution contained (mM): 150 NaCl, 1 EGTA, 2 CaCl2, 10 HEPES, pH 7.2. Internal solutions contained (mM): 120 CsGlut or CsAsp, 10 HEPES, 4 ATP, pH 7.2 (CsOH) with free [Ca2+] ~10 nM (0.1 CaCl2, 1.1 EGTA). Intracellular solutions contained Cs+ to decrease possible contamination of Cl- outward current (inward flux) by outward K+ currents (outward flux). Chemicals were obtained from Fisher Scientific or Sigma Chemical Co. The osmolarities of all solutions were determined immediately before recording with a vapor pressure osmometer (Wescor Inc.) and were adjusted by the addition of sucrose, as required.
Recording.
The current was activated by challenging cells with a transmembrane hyposmotic gradient between the intracellular and extracellular solutions. The gradient was created by membrane rupture. Current development was monitored by 500-ms linear voltage ramps from a holding potential of -60 to +60 mV at an interpulse interval of 10 s. Normal cellular current convention is used when referring to the direction of current; i.e., outward current refers to inward Cl- ion flow. Ionic currents were measured using the whole cell configuration of the standard patch clamp technique (, using the above recording solutions. Fire polishing was necessary for N51A glass to create high resistance seals. Part of the experiments were performed using SGI glass from Richland Glass Co. These pipettes generated high resistance seals without fire polishing. NaCl agar bridge was used for a reference electrode. Currents were recorded using an EPC9 amplifier (HEKA Electronik) and accompanying acquisition and analysis software (Pulse & PulseFit; HEKA Electronik) running on a Macintosh Quadra 700 or PowerCenter 150. Pipette and whole-cell capacitance was automatically compensated. Whole cell capacitance and series resistance were monitored throughout the recording. In recordings using cells with large current amplitudes (>500 pA), series resistance compensation (95% compensation with a 100-µs lag) was used. Occasionally, a lower percentage compensation was required to prevent current oscillation. Cells exhibiting small current amplitudes (<500 pA) did not require series resistance compensation. In these cases, the voltage error involved was <5%.
Statistical Analysis
Statistical analysis of the data was performed using a standard two-sample Student's t test assuming unequal variances of the two data sets. Statistical significance was determined using a two-tail distribution assumption and was set at 5% level (P < 0.05).
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RESULTS |
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Exposure to MßCD:Cholesterol Modulates Cholesterol Level in Endothelial Cells
Recent studies have demonstrated that cyclic oligosaccharides ß-cyclodextrins provide a precise and reproducible method for modulating membrane cholesterol content in several cell types (
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Exposure of BAEC to MßCD saturated with cholesterol for time periods between 30 and 120 min resulted in a gradual increase of cholesterol content in these cells (Fig 1 A). A 45% increase that was observed after 120 min is similar to the degree of cholesterol enrichment in endothelial cells exposed to ßvery low density lippoprotein (ß-VLDL) (
Modulation of Cholesterol Content Affects Activation of VRAC
Cholesterol enrichment with saturated MßCD.
Activation of VRAC was tested by challenging the cells either with a mild osmotic gradient (extracellular:intracellular osmotic ratio of 0.85) or with a strong osmotic gradient (0.70 extracellular:intracellular osmotic ratio). Osmotic gradients were maintained throughout the experiment. Enrichment of the cells with cholesterol resulted in suppression of VRAC activation when the cells were challenged with a mild osmotic gradient (Fig 2A and Fig B), but had no effect on VRAC activation when the cells were challenged with a strong osmotic gradient (Fig 2C and Fig D).
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Fig 2 A shows typical VRAC currents that developed in a cell that was exposed to MßCD:cholesterol solution for 120 min (bottom) and in a control cell (top) when both cells were challenged with a mild osmotic gradient and recorded on the same day. Currents that developed in response to a strong osmotic gradient are shown in Fig 2 C. All currents were elicited by voltage ramps from a holding potential of -60 to +60 mV and recorded 50, 200, 350, 500, and 650 s after challenging the cells with an osmotic gradient. Amplitudes of the currents elicited by the same voltage ramps increase as VRAC develops. An outward rectification of current traces results from an endogenous rectification typical for VRAC currents and from a nonsymmetric anion composition of the recording solutions.
The experimental conditions allow recording of VRAC with minimal contamination from cation currents (see METHODS). Therefore, activation of VRAC is measured as an increase in outward current amplitude (net inward Cl- flux) under hyposmotic conditions. Average time courses of VRAC development in cells exposed to saturated MßCD:cholesterol solution and in control cells are compared in Fig 2B and Fig D. The currents are normalized for cell capacitance to eliminate the variability of VRAC amplitudes that may result from the differences in the sizes of individual cells. In all our experiments, VRAC activity in cells exposed to MßCD:cholesterol (or cholesterol-free MßCD) solutions was compared with VRAC activity in control cells recorded on the same day. Since stable VRAC recording could be achieved only from four to five cells a day, in a typical experiment the currents were recorded from two to three cells enriched with cholesterol and from two to three control cells. The time courses of VRAC development were then averaged over at least three experimental days. This protocol allowed us to minimize the variability in VRAC activity between the experiments that could arise from uncontrollable fluctuations in the cell culture conditions.
Similar to our earlier observations (
Membrane cholesterol depletion. To test whether the suppression of VRAC activation by MßCD:cholesterol is due to cholesterol enrichment and not to an effect of MßCD itself, current activation was determined in cells exposed to cholesterol-free MßCD. Exposure of cells to cholesterol-free MßCD for 120 min resulted in a marked increase in VRAC amplitudes that developed in response to a mild osmotic gradient (Fig 3A and Fig B). A few cells in this experimental group developed currents in the 300 pA/pF range, currents that we never observed in control cells recorded on the same days when challenged with a mild osmotic gradient. Exposure to empty MßCD, however, had no effect on VRAC currents that developed in response to a strong osmotic gradient (Fig 3C and Fig D), indicating that a decrease in membrane cholesterol increases the VRAC sensitivity to the osmotic challenge.
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Fig 4 shows mean peak VRAC current densities measured in cells challenged with a mild osmotic gradient after the cells were exposed to MßCD:cholesterol or to empty MßCD solutions for variable periods of time (30120 min). Mean peak currents decrease gradually with an increase in cell cholesterol content. Linear regression analysis yielded a correlation coefficient of 0.98. Enhancement of VRAC activation in cells exposed to empty MßCD confirmed that VRAC activation is sensitive to the cholesterol content of the membrane. The opposite effects of saturated MßCD:cholesterol complexes and of cholesterol-free MßCD on VRAC activation exclude the possibility that suppression of VRAC development in cells enriched with cholesterol is due to an effect of the MßCD alone.
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Cholesterol-enriched lipid dispersions.
Dependence of VRAC activation on cholesterol content of the membrane was further confirmed using an alternative method to enrich the cells with cholesterol. Cholesterol-enriched lipid dispersions with a cholesterol:phospholipid molar ratio of 2:1 have been previously used to study the effects of cholesterol enrichment on function of smooth muscle cells (
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Cells that were exposed to cholesterol-enriched lipid dispersions developed much smaller currents than cells that were exposed to control lipid dispersions (Fig 5). There was no difference in VRAC activity between cells that were exposed to 0:1 and 1:2 lipid dispersions, but cells that were not exposed to any lipid dispersions developed significantly larger currents (Fig 5, inset). One hypothesis to explain this observation is that the cell membrane equilibrates with liposomal lipids during the relatively long exposure (48 h) and that leads to an alteration of cholesterol distribution in the cellular membranes. Since cholesterol is distributed nonrandomly in the plasma membranes (
Modulation of the Membrane Cholesterol Content Has No Effect on VRAC Anion Selectivity
Earlier studies have shown that VRAC channels are permeable not only to inorganic anions but organic anions, such as glutamate and aspartate (
where Paa/PCl is the ratio of the permeability of the relevant amino acid to that of Cl-. A lack of cholesterol effect on the anion permeability ratios suggests that the basic pore properties of the channel are not affected by the changes in membrane cholesterol.
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Modulation of the Membrane Cholesterol Content Has No Effect on VRAC Inactivation Properties
Voltage-dependent inactivation is a hallmark of volume-regulated anion current in a variety of cell types (
Voltage-dependent inactivation of VRAC in cells exposed either to MßCD:cholesterol complexes, cholesterol-free MßCD, as well as control cells are shown in Fig 7. A two-pulse voltage protocol with a conditioning pulse ranging from -60 to +140 mV followed by a test pulse to +100 mV was used to determine the voltage sensitivity and kinetic properties of the inactivation. Voltage-dependent inactivation is apparent from the accelerated decay of the anion current at depolarization more positive than +80 mV, as shown in Fig 7 A. The inactivation ratio was defined as the ratio between the amplitude of a test pulse delivered after a conditioning prepulse to the amplitude of a control test pulse (Fig 7 B). The steepness of the decrease of the inactivation ratio provides a measure of the amount of charge that has to move for a channel to change its conformation from an open to an inactivated state. A similarity between the inactivation curves in cells enriched with, or depleted from, cholesterol suggests that changes in cholesterol content do not affect the transition between the open and inactivated states of the channel. A differential effect of membrane cholesterol on activation and inactivation of the same channels has been reported previously for N-type calcium channels (
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Modulation of Cholesterol Content Has No Effect on Membrane Capacitance
Earlier studies have shown that a two- to threefold increase of membrane cholesterol results in a 1020% increase in membrane bilayer thickness in aortic smooth muscle cells (
In the present study, we have evaluated the thickness of endothelial membranes in cholesterol-depleted and cholesterol-enriched cells by measuring electrical capacitance of the membrane. Since the specific capacitance of the membrane is inversely proportional to its width (
where is the dielectric constant of the membrane and
0 is the polarizability of the free space. In this case, a 20% increase in membrane thickness may result in up to a 20% decrease in specific membrane capacitance. In the present study, however, changes in cell cholesterol had no effect on membrane capacitance of endothelial cells (Fig 8 A), suggesting that thickness of the membrane was not altered significantly.
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Fig 8 B shows that endothelial cell capacitance did not increase when the cells were challenged osmotically, indicating that there is no increase in membrane area during cell swelling. This observation is in agreement with our earlier studies in B-lymphocytes (
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DISCUSSION |
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The main finding of this study is that VRAC activation in endothelial cells is modulated by the cholesterol content of the membrane when the cells are challenged with a mild osmotic gradient, but not when the cells are challenged with a strong osmotic gradient. These observations provide direct evidence that the lipid environment of the plasma membrane plays a role in the regulation of VRAC. The effect of cholesterol is specific for VRAC activation in the sense that elevation of membrane cholesterol reduces the steady state (plateau) level of VRAC current density without having any effect on VRAC ion selectivity or its inactivation properties.
One mechanism that may be responsible for the suppression of VRAC activity in response to the elevation of membrane cholesterol is a shift in the equilibrium between the open and closed states of the channel towards the closed state. Alternatively, it may alter the basic pore properties of the channel, decreasing the single-channel conductance. Earlier studies have shown that an increase in membrane cholesterol content reduced the open probability of Ca2+-dependent K+ channels (
To determine whether VRAC suppression by an increase in membrane cholesterol results from a shift in the equilibrium between VRAC conformational states or from a decrease in its single channel conductance, we have compared the effects of membrane cholesterol on VRAC activation in cells challenged with mild or strong osmotic gradients. The rationale of this approach is that if elevation of membrane cholesterol affects the equilibrium between the channel conformational states, then membrane cholesterol is expected to shift the sensitivity of VRAC to osmotic stimuli so that the channel becomes less sensitive to the osmotic challenge. In this case, the saturating level of stimulus is expected to invoke the same responses at low and high membrane cholesterol. By contrast, if cholesterol decreases VRAC single channel conductance, then VRAC activity is expected to be suppressed at all levels of the osmotic stimulus. Our study shows that an increase in membrane cholesterol suppresses VRAC activation when the cells are challenged with a mild osmotic gradient (0.85 osmotic ratio), whereas it had no effect on VRAC development when the cells were challenged with a strong osmotic gradient (0.70 osmotic ratio). These observations suggest that increasing membrane cholesterol affects the equilibrium between the open and closed states of VRAC channel rather than its single channel conductance. A lack of cholesterol effect on glutamate-:Cl- and aspartate-:Cl- permeability ratios supports the conclusion that membrane cholesterol content has no effect on the basic pore properties of VRAC and that its effect on VRAC activity is through a shift in the equilibrium between the channel conformational states.
How can changes in membrane cholesterol shift the equilibrium between the closed and open states of the channel? We suggest that suppression of VRAC by an increase in membrane cholesterol is due to an increase in membrane deformation energy that is associated with the transition between the closed and open states of the channel. Membrane deformation energy has been described as a function of three major components: (a) a compressionexpansion component that depends on membrane thickness, (b) a splaydistortion component that depends on the orientation of the phospholipid hydrocarbon chains of the lipid molecules, and (c) a surface tension component that depends on the density of the polar head groups along the surface of the membrane (T/(
A/A), where
T is a change in membrane tension accompanied by a relative increase of membrane area of
A/A, is considered a measure of membrane stiffness (
Is it possible to determine which of the mechanical properties of the membrane bilayer that are affected by membrane cholesterol content are responsible for the effect of cholesterol on VRAC activation? A complete separation of different mechanical properties is not likely possible because of the interdependence between these properties (
Alternatively, membrane cholesterol may affect the intrinsic activation energy of the VRAC protein through specific sterolprotein interactions. In many cases, there is no strong specificity between intrinsic proteins and surrounding lipids (
We propose a model for the role of membrane deformation energy in the mechanism by which VRAC channels may sense osmotic gradients across the plasma membrane. We suggest that cell swelling may alter membrane deformation energy that is associated with VRAC activation by altering the physical properties of the membrane bilayer. Specifically, membrane tension may alter the thickness of the membrane, consequently modulating a hydrophobic mismatch between the channel hydrophobic exterior and the thickness of the bilayer hydrophobic core (
Elevation of plasma cholesterol levels is associated with an increased risk of atherosclerosis (
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Footnotes |
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1 Abbreviations used in this paper: BAEC, bovine aortic endothelial cells; FC, unesterified (free) cholesterol; MßCD, methyl-ß-cyclodextrin; PL, unesterified egg phosphatidylcholine; VRAC, volume-regulated anion current.
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Acknowledgements |
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We thank Dr. Sarah Garber for her support during the initial stages of these experiments and for many interesting and helpful discussions. We thank Drs. Peter F. Davies, Olaf S. Andersen, John M. Russell, Michal Bental-Roof, and Michael M. White for critical reading of the manuscript. We are grateful to Ms. Liz Cannon for excellent technical assistance in preparing lipid dispersions and lipid measurements and to Ms. Rebecca Riley for her help with cell culture.
This work was supported by National Institutes of Health grants NIDDK47762 (S. Garber), HL 30496 (T.N. Tulenko), Program Project Grant HL 22633 (G.H. Rothblat), and Training Grant HL 07443 (A.E. Christian).
Submitted: 11 November 1999
Revised: 3 February 2000
Accepted: 7 February 2000
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