Abstract
Despite potently inhibiting the nociceptive voltage-gated sodium (Nav) channel, Nav1.7, 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 Nav1.7 IC 50 by a yet undefined mechanism. Nav channels are structurally related to voltage-gated calcium (Cav) channels, Cav1 and Cav2. 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 Cav1.2, Cav1.3, Cav2.1, and Cav2.2 mediated currents in HEK293 cells, with no inhibition of Cav2.3. As a major DRG I Ca component, Cav2.2 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 Cavs 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.
Highlights
IntroductionPhysiological pain (acute, nociceptive) functions as early warning to protect the organism from injury
Physiological pain functions as early warning to protect the organism from injury
It was shown that genetic deletion of SCN9A in both mice and humans, that the absence of functional Nav1.7 but not Nav1.8, increases endogenous opioid receptor (OR) analgesia via upregulation of the enkephalin precursor Penk mRNA, which could be inhibited by the OR antagonist, naloxone (Minett et al, 2015). μ-Theraphotoxin Pn3a (μ-TRTX-Pn3a), a three disulphide bridged, 35 amino acid peptide isolated from the venom of the South American tarantula Pamphobeteus nigricolour, is a selective and potent Nav1.7 channel inhibitor (Deuis et al, 2017)
Summary
Physiological pain (acute, nociceptive) functions as early warning to protect the organism from injury. Loss-of-function mutation of the SCN9A gene that codes for Nav1.7 leads to congenital insensitivity to pain (Cox et al, 2006), whereas SCN9A gain-of-function mutations causes paroxysmal extreme pain disorder and primary erythromelalgia (Dib-Hajj et al, 2008) These observations lead to increased interest in discovery of a highly selective and potent inhibitors of Nav1.7 to reduce the side effects seen in pan Nav channel inhibitors. Co-administration of Pn3a (1 mg/kg) with sub-therapeutic doses of opioids results in significant analgesia/anti-allodynia advocating for synergistic analgesic effects in rodent models of pain (Deuis et al, 2017; Mueller et al, 2019) In these reports, opioid receptor (OR) modulators naloxone and oxycodone did not alter Nav1.7 currents, nor Pn3a affected μ-/δ-/κ- OR mediated signaling (Mueller et al, 2019).
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