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

摘要: Metabotropic glutamate receptor (mGluR) activation has been extensively studied under steady-state conditions. However, at central synapses, mGluRs are exposed to brief submillisecond transients and may not reach steady-state. The lack of information on the kinetics mGluR impairs accurate predictions their operation during synaptic transmission. Here, we report experiments designed investigate in real-time. We inserted either CFP or YFP into second intracellular loop mGluR1β. When these constructs were coexpressed PC12 cells, application induced a conformational change that could be monitored, using fluorescence resonance energy transfer (FRET), with an EC50 7.5 μM. FRET response was mimicked by agonist DHPG, abolished competitive antagonist MCPG, partially inhibited mGluR1-selective allosteric modulators. These results suggest reports active conformations mGluR1 dimers. solution exchange cell membrane optimized for voltage-clamped cells recording current co-application 30 mM potassium. applied increasing concentrations up 2 mM, time course decreased minimum approximately 10 ms, whereas deactivation remained constant (∼50 ms). During long-lasting applications, no desensitization observed. In contrast, observed robust sensitization developed over 400 ms. Activation, deactivation, courses amplitudes used derive kinetic scheme rate constants, from which inferred frequency dependence non-steady-state conditions, as occurs