Fast kinetics of mGluR1 activation measured by an optical method

Metabotropic glutamate receptor (mGluR) activation has been extensively studied in 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 regarding the kinetics of mGluR activation impairs accurate predictions of the total amount of their activation during synaptic transmission. Here, we describe the time course of mGluR1β activation in order to provide a hypothetic kinetic scheme for mGluR1. To monitor activation more directly than by measuring the delayed downstream activation of second messengers, we designed two constructs which enable fluorimetric measurement of intracellular conformational changes induced by extracellular glutamate binding. These constructs are the native mGluR1β into which either CFP or YFP were inserted. When coexpressed in PC12 cells, fluorescence imaging showed that most mGluR1β-CFP and mGluR1β-YFP were targeted to the cytoplasmic membrane. Application of glutamate induced a change in fluorescence resonance energy transfer (FRET) between CFP and YFP as a consequence of conformational changes of the mGluR1β-CFP- mGluR1β-YFP dimers. The FRET response saturated for ∼ 200 μM glutamate (Δratio yellow/cyan = 0.79 ± 0.22%, n = 7) and had an EC₅₀ of ∼ 5 μM, similar to that reported for native mGluR1. The response to 15 μM glutamate was abolished by the mGluR1 competitive antagonist MCPG (1 μM). The group I mGluR agonist DHPG induced a FRET response 73.9 ±.2% weaker than the response to glutamate but with a similar EC₅₀ (n = 5). These data support the use of the FRET response as an index of the conformational change that is normally associated with activation of the native mGluR1. Glutamate was applied onto cells with a fast solution exchange system. Solution exchange at the cell membrane was monitored as K⁺ currents evoked by co-application of 34 mM K⁺ in patch-clamped cells. When fast solution exchange was achieved, the kinetics of the FRET response evoked by glutamate application were determined. No delay between the onsets of the solution exchange and the FRET response was detected. The activation could be fitted by a simple exponential with τ values of (in ms) 121 ± 11.8 (n=7), 69.3 ± 5.4 (n =7), 50.7 ± 2.3 (n =6), 32.4 ± 2.4 (n=14) ,14.1 ± 2.2 (n=7) for 5μM, 10μM, 20μM, 200μM and 2μM glutamate application respectively. The deactivation could also be fitted by a simple exponential with a τ value of 49.8 ± 2ms (n=4). Finally, no desensitization was observed. On the contrary, we observed a sensitization of 24.4 ± 0.9% (n=3) following 10s of 200 μM glutamate application. We derive from these data a kinetic scheme for mGluR1 that should help to better understand mGluR1 function at central synapses.