Effect of Hydrophobic Surfactant Proteins SP-B and SP-C on the Permeability of Phospholipid Membranes

Abstract

Pulmonary surfactant is a complex mixture of lipids and proteins whose main function is to reduce surface tension at the alveolar air-liquid interface in order to avoid alveolar collapse at the end of expiration and facilitate the work of breathing. It is composed by around 90% lipids and 8-10% specific proteins, including the hydrophobic SP-B and SP-C. In this study, we have analyzed the effect of hydrophobic surfactant proteins on the permeability of phospholipid membranes by two different approaches: fluorescence microscopy of giant vesicles (GV) and electroconduction in planar lipid membranes. The effect of surfactant proteins on the permeability of GV membranes was assessed under the microscope using the fluorescent water-soluble probes FM®1-43 and calcein. Membrane-sensitive FM®1-43 only labels the external leaflet of membranes, and calcein emits green fluorescence in aqueous media. Neither can permeate through pure lipid membranes. In the presence of physiological amounts of SP-B and SP-C, giant oligolamellar POPC vesicles incorporated FM®1-43 in every single membrane when added to the external medium and were also permeable to calcein. These results suggest the existence of direct connections between aqueous compartments of GV in the presence of these proteins. On the other hand, planar lipid membranes (PLM) have been widely used to study ionic permeation through phospholipid bilayers mediated by membrane proteins. Permeability of bilayers incorporating small amounts of hydrophobic surfactant proteins has been analyzed in PLMs prepared by the dual monolayer technique. Conductance and selectivity experiments as well as noise analysis of the signals were performed. All our measurements indicate the formation of channel-like structures with defined properties in terms of ionic conductance and selectivity. Possible implications of membrane-permeabilizing structures in the biological context of the pulmonary surfactant system will be discussed.