Optical control of glutamate receptors using genetically encoded unnatural amino acids

谷氨酸受体是脑内快速兴奋性突触传递所必需的。关于谷氨酸受体的生物发生、运输、药理学和结构有相当多的数据。不幸的是，大多数相关的测量都是在几分钟、几小时甚至几天内完成的，因此与突触传递的快速毫秒级时间尺度相比非常慢。特别是，AMPA型谷氨酸受体的激活状态和一个矛盾的谷氨酸结合脱敏状态的动力学信息是缺乏的。我们的团队最近利用最先进的化学和分子生物学技术，在哺乳动物细胞中赋予AMPA型谷氨酸受体遗传编码的光驱动失活。这一技术进步使我们能够在毫秒级的正常快速激活过程中捕获受体。我们建议使用光活性氨基酸交联剂，与电生理记录和计算建模相结合，建立开放通道，激活状态的膜结构域的第一个地图，并揭示在脱敏状态的细胞外结构域的地形。一个互补的目标是将生物物理学的见解转化为控制光突触传递的工具。谷氨酸受体亚基的遗传编码携带光敏交联剂将使我们能够在活神经元中将受体共价转化为可激活或非活性形式。这种方法将使理性的干扰兴奋性传输到秒的境界，与亚细胞空间分辨率，绕过缓慢的稳态过程。通过这项工作，我们希望证明内源性信号传导组分的快速光控制的一般原理，以探测信号传导途径，作为对已建立的光遗传学方法的补充。

Glutamate receptors are essential for fast excitatory synaptic transmission in brain. Considerable data is available about glutamate receptor biogenesis, trafficking, pharmacology and structure. Unfortunately, most of the relevant measurements have been made over minutes, hours or even days, and were hence very slow compared to the rapid, millisecond timescale of synaptic transmission. Particularly, for AMPA-type glutamate receptors, information about the dynamics of the activated state and a paradoxical glutamate-bound desensitised state are lacking. Our group recently exploited state-of-the-art chemical and molecular biology to endow an AMPA-type glutamate receptor with genetically-encoded, light-driven inactivation in mammalian cells. This technical advance enabled us to trap receptors during normal rapid activation with millisecond timing. We propose to use photo-active amino acid crosslinkers, in concert with electrophysiological recording and computational modelling, to build the first map of the membrane domains in the open-channel, activated state, and to uncover the topography of the extracellular domains in the desensitised state. A complementary aim is to translate biophysical insights into tools to control synaptic transmission with light. Genetic encoding of glutamate receptor subunits harbouring photoactive crosslinkers will allow us convert receptors covalently between into activatable or inactive forms, in live neurons. This approach will bring rational interference with excitatory transmission into the realm of seconds, with subcelllular spatial resolution, bypassing slow homeostatic processes. With this work we expect to prove the general principle of fast photocontrol of endogenous signalling components to probe signalling pathways, as a complement to established optogenetic methods.