Nicotinic postsynaptic membranes from Torpedo: sidedness, permeability to macromolecules, and topography of major polypeptides.

Abstract

Experiments were conducted to examine the topographic arrangement of the polypeptides of the acetylcholine receptor (AcChR) and the nonreceptor M, 43,000 protein in postsynaptic membranes isolated from Torpedo electric organ. When examined by electron microscopy, >85% of vesicles were not permeable to ferritin or lactoperoxidase (LPO) . Exposure to saponin was identified as a suitable procedure to permeabilize the vesicles to macromolecules with minimal alteration of vesicle size or ultrastructure . The sidedness of vesicles was examined morphologically and biochemically. Comparison of the distribution of intramembrane particles on freeze-fractured vesicles and the distribution found in situ indicated that >85% of the vesicles were extracellular-side out. Vesicles labeled with a-bungarotoxin (a-BgTx) were reacted with antibodies against a-BgTx or against purified AcChR of Torpedo. Bound antibodies were detected by the use of ferritin-conjugated goat anti-rabbit antibody and were located on the outside of >99% of labeled vesicles . Similar results were obtained for normal vesicles or vesicles exposed to saponin. Quantification of the amount of [3 H]-a-BgTx bound to vesicles before and after they were made permeable with saponin indicated that <5% of aBgTx binding sites were cryptic in normal vesicles . It was concluded that >95% of postsynaptic membranes were oriented extracellular-side out.LPO-catalyzed radioiodinations were performed on normal and saponin-treated vesicles and on vesicles from which the Mr (relative molecular mass) 43,000 protein had been removed by alkaline extraction . In normal vesicles, polypeptides of the AcChR were iodinated while the Mr 43,000 protein was not. In vesicles made permeable with saponin, the pattern of labeling of AcChR polypeptides was unchanged, but the M, 43,000 protein was heavily iodinated. The relative iodination of AcChR polypeptides was unchanged in membranes equilibrated with agonist or with a-BgTx or after alkaline-extraction. It was concluded that the M, 43,000 protein is present on the intracellular surface of the postsynaptic membrane and that AcChR polypeptides are exposed on the extracellular surface.Analysis ofthe mechanism of permeability control by nicotinic cholinergic receptors is facilitated by the isolation ofthe plasma membrane of the innervated Torpedo electrocyte surface, which is highly enriched in nicotinic receptors (for a review, see reference 1). The nicotinic receptor (AcChR), which constitutes -r50% of the protein in the purified postsynaptic membrane, is composed of polypeptides of apparent molecular weights

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