Oligomeric forms of the membrane-bound acetylcholine receptor disclosed upon extraction of the M(r) 43,000 nonreceptor peptide

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Abstract
Oligomeric forms of the acetylcholine receptor are directly visualized by EM in receptor-rich membranes from Torpedo marmorata. The receptor structures are quantitatively correlated with the molecular species identified only after detergent solubilization, and related to the polypeptide composition of the membranes and changes in it. Structural identification is made possible by the increased fragility of the membranes after extraction of nonreceptor peptides and their subsequent disruption upon drying onto hydrophilic carbon supports. Receptor particles in native membranes depleted of nonreceptor peptides appear as single units of 7-8 nm, and double and multiple aggregates of them. Particle doublets having a main-axis diameter of 19 .+-. 3 nm predominate in these membranes. Linear aggregates of particles similar to those observed in rotary replicas of quick-frozen fresh electrocytes are present in the alkaline-extracted membranes. Chemical modifications of the thiol groups shift the distribution of structural species. Dithiothreitol reduction, which renders almost exclusively the 9S, monomeric receptor form, results in the observation of the 7-8 nm particle in isolated form. The proportion of doublets increases in membranes alkylated with N-ethylmaleimide. Treatment with 5,5''-dithio-bis-(nitrobenzoic acid) increases the proportion of higher oligomeric species, and particle aggregates (n = oligo) predominate. The nonreceptor .nu.-peptide (doublet of MW 43,000) appears to play a role in the receptor monomer-polymer equilibria. Receptor protein and .nu.-peptide co-aggregate upon reduction and reoxidation of native membranes. In membranes protected ad initio with N-ethylmaleimide, only the receptor appears to self-aggregate. The .nu.-peptide cannot be extracted from these alkylated membranes, though it is easily released from normal, subsequently alkylated or reduced membranes. A stabilization of the dimeric species by the nonreceptor .nu.-peptide is suggested by these experiments. Monospecific antibodies against the .nu.-peptide are used in conjunction with rhodamine-labeled anti-antibodies in an indirect immunofluorescence assay to map the vectorial exposure of the .nu.-peptide. When intact membranes, .nu.-peptide depleted and holey native membranes (treated with 0.3% saponin) are compared, maximal labeling is obtained with the latter type of membranes, suggesting a predominantly cytoplasmic exposure of the antigenic determinants of the .nu.-peptide in the membrane. The influence of the .nu.-peptide in the thiol-dependent interconversions of the receptor protein and the putative topography of the peptide are analyzed in light of the results.

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