Vm modulates mGlu5-mediated Gq/11activation
We then assessed the influence of Vm on the activation by mGlu5 receptor of canonical effectors, starting with the Gq/11 activation. We monitored Gq/11activation by mGlu5 using Effector Membrane Translocation Assay (EMTA) ebBRET in single-cell BRET imaging (, Figure 4 ) and cell population (Figure Supp. 3 ). HEK293T cells were transfected with mGlu5, Gq/11, p63hRhoGEF-RlucII and rGFP-CAAX coding plasmids. Gq/11 activation triggers the recruitment of its cytosolic effector, p63RhoGEF-RlucII, to the plasma membrane, which is reported by an increase in ebBRET with the plasma membrane-anchored rGFP-CAAX. The ebBRET signal is therefore a measure of Gq/11 activation (, Figure 4A ). Of note, the ebBRET signal was not affected by Vdepol compared to Vrest, validating the use of this biosensor to assess the influence of Vm on mGlu5-induced Gq/11 activation (Figure Supp 1C ). Application of DHPG (100µM) induced a slight but significant increase in ebBRET at Vrest and Vdepol (Figure 4B and4C ). Paired measurement of the DHPG net effect from individual cell ebBRET values revealed a mean DHPG-induced increase of 97 ± 9.9 milliBRET units at Vrest, and only 22.8 ± 6.2 at Vdepol (Figure 4D ). These findings indicate a significant reduction of mGlu5 receptor-induced Gq/11activation at Vdepol. Similar results were obtained with ebBRET measurement in cell populations, where the mGlu5 agonist-induced increase of ebBRET was significantly reduced by Vdepol(agonist-induced ebBRET increase was reduced by 24.8 ± 7.2 % at Vdepol compared to Vrest, Figure Supp 3A ). The same protocol applied to AT1 receptor showed no influence of Vm on AT1 receptor agonist-induced Gq/11-coupling (Figure Supp 3B ), suggesting once again that Vdepol specifically affects mGlu5 receptor signaling.