Isolated-patch recording from liposomes containing functionally reconstituted chloride channels from Torpedo electroplax

Abstract

Small unilamellar vesicles formed from purified phospholids by detergent/dialysis methods may be enlarged to 30-microns diameter by freezing and thawing. Very-high-resistance seals were formed by applying a glass micropipette to the surface of these large liposomes, and single bilayer "patches" of membrane were isolated from the liposome surface while remaining sealed to the micropipette. The exogenous channel-forming peptides gramicidin and alamethicin induced characteristic single-channel fluctuation behavior in these excised patches held under voltage-clamp conditions. Large liposomes were formed from the small unilamellar vesicles made from cholate extracts of Torpedo electroplax plasma membrane vesicles. Isolated patches formed from these reconstituted membranes displayed current fluctuations due to single voltage-gated Cl- channels from non-innervated-face membranes; the properties of these Cl- channels are identical to those observed in planar bilayer membranes after direct insertion from native membranes. This liposome-patch method combines the advantages of membrane protein incorporation into liposomes with high-resolution electrical recording methods and may provide a generally applicable approach to the study of integral membrane channel proteins after solubilization and reconstitution.