Reconstitution of a Functional Acetylcholine Receptor

摘要: The ‘functional’ state of the acetylcholine receptor protein has been followed during reconstitution with fluorescent agonist [1-(5-dimethylaminonaplithalene)-sulfonamido]-n-hexanoic acid-β-N-trimethylammonium bromide ethyl ester (Dns-C6-Cho) and rapid-mixing techniques. Under appropriate conditions, a majority sites can be recovered in low-affinity state(s) for Dns-C6-Cho, similar to that found native membrane-bound receptor. This slowly interconverted high-affinity after rapid mixing agonist, non-competitive channel blockers, like local anesthetics, still regulate this transition an allosteric manner. Several experimental conditions commonly used solubilization its purification presence sodium cholate result failure reconstitution: soluble is stabilized which no longer agonists or anesthetics. On other hand, it demonstrated if concentration lipids remains elevated cholate, (9 S) form obtained shows most characteristic properties particular slow interconversion effect anesthetics on transition; furthermore, these manipulated ad libitum submitted, instance, column filtrations sucrose gradient centrifugations detergent, without losing conformational transitions. After elimination detergent yields reconstituted presents binding identical those leads formation vesicles exhibit carbamylcholine-sensitive ion fluxes. A necessary sufficient condition functional therefore conservation, lipids, detergent-soluble form.