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.