Transient permeabilization induced osmotically in membrane vesicles from Torpedo electroplax: a mild procedure for trapping small molecules

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Abstract
During hypoosmotic stress, membrane vesicles enriched in acetylcholine receptors from the electric organ of T. californica become more permeable to external tracer molecules. When vesicles are immersed in 3 volumes of water containing 22Na+, 50-70% of 22Na+ equilibration is attained within 90 s. 22Na+ uptake is greatly diminished only 6-10 s after an osmotic shock and vesicle resealing is completeled within 15 s. Also, 90 s after osmotic shock, efflux rates are comparable to those of native vesicles, which indicates the vesicles have resealed. During osmotic shock the entry of molecules into the vesicles increases with the strength of the osmotic shock and depends on the size of the permeant. With a given strength of osmotic shock, the large molecule [3H]inulin (Mr 5000) is taken up less than the smaller molecules 22Na+ and [3H]sucrose. .alpha.-Bungarotoxin binding latency of the vesicles is not affected by osmotic shock. The sideness of the vesicles apparently remains unchanged. The acetylcholine receptors in the vesicles remain functional after osmotic shock; e.g., 90 s after 22Na+ and [3H]sucrose are loaded into vesicles by osmotic shock, only 22Na+ is released by dilution in a buffer containing carbamoylcholine (carbamylcholine) 22Na+ influx into previously shocked vesicles is specifically stimulated by carbamoylcholine. These stimulations in the shocked vesicles are blocked by d-tubocurarine or .alpha.-bungarotoxin, and they are desensitized by preincubation with carbamoylcholine. Using osmotic shock to load molecular probes into these membrane intact vesicles may provide a powerful tool for studying the inner surfaces of the intact vesicles.