Vm affects mGlu5 gating of TRPC6 channels
Transient Receptor Potential Channels (TRPC) are well known effectors of GPCRs intracellular secondary messengers. Typically, TRPC6 opening is triggered by the PLC signaling cascade that triggers formation of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3, ). In particular, mGlu5 receptor induces TRPC6-dependent Ca2+ influx . We then further tested the influence of Vm on the ability of mGlu5 to gate TRPC6 channels. For this purpose, we co-transfected mGlu5-Venus and TRPC6-tomato in HEK293T cells and recorded whole-cell currents induced by DHPG (100µM), in voltage-clamp experiments (Figure 5) . In cells co-expressing mGlu5 and TRPC6 (Figure 5B ), but not in cells expressing mGlu5 alone (Figure 5A ), DHPG applied at a holding potential of -80mV triggered a large inward current with similar kinetics and amplitude than previously reported in HEK293T cells . The current-voltage relationship (I/V curve, Figure 5C ) also displayed typical TRPC6 permeation and rectification properties . Given that the ions flow through open channels is governed by the membrane potential, we could not simply compare the current density generated and recorded by activation of mGlu5 at different potentials. We therefore perfused the mGlu5 agonist at different holding potentials (either -80 mV or -20 mV) and then rapidly recorded the currents generated over the full range of Vm ramping (from -80mV to +60mV in 100ms, Figure 5C ). When DHPG was applied at a holding potential of -20mV instead of -80 mV, the current amplitude recorded with the same voltage ramp protocol was strongly reduced. For example, we recorded a mean inward current density of 46.65 ± 10.32 pA/pF at -80 mV triggered by DHPG application at -80mV, versus 17.69 ± 6.36 pA/pF at -80 mV when DHPG was applied at -20mV (top insert, Figure 5C ). Importantly, these two protocols triggered the same I/V curve in non-stimulated cells, excluding a holding potential-dependent artefact during the recordings (Figure Supp 4 ). These results confirm that mGlu5 is more active at resting potentials compared to depolarized potentials, which impacts TRPC6 gating by mGlu5 downstream effectors.