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.