CBD3 compounds inhibit total Ca2+ currents in
sensory neurons
The calcium imaging results do not discriminate between small, medium
and large diameter DRG neurons, therefore we next dissected the effects
of the top compounds on a subpopulation of small-to-medium diameter
nociceptive neurons (Basbaum, Bautista, Scherrer & Julius, 2009) since
large-diameter DRG neurons in normal conditions transduce mechanical and
proprioceptive information. The medium and small DRG neurons belong to
lightly myelinated Aδ and unmyelinated C fibers, respectively. These
primary afferent fibers are necessary for pain transmission since they
send nociceptive information to the dorsal horn of the spinal cord
(Basbaum, Bautista, Scherrer & Julius, 2009); thus, we performed
whole-cell patch-clamp recordings in small-to-medium diameter DRG
neurons from female rats to electrophysiologically validate the nine top
compounds that blocked Ca2+ influx.
DRGs were treated overnight with 20 µM of test compounds or vehicle
(0.1% DMSO). Inward current through Ca2+ channels was
carried by Ba2+ and will be referred to it as
Ca2+ currents. From a holding potential of -90 mV,
200-ms depolarization steps from -70 to +60 mV in 10 mV increments, we
elicited a family of Ca2+ currents (Figure
2B ). We next obtained the peak current density and normalized it to the
control group (Figure 2C ) and found that CBD3063, 3065 and 3074
significantly reduced total Ca2+ currents when
compared to cells treated with 0.1% DMSO (DMSO: 1.00 ± 0.06 pA/pF;
CBD3063: 0.51 ± 0.04 pA/pF; CBD3065: 0.52 ± 0.07 pA/pF; CBD3074: 0.58 ±
0.06 pA/pF).
Of the three compounds that inhibited Ca2+ currents,
CBD3063 and CBD3065 represent unique chemotypes (Table 1 ) and
were selected for additional characterization. However, in preliminary
tests, CBD3065 did not exhibit analgesic properties (data not shown).
Thus, CBD3065 was not further pursued. We measured current
density–voltage relationships and observed that incubation with 20 µM
of CBD3063 significantly decreased Ca2+ current
density from 0 to 30 mV (Figure 3A, B ). Furthermore, at peak
current density (+10 mV; Figure 3C ), the reduction in
Ca2+ currents imposed by CBD3063 was
~46.54% when compared to cells treated with 0.1% DMSO
(DMSO: -93.23 ± 10.40 pA/pF; CBD3063: -49.84 ± 6.17 pA/pF). Inspection
of voltage–dependence of activation revealed no difference in the half
activation potential and slope factors between groups (Figure
3D and Table 2 ). We also assessed steady–state inactivation
kinetics of the channels at multiple test potentials by measuring the
fraction of current remaining at +10 mV. As seen in Figure 3Dand Table 2 , our results revealed no significant differences in
half inactivation potential and slope factors between the conditions.
Collectively, our findings indicate that our top compound – CBD3063 –
inhibits Ca2+ currents in small-to-medium diameter
(i.e., presumptively nociceptive) sensory neurons.