Introduction
G-protein coupled receptors (GPCRs) represent a class of 7-transmembrane receptors that are involved in numerous physiological processes and remain the most extensively targeted protein family by approved drugs . Recent research has unveiled that the activity of certain GPCRs can be modulated by the membrane voltage (Vm). In neurons, where Vm undergoes permanent changes, this emerging property could significantly impact the functioning of neurotransmitter-activated GPCRs and their role as modulators of synaptic transmission. Indeed, Vm has been shown to positively or negatively regulate the function of several GPCRs, about thirty out of the thousand members of this big family, including acetylcholine , purine , opioid , dopamine , and prostanoid receptors . Although the structural mechanisms that underlie the sensitivity of GPCRs to Vm are still being elucidated , functional studies using site-directed mutagenesis of the Vm sensor suggest that GPCR activity can be affected, leading to impaired neurotransmitter release or synaptic plasticity and behavior .
Glutamate is the primary excitatory neurotransmitter in the brain that binds to both ionotropic (AMPA, NMDA, Kainate receptors) and metabotropic glutamate (mGlu) receptors. The mGlu receptor family, comprising eight G protein-coupled receptors (mGlu1-8), has been extensively studied for their modulatory role in synaptic transmission and plasticity . Preclinical and clinical studies have targeted these receptors in various neurological disorders, such as Autism, Fragile X syndrom, Schizophrenia, Parkinson’s, and Alzheimer’s disease . So far, only one study, in the Xenopus oocytes expression system, suggests a direct sensitivity of some mGlu receptors to Vm (mGlu1 and 3, ). Of note, membrane potential changes seem to play a synergistic role in neurons on signaling events mediated by mGlu5 and mGlu7 receptors, but without any evidence of a direct action of Vm on the receptor itself. Yet, due to their synaptic location, these receptors are permanently exposed to membrane voltage fluctuations. Therefore, demonstrating the sensitivity of mGlu receptors to Vm could provide insights into their role depending on the state of neuronal activity. In this study, we have selected the postsynaptic mGlu5 receptor as a model receptor, known for its role as neuromodulator of synaptic transmission. mGlu5 is also a key trigger in the induction of synaptic plasticity, working in concert with ionotropic NMDA receptors through physical and functional crosstalk . Interestingly, NMDA receptors are a prototype of receptors whose activity is regulated by the membrane potential. As a detector of neuronal activity coincidence, NMDA activation is limited to synapses whose pre- and postsynaptic elements are activated simultaneously . Thus, a sensitivity of the mGlu5 receptor to Vm could similarly specify the identity of synapses on which mGlu5 exerts its functional effects depending on the neuron’s activity history.
In this report, we explore the intricate signaling pathways associated with the conformational change of postsynaptic mGlu5 receptor induced by glutamate binding. This conformational change leads to the activation of Gq/11-type proteins, the activation of the PLC-PKC pathway, and the subsequent release of Ca2+ from internal stores through IP3 receptors , ultimately fine-tuning synaptic transmission by gating channels and ionotropic receptors, including NMDA , AMPA , and transient receptor potential canonical (TRPC) channels . Specifically, we investigate the impact of Vm on each of the canonical signaling events of the mGlu5 receptor using a variety of biosensors and patch clamp recordings. Our results demonstrate that Vm modulates mGlu5 receptor activation and its downstream signaling, with a depolarization of the membrane favoring the inactive conformation of mGlu5 and leading to a decrease in mGlu5-induced Gq/11 activation, Ca2+signaling, TRPC6 gating, and NMDA receptor facilitation. Interestingly, our data reveals that the mGlu5 receptor functions optimally at potentials close to the resting potential of the cells.