Optical control of glutamate receptors using genetically encoded unnatural amino acids

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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.