The mGlu5 receptor functions optimally at the resting potential of
cells.
We observed that each of the receptor signaling steps we studied were
affected by the membrane potential, and our results indicate that the
optimal function of the receptor occurs when the cell is at rest.
A sensor that monitored the distance between the extracellular domains
of the receptor’s protomers within the mGlu5 homodimer over time allowed
us to determine the activation state of the receptor when bound to an
agonist. FRET measurements showed that depolarization of the membrane
favored an inactive-like conformation of the mGlu5 receptor, which
increased the potency of its orthosteric competitive antagonist. These
findings were consistent with a previous study demonstrating that
glutamate binding to the mGlu3 receptor could be inhibited by membrane
depolarization . Although this sensor provided information only about
the extracellular domains of the receptor, this is a reliable indicator
of the activation state of mGlu receptors . The conformational change of
the extracellular domains of the receptor may be induced by a
conformational change of the seven transmembrane domains (7TM) due to
depolarization, as the 7TM are the closest elements of the receptor to
the electric field (see discussion below). This hypothesis is supported
by structural studies showing that activation of the 7TM stabilize the
active conformation of the extracellular domains of the mGlu5 receptor .
The mGlu5 receptor’s ability to activate Gq is
diminished under depolarizing conditions. The Gq protein
activation sensor relies on the translocation of Gqprotein effector, p63RhoGEF, to the membrane upon activation of a GPCR.
The BRET signal observed between p63RhoGEF bound to RlucII and
membrane-targeted rGFP through a CAAX sequence corresponds well with
Gq protein activation . The translocation of the
Gq effector to the membrane appears to be
voltage-dependent only when the mGlu5 receptor is activated. In the
exact same experimental conditions, no effect was observed on activation
of the AT1 receptor. Nevertheless, this receptor activates the same
pathway as the mGlu5 receptor , and therefore serves as a rigorous
control to emphasize the specificity of Vm action on
mGlu5 receptor signaling. Activation of Gq proteins by
mGlu5 receptors induces IP3 production and triggers a significant
release of Ca2+ from intracellular stores into the
cytosol, resulting in typical Ca2+ oscillations
previously reported to be caused by PKC-induced phosphorylation and
desensitization of mGlu5 receptor . Depolarization reduced the release
of Ca2+ from intracellular stores generated by mGlu5
receptor activation. Not only did the number of cells generating
Ca2+ oscillations decrease following mGlu5 receptor
stimulation, but also the frequency of oscillations per oscillating cell
was significantly reduced by membrane depolarization. In contrast,
Vm did not influence the Ca2+ response
initiated by activation of the AT1 receptor, which is also known to
produce similar Ca2+ release . These findings indicate
that depolarization specifically impairs Ca2+ release
induced by the mGlu5 receptor.
Activation of Gq-protein by mGlu5 receptors induces
production of PIP2 and DAG, which in turn triggers the opening of TRPC
channels . Co-expression of TRPC6 channels in cells resulted in the
inward current being triggered by the activation of mGlu5 receptors,
with typical current kinetics, amplitude, and rectification properties ,
indicating that these channels are gated by mGlu5 receptor activation.
By varying the imposed potentials for a few milliseconds,
current-potential curves were used to determine the channel conductance.
Stimulation of mGlu5 receptors at a holding potential of -20 mV instead
of -80 mV significantly decreased the conductance of TRPC6. These
findings suggest that membrane depolarization inhibits the ability of
the mGlu5 receptor to open TRPC6 channels.
Other channels are controlled by the mGlu5 receptor, including the
ionotropic glutamate receptors of the NMDA type , which also play a
fundamental role in the induction of synaptic plasticity. The intricate
cross-talk between mGlu5 and NMDA receptors defines functional neuronal
networks, which evolve through complex mutual regulations that are
influenced by the dynamics of synaptic protein complexes and the context
of neuronal activity . These regulations can lead to either potentiation
or inhibition of their activations. Potentiation of NMDA receptor
activity by mGlu5 receptors has been shown to depend on the
Gq-PKC-Src signaling pathway in various neuronal
contexts . Our experimental results confirm this finding, as we observed
a significant increase in NMDA current upon mGlu5 stimulation. However,
we also found that this facilitation is dependent on the holding
potential of the neuron and decreases with depolarizing membrane
potential. These results suggest that the activity of mGlu5 receptors is
lower at depolarized potentials, limiting the facilitation of NMDA to
potentials close to the resting potential of neurons. In addition, our
findings reveal a permissive role of the mGlu5 receptor on NMDA receptor
activation even under physiological concentrations of
Mg2+ at resting potential, further expanding the role
of NMDA receptors in synaptic plasticity.