The external membrane leaflet plays a key role in the organization of the cell plasma membrane as a mosaic of ordered microdomains enriched in sphingolipids and cholesterol and of fluid domains. In this study, the thermotropic behavior and the topology of bilayers made of a phosphatidylcholine/sphingomyelin mixture, which mimicks the lipid composition of the external leaflet of renal brush-border membranes, were examined by differential scanning calorimetry and atomic force microscopy. In the absence of cholesterol, a broad phase separation process occurred where ordered gel phase domains of size varying from the mesoscopic to the microscopic scale, enriched in sphingomyelin, occupied half of the bilayer surface at room temperature. Increasing amounts of cholesterol progressively decreased the enthalpy of the transition and modified the topology of membranes domains up to a concentration of 33 mol % for which no membrane domains were detected. These results strongly suggest that, in membranes highly enriched in sphingolipids like renal and intestinal brush borders, there is a threshold close to the physiological concentration above which cholesterol acts as a suppressor rather than as a promoter of membrane domains. They also suggest that cholesterol depletion does not abolish the lateral heterogenity in brush-border membranes.According to the current view, the plasma membrane of eucaryotic cells is organized in an in-plane mosaic of microdomains (1, 2). Rafts correspond to a category of microdomains, enriched in sphingolipids (SPL) 1 and cholesterol (Chl), which play a key role in the expression and regulation of the plasma membrane functions (3, 4). This conclusion was reached essentially through the use of two experimental procedures, the low temperature non-ionic detergent extraction (2) and the Chl depletion of cells (5, 6). The resistance to low temperature, non-ionic detergent extraction of numerous membrane proteins is associated to a liquid ordered (L o ) or to a gel ordered (L ␤ ) state of membrane lipids, which strongly suggests that the physical state of these membrane lipids is of primary importance in the formation of the membrane microdomains mosaic (7,8). Formation of the L o phase, or more precisely of the fluid liquid ordered L o␣ and gel liquid ordered L o␤ phases (9), depends on the presence of Chl (10, 11). SPL also appear to be determinant for the existence of eucaryotic plasma membrane rafts (3, 4), and this could be explained by the preferential interaction of Chl with SPL rather than with the other phospholipid species in natural phospholipid-Chl mixtures (10, 12, 13). Because SPL are essentially localized on the external leaflet of the plasma membrane (14), this strongly suggests that this membrane leaflet plays a crucial role in the existence of microdomains.Renal brush-border membranes (BBM), which constitute the apical membrane of the proximal tubule epithelial cells, are highly ordered structures, as shown by fluorescence polarization and ESR data (15). Their glycerophospholipid GPL...

The plasma membrane outer leaflet plays a key role in determining the existence of rafts and detergent-resistant membrane domains. Monolayers with lipid composition mimicking that of the outer leaflet of renal brush border membranes (BBM) have been deposited on mica and studied by atomic force microscopy. Sphingomyelin (SM) and palmitoyloleoyl phosphatidylcholine (POPC) mixtures, at molar ratios varying from 2:1 to 4:1, were phase-separated into liquid condensed (LC) SM-enriched phase and liquid expanded (LE) POPC-enriched phase. The LC phase accounted for 33 and 58% of the monolayers surface for 2:1 and 4:1 mixtures, respectively. Addition of 20-50 mol % cholesterol (Chl) to the SM/POPC (3:1) mixtures induced marked changes in the topology of monolayers. Whereas Chl promoted the connection between SM domains at 20 mol %, increasing Chl concentration progressively reduced the size of domains and the height differences between the phases. Lateral heterogeneity was, however, still present at 33 mol % Chl. The results indicate that the lipid composition of the outer leaflet is most likely responsible for the BBM thermotropic transition properties. They also strongly suggest that the common maneuver that consists of depleting membrane cholesterol to suppress rafts does not abolish the lateral heterogeneity of BBM membranes.