CBD3063 selectively decreases Cav2.2 (N-type)
Ca2+ currents in sensory neurons
We next asked if the CBD3063-mediated decrease in Ca2+currents was due to our target Cav2.2 (N-type) channels.
To test the minimum concentration needed to inhibit N-type
Ca2+ channels’ function, small-to-medium sized DRG
neurons were treated overnight with 2, 20 and 50 µM of CBD3063 or
control (0.1% DMSO). The next day, whole-cell patch-clamp recordings
were performed in the presence of a cocktail of blockers of all other
subtypes of calcium channels present in DRG neurons, thus isolating the
N-type currents. When compared to DMSO-treated cells, 2 µM of CBD3063
did not modify the activity of these channels. On the contrary, 20 and
50 µM of CBD3063 significantly reduced peak current density, thus, 20 µM
was sufficient to decrease N-type currents and used for further
experimentation (DMSO: 1.00 ± 0.07; 2 µM CBD3063: 0.93 ± 1.07; 20 µM
CBD3063: 0.74 ± 0.04; 50 µM CBD3063: 0.65 ± 0.06; Figure 5A ). A
significant decrease in N-type currents (Figure 5B ) and current
density at 0 and +10 mV (Figure 5C ) was observed in cells
incubated with 20 µM of CBD3063. At peak current density (+10 mV;Figure 5D ), CBD3063 decreased N-type currents by
~33.5% when compared to the control group (DMSO:
-104.00 ± 11.32 pA/pF; CBD3063: -69.18 ± 6.54 pA/pF). Plotting
voltage–dependence of activation and steady–state inactivation curves
revealed no differences in half activation or inactivation potentials
and slope factors between these two conditions (Figure 5E andTable 2 ). These data confirm that targeting the
CRMP2–Cav2.2 interaction with CBD3063 decreases current
influx through N-type Ca2+ channels.