Graphical Abstract
mGlu5 receptor function is optimal at resting cell potential. Membrane depolarization stabilizes an inactive-like conformation of mGlu5 receptor and decreases agonist-induced Gq protein activation, Ca2+ release from intracellular stores and gating of NMDA and TRPC6 channels.
Figure 1. Voltage regulates mGlu5 agonist-induced Ca2+ release. HEK cells stably expressing GCaMP6s also transiently expressed mGlu5 (B-F) or AT1 (G) receptors. A)Scheme of GCaMP6s fluorescent Ca2+ sensor - adapted from . GCaMP6s is composed of a circularly permuted GFP, a calmodulin (CaM) and a peptide from smooth-muscle myosin light-chain kinase (RS20). B) DHPG (100µM)-induced fluorescence fluctuation in cells expressing (black and blue) or not (grey) mGlu5 receptor, in absence (grey and black) or presence (blue) of the mGlu5 NAM, MPEP (10µM); Representative illustration. C) Peak of fluorescence fluctuation induced by quisqualate (10µM) in isotonic extracellular solutions containing increasing KCl concentrations ([KCl] in mM), normalized to fluorescence fluctuations recorded with the 3mM [KCl] solution. Data are mean ± SEM of n = 6 triplicates from independent experiments. Statistics: Kruskal-Wallis test, D) Membrane potential measured in the current clamp whole-cell configuration with extracellular solutions containing either 3mM (black) or 100mM (red) KCl. Data are mean ± SEM of n = 10 cells from 3 independent experiments. Statistics: Friedman test E) Dose-response curves of DHPG-induced fluorescence fluctuation with KCl 3mM (black, Vrest) and 100mM (red, Vdepol), normalized to Vrest peak of each experiment. Data are mean ± SEM of n = 3 independent experiments. F and G)Quisqualate (10µM, F) or Angiotensine II (1µM, G) -induced fluorescence fluctuation in KCl 3mM (Vrest) and 100mM (Vdepol) solutions. Left, representative illustration; Right, mean ± SEM of n = 7 to 14 triplicates from independent experiments; Statistics: one sample Wilcoxon test.
Figure 2. Voltage regulates mGlu5 agonist-induced Ca2+ oscillations. A) Representative field of GCaMP6s expressing cells in epifluorescence microscopy - Scale = 10 µm.B) Quisqualate (10µM)-induced fluorescence fluctuation at Vrest (left, black) and Vdepol (middle, red), for individual field of view (thin) and average (thick) aligned to the peak of response, and quantification of the mean ± SEM of n = 14 to 21 independent experiments (right, Statistics: Mann-Whitney test)C) Fluorescence fluctuation recorded in representative oscillating, single spike and silent cells at Vrest and Vdepol in response to quisqualate (10µM) with proportion of cells in each category displayed in a circular diagram, n = 403 cells for Vrest and 324 cells for VdepolD) Percentage of oscillating cells (> 2 oscillations) at Vrest and Vdepol. n = 324 to 403 cells from 14 to 18 independent experiments. Statistics: N-1 χ² test, p = 0.0001. E) Global frequency of fluorescence oscillations induced by quisqualate (10µM) application at Vrest and Vdepol. Data are mean ± SEM of n = 280 to 408 cells. Statistics: unpaired t-test F)Representative illustrations (left) and violin plots with mean indicated by a dotted line (right) of fluorescence oscillations frequency induced by Vrest or Vdepol imposed after quisqualate (10µM) application (n = 21 to 35 cells - Statistics: unpaired t-test)
Figure 3. Voltage tunes mGlu5 receptor activation probability .A) Scheme of the TR-FRET sensor of mGlu5 conformation with SNAP-Lumi4-Tb (purple) and SNAP-fluoroscein (green) linked on SNAP-tag-mGlu5 VFT domains. The FRET signal is inversely proportional to the number of receptors in an active-like conformation. B)TR-FRET intensity at Vrest with and without quisqualate 10µM (left) and quisqualate (10µM) effect at Vrest and Vdepol (right). Data are mean ± SEM of n = 10 independent experiments. Statistics: Wilcoxon test. C)Dose-response curve of LY341495 mGlu antagonist applied at Vrest or Vdepol. Data are mean ± SEM of n = 4 independent experiments normalized to the maximum TR-FRET of Vdepol.
Figure 4. Voltage modulates mGlu5-mediated Gq activation. A)Scheme of EMTA ebBRET sensor of Gq activation . When Gq is activated by mGlu5, p63-RhoGEF-RlucII is recruited to the membrane where it interacts with rGFP-CAAX. B and C) Representative illustrations (B) and mean ± SEM (C) of BRET signals measured before (CT) and after (DHPG, 100µM) stimulation at Vrest or Vdepol. N= 86 to 97 cells from more than 3 independent experiments; statistics: Paired t-test. D) Single cell BRET measurement of DHPG net effect; statistics: Unpaired t-test.
Figure 5. Voltage controls mGlu5 gating of TRPC6 channels. Aand B) - Whole-cell patch clamp current induced by DHPG (100µM) on HEK293T cells expressing mGlu5-Venus alone (A) or with TRPC6-tomato (B). C) mGlu5-Venus/TRPC6-tomato co-transfected cells were held at -80mV (black) or -20mV (red) during DHPG (100µM) application. Current-voltage relationship was then recorded at the maximum response induced by DHPG (100µM) for potentials ranging from -80mV to +60mV in 100ms (bottom). Inset: Mean ± SEM current density at -80mV. Data are mean ± SEM of n = 9 cells per condition from 4 independent experiments, Statistics: Mann-Whitney test.
Figure 6. Voltage regulates mGlu5 gating of NMDA receptor in hippocampal neurons. A) Representative current and mean density induced by DHPG (50µM, left) or NMDA (30µM, right) at -80mV. B - D)Representative currents (B), mean current density and INMDA+DHPG/INMDA current ratio (C), and percentage of DHPG-induced NMDA current potentiation (D) measured in presence of NMDA (30µM) alone or co-applied with DHPG (50µM) at -80mV (black) and -40mV (red) holding potential. Measures of INMDA (blue) and INMDA+DHPG (black) used for quantification in C and D are indicated by arrows in B. In D, paired measurement of INMDA + DHPG / INMDA were performed subsequently at -80mV or -40mV, in a random order, on the same neuron. C and D data are mean ± SEM of n = 10 to 15 neurons from at least 4 independent experiments; Statistics: Wilcoxon matched-pairs signed rank test.