The E1015K variant in the synprint region of the CaV2.1 channel alters channel function and is associated with different migraine phenotypes.

Mutations in the CACNA1A gene, which encodes the pore-forming α1A subunit of the CaV2.1 voltage-gated calcium channel, cause a number of human neurologic diseases including familial hemiplegic migraine. We have analyzed the functional impact of the E1015K amino acid substitution located in the "synprint" domain of the α1A subunit. This variant was identified in two families with hemiplegic migraine and in one patient with migraine with aura. The wild type (WT) and the E1015K forms of the GFP-tagged α1A subunit were expressed in cultured hippocampal neurons and HEK cells to understand the role of the variant in the transport activity and physiology of CaV2.1. The E1015K variant does not alter CaV2.1 protein expression, and its transport to the cell surface and synaptic terminals is similar to that observed for WT channels. Electrophysiological data demonstrated that E1015K channels have increased current density and significantly altered inactivation properties compared with WT. Furthermore, the SNARE proteins syntaxin 1A and SNAP-25 were unable to modulate voltage-dependent inactivation of E1015K channels. Overall, our findings describe a genetic variant in the synprint site of the CaV2.1 channel which is characterized by a gain-of-function and associated with both hemiplegic migraine and migraine with aura in patients.

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

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.

Pharmacological characterisation of the highly NaV1.7 selective spider venom peptide Pn3a.

Human genetic studies have implicated the voltage-gated sodium channel NaV1.7 as a therapeutic target for the treatment of pain. A novel peptide, µ-theraphotoxin-Pn3a, isolated from venom of the tarantula Pamphobeteus nigricolor, potently inhibits NaV1.7 (IC50 0.9 nM) with at least 40-1000-fold selectivity over all other NaV subtypes. Despite on-target activity in small-diameter dorsal root ganglia, spinal slices, and in a mouse model of pain induced by NaV1.7 activation, Pn3a alone displayed no analgesic activity in formalin-, carrageenan- or FCA-induced pain in rodents when administered systemically. A broad lack of analgesic activity was also found for the selective NaV1.7 inhibitors PF-04856264 and phlotoxin 1. However, when administered with subtherapeutic doses of opioids or the enkephalinase inhibitor thiorphan, these subtype-selective NaV1.7 inhibitors produced profound analgesia. Our results suggest that in these inflammatory models, acute administration of peripherally restricted NaV1.7 inhibitors can only produce analgesia when administered in combination with an opioid.

Conotoxins That Could Provide Analgesia through Voltage Gated Sodium Channel Inhibition.

Chronic pain creates a large socio-economic burden around the world. It is physically and mentally debilitating, and many suffers are unresponsive to current therapeutics. Many drugs that provide pain relief have adverse side effects and addiction liabilities. Therefore, a great need has risen for alternative treatment strategies. One rich source of potential analgesic compounds that has immerged over the past few decades are conotoxins. These toxins are extremely diverse and display selective activity at ion channels. Voltage gated sodium (NaV) channels are one such group of ion channels that play a significant role in multiple pain pathways. This review will explore the literature around conotoxins that bind NaV channels and determine their analgesic potential.