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 _v 1.7, 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 _v 1.7 IC ₅₀ by a yet undefined mechanism. Na _v channels are structurally related to voltage-gated calcium (Ca _v ) channels, Ca _v 1 and Ca _v 2. These channels mediate the high voltage-activated (HVA) calcium currents ( I _Ca ) 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 _Ca and 60-80% of Ca _v 1.2, Ca _v 1.3, Ca _v 2.1, and Ca _v 2.2 mediated currents in HEK293 cells, with no inhibition of Ca _v 2.3. As a major DRG I _Ca component, Ca _v 2.2 inhibition by Pn3a ( IC ₅₀ = 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 _Ca 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 _v s at previously reportedly antinociceptive concentrations in animal pain models. We show additive modulation of DRG HVA- I _Ca 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 _Ca at high Pn3a doses, or through additive inhibition by low Pn3a and mild OR activation.
Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
(Copyright © 2021 McArthur, Munasinghe, Finol-Urdaneta, Adams and Christie.)