Watty A, Methfessel C, Hucho F
Institut für Biochemie, Fachbereich Chemie, Freie Universität Berlin, Thielallee 63, D-14195 Berlin, Germany.
Proc Natl Acad Sci U S A. 1997 Jul 22;94(15):8202-7. doi: 10.1073/pnas.94.15.8202.
Receptor activity can be described in terms of ligand-induced transitions between functional states. The nicotinic acetylcholine receptor (nAChR), a prototypic ligand-gated ion channel, is an "unconventional allosteric protein" which exists in at least three interconvertible conformations, referred to as resting (low agonist affinity, closed channel), activated (open channel), and desensitized (high agonist affinity, closed channel). Here we show that 3,3'-dimethyl suberimidate (DMS) is an agonistic bifunctional cross-linking reagent, which irreversibly "freezes" the nAChR in a high agonist affinity/closed-channel state. The monofunctional homologue methyl acetoimidate, which is also a weak cholinergic agonist, has no such irreversible effect. Glutardialdehyde, a cross-linker that is not a cholinergic effector, fixes the receptor in a low-affinity state in the absence of carbamoylcholine, but, like DMS, in a high-affinity state in its presence. Covalent cross-linking thus allows us to arrest the nAChR in defined conformational states.
受体活性可以用配体诱导的功能状态转变来描述。烟碱型乙酰胆碱受体（nAChR）是一种典型的配体门控离子通道，是一种“非常规变构蛋白”，它至少存在三种可相互转换的构象，分别称为静息态（低激动剂亲和力，通道关闭）、激活态（通道开放）和脱敏态（高激动剂亲和力，通道关闭）。在这里，我们表明3,3'-二甲基辛二亚氨酸酯（DMS）是一种激动性双功能交联剂，它能不可逆地将nAChR“冻结”在高激动剂亲和力/通道关闭状态。单功能同系物乙酰甲脒，它也是一种弱胆碱能激动剂，没有这种不可逆作用。戊二醛是一种不是胆碱能效应器的交联剂，在没有氨甲酰胆碱的情况下将受体固定在低亲和力状态，但与DMS一样，在有氨甲酰胆碱的情况下将其固定在高亲和力状态。因此，共价交联使我们能够将nAChR捕获在特定的构象状态。
