Individual Leaflets of a Membrane Bilayer Can Independently Regulate Permeability

Abstract

Water rapidly crosses most membranes, but only slowly crosses apical membranes of barrier epithelia such as bladder and kidney collecting duct, a feature essential to barrier function. How apical membrane structure reduces permeabilities remains unclear. Cell plasma membranes contain two leaflets of distinct lipid composition; the role of this bilayer asymmetry in membrane permeability is unclear. To determine how asymmetry of leaflet composition affects membrane permeability, effects on bilayer permeation of reducing single leaflet permeability were determined using two approaches: formation of asymmetric bilayers in an Ussing chamber, with only one of two leaflets containing cholesterol sulfate, and stabilization of the external leaflet of unilamellar vesicles with praeseodymium (Pr 3؉ ). In both systems, permeability measurements showed that each leaflet acts as an independent resistor of water permeation. These results show that a single bilayer leaflet can act as the barrier to permeation and provide direct evidence that segregation of lipids to create a low permeability exofacial leaflet may act to reduce the permeability of barrier epithelial apical membranes.Epithelial cells generate and maintain apical membrane bilayers made up of leaflets of distinct composition by mechanisms involving asymmetric biosynthesis in the Golgi, oriented insertion into the plasma membrane, and the activity of ATPdriven phospholipid flippases (1-6). In several epithelia, such as those of the stomach, kidney collecting duct and thick ascending limb, and mammalian bladder, these apical membranes exhibit exceptionally low permeabilities to water, small nonelectrolytes, protons, and ammonia. These low permeabilities are critical to the barrier function of these epithelia (7-11). The structural features responsible for the low permeabilities of these apical membranes remain poorly defined as is the physiological role of the bilayer asymmetry observed in these membranes. The present studies examine the role of bilayer asymmetry in governing the permeability of membranes to water by measuring permeabilities across artificial symmetric and asymmetric membranes. The results show that a single leaflet of a membrane bilayer can act as a barrier to water flux and provide strong evidence that each leaflet acts as an independent resistor to permeation.

Reconstituted Aquaporin 1 Water Channels Transport CO2 across Membranes

“…Before flux measurements, extravesicular CF was removed by passing the liposomes through a Sephadex G-50 column as described previously (11). Previous studies have demonstrated that this approach forms unilamellar vesicles (5,12); median vesicular size was determined by quasielastic light scattering (5,11).…”

“…Vesicles were prepared by heating to 40°C and then cooling on ice three times, followed by 20 serial extrusions of the mixture through a 0.1-m pore polycarbonate filter using an Avanti mini-extruder (Avanti Polar Lipids, Inc., Alabaster, AL) warmed on a heating block to ϳ50°C as described (5,10,11). Before flux measurements, extravesicular CF was removed by passing the liposomes through a Sephadex G-50 column as described previously (11). Previous studies have demonstrated that this approach forms unilamellar vesicles (5,12); median vesicular size was determined by quasielastic light scattering (5,11).…”

Reconstituting the Barrier Properties of a Water-tight Epithelial Membrane by Design of Leaflet-specific Liposomes

“…Water Permeability Measurements-Osmotic water permeability (P f ) was measured at 25 C as described (3,18,20). All other permeabilities were measured at 25 C also.…”

“…These experiments revealed an extremely low water permeability for exofacial liposomes of 2.4 Ϯ 0.4 ϫ 10 Ϫ4 cm/s, whereas cytoplasmic liposomes were 18.1 times more permeable with a P f ϭ 4.4 Ϯ 0.3 ϫ 10 Ϫ3 cm/s (p Ͻ 0.05). Previous work from our laboratory has shown that the leaflets in a bilayer each offer independent resistances to water and solute permeation (18,19). From Equation 1 we can calculate a single leaflet permeability from the exofacial liposomes and a single leaflet permeability for the cytoplasmic liposomes (both symmetric bilayers) and combine them to arrive at a theoretical permeability for an MDCK apical membrane.…”

“…Because the lipid structure of the MDCK cell apical membrane is well defined, we aimed to reconstitute the low permeability properties of the MDCK type 1 cell, in an effort to understand the determinants that contribute significantly to this membrane's barrier properties (17). We also wished to further validate our earlier findings that each leaflet in a phospholipid bilayer offers an independent resistance to permeation (18,19). This hypothesis can be summarized by the following equation, in which the resistance offered by each leaflet is equivalent to the reciprocal of the permeability.…”