McCrea P D, Popot J L, Engelman D M
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511.
EMBO J. 1987 Dec 1;6(12):3619-26. doi: 10.1002/j.1460-2075.1987.tb02693.x.
Current folding models for the nicotinic acetylcholine receptor (AChR) predict either four or five transmembrane segments per subunit. The N-terminus of each subunit is almost certainly extracellular. We have tested folding models by determining biochemically the cellular location of an intermolecular disulfide bridge thought to lie at the delta subunit C-terminus. Dimers of AChR linked through the delta-delta bridge were prepared from Torpedo marmorata and T.californica electric organ. The disulfide's accessibility to hydrophilic reductants was tested in a reconstituted vesicle system. In right-side-out vesicles (greater than 95% ACh binding sites outwards), the bridge was equally accessible whether or not vesicles had been disrupted by freeze--thawing or by detergents. Control experiments based on the rate of reduction of entrapped diphtheria toxin and measurements of radioactive reductant efflux demonstrated that the vesicles provide an adequate permeability barrier. In reconstituted vesicles containing AChR dimers in scrambled orientations, right-side-out dimers were reduced to monomers three times more rapidly than inside-out dimers, consistent with the measured rate of reductant permeation. These observations indicate that in reconstituted vesicles the delta-delta disulfide bridge lies in the same aqueous space as the ACh binding sites. They are most easily reconciled with folding models that propose an even number of transmembrane crossing per subunit.
目前烟碱型乙酰胆碱受体(AChR)的折叠模型预测每个亚基有四个或五个跨膜片段。几乎可以肯定每个亚基的N端位于细胞外。我们通过生物化学方法确定了一个被认为位于δ亚基C端的分子间二硫键的细胞位置,以此来测试折叠模型。通过电鳐和加州电鳐的电器官制备了通过δ-δ桥连接的AChR二聚体。在重构的囊泡系统中测试了二硫键对亲水性还原剂的可及性。在外翻囊泡(超过95%的ACh结合位点向外)中,无论囊泡是否通过冻融或去污剂破坏,该桥对还原剂的可及性是相同的。基于被困白喉毒素的还原速率和放射性还原剂流出量测量的对照实验表明,囊泡提供了足够的渗透屏障。在含有随机取向的AChR二聚体的重构囊泡中,外翻二聚体还原为单体的速度比内翻二聚体快三倍,这与测量的还原剂渗透速率一致。这些观察结果表明,在重构囊泡中,δ-δ二硫键与ACh结合位点位于相同的水相空间。它们最容易与每个亚基有偶数个跨膜穿越的折叠模型相符合。