Heidmann T, Sobel A, Popot J L, Changeux J P
Eur J Biochem. 1980 Sep;110(1):35-55. doi: 10.1111/j.1432-1033.1980.tb04839.x.
The 'functional' state of the acetylcholine receptor protein has been followed during reconstitution with the fluorescent agonist [1-(5-dimethylaminonaphthalene)-sulfonamidol]-n-hexanoic acid-beta-N-trimetylammonium bromide ethyl ester (Dns-C6-Cho) and rapid-mixing techniques. Under appropriate conditions, a majority of the acetylcholine receptor sites can be recovered in a low-affinity state(s) for Dns-C6-Cho, similar to that found with the native membrane-bound receptor. This state can be slowly interconverted to a high-affinity state after rapid mixing with the agonist, and the non-competitive channel blockers, like the local anesthetics, still regulate this transition in an allosteric manner. Several experimental conditions commonly used for the solubilization of the receptor and for its purification in the presence of sodium cholate result in the failure of reconstitution: the soluble receptor protein is stabilized in a low-affinity state which can no longer be interconverted to a high-affinity state in the presence of agonists or local anesthetics. On the other hand, it is demonstrated that if the concentration of lipids remains elevated in the presence of sodium cholate, a soluble (9-S) low-affinity form of the receptor protein can be obtained which shows most of the characteristic properties of the membrane-bound receptor and in particular the slow interconversion to the high-affinity state and the effect of local anesthetics on this transition; furthermore, in these conditions the soluble protein can be manipulated ad libitum and submitted, for instance, to column filtrations and sucrose gradient centrifugations in the presence of detergent, without losing its characteristic conformational and allosteric transitions. After elimination of the detergent this form yields a reconstituted receptor which presents binding properties identical to those of the native membrane-bound receptor and leads to the formation of vesicles which exhibit carbamylcholine-sensitive ion fluxes. A necessary and sufficient condition for functional reconstitution is therefore the conservation, in the presence of lipids, of the allosteric properties of the receptor protein in its detergent-soluble form.
利用荧光激动剂[1-(5-二甲基氨基萘)-磺酰胺基]-正己酸-β-N-三甲基溴化铵乙酯（Dns-C6-Cho）和快速混合技术，研究了乙酰胆碱受体蛋白在重组过程中的“功能”状态。在适当条件下，大多数乙酰胆碱受体位点可恢复到对Dns-C6-Cho的低亲和力状态，类似于天然膜结合受体的状态。与激动剂快速混合后，这种状态可缓慢转变为高亲和力状态，并且非竞争性通道阻滞剂，如局部麻醉剂，仍以变构方式调节这种转变。几种常用于受体溶解及其在胆酸钠存在下纯化的实验条件会导致重组失败：可溶性受体蛋白稳定在低亲和力状态，在激动剂或局部麻醉剂存在下不再能转变为高亲和力状态。另一方面，已证明如果在胆酸钠存在下脂质浓度保持升高，则可获得受体蛋白的可溶性（9-S）低亲和力形式，其显示出膜结合受体的大多数特征特性，特别是向高亲和力状态的缓慢转变以及局部麻醉剂对这种转变的影响；此外，在这些条件下，可溶性蛋白可随意操作，并例如在去污剂存在下进行柱过滤和蔗糖梯度离心，而不会丧失其特征性的构象和变构转变。去除去污剂后，这种形式产生重组受体，其具有与天然膜结合受体相同的结合特性，并导致形成对氨甲酰胆碱敏感的离子通量的囊泡。因此，功能重组的必要和充分条件是在脂质存在下，受体蛋白去污剂可溶形式的变构特性得以保留。
