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.