Ionotropic glutamate receptors are ligand-gated ion channels that mediate excitatory synaptic transmission in the central nervous system. Desensitization of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid subtype after glutamate binding appears critical for brain function and involves rearrangement of the ligand binding domains (LBDs). Recently, several full-length structures of ionotropic glutamate receptors in putative desensitized states were published. These structures indicate movements of the LBDs that might be trapped by cysteine cross-links and metal bridges. We found that cysteine mutants at the interface between subunits A and C and lateral zinc bridges (between subunits C and D or A and B) can trap freely desensitizing receptors in a spectrum of states with different stabilities. Consistent with a close approach of subunits during desensitization processes, the introduction of bulky amino acids at the A-C interface produced a receptor with slow recovery from desensitization. Further, in wild-type GluA2 receptors, we detected the population of a stable desensitized state with a lifetime around 1 s. Using mutations that progressively stabilize deep desensitized states (E713T and Y768R), we were able to selectively protect receptors from cross-links at both the diagonal and lateral interfaces. Ultrafast perfusion enabled us to perform chemical modification in less than 10 ms, reporting movements associated to desensitization on this timescale within LBD dimers in resting receptors. These observations suggest that small disruptions of quaternary structure are sufficient for fast desensitization and that substantial rearrangements likely correspond to stable desensitized states that are adopted relatively slowly on a timescale much longer than physiological receptor activation.

中文翻译：

---

AMPA受体脱敏时间尺度和构象空间的测量

离子型谷氨酸受体是配体门控离子通道，在中枢神经系统中介导兴奋性突触传递。谷氨酸盐结合后 α-氨基-3-羟基-5-甲基-4-异恶唑丙酸亚型的脱敏似乎对大脑功能至关重要，并涉及配体结合域 (LBD) 的重排。最近，发表了几种处于假定脱敏状态的离子型谷氨酸受体的全长结构。这些结构表明可能被半胱氨酸交联和金属桥捕获的 LBD 的运动。我们发现亚基 A 和 C 以及侧锌桥（亚基 C 和 D 或 A 和 B 之间）之间界面处的半胱氨酸突变体可以在具有不同稳定性的一系列状态中自由捕获脱敏受体。与脱敏过程中亚基的紧密接近一致，在 AC 界面引入大量氨基酸产生了一个从脱敏中缓慢恢复的受体。此外，在野生型 GluA2 受体中，我们检测到稳定脱敏状态的种群，寿命约为 1 秒。使用逐渐稳定深度脱敏状态（E713T 和 Y768R）的突变，我们能够选择性地保护受体免受对角线和横向界面的交联。超快灌注使我们能够在不到 10 毫秒的时间内进行化学修饰，报告静息受体中 LBD 二聚体在这个时间尺度上与脱敏相关的运动。