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.