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首页> 外文期刊>Anesthesiology >Identification of two mutations (F758W and F758Y) in the N-methyl-D-aspartate receptor glycine-binding site that selectively prevent competitive inhibition by xenon without affecting glycine binding
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Identification of two mutations (F758W and F758Y) in the N-methyl-D-aspartate receptor glycine-binding site that selectively prevent competitive inhibition by xenon without affecting glycine binding

机译：鉴定N-甲基-D-天冬氨酸受体甘氨酸结合位点中的两个突变（F758W和F758Y），这些突变可选择性地阻止氙的竞争性抑制而不影响甘氨酸的结合

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摘要

Background: Xenon is a general anesthetic with neuroprotective properties. Xenon inhibition at the glycine-binding site of the N-Methyl-D-aspartate (NMDA) receptor mediates xenon neuroprotection against ischemic injury in vitro. Here we identify specific amino acids important for xenon binding to the NMDA receptor, with the aim of finding silent mutations that eliminate xenon binding but leave normal receptor function intact. Methods: Site-directed mutagenesis was used to mutate specific amino-acids in the GluN1 subunit of rat NMDA receptors. Mutant GluN1/GluN2A receptors were expressed in HEK 293 cells and were assessed functionally using patch-clamp electrophysiology. The responses of the mutant receptors to glycine and anesthetics were determined. Results: Mutation of phenylalanine 758 to an aromatic tryptophan or tyrosine left glycine affinity unchanged, but eliminated xenon binding without affecting the binding of sevoflurane or isoflurane. Conclusions: These findings confirm xenon binds to the glycine site of the GluN1 subunit of the NMDA receptor and indicate that interactions between xenon and the aromatic ring of the phenylalanine 758 residue are important for xenon binding. Our most important finding is that we have identified two mutations, F758W and F758Y, that eliminate xenon binding to the NMDA receptor glycine site without changing the glycine affinity of the receptor or the binding of volatile anesthetics. The identification of these selective mutations will allow knock-in animals to be used to dissect the mechanism(s) of xenon's neuroprotective and anesthetic properties in vivo.
机译：背景：氙气是一种具有神经保护特性的全身麻醉剂。 N-甲基-D-天冬氨酸（NMDA）受体的甘氨酸结合位点的氙气抑制介导氙气神经保护免受体外缺血性损伤。在这里，我们确定了对于氙与NMDA受体结合重要的特定氨基酸，目的是寻找消除氙结合但保持正常受体功能完整的沉默突变。方法：采用定点诱变法突变大鼠NMDA受体GluN1亚基中的特定氨基酸。突变的GluN1 / GluN2A受体在HEK 293细胞中表达，并使用膜片钳电生理学对其功能进行评估。确定了突变体受体对甘氨酸和麻醉药的反应。结果：苯丙氨酸758突变为芳香族色氨酸或酪氨酸使甘氨酸亲和力保持不变，但消除了氙气结合，而不会影响七氟醚或异氟烷的结合。结论：这些发现证实氙与NMDA受体GluN1亚基的甘氨酸位点结合，并表明氙与苯丙氨酸758残基的芳香环之间的相互作用对于氙的结合很重要。我们最重要的发现是我们确定了两个突变F758W和F758Y，它们消除了氙与NMDA受体甘氨酸位点的结合，而没有改变受体的甘氨酸亲和力或挥发性麻醉剂的结合。这些选择性突变的鉴定将允许敲入的动物用于在体内解剖氙气的神经保护和麻醉特性的机制。

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