Abstract
Metabotropic glutamate receptors (mGluR) regulate neuronal excitability and synaptic transmission by sensing L-glutamate - the major excitatory neurotransmitter in the central nervous system. Their crucial role for synaptic function makes them attractive targets for the treatment of numerous neurological and psychiatric diseases including for instance anxiety, depression, schizophrenia and addiction. To explore mGluR activation, we monitored its conformational changes using single molecule two and three color FRET with submillisecond resolution. We established multiple, minimally invasive conformational FRET sensors through bioorthogonal double labeling of two reactive and different non-canonical amino acids after incorporation by geneticcode expansion. These sensors report on the initial step of mGluR2 activation, including the reorientation of the upper and the lower lobes of the venus flytrap domain (VFT) in an intersubunit fashion and its closure in an intrasubunit fashion. By studying ligand induced conformational changes on single receptors, we show that the natural full agonist glutamate is sufficient to close the VFTs but a synergy with synthetic positive allosteric modulators or the G protein, is required for full activation. Our study highlights the power of minimally invasive, ncAA-based, bioorthogonal labeling to dissect domain-specific conformational rearrangements of single GPCRs using smFRET.