The N-type voltage-gated Ca²⁺ channel Ca_v2.2 is expressed predominantly at presynaptic neuronal terminals throughout the central and peripheral nervous systems, where it is critical for neurotransmitter release. Like the other members of the Ca_v family, Ca_v2.2 is a hetero-oligomeric channel comprising the core pore-forming α1B subunit, which determines the main biophysical and pharmacological properties of the channel, and typically three auxiliary subunits.

Alternative splicing is an essential mechanism used extensively in the mammalian nervous system to increase the level of diversity that can be achieved by a set of genes [26]. Two Ca_v2.2 splice variants have been reported to occur predominantly in the central and peripheral nervous system [27]. The Ca_v2.2 splice variant 37a is of particular interest, as it replaces the usual variant 37b in a specific subset of rat nociceptive neurons, and may thus represent a potential therapeutic target [28,29,30]. Additional splice variants, named Δ1 and Δ2, lack large parts of the domain II–III linker region including the synaptic protein interaction site, have been isolated from human neuroblastoma cells and brain cDNA libraries [31]. Clinically important, the Δ1 channel was less sensitive to inhibition by both ω-conotoxin MVIIA and GVIA than either the Δ2 variant or the full-length construct [31]. However, since a human splice variant that is only expressed in pathological pain states or in nociceptive pathways has not been identified to date, targeting of Ca_v splice variants as an analgesic strategy remains to be validated in humans.

While it is now appreciated that ω-conotoxins represent some of the most selective known inhibitors of the neuronal Ca_v2.2 isoform, and many of the key residues involved in binding have been identified, the precise peptide binding determinants and binding sites on Ca_v channels are not clearly identified. It is generally accepted that ω-conotoxins act as pore blockers [11,32], although the binding site has been mapped primarily to the external vestibule of the channel in the domain III pore-forming S5–S6 region (see [9] for docking model). In addition, inhibition of Ca_v2.2 by ω-conotoxins can be notably more complex than would be expected from simple pore blockers sharing a homologous binding site. While some of the best-characterized ω-conotoxins, such as GVIA and MVIIA, bind nearly irreversibly to Ca_v2.2, reversibility can be induced by voltage protocols which take advantage of the preferential binding of ω-conotoxin to the inactivated rather than resting state of Ca_v2.2 [33].

The large putative extracellular loop between IIIS5 and IIIH5 has been shown to be critically important for the block of Ca_v2.2 by the extensively studied peptide inhibitor of Ca_v2.2, ω-conotoxin GVIA (Figure 1). In particular, residues Gln1327, Glu1334, Glu1337, Gln1339 [11] and Gly1326 and Glu1332 of this region were identified as being important for blockage by GVIA [34]. The latter group proposed that Gly1326 may form a barrier that controls access of peptide toxins to the outer vestibule of the channel pore and stabilizes the toxin-channel interaction [34].In addition, the complex between MVIIA or GVIA toxins and Ca_v2.2 has been proposed to involve a central aromatic residue: tyrosine in the peptide, which is critical for high affinity interactions (Figure 3), plus lateral basic residues that form salt bridges with Glu1332 and perhaps Glu1334 and Glu1337 on the channel [34].

However, voltage-dependent reversibility varies significantly between ω-conotoxins, with CVIE and CVIF showing voltage-dependent reversal particularly in the presence of α2δ1 and β subunits, while reversibility of block by GVIA and MVIIA are only weakly influenced by co-expression with the subunits α2δ1 and β2a or β3 subunit [16,35]. Residues identified to contribute to this reversibility of ω-conotoxin block include in particular Gly1326, as well as intracellular domain II-III linker regions (Figure 1) [31,32].

While the pore-forming α1 subunit determines the main electrophysiological and pharmacological properties of Ca_v channels, auxiliary α2δ and β-subunits can modify channel gating properties and thus have a significant influence on calcium channel function [35,36]. To date, four auxiliary α2δ1–4 subunits, consisting of extracellular disulfide-linked α2δ dimers of 170 kDa, and four auxiliary β1–4 subunits [37] forming a 55 kDa cytoplasmic complex with the α1 subunit, have been identified. In addition, a 33 kDa γ subunit comprising four transmembrane segments was first found as a component of skeletal muscle Ca_v channels [38], and its related isoforms are expressed in heart and brain (for review see [14,22]). The presence or absence of the auxiliary subunits modulate the α1 subunit function and play an important functional role, modifying and regulating the kinetic as well as pharmacological properties of Ca_v channels [16,35,39].

Ca_v2.2 is expressed in a common pathway downstream from the large variety of receptors that mediate pain responses, thus, inhibition of this Ca_v subtype can mediate analgesia [65,66]. Although different types of calcium channels are found in nociceptive pathways, Ca_v2.2 is particularly important in controlling signaling in nociceptive pathways such as the ventral and dorsal horn of the spinal cord and dorsal root ganglion (DRG) neurons, especially along the dendrites and at presynaptic terminals where it contributes critically to neurotransmitter release [67]. As a consequence of the change in membrane potential that occurs in response to a painful peripheral stimulus, Ca_v2.2 channels open, resulting in an increase in intracellular calcium. This in turn triggers synaptic vesicle release of neurotransmitters such as glutamate, substance P and CGRP (Calcitonin Gene Related Peptide), which activate post-synaptic receptors in the membrane of spinothalamic neurons and nerve terminals localized in the dorsal horn of the spinal cord [67,68] (Figure 2), allowing the propagation of pain signals.

Several lines of evidence support Ca_v2.2 as an important pain target. Studies of Ca_v2.2 knock-out mice have shown that these animals, in contrast to Ca_v2.1 knock-out mice, had normal CNS (central nerve system) and motor function, but were resistant to development of neuropathic pain in a spinal nerve ligation model, and were insensitive to formalin-induced or visceral pain [69,70]. Furthermore, morphine, an opioid analgesic used for many years as the first option to treat severe pain, indirectly modulates Ca_v2.2 channels. Binding of morphine to μ-opioid receptors leads to inhibition of Ca_v2.2 through Gβγ-mediated signaling that reduces the ability of DRG sensory neurons to propagate pain signals centrally [67,71].

In addition, the α2δ1 auxiliary subunit of the Ca_v2.2 channels has been reported to be up-regulated in pain states in the dorsal root ganglion (DRG) and spinal dorsal horn after nerve injury [43,44,72,73], suggesting an involvement of this subunit with pathophysiological mechanisms of pain [73]. Although as discussed above, other mechanisms may underlie the involvement of the α2δ subunit with pain; studies using transgenic mice have found that the pro-algesic effects of α2δ subunits are mediated at least partially by enhancing Ca_v2.2 activity in sensory neurons [73]. In this study the author suggested it occurred possibly through enhanced formation of the functional Ca_v complex in lipid raft micro domains, as well as through hyper-excitability in dorsal horn neurons in response to peripheral stimulation [73].

Lastly, in 2004 the Ca_v2.2 blocker peptide ω-conotoxin MVIIA or ziconotide (Prialt^®), was approved for the treatment of severe chronic pain associated with cancer, AIDS and neuropathies. Intrathecal injection (delivered directly to the spinal cord) of this synthetic peptide has proved effective against both neuropathic and inflammatory pain in laboratory animals and man [15,18,69,74,75], although associated side effects limit its application. Importantly, ziconotide acts synergistically with opioid analgesics without inducing tolerance or addiction [19].

Cone snails comprise over 500 species of marine predatory gastropods that are mostly found on or near coral reefs in tropical and subtropical waters, including the coastal waters of Australia. They produce a highly complex mixture of venom peptides which have evolved for prey capture and defense (for review see [1,9]). This includes the ω-conotoxins, small disulfide-rich peptides with defined activity at mammalian Ca_v isoforms.

The ω-conotoxins belong to the O-superfamily, which also includes the δ-conotoxins (inhibit the fast inactivation of the voltage gated Na⁺ channels), µO-conotoxins (inhibit voltage-gated Na⁺ currents) and κ-conotoxins (interact with K⁺ channels). To date, ω-conotoxins targeting mammalian Ca_v isoforms have only been isolated from piscivorous cone snails, where they are likely to have evolved as part of the “motor cabal” leading to rapid flaccid paralysis of their fish prey [76]. In contrast, the few ω-conotoxins isolated from mollusc-hunting species to date (PnVIA and PnVIB) have been found to be inactive at mammalian Ca_v channels, suggesting distinct phylum-selective pharmacology, consistent with their sequence diversity (for review see: [9]).

The ω-conotoxin family comprises peptides ranging from 24 to 30 amino acids in length. As seen for the snake and spider venom toxins, the relatively low sequence homology among ω-conotoxins suggests that the overall three-dimensional structure and charge distribution underpin their interaction with Ca_v2.2 channels (Table 2, Figure 3). Although the remainder of the amino acids shows no absolute sequence conservation, positions 2 and 25 are always occupied by either a lysine (K) or an arginine (R), and the most active forms have a tyrosine (Y) at position 13. Moreover, a high proportion of residues contain hydroxyl moieties, which is accentuated in many of the ω-conotoxins by the substitution of γ-hydroxyproline for proline [77]. Disulphide bonds fold the peptide into a highly conserved cysteine framework pattern (C-C-CC-C-C) (Table 2, Figure 3) that contributes to tertiary structure stabilization. This configuration defines the canonical ω-conotoxin fold, which comprises a triple-stranded β-sheet/inhibitory cysteine-knot framework (Figure 3) [78]. A unique feature of conotoxins is their high degree of post-translational modification [79], and several ω-conotoxin have stability enhanced naturally through the use of these post-translational modifications (PTMs) [1,80]. This mechanism may limit potential degradation by carboxylases, which would otherwise rend the peptide biologically inactive. In addition, a C-terminal amide and the relative abundance of basic residues within ω-conotoxins class gives them an overall net positive charge, which presumably assists in their complementary binding to Ca_v2.2 channels [81].

The binding determinants for the high affinity interaction of ω-conotoxins with Ca_v2.2 have been proposed to rely on a two-point pharmacophore formed by the highly conserved Y13 (tyrosine) and K2 (lysine) [8,85,86,93,94]. However, Y13 and K2 are also often conserved in ω-conotoxins with activity at Ca_v2.1. Thus, residues contributing to selectivity at Ca_v2.2 over Ca_v2.1 are less clear. Intriguingly, the two ω-conotoxins that display most sequence homology, MVIIA and MVIIC, target quite different Ca_v subtypes (Ca_v2.2 and Ca_v2.1, respectively), whereas conversely, ω-conotoxins GVIA and MVIIA inhibit the same Ca_v subtype (Ca_v2.2), despite significantly lower sequence homology (Table 2, Figure 4). An extensive study assessing loop splice hybrids found that selectivity of MVIIA and MVIIC for Ca_v2.2 and Ca_v2.1 respectively, is controlled in a concerted manner by residues of loop 2 and 4 (Figure 3) [81]. Peptides with homogeneous combinations of loop 2 and 4 display clear selectivity while those with heterogeneous combinations of loops 2 and 4, are less discriminatory (Figure 3A−B) [81]. CVID is notable for its high potency at Ca_v2.2 and low potency at Ca_v2.1, making this peptide the most Ca_v2.2-selective peptide described to date [8,90].

Importantly, it has been reported that the affinity of ω-conotoxins is often profoundly affected by the presence of auxiliary subunits, in particular α2δ and β subunits [16,35,95,96,97,98]. However, the degree to which co-expression of auxiliary subunits affects ω-conotoxin potency can vary significantly, with MVIIA, GVIA, CVID and CVIF being particularly susceptible to affinity reductions in the presence of α2δ1 subunits, while the potency of CVIE is affected to a lesser degree by co-expression with auxiliary subunits [16,35]. Such pharmacological effects can have profound implications for the therapeutic potential of ω-conotoxins, as α2δ1 subunit expression is increased in dorsal root ganglion and spinal cord in several animal models of neuropathic pain [1,16,99].

Spiders, the most species-rich family of terrestrial venomous predators, have evolved highly complex venoms to assist with prey capture ([105], for review see [106]). Like cone snails, spiders have evolved a myriad of peptide venom components with activity at voltage-gated ion channels including Ca_v2.2 (for review see [106,107]).

Interestingly, spider toxins have provided some of the most subtype-selective Ca_v2.1 and Ca_v2.3 inhibitors known to date [108,109]. However, in contrast to conotoxins, which are notable for their selectivity for Ca_v2.2 in particular (for review see [9]), relatively few spider venom peptides are active at Ca_v2.2, and even fewer show selectivity for Ca_v2.2 over other Ca_v isoforms (see Table 2 and Table 3) [4,5].

Spider venom peptides with activity at Ca_v2.2 have to date only been described from Haplopelma huwenum, Agelenopsis aperta, Phoneutria nigriventer, Phoneutria reidyi and Segestria florentina [7,110,111,112,113,114,115,116,117,118,119,120,121,122,123]. These Ca_v2.2 inhibitors are structurally diverse and can share common structural motifs with the ω-conotoxins (Figure 3, Figure 4, Figure 5), with 3 disulfide bonds forming an inhibitory cysteine knot, or have as many as 7 disulfide bonds, as is the case for ω-ctenitoxin-Pn3a [113,119]. The majority of these peptides have little selectivity for Ca_v2.2 and displays activity at Ca_v1, Ca_v2.1 and Ca_v2.3 isoforms. For example, ω-agatoxin-Aa3a is equipotent at Ca_v1 and Ca_v2.2, and ω-ctenitoxin-Pn2a from Phoneutria nigriventer blocks voltage-gated calcium channels Ca_v2.1, Ca_v2.3, Ca_v1 and Ca_v2.2 in decreasing order of potency [119]. Some spider venom peptides, such as the theraphotoxins Hh1a-d, even inhibit the related Na_v channels and are designated as ω-/µ-toxins based on this pharmacological profile [110]. In contrast, ω-segestritoxin-Sf1a, ω-agatoxin-Aa2a and ω-theraphotoxin-Hh1a or huwentoxin-10 preferably inhibit vertebrate Ca_v2.2, although it is unclear whether their selectivity can match the more than 10,000-fold preference for Ca_v2.2 over Ca_v2.1 exhibited by some conotoxins [8,113,115,123]. Given the similar overall structure shared by cone snail and spider venom peptides, these divergent selectivity profiles are surprising. However, as illustrated by conopeptides, small differences in structure may result in profound effects on Ca_v selectivity. Specifically, conotoxins MVIIA, MVIIC, GVIA and CVID share a high degree of sequence similarity and are structurally closely related. Nonetheless, MVIIC is a selective Ca_v2.1 inhibitor, while MVIIA, GIVA and CVID are highly selective Ca_v2.2 blockers [8].

Though spider toxins are generally accepted to act as gating modifiers at Ca_v channels, relatively little is known about the mechanism of action of Ca_v2.2 block. For example, ω-ctenitoxin-Pn4a was shown to decrease peak Ca_v2.2 current with little effect on voltage-dependence of activation [120], and Ca_v2.2-specific inhibitors from spider venoms such as ω-segestritoxin-Sf1a were able to displace radiolabelled ω-conotoxins (Table 3) [123], suggesting that these peptides may act like pore blockers rather than gating modifiers at Ca_v2.2. Importantly, little is known about the influence of Ca_v auxiliary subunits on inhibition by spider venom peptides. Given that the pharmacology of cone snail toxins is known to be affected by auxiliary subunits [16,35,124], future studies should include characterising the effect of auxiliary subunits on inhibition of Ca_v2.2 by spider venom peptides.

While the analgesic potential of Ca_v2.2-selective peptides from spider venom has not been assessed extensively, several studies report efficacy with little side effects in various animal models of pain. ω-ctenitoxin-Pn4a (PnTx3–6, Phα1β, neurotoxin 3–6), a toxin from the spider Phoneutria nigriventer which non-selectively inhibits neuronal Ca_v with a rank order of potency of Ca_v1.2 > 2.2 > 2.1 > 2.3 [119,120,126,127], elicited prolonged analgesia after intrathecal administration in an animal model of incisional pain [128,129]. ω-ctenitoxin-Pn4a and had no effect on mean arterial blood pressure, heart rate or gross neuronal performance, suggesting that Ca_v inhibitors from spider venoms could also find therapeutic application for the treatment of pain [128,129]. Another non-selective toxin from Phoneutria nigriventer, ω-ctenitoxin-Pn2a (rank order of potency: Ca_v2.1 > 2.3 > 1 > 2.2) [118], showed prevalent antinociceptive effects in neuropathic pain models and did not cause adverse motor effects efficacious doses [130]. However, as Ca_v2.1 and Ca_v3 have been proposed as analgesic targets in their own right [131,132], the contribution of non-Ca_v2.2 channels to these observed in vivo effects remains to be determined.

Kurtoxin, a 63-residue peptide isolated from the venom of the scorpion Parabuthus transvaalicus is related to the α-scorpion toxins, a family of toxins that slow inactivation of Na_v channels. While kurtoxin has shown selectivity for heterologous expressed Ca_v3 or T-type calcium channels [137], activity at Ca_v2.2 has been described in rat sympathetic and thalamic neurons [135]. In contrast to ω-conotoxins with activity at Ca_v2.2 channels, scorpion toxins with activity at Ca_v channels, including kurtoxin, act as gating modifiers. Thus, selectivity differences observed in overexpression systems compared to neurons may be due to the influence of auxiliary subunits, although this has not been assessed to date.

Calcicludine (60 residues, 3 disulfide bonds) and calciseptine (60 residues, 4 disulfide bonds) were isolated from the venom of the black mamba, Dendroaspis polyepsis, and the green mamba, Dendroaspis angusticeps, respectively. While calciseptine inhibits L-type calcium channels and has no activity at Ca_v2.2 [138], calcicludine is less selective and potently inhibits all high-voltage-activated calcium channels, including Ca_v2.2 [139].

Recently, two peptides with activity at neuronal Ca_v channels were isolated from the venom of the centipede Scolopendra subspinipes mutilans. ω-SLPTX-Ssm1a, an 83 residues peptide containing 7 cysteines was found to act as an activator of Ca_v channels [140], while a smaller peptide, ω-SLPTX-Ssm2a (54 residues) was shown to inhibit Ca_v channels expressed in DRG neurons [140]. However, the Ca_v subtype selectivity of these peptides is currently unknown but it may include Ca_v2.2, which is expressed in peripheral sensory neurons.

The predatory assassin bugs (Hemiptera: Reduviidae) contain a complex mixture of small and large peptides in its toxic saliva which is used to immobilize and pre-digest their prey, and for defense against predators [141]. Three novel peptide toxins, named Ado1, Ptu1 and Iob1, isolated from three species of assassin bugs (P. turpis, A. dohrni, and I. obscurus), were biologically active in electrophysiological assays using BHK-N101 cells stably expressing rabbit Ca_v2.2, β_1A subunit, and α2δ subunit [141]. Ptu1 binds reversibly to Ca_v2.2 with lower affinity than ω-conotoxin MVIIA. Ptu1 lacks most of the residues shown to be important for ω-conotoxin binding to the N-type calcium channel, including equivalents of Tyr13 or Lys2 (Figure 4 and Figure 5). This peptide belongs to the inhibitory cysteine knot structural family (ICK) that consists of a four-loop Cys scaffold forming a compact disulfide-bonded core [142]. Thus, as for the other venom peptides compared here, the structure of Ptu1 aligned with related Ca_v2.2 blockers MVIIA and GVIA, indicating that a common functional motif can be supported by a common three-dimensional structure despite the lack of sequence homology (Figure 5).