Vm tunes mGlu5 receptor probability of activation
The reduced ability of mGlu5 receptor to induce Ca2+release from intracellular stores at Vdepol may be attributed to a decrease in the expression of receptors at the plasma membrane and/or a diminished activation of these receptors under depolarized conditions. To investigate the first hypothesis, we measured the receptor cell surface expression using an enhance bystander Bioluminescence Resonance Energy Transfer (ebBRET) biosensor. ebBRET is based on naturally interacting chromophores luciferase (RLuc) and green fluorescent protein (rGFP) from Renilla reniformis , enabling to quantify the cell surface expression of an Rluc-tagged protein expressed with the rGFP anchored at the plasma membrane by a CAAX sequence (Figure Supp. 2A ). In mGlu5-RlucII and rGFP-CAAX co-transfected cells, incubation of MPEP for 5 to 30 minutes resulted in an expected increase in receptor expression at the cell surface, as reported by an increase in ebBRET (Figure Supp. 2B ). This control experiment validated the use of the ebBRET biosensor. However, we did not observe any ebBRET variation induced by shorter (2 min) applications of MPEP or quisqualate, nor by Vdepol alone or combined with these ligands (Figure Supp. 2C ). Therefore, this experiment ruled out any potential effect of Vdepol on the receptor cell surface expression, in these experimental conditions.
To investigate the proportion of active and inactive mGlu5 receptors at the cell surface, we then used a Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) biosensor, reporting structural dynamics and drug action at the mGlu5 receptor . The conformational changes of the extracellular ligand-binding domains (ECDs) of mGlu5 dimers are associated with receptor activation: SNAP-mGlu5 (ST-mGlu5) labeled with the Lumi4-Tb donor and the fluorescein acceptor, display a high FRET signal in the absence of ligand or in presence of the antagonist and a low FRET signal in the presence of glutamate or other orthosteric agonists (Figure 3A ). Any approach expected to stabilize the active conformation of the effector domain increases the agonist potency in stabilizing the active ECDs conformation . Hence, TR-FRET measurements allowed to determine the proportion of active and inactive ST-mGlu5 receptors, depending on Vm. Importantly, labelling mGlu5 receptors separately on distinct cell populations, we first controlled that neither the fluorescein nor the Lumi4-Tb emissions were affected by Vdepol (Figure Supp 1B ), ruling out any effect of Vdepol on the performance of the biosensor. In ST-mGlu5 expressing cells co-labeled with Lumi4-Tb and fluorescein (Figure 3B ), quisqualate (10µM) induced a decrease in TR-FRET at Vrest (-17.2 ± 4.7%), reporting an agonist-induced increase of mGlu5 receptors in an active-like conformation. However, TR-FRET intensities measured in presence of quisqualate were significantly increased at Vdepol (+7.0 ± 2.6%) compared to Vrest, indicating that Vdepol reduced the ability of the agonist to trigger conformational activation of the receptor (Figure 3B ). This lower efficiency of quisqualate at Vdepol to decrease the TR-FRET reports a higher proportion of mGlu5 receptors in an inactive-like conformation at Vdepol compared to Vrest. Consistently, we noticed a shift in IC50 of the LY341495 mGlu competitive antagonist, from 31.28 µM at Vrest to 6.75 µM at Vdepol(Figure 3C ), demonstrating a Vm effect on ligand affinity for the mGlu5 receptor orthosteric pocket. These results demonstrate the influence of Vm on the conformation of the receptor. Vdepol stabilizes the inactive-like conformation of the receptor, increasing the antagonist potency.