Ion dependence of mGluRs activity has been

Ion dependence of mGluRs activity has been previously reported, notably to Ca2+ and Cl− (Kuang and Hampson, 2006). While mGlu1 and mGlu3 receptors were demonstrated to be sensitive to Ca2+, Cl− modulation was reported for all mGluRs, with a lesser extent for the mGlu2 receptor (DiRaddo et al., 2014; Jiang et al., 2014; Tora et al., 2015; Vafabakhsh et al., 2015). Of note, the cooperativity between an amino 12-O-tetradecanoyl phorbol-13-acetate ligand and ions to stabilize the active conformation of the receptor is also observed in another receptor from the GPCR class C, the Ca2+ sensing receptor. This receptor is activated concomitantly both by Ca2+ ions and an l-amino acid, such as L-Phe, that binds in a pocket corresponding to the glutamate binding pocket in mGluRs (Zhang et al., 2014). Here, we showed that the mGlu3 receptor is particularly strongly potentiated by Cl− ions as compared to other mGluRs. This strong positive cooperativity between Cl− ions and glutamate occurs through a unique strengthened interaction network between the two lobes of the extracellular domain of the mGlu3 receptor. This network acts as a “Cl−-lock” that dramatically reinforces glutamate binding and function. Previously, both Ca2+ and Cl− ions were proposed to display agonist activity on the mGlu3 receptor (DiRaddo et al., 2015; Vafabakhsh et al., 2015). However, in our experiments, Cl−per se does not display direct agonism, as revealed by the glutamate insensitive mGlu3 mutant T174A, which is not activated by Cl−. We propose that what has been interpreted previously as Cl− direct agonism in very sensitive assays may correspond in fact to the potentiation by Cl− ions of the activity induced by residual ambient glutamate. Indeed, cells are constitutively releasing glutamate in extracellular media. We have recorded concentrations ranging from 0.6 to more than 5 μM. These glutamate concentrations are in line with those measured in another recent publication (Doornbos et al., 2018). Thus, Cl− ions are pure positive allosteric modulators of the mGlu3 receptor. It is unlikely to observe the occurrence of rapid Cl-concentration changes compatible with dynamic modulation of mGlu3 receptor activity. Short-term variations occur during transport or synaptic activities where rapid Cl− fluxes through GABA or glycine-gated ion channels modify Cl-concentrations (Staley et al., 1995). We have previously evaluated the discrete variations of Cl− concentrations in the synaptic cleft during GABAergic events in physiological or pathological conditions (Tora et al., 2015). We found that multiple synaptic events can modify synaptic Cl− concentrations up to 25 mM. Long-term changes in Cl− concentrations can be observed during maturation (Ben-Ari et al., 2007), modifications of extracellular matrix (ECM) and pathologies (De Koninck, 2007; Kaila et al., 2014). Interestingly, fast extracellular Cl-changes due to synaptic activity and slow changes could be additive. Thus, in particular conditions, such as anoxia where variations of Cl-exceeding 30 mM can be observed (Jiang et al., 1992), Cl-variations could reach up to 55 mM. However, given the potency of Cl− on the mGlu3 receptor (∼30 mM) as compared to the physiological extracellular Cl− concentration in mature CNS (∼120 mM), the potentiation of mGlu3 receptor activity by Cl− ions is probably mostly constitutive. Other members of the mGluR family could be dynamically regulated by Cl−, such as the presynaptic mGlu4 receptor (Tora et al., 2015). Indeed, unlike the mGlu3 receptor, mGlu4 presents characteristics that makes it more susceptible to be actively regulated by discrete Cl− variations. First, this receptor is expressed in GABAergic synapses where rapid Cl-variations occur following opening of GABAA receptors. Second, the potency of Cl− on mGlu4 receptoris close to the physiological Cl-concentration (∼80 mM) and presents a steep relationship between Cl− and glutamate-induced activity (with an NHill ∼6). This means that small changes in Cl− concentration yield to large changes in response to glutamate. On the other hand, dramatic changes in Cl− homoeostatis occur during CNS maturation (Ben-Ari et al., 2007). We can hypothesize that mGlu3 receptors should shift from a “dynamic” mode of glutamate response during the early stages to the highly-glutamate sensitive and sustained response mode described in the present study in mature stages. This should affect its biological functions in early stages, such as the crucial control of mGlu5-mediated neuroprotection described in Di Menna et al. (Di Menna et al., 2018). One can hypothesize that when extracellular Cl− concentrations are low in premature stages, mGlu3 receptor should be able to signal and regulate the release of neurotrophic factors from astrocytes, given its high Cl− sensitivity. However, in the absence of specific data this remains purely speculative and further investigations will be needed to evaluate the impact of Cl− on mGluR function in the premature brain. In the mature brain, while it is unlikely to observe the occurrence of Cl− concentrations changes compatible with a significant modulation of mGlu3 receptor activity, a Cl-mediated rapid adaptation of the regulatory role of some mGluRs, such as mGlu4 receptors present on GABAergic terminals, could be observed.