Correspondence to: Warren G. Hill, Laboratory of Epithelial Cell Biology, Renal-Electrolyte Division, University of Pittsburgh School of Medicine, A1222 Scaife Hall, 3550 Terrace Street, Pittsburgh, PA 15261. Fax:(412) 624-5009 E-mail:whill{at}pitt.edu.
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Abstract |
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To understand how plasma membranes may limit water flux, we have modeled the apical membrane of MDCK type 1 cells. Previous experiments demonstrated that liposomes designed to mimic the inner and outer leaflet of this membrane exhibited 18-fold lower water permeation for outer leaflet lipids than inner leaflet lipids (Hill, W.G., and M.L. Zeidel. 2000. J. Biol. Chem. 275:3017630185), confirming that the outer leaflet is the primary barrier to permeation. If leaflets in a bilayer resist permeation independently, the following equation estimates single leaflet permeabilities: 1/PAB = 1/PA + 1/PB (Eq. l), where PAB is the permeability of a bilayer composed of leaflets A and B, PA is the permeability of leaflet A, and PB is the permeability of leaflet B. Using Equation 1 for the MDCK leafletspecific liposomes gives an estimated value for the osmotic water permeability (Pf) of 4.6 x 10-4 cm/s (at 25°C) that correlated well with experimentally measured values in intact cells. We have now constructed both symmetric and asymmetric planar lipid bilayers that model the MDCK apical membrane. Water permeability across these bilayers was monitored in the immediate membrane vicinity using a Na+-sensitive scanning microelectrode and an osmotic gradient induced by addition of urea. The near-membrane concentration distribution of solute was used to calculate the velocity of water flow (Pohl, P., S.M. Saparov, and Y.N. Antonenko. 1997. Biophys. J. 72:17111718). At 36°C, Pf was 3.44 ± 0.35 x 10-3 cm/s for symmetrical inner leaflet membranes and 3.40 ± 0.34 x 10-4 cm/s for symmetrical exofacial membranes. From Equation 1, the estimated permeability of an asymmetric membrane is 6.2 x 10-4 cm/s. Water permeability measured for the asymmetric planar bilayer was 6.7 ± 0.7 x 10-4 cm/s, which is within 10% of the calculated value. Direct experimental measurement of Pf for an asymmetric planar membrane confirms that leaflets in a bilayer offer independent and additive resistances to water permeation and validates the use of Equation 1.
Key Words: MDCK cells, apical membrane, fluidity, cell membrane permeability, barrier function
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INTRODUCTION |
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The process of water permeation across lipid membranes has significant implications for cellular physiology and homeostasis (
Although the precise structural features of barrier membranes responsible for reducing water and solute fluxes are not entirely clear, some common features have emerged in recent studies. Water permeability has been shown to correlate strongly with membrane fluidity (
To understand the functional consequences of lipid asymmetry and the role it might play in creating a barrier to water permeation, we have previously modeled the inner and outer leaflet of the MDCK Type 1 apical membrane. This membrane has been shown to have extremely low permeabilities for water and solutes (
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MATERIALS AND METHODS |
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Lipids
The following lipids from Avanti Polar Lipids Inc. were used to construct planar lipid membranes that would reconstitute the MDCK apical membrane: bovine heart phosphatidylethanolamine (Cat. No. 830025), brain phosphatidylserine (Cat. No. 830032), bovine liver phosphatidylinositol (Cat. No. 830042), bovine heart phosphatidylcholine (PC,* Cat. No. 830052), cholesterol (Cat. No. 700000), egg sphingomyelin (Cat. No. 860061), and brain cerebrosides (Cat. No. 131303). The lipid composition of the inner and outer leaflets are shown in Table 1. Justification for these compositions may be found in
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Membrane Formation
Planar bilayer membranes were built by the monolayer apposition technique across a 150250-µm hole in a septum (thickness 25 µm) separating two compartments of a Teflon chamber (
An osmotic gradient was created by addition of urea (Merck) to one side of the membrane only. Because the urea permeability of these membranes was three orders of magnitude smaller than the water permeability (1.6 x 10-6 vs. 4.4 x 10-3 cm/s for cytoplasmic and 3.10-8 vs. 2.4 x 10-4 cm/s for exofacial [
Water Flux Measurements
Transmembrane water flux leads to solute concentration changes in the immediate vicinity of the membrane. Water passing through the membrane dilutes the solution it enters and concentrates the solution it leaves (t (
t was obtained by fitting the sodium concentration profiles to the equation:
where x is the distance from the membrane. D, a, and Cs denote the diffusion coefficient, the stirring parameter, and the solute concentrations at the interface, respectively. Subsequently, t allows us to determine the membrane water permeability, Pf:
where , Cosm, and Vw are the osmotic coefficient, the near-membrane concentration of the solute used to establish the transmembrane gradient, and the partial molar volume of water, respectively (
The sodium concentration distribution in the immediate membrane vicinity was measured using a scanning ion-sensitive microelectrode (Fig 1). The latter was moved perpendicular to the surface of the membrane by a hydraulic microdrive manipulator (Narishige). The touching of the membrane was indicated by a steep potential change (
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To make the electrode, an aluminosilicate glass capillary tube (Science Products GmbH) was pulled in two stages and silanized by bis-(dimethylamino)-dimethylsilane (Fluka). After bending the tip (outside diam 2 µm) perpendicular to the rest of the barrel, ion sensitivity was achieved by filling the tip with an ionophore cocktail (sodium ionophore II cocktail A; Fluka) according to the procedure described by
Voltage sampling between microelectrode and reference electrode, both of which were placed at the trans side of the bilayer membrane, was performed by a digital multimeter connected to an impedance converter (model AD 549; Analogue Devices). Data was transferred to a personal computer via an RS232 interface.
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RESULTS |
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Planar bilayers, both symmetrical and asymmetrical, formed spontaneously across a small diameter aperture (200 µm) in an apparatus shown schematically in Fig 1. Water flux across membranes created in this way was induced by urea addition to the cis side of the membrane. During the experiment, membrane capacitance did not change and was equal to 0.82 ± 0.07 µF/cm2 for both symmetrical and asymmetrical bilayers.
Water flux across the membrane in response to the osmotic gradient and retention of sodium ions in the trans compartment resulted in an increasing sodium concentration in the immediate membrane vicinity. Representative sodium concentration distributions measured adjacent to a membrane constructed from cytoplasmic lipids are shown in Fig 2 A. The higher the osmotic gradient imposed, the higher the degree of near-membrane polarization. The transmembrane water flux densities (Jw) were proportional to the effective osmotic gradient (Fig 2 B). To calculate the latter, the urea gradient was corrected for dilution. Because the diffusion coefficients of urea and sodium are very close, it was assumed that the ratios of their near-membrane and bulk concentrations are identical (
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Similar measurements in the immediate vicinity of membranes formed from exofacial lipids and for asymmetrical bilayers built from an exofacial and a cytoplasmic leaflet revealed a very small polarization of sodium ions making measurements difficult. This was due to the very low water permeation rate across these "tight" membranes. Facilitating water permeation by increasing the temperature to 36°C resulted in well pronounced and reproducible concentration profiles (Fig 3 A, curve 1). With a Pf of only 3.4 ± 0.4 x 10-4 cm/s, the membranes composed of exofacial lipids revealed the lowest permeability coefficient of the three membranes tested (Fig 3 B for combined data). In contrast, the water permeability of cytoplasmic bilayers was 10-fold higher at 34.4 ± 3.5 x 10-4 cm/s (Fig 3 A, curve 2). As predicted, Pf of asymmetrical membranes was not simply the arithmetical mean of the leaflet permeabilities and, indeed, was very close to the concentration curve shown for a symmetric exofacial bilayer (Fig 3 A, curve 3). Calculation of Pf from the experimental profile revealed a value of 6.7 ± 0.7 x 10-4 cm/s that was close to the permeability of exofacial membranes (Fig 3 B).
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To validate the permeability measurements under different experimental geometries, sodium ion dilution adjacent to the cis membranewater interface was measured, and the direction of the water flux across the bilayer was changed (by altering the relative position of the cytoplasmic and exofacial monolayers). Identical water permeabilities were obtained under all experimental conditions (data not shown).
The polarization of sodium ions was studied at different temperatures (Fig 4 A). These experiments permitted calculation of the activation energy (Ea) of transmembrane water transport. The activation energy derived from the slope of the plot in Arrhenius coordinates for cytoplasmic lipids was equal to 14.7 ± 1.5 kcal/mol (Fig 4 B). Similar experiments with symmetrical exofacial membranes revealed an Ea of 22.1 ± 3.0 kcal/mol. Hence, membranes with tighter packing and lower fluidity due to the presence of significant quantities of cholesterol and sphingolipid, have a significantly higher Ea for water permeation. Estimates of water permeability at 25°C for these membranes yield values of 1.36 x 10-3 cm/s and 1.0 x 10-4 cm/s for cytoplasmic and exofacial bilayers, respectively. These compare favorably with previously measured permeabilities in liposomes of 4.4 x 10-3 and 2.4 x 10-4 cm/s, respectively (
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DISCUSSION |
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Much of our current understanding of membrane permeation processes has been achieved by studying the permeant behavior of water across simple model lipids like dipalmitoylphosphatidylcholine (DPPC). An understanding of permeant behavior in cellular membranes is, however, a much more difficult challenge. Cell membranes are extremely complex in both their composition and their structure. In addition to lipids that exhibit variations in head groups, acyl chain lengths, cholesterol concentrations, and degrees of saturation, membranes also exhibit bilayer asymmetry, phase discontinuities, membrane-embedded proteins, and may have their permeation properties affected by extracellular matrix and/or mucus. In these experiments, our aim was to use a novel technique for measuring water permeability across planar bilayers (
Many, if not all cells, are now thought to exhibit differences in the lipid composition of individual leaflets in their plasma membranes (
Previous studies that have attempted to examine bilayer asymmetry have had limitations. Cholesterol sulfate (CS), which has the advantage of very slow translocation rates from one side of the bilayer to the other, was used to dope one or both sides of planar diphytanylphosphatidylcholine (DPhPC) membranes. Adding CS to both sides of a DPhPC bilayer reduced diffusive water permeability from 14.9 x 10-4 to 5.4 x 10-4 cm/s. Adding it to one side of the bilayer to induce lipid asymmetry reduced membrane permeability to 8.9 x 10-4 cm/s (
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(1) |
where PAB is the permeability of a bilayer composed of leaflets A and B, PA is the permeability of leaflet A, and PB is the permeability of leaflet B. Unfortunately, the differences between DPhPC bilayers and DPhPC-CS bilayers were modest, and so alternative interpretations were possible. Indeed, the data are equally well explained by an arithmetic mean, which would suggest that the overall permeability of the bilayer is an average of the permeabilities offered by each leaflet. Bilayer asymmetry also has been artificially induced in DPPC liposomes by addition of the trivalent rare earth metal praseodymium (
We have now used lipid mixtures that reflect the composition of the MDCK apical membrane inner and outer leaflets and created symmetric and asymmetric planar bilayers from them. By the use of a Na+-sensitive microelectrode, which could be positioned within the unstirred layer next to the membrane surface (Fig 1), it was possible to measure an osmotically induced water flux which was proportional to the size of the osmotic gradient imposed (Fig 2). This allowed us to test the validity of Equation 1 as a description for water permeation behavior directly. Measurements of water permeability across symmetric and asymmetric bilayers of MDCK lipids demonstrated a 10-fold difference between inner and outer leaflet membranes at 36°C (Fig 3 B). An asymmetric membrane composed of an inner and an outer leaflet shows a Na+ polarization profile far closer to that of outer leaflet lipids than inner leaflet lipids, and this is reflected in a Pf that is only twofold higher (6.7 x 10-4 cm/s compared with 3.4 x 10-4 cm/s). These data indicate that most of the resistance offered by the MDCK apical membrane to water permeation is contributed by the outer leaflet that is enriched in cholesterol and sphingolipids. Application of Equation 1 to the Pf values calculated from curves 1 and 2 (Fig 3 B) results in an estimated permeability for the MDCK membrane of 6.2 x 10-4 cm/s, which is extremely close to the measured value of 6.7 ± 0.7 x 10-4 cm/s. We conclude that this data validates the use of Equation 1 for describing membrane permeation behavior, and that individual leaflets of a bilayer do indeed offer independent and additive resistances to water permeation. In addition, we believe the results confirm the conclusions reached in earlier studies (
The Ea for water flux across cytoplasmic lipids is similar to that seen for membranes formed from Escherichia coli lipids, as derived from microelectrode measurements on planar membranes (10 to
21 kcal/mol (for a review see
These results demonstrate that outer leaflets act as the major barrier to permeation across barrier apical membranes. In cells specialized to serve as barriers to water flux, such as the collecting duct in the absence of antidiuretic hormone, the thick ascending limb of Henle, the bladder, and the stomach, the process by which the cell achieves barrier function appears to be the segregation of specific lipids into the outer leaflet of the apical membrane. From studies examining the effects of lipid composition on permeabilities (
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Footnotes |
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* Abbreviations used in this paper: CS, cholesterol sulfate; DPPC, dipalmitoylphosphatidylcholine; DPhPC, diphytanylphosphatidylcholine; Ea, activation energy; PC, phosphatidylcholine.
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Acknowledgements |
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We thank Dr. John Mathai for useful discussions.
This work was supported by a Research Fellowship from the National Kidney Foundation (to W.G. Hill) and by the National Institutes of Health Grant DK43955. Financial support of the Deutsche Forschungsgemeinschaft (Po 533/2-3 and Po 533/7-1) is gratefully acknowledged.
Submitted: 14 June 2001
Revised: 14 August 2001
Accepted: 14 August 2001
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