Optical measurement of mGluR1 conformational changes reveals fast activation, slow deactivation, and sensitization

Abstract

Metabotropic glutamate receptor (mGluR) activation has been extensively studied under steady-state conditions. However, at central synapses, mGluRs are exposed to brief submillisecond glutamate transients and may not reach steady-state. The lack of information on the kinetics of mGluR activation impairs accurate predictions of their operation during synaptic transmission. Here, we report experiments designed to investigate mGluR kinetics in real-time. We inserted either CFP or YFP into the second intracellular loop of mGluR1␤. When these constructs were coexpressed in PC12 cells, glutamate application induced a conformational change that could be monitored, using fluorescence resonance energy transfer (FRET), with an EC50 of 7.5 M. The FRET response was mimicked by the agonist DHPG, abolished by the competitive antagonist MCPG, and partially inhibited by mGluR1-selective allosteric modulators. These results suggest that the FRET response reports active conformations of mGluR1 dimers. The solution exchange at the cell membrane was optimized for voltageclamped cells by recording the current induced by co-application of 30 mM potassium. When glutamate was applied at increasing concentrations up to 2 mM, the activation time course decreased to a minimum of approximately 10 ms, whereas the deactivation time course remained constant (ϳ50 ms). During long-lasting applications, no desensitization was observed. In contrast, we observed a robust sensitization of the FRET response that developed over approximately 400 ms. Activation, deactivation, and sensitization time courses and amplitudes were used to derive a kinetic scheme and rate constants, from which we inferred the EC50 and frequency dependence of mGluR1 activation under non-steady-state conditions, as occurs during synaptic transmission.he amino acid L-glutamate, the main neurotransmitter in the CNS, is referred to as a fast neurotransmitter because it mediates fast excitatory neurotransmission by activating ionotropic glutamate receptors (i.e., NMDA, AMPA, and kainate receptors). It also targets metabotropic glutamate receptors (mGluRs, 8 types) that belong to the class C G protein-coupled receptor (GPCR) subfamily. As opposed to ionotropic glutamate receptors, mGluRs mediate slower effects of glutamate, downstream of G protein activation (1). In addition, mGluRs have often been considered to play a role as extrasynaptic receptors because they are not located within the synaptic cleft (2), and their affinity for glutamate may be high enough (3) to detect glutamate concentration in extrasynaptic locations. Their extrasynaptic localization appears consistent with the slow effects of their activation because glutamate transients are expected to be less sudden at extra-and perisynaptic sites than opposite to the release site. However, even at extrasynaptic locations, glutamate transients evoked by synaptically released glutamate occur within milliseconds, as shown by recording of glutamate transporter-mediated currents in glial cells that wrap around synapses (4, 5...

Interdomain movements in metabotropic glutamate receptor activation

“…Indeed, in the proposed resting orientation observed with the soluble VFTs, the CRDs are far apart, as will then be the 7TM domains. However, such proposed conformation of the dimer is not compatible with FRET data obtained when CFP and YFP are fused to the intracellular loops of the receptor (17,36). Indeed these data revealed a close proximity between the 7TM domains, closer that what is expected on the basis of the resting orientation of the VFTs.…”

Spatial and Temporal Aspects of Signaling by G-Protein–Coupled Receptors

“…Overall, the transitions of GPCRs into truly active conformations appear to be incomplete under physiologic conditions, and stabilization by binding to a G-protein or to a (b-)arrestin appears to be required in order to produce a fully active receptor (Elgeti et al, 2013;Manglik et al, 2015). Activation of nonrhodopsin receptors has been followed mostly using fluorescence resonance energy transfer-based sensors and rapid application of agonists by superfusion; this has led to reported activation times down to 10 milliseconds (Marcaggi et al, 2009), but mostly in the range of 30-80 milliseconds (Vilardaga et al, 2003;Lohse et al, 2008Lohse et al, , 2012. Levitz et al (2013) used a tethered, light-switchable agonist to activate a metabotropic glutamate receptor, but did not determine the speed of activation of the receptor.…”

Distinct roles of metabotropic glutamate receptor dimerization in agonist activation and G-protein coupling

“…It has been shown that a relative movement at the level of the VFTs, the CRDs (9,20,46), and the 7TMs of the two subunits (19,21,22) occurs during activation of class C GPCRs. More recently, we documented that the change in conformation of one of the 7TM domains within the dimer occurs after the relative movement between the protomers (19), consistent with a concerted activation mechanism that results from dimer rearrangement.…”

“…Structural as well as mutagenesis studies demonstrated a large change in the relative position of the VFTs is associated with dimer activation (9,14,15,20), but this is not consistent with all structures available (8). At the 7TM level, a movement between the two subunits has also been demonstrated using FRET studies (19,21,22). It remains to be determined whether such changes are only associated with the activation process, result as a consequence of other more critical steps, or represent the unique driving force for 7TM activation.…”