Spider Venom Peptide Pn3a Inhibition of Primary Afferent High Voltage-Activated Calcium Channels

Despite potently inhibiting the nociceptive voltage-gated sodium (Na_v) channel, Na_v1.7, µ-theraphotoxin Pn3a is antinociceptive only upon co-administration with sub-therapeutic opioid agonists, or by itself at doses >3,000-fold greater than its Na_v1.7 IC by a yet undefined mechanism. Na_v channels are structurally related to voltage-gated calcium (Ca_v) channels, Ca_v1 and Ca_v2. These channels mediate the high voltage-activated (HVA) calcium currents (I ) that orchestrate synaptic transmission in nociceptive dorsal root ganglion (DRG) neurons and are fine-tuned by opioid receptor (OR) activity. Using whole-cell patch clamp recording, we found that Pn3a (10 µM) inhibits ∼55% of rat DRG neuron HVA-I and 60-80% of Ca_v1.2, Ca_v1.3, Ca_v2.1, and Ca_v2.2 mediated currents in HEK293 cells, with no inhibition of Ca_v2.3. As a major DRG I component, Ca_v2.2 inhibition by Pn3a (IC = 3.71 ± 0.21 µM) arises from an 18 mV hyperpolarizing shift in the voltage dependence of inactivation. We observed that co-application of Pn3a and µ-OR agonist DAMGO results in enhanced HVA-I inhibition in DRG neurons whereas co-application of Pn3a with the OR antagonist naloxone does not, underscoring HVA channels as shared targets of Pn3a and opioids. We provide evidence that Pn3a inhibits native and recombinant HVA Ca_vs at previously reportedly antinociceptive concentrations in animal pain models. We show additive modulation of DRG HVA-I by sequential application of low Pn3a doses and sub-therapeutic opioids ligands. We propose Pn3a's antinociceptive effects result, at least in part, from direct inhibition of HVA-I at high Pn3a doses, or through additive inhibition by low Pn3a and mild OR activation.