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Block of Voltage-Gated Calcium Channels by Peptide Toxins

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Introduction

Structurally, the calcium channels are closely related to sodium channels, with the main structurally significant difference being the positioning and nature of the residues that line the selectivity filter in the pore of the channel. There are at least six pharmacologically distinct calcium channels types, including L-, N-, P/Q-, T, and R-type calcium channels (Table 1).1 Within each group are multiple subtypes that are presently less easy to distinguish pharmacologically. In the nervous system, several types of ion channels may contribute to processes such as neurotransmitter release, with the ratio and role for each type varying among different nervous tissues2 (see Table 1). A number of different peptide toxins from marine snails (conotoxins) and spiders (agatoxins, grammotoxin and DW13.3 Filistata hibernalis) are known to potently inhibit the activities of specific voltage-dependent calcium channels.3,4 The conotoxins are usually smaller in size comprising around 22—30 amino acid residues and are believed to act by physically blocking the pore of the channel.5 Spider toxins such as ω-Aga IVA6 typically larger consisting of 60—90 residues and ω-grammotoxin7 around 30—40 residues affect channel gating.8

Table 1. Subunit composition and function of Ca2+ channel types.

Table 1

Subunit composition and function of Ca2+ channel types.

The diversity of peptide toxins provides the possibility for selective modulation of nerve function that may allow the selective treatment of diseases such as pain and stroke. The first examples of calcium channel inhibitors being useful therapeutically are the ω-conotoxins MVIIA (Ziconotide or Prialt, Elan) and ω-CVID (AM336, AMRAD) which target the N-type calcium channel and are currently in clinical trials for chronic pain. ω-Conotoxins are a large family of structurally related peptides with a wide range of specificities for different subtypes of the VSCC (Table 2).9-18 Their high selectivity has made them enormously valuable as physiological tools. A remarkable feature of the six cysteine/four-loop framework exemplified by the ω-conotoxins is the presence of a cystine knot within the structures. This motif consists of an embedded loop in the structure formed by two of the disulfide bonds and their connecting backbone segments. This loop is penetrated by the third disulfide bond in a remarkable example of nature's engineering designs. Although the structure of the core of ω-conotoxins is rigid due to the knotted disulfide structure, NMR studies have revealed conformational flexibility in the exposed loops that might influence receptor binding. This review will describe structural aspects of peptide toxins from cone snails as well as those from snakes and spiders that selectively inhibit different voltage-sensitive calcium channels (VSCCs). Several excellent general reviews describing peptide toxin interactions with voltage-gated ion channels including calcium channels have been published recently.19-21

Table 2. Sequences of native ω-Conotoxins from different Conus species.

Table 2

Sequences of native ω-Conotoxins from different Conus species.

L-Type VSCC Antagonists

Toxins from several species of snails and snakes block the L-type VSCC. For example, ω-conotoxin TxVII from the mollusc hunting Conus textile targets dihydropyridine-sensitive L-type calcium channels.17 TxVII is similar to other ω-conotoxins, including GVIA, MVIIA and CVID, having 26 residues including six Cys which form three disulphide bonds (Table 2). Thus TxVII shares a similar four-loop framework with the other ω-conotoxins (fig. 1). However, TxVII is negatively charged and has a large hydrophobic patch unlike other ω-conotoxins that target N-and P/Q-type VSCCs which are highly positively charged (+5 to +6), These positive charges contribute to N-type VSCC binding, with Lys2 being the most critical charge22-24 The conserved Lys2 is the only positively charged residue in TxVII and there are several negative charges in the proximity of Lys2. It is also missing a Tyr in position 13, which has been shown by several groups to be crucial for activity at the N-type VSCC.22,25,26 Instead TxVII has a hydrophobic Leu13. The hydrophobic patch in TxVII, which includes Phe11 and Trp26, is not found in other ω-conotoxins and could be part of the face that interacts with the L-type VSCC. These differences presumably act in concert to explain why TxVII does not bind to the N-type VSCC, despite having the consensus structure of ω-conotoxins. Interestingly, TxVII reversibly inhibits snail but not rat L-type currents,27 indicating that it has evolved to selectively inhibit calcium channels found in its prey. The sequence of TxVII resembles δ-conotoxin TxVIA, an inhibitor of sodium channel inactivation and despite the fact that they act on different ion channels, they share the common Cys four loop framework (fig. 2).28,29 Conotoxins with this conserved cysteine framework highlight the cysteine knots as an excellent structural scaffold from which to present different side chains to achieve diverse biological activities.27

Figure 1. Three different ω-conotoxins that inhibit different voltage-sensitive calcium channels.

Figure 1

Three different ω-conotoxins that inhibit different voltage-sensitive calcium channels. Ribbon diagram and sequnence alignments for A) L-type VSCC inhibitor TxVII from C. textile, B) P/Q-type VSCC inhibitor MVIIC from C. magus and C) N-type VSCC (more...)

Figure 2. Superimposition of solution structures of A) ω-conotoxin TxVII, a L-type calcium channel inhibitor from Conus textile and B) δ-conotoxin TxVIA, a sodium channel inhibitor.

Figure 2

Superimposition of solution structures of A) ω-conotoxin TxVII, a L-type calcium channel inhibitor from Conus textile and B) δ-conotoxin TxVIA, a sodium channel inhibitor. Disulfides are indicated in orange and corresponding residues are (more...)

Recently a conotoxin member of the Contryphan family, glacontryphan-M from C. marmoreus was reported to be the first contryphan peptide containing two γ-carboxylatedglutamic to be a selective L-type calcium channel blocker.30 Interestingly the NMR solution structure of glacontryphan-M superimposes with the backbone of Contryphan-R31 and Contryphan-Vn.32,33 Similar to Contryphan-Vn, which was recently reported to be a Ca2+-dependent K+ channel inhibitor, glacontryphan-M requires calcium binding to the N-terminal of the peptide to be active at the L-type calcium channel. The binding of calcium to the peptide induces a structural perturbation in the Gla-containing part of the peptide believed to be important for the activity of the peptide.

The venoms from the snakes of the families Elapidae and Hydrophidae contain a variety of toxins with distinct pharmacologies. A large portion of these toxins are either neurotoxins34 that act postsynaptically to block the nicotinic acetylcholine receptor causing flaccid paralysis of the muscle,34 or cytotoxins35 which appear to change membrane permeability and/or membrane organisation. These two very distinct pharmacological classes share a similar structural motif, namely the three-fingered architecture, where four conserved cysteine pairs are organised as a “disulfide β-cross”.36,37 A third class of postsynaptically active toxins have been isolated from the venom of the mamba snakes (Dendroaspis spp). Even though they also have a three-fingered architecture, they show no neurotoxic or cytotoxic activity. Their homology and immunological properties have led to further subdivision into four subgroups32,39 of which two have been pharmacologically characterised. Toxins of subgroup I are potent inhibitors of acetylcholine esterases,40 whereas subgroup III contains toxins such as FS2 from Dendroaspis polyepis polyepis,38 C10S2C2 from D. augusticepceps (green mamba)39 and calciseptine from the venom of D. p. polyepis. Calciseptine and FS2 differs only in two residue and have similar blocking effect at the L-type VSCC41 (fig. 3).

Figure 3. Three different L-type calcium channel blockers from different venomous species.

Figure 3

Three different L-type calcium channel blockers from different venomous species. A) TxVII, a 26 residue peptide from the cone snail C. textile; B) Calcicludine (CaC), a 60 residue peptide from the green mamba snake Dendroaspis augusticeps and C) FS2, (more...)

Calciseptine, a 60 residue peptide, has been found to be an antagonist at heart L-type calcium channels, but acts as an agonist at frog and mammalian skeletal muscle L-type calcium currents.42 Like ω-agatoxin IIIA from the spider venom,43 calciseptine blocks the L-type VSCC with weak efficacy and low specificity.44,45 Flanking proline residues have been proposed to bracket the L-type calcium channel binding site of calciseptine and FS2.46,47 In calciseptine they are spaced four residues apart (P-42 TAMWP-47) to present this series of residues in a well-defined loop in the two proteins. Albrand et al (1995)48 also proposed that Trp contributes to binding to the L-type channel. In support, an eight residue long peptide containing the P-42 TAMWP-47 motif (L-calchin) retained the parent peptide activity at the L-type calcium channel, but with lower potency.46

Calcicludine (CaC), a 60 amino acid peptide isolated from the green mamba venom (Dendroaspis augusticeps), irreversibly inhibits L-type calcium channels (IC50 ∼90 nM), without affecting the voltage-dependence or kinetics of the current. However, even at saturating toxin concentration, block was incomplete (∼60%), indicating either a partial occlusion of the pore or an effect on channel gating. Recent data suggest that CaC interacts with multiple transmembrane domains of the L-type VSCC to mediate either a partial occlusion of the conductance pathway or a reduction in the steady-state open probability of the L-type VSCC.45 CaC block is both tissue and species dependent.45 Structurally, CaC is unrelated to calcium channel blocking peptides isolated from marine snails, spiders or other snakes. Instead it exhibits structural homology to a number of protease inhibitors such as the bovine pancreatic trypsin inhibitor (BPTI)49,50 and the dendrotoxin family of snake toxins that inhibit the K+ channel.51 The secondary structural features shared by these three peptides include a short 3—10 helix, a hairpin β-sheet twisted 180° and a regular α-helix.51 Closer examination of the secondary structure of dendrotoxin-I and CaC they differ at the N-terminus. N-terminal chimeras between CaC and dendrotoxin indicate that differences in distribution of N-terminal cationic charges underlies their L-type calcium channel or potassium channel preferences51,52 (fig. 4). Interestingly, three of the four prolines in the CaC are in the N-terminal region, which also includes a tryptophan previously identified to be involved in TxVII,17,28 calcispetine and FS2,47 affinity for the L-type VSCC. However, the precise role for these residues in CaC remains to be elucidated.

Figure 4. Ribbon diagrams of three venom peptides with low sequence homology but high structural homology.

Figure 4

Ribbon diagrams of three venom peptides with low sequence homology but high structural homology. A) Bovine pancreatic trypsine inhibitor (BPTI) in pink, B) Calcicludine (CaC), a L-type calcium channel inhibitor from the green mamba snake, Dendroaspis (more...)

DW13.3 is a74 residue peptide isolated from the venom of the spider Filistata hibernalis that contains 12 cysteines.53 It causes a potent and reversible inhibition of Cav2.1, Cav2.2, Cav1.2 and Cav2.3 expressed in Xenopus oocytes with IC50 values of 4.3, 14.4, 26.8 and 96.4 nM, respectively. The dose-response data indicate that the peptide binds in a 1:1 manner, producing different extents of block at saturating concentrations, being most effective at Cav1.2. Structurally, DW13.3 differs from other calcium channel peptide toxins, but it has the same broad specificity across different voltage-sensitive calcium channel subtypes as that of ω-Aga IIIA, a 76 residue peptide isolated from the funnel web spider A. aperta.54-56 Despite the lack sequence homology, they both have an overall positive charge and a hydrophobic core of 12 cysteine residues forming six disulfide bridges. These six disulfide bonds again appear to maintain a conserved three-dimensional fold from which to present amino acids side chains in specific orientations.

N-Type VSCC Antagonists

Toxins that selectively target the N-type voltage-sensitive calcium channel have mainly been isolated from cone snails (ω-conotoxin), whereas a number of nonselective inhibitors for this channel have been found in both snake and spider venoms. ω-Conotoxin inhibitors have been identified from a number of different fish hunting cone snails, including GVIA from Conus geographus,57,58 MVIIA from Conus magus,59 CVID from Conus catus16 and CnVIIA from Conus consors15 (see Table 2). All ω-conotoxins identified to date are 25—27 amino acid residues long but specific variations in primary sequence can be accommodated without affecting potency, selectivity or the structural core of the peptide (fig. 5). As found for snake and the spider toxins, the lack of sequence homology among ω-conotoxins suggests that the overall three-dimensional structure and charge distribution underpin their interaction with the N-type VSCC. Interestingly, the two ω-conotoxins that display most sequence homology (MVIIA and MVIIC) (Table 2) target quite different VSCCs (Cav2.2 and Cav2.1, respectively). Whereas ω-conotoxins GVIA and MVIIA inhibit the same calcium channel subtype despite remarkably low sequence homology (Table 2).

Figure 5. Sequence alignment and ribbon diagrams for selective N-type voltage-sensitive calcium channel blockers from three different cone snail species, A) CVID from C.

Figure 5

Sequence alignment and ribbon diagrams for selective N-type voltage-sensitive calcium channel blockers from three different cone snail species, A) CVID from C. catus, B) GVIA from C. geographus and C) MVIIA from C. magus. Disulfide bond connectivity (yellow), (more...)

All the ω-conotoxins are characterised by a common cysteine scaffold that stabilises the four-loop frame work (fig.5). This configuration defines the canonical ω-conotoxin fold, which comprises a triple-stranded β-sheet/inhibitory cysteine-knot frame work 60,61 that has also been found in peptides such as PVIIA (a K+-channel blocker from Conus. purpurascens,62,63 and the cyclic cysteine knot peptide Kalata.64 Interestingly, independent of sequence or structural homology, the affinity of all ω-conotoxins for the N-type (Cav2.2) calcium channel is reduced by the presence of the α2δ auxilary subunit.65

Structure Activity Studies at the N-Type VSCC

Only limited information is available on the location of the ω-conotoxin binding site on the N-type calcium channel protein. By using a chimeric approach, Ellinor et al5 showed that the outer vestivule of the pore region in the third domain of the N-type calcium channel α1 subunit contained amino acid residues that appeared to contribute to conotoxing block. Additional residues in this region were implicated by Feng et al.66 However, it is likely that other parts of the N-type calcium channel are involved in the docking of the blocking molecules.

Several groups have undertaken extensive studies of the structure-activity relationship (SAR) of ω-conotoxins to identify all residues important for binding to the N-type calcium channel (for review see ref. 67). These SAR investigations have mainly consisted of alanine scans and then further probing of positions found to influence binding. Initially these studies were followed by CD-spectroscopy, but more recently NMR studies have been used to identify structural perturbations that may have accompanied residue replacements.

The first and most important residue identified to be crucial for ω-conotoxin binding to the N-type VSCC was the conserved Tyr13. Replacing the Tyr13 with Phe and the binding drops affinity by 2—3 orders of magnitude in GVIA,22 MVIIA26 and CVID,68 and Ala replacement decreased binding another order of magnitude.22, 25 The orientation of Tyr13 is also important, since D-Tyr13 analogues of MVIIA (N-type specific) and MVIIC (P/Q-type selectivie) had 3—4 orders of magnitude lower affinity than the native conotoxin.69 In these analogues the shape of loop 2 was severely distorted. The decrease in binding of D-Tyr13-MVIIA for the N-type calcium channel appears to stems from the loss of either (i) a hydrogen bond from the hydroxyl group on Tyr13, (ii) a hydrophobic interaction from Leu11 and/or (iii) a putative electrostatic interaction from Arg10.69

Lys2 is another conserved residue in N-type selective ω-conotoxins that contributes to activity. Replacing Lys2 with an alanine in either GVIA,23- 25 or MVIIA23, 26 dramatically reduces affinity. Arginine and ornithine can effectively replace lysine, but bulkier aromatic residues hinder binding.70 Leucine and norleucine also reduce binding affinity, indicating that side chain length and charge at position 2 contribute to GVIA binding.70 Lys2 may interact with the VSCC in other ω-conotoxins including MVIIA, CVID and CnVIIA. Alternatively, the positive charge in these latter peptides could interact with Asp14 in loop 2 to provide a structural contribution.

Another residue conserved accross ω-conotoxins that target the N-type calcium channel is glycine 5. One study showed that replacing the glycine with the more sterically hindered alanine resulted in difficulty oxidising the peptide and a nonnative disulfide connectivity dominating the oxidised products. This is not surprising since the glycine is an integral part of the type II β-turn that extends across residue 3—6 in the ω-conotoxins, and fixing the Φ-angle in a negative position is most likely unfavourable for the native conformation.25 However, replacing the glycine with a D-alanine, does not hinder folding in either GVIA25 or CVID (Lewis et al, unpublished data) and these peptides retain activity at the N-type calcium channel.

Loop Integrity in ω-Conotoxins

A study by Nielsen et al71 of 14 loop splice hybrids of MVIIA (N-type inhibitor) and MVIIC (P/Q-type inhibitor) revealed that loop 2 and loop 4 make the greatest contribution to subtype selectivity, whereas loops 1 and 3 contributions are negligible. 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.71 The recently published structure of CVID16 shows that the orientation of loop 2 in relation to loop 4 in this peptide is quite different from previously published structures of ω-conotoxins. Two additional hydrogen bonds were identified in CVID that position loop 4 closer to loop 2 and introduce a “kink”16 which is not seen in either GVIA72- 76 or MVIIA.77- 79 GVIA, having a slightly larger loop 4 instead shows preference for a “kink” in the opposite direction.76

Other N-Type VSCC Inhibitors

Peptides belonging to the inhibitory cysteine knot family that inhibit N-type calcium channels have been identified in assassin bug venom. Three novel peptides named Ado1, Ptu1 and Iob1 have been isolated80 and the solution structure of Ptu1 determined by NMR.81 Despite the fact that these peptides have low sequence homology with the ω-conotoxins, they have a similar four-loop framework. The three peptides have quite high sequence homology, despite coming from different Reduviiae genera. Ptu1 and Ado1 are basic peptides, whereas Iob1 is neutral. All the three peptides have one or more aspartic acids in the N-terminal region, which is homologous to PnVIB from C. pennaceus.82 CD spectroscopy indicated that Ptu1 has less β-sheet than ω-conotoxins.80 The solution structure confirmed this, with the only secondary structure being a β-sheet region comprising two antiparallel β-strands.81 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 Lys2.80 Despite these differences, Ptu1 shares pharmacological specificity reminiscent of MVIIA and GVIA, with 300 nM Ptu1 inhibiting ∼50% of Ba2+ current through N-type VSCCs expressed in BHK cells. However, neither Ado1 nor Ptu1 displace binding of125 I-GVIA to N-type calcium channel in rat brain membrane (personal communication).

The spider toxins ω-grammotoxin-SIA8 isolated from the Chilean tarantula Grammostola spatulata or Phrixotrichus spatulata, ω-agatoxinIIIA83 from the funnel web spider Agelenopsis aperta 83 and Huwentoxin-I isolated from the chinese spider Selenocosmia huwena 84 have also been found to block N-type (and P/Q-type) VSCCs. NMR solution of the two peptides ω-grammotoxin-SIA85 and Huwentoxin-I,86 indicates that these toxins share the ‘inhibitory cysteine knot’ motif characteristic for the ω-conotoxins. Both peptides contain the antiparallel triple stranded β-sheet stabilised by the cysteine knot.

P/Q-Type VSCC Antagonists

ω-Conotoxins

ω-Conotoxins MVIIC and MVIID from C. magus, and SVIB from Conus striatus preferentially target the P/Q-type calcium channel.87 Despite this difference in selectivity, they possess the consensus inhibitory cysteine knot structure found in ω-conotoxins selective for the N-type calcium channel.71 To start to understand VSCC subtype differentiation among ω-conotoxins, we compared the structure of MVIIA to SVIB and a synthetic hybrid, SNX-202, which has altered specificity for both VSCC subtypes.79 The secondary structures of the three peptides are almost identical, consisting of a triple-stranded β-sheet and several turns. The three-dimensional structures of SVIB and MVIIA are likewise quite similar, but subtle differences including a change in the relative orientation between loops 2 and 4 are likely to underlie the selectivity differences among the peptides.

From the above structural studies and a large number of other studies of molecules within this family, it is apparent that the ω-conotoxins form a consensus structure despite differences in sequence and VSCC subtype specificity. This indicates that the ω-conotoxin macrosites for the N/P/Q-subfamily of VSCCs are most likely related, with specificity for receptor targets being conferred by the positions of functional sidechains on the surface of the peptides. Structural studies of the type described above are likely to lead to the development of second-generation analogues which may overcome some of the side-effects associated with intrathecal delivery of ω-conotoxins such as MVIIA.

Spider Toxins

ω-Aga-IVA and ω-Aga-IVB were the first spider peptides identified to specifically inhibit the P-type calcium current in the brain. These toxins block P-type channel activity by antagonizing activation gating and likely act at a region other than the pore.8 Indeed, Bourinet et al6 showed that insertion of two amino acid residues (proline and asparagine) through alternate splicing in the extracellular domain IV S4-S4 linker region dramatically reduces ω-Aga-IVA sensitivity of transiently expressed α1A (Cav2.1) calcium channels. These residues thus act as a molecular switch between P-type and Q-type calcium channel phenotypes with regard to toxin sensitivity, and indicate a site of action outside of the pore region. Unlike the very poorly reversible pore blocking conotoxins, block by ω-Aga-IVA (and ω-grammotoxin SIA) can be reversed by application of strong membrane depolarisations which are thought to dislodge these toxins from their sites of action.8

The solution structure of ω-Aga-IVB shows that apart from a long and disordered N- and C-terminal, the peptide has a well-defined core which again is structurally stabilised with a network of disulfide bonds. This configuration produces a structure strikingly similar to the solution structure of MVIIC (fig. 6).88 One face of the molecule has a cluster of basic residue that has been suggested to be involved in the binding interaction with the channel.89,90

Figure 6. Comparison of two P/Q-type calcium channel inhibitors from a snail and a spider.

Figure 6

Comparison of two P/Q-type calcium channel inhibitors from a snail and a spider. A) MVIIC from the fish hunting cone snail C. magus, and B) ω-Aga IVB from the funnel web spider Agenelopsis aperta/. Peptides are superimposed over the heavy backbone (more...)

R-Type VSCCC Antagonists

The R-type calcium channel is the least defined of the six identified calcium channels to date. This has partly been due to the lack of selective inhibitors. However, recently a 41 amino acid peptide (SNX-482) was isolated from the venom of the African tarantula, Hysteraocrates gigas.91 SNX-482 was found to be structurally homologous to the spider toxins grammatoxin SIA8 and hanatoxin92 that block calcium and potassium channels, respectively by affecting channel gating. However, SNX-482 was found to completely and irreversibly inhibit expressed R-type calcium channel current with an IC50 ∼20 nM91 and is now being used as a tool93 to unravel the physiological role of the α1E calcium channel, which is widely distributed in the brain.94-96 Bourinet et al97 confirmed that SNX-482 is indeed a gating-modifer reminiscent of grammotoxin SIA, ω-Aga-IVA and hanatoxin, and can be reversed by strong membrane depolarization. They also showed, using chimeric calcium channel α1 subunits, that the toxin needs to interact with both domain III and IV of the α1E subunit to influence channel gating.97 SNX-482 applied to L-type calcium channels caused an incomplete block (∼25%) at 200 nM.97 Despite the lack of selectivity at the R-type calcium channel, SNX-482 is useful in further characterising the role of an important component of this poorly characterised calcium channel.

T-Type Antagonists

Since the cloning of the T-type calcium channel98-100 it has been possible to screen this channel for selective inhibitors. Kuroxin, identified by Chuang and coworkers (1998) in the venom of the South African scorpion Parabuthus transvaalicus, inhibits the T-type current by modifying channel gating.101 This 63 amino acid residues peptide has eight Cys and is most likely a member of the cysteine knot family of peptides. Comparing kurtoxin with other previously described toxins suggests that it is closely related to the α-scorpion toxins, a family of toxins that slow inactivation of sodium channels, but are also known as gating modifiers.101,102 While kurtoxin acts on the low-threshold α1G and α1H calcium channels, it also partially inhibits the high-threshold N-type, P-type and L-type currents103 in rat peripheral and central neurones. Despite lack of selectivity, kurtoxin is a promising tool for functional and structural studies of this low-threshold calcium current.

Conclusions and Future Prospects

Peptide toxins that selectively block voltage sensitive calcium channels have contributed enormously to our understanding of the role of specific calcium channels in normal and pathological conditions. Several members of the ω-conotoxin class of calcium channel blockers are currently in clinical trials for chronic pain. While they are particularly efficacious when delivered intrathecally, side effects may limit their usage. Future advances will come with the discovery of new probes for VSCC subtypes, particularly those that are more selective for pain pathways and disease states.

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