Open Access This article is
- freely available
Marine Drugs 2006, 4(3), 215-227; doi:10.3390/md403215
New Conotoxin SO-3 Targeting N-type Voltage-Sensitive Calcium Channels
Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China
School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China
Beijing Institute of Biotechnology, Beijing 100071, China
Author to whom correspondence should be addressed: Tel. (86)-10-66931625; Fax (86)-10- 68211656
Received: 10 March 2006 / Accepted: 30 March 2006 / Published: 6 April 2006
Selective blockers of the N-type voltage-sensitive calcium (CaV) channels are useful in the management of severe chronic pain. Here, the structure and function characteristics of a novel N-type CaV channel blocker, SO-3, are reviewed. SO-3 is a 25- amino acid conopeptide originally derived from the venom of Conus striatus, and contains the same 4-loop, 6-cysteine framework (C-C-CC-C-C) as O-superfamily conotoxins. The synthetic SO-3 has high analgesic activity similar to ω-conotoxin MVIIA (MVIIA), a selective N-type CaV channel blocker approved in the USA and Europe for the alleviation of persistent pain states. In electrophysiological studies, SO-3 shows more selectivity towards the N-type CaV channels than MVIIA. The dissimilarity between SO-3 and MVIIA in the primary and tertiary structures is further discussed in an attempt to illustrate the difference in selectivity of SO-3 and MVIIA towards N-type CaV channels.
Keywords:ω-conotoxins; SO-3; MVIIA; voltage-sensitive calcium channels; N-type calcium channel blockers; pain
Conotoxins are a diverse set of peptide neurotoxins secreted by the venomous marine snails of the genus Conus for prey capture and other biological purposes. It is estimated that more than 50,000 distinct active peptides are present in Conus venoms, most of which can selectively target a specific subtype of voltage-gated ion channel, ligand-gated ion channel, or G-protein-coupled receptor. Due to their highly pharmacological potency and target selectivity, Conus peptides have attracted extensive attention with their potentials to be developed as new research tools in the field of neuroscience and novel medications in clinic for neurological diseases [1–3]. Several conopeptides and their related compounds with potential clinical applications are in the early stage of development. Recently, a synthetic conopeptide, ω-conotoxin MVIIA (MVIIA), has been approved in the USA and Europe for the management of severe resistant pain. This peptide has been given a generic name, Ziconotide, and a commercial name, Prialt, by its developer, Elan Pharmaceuticals [4,5]. MVIIA is a selective blocker of the N-type voltage-sensitive calcium (CaV) channels, with a 25-amino acid sequence identical to that of the parent venom constituent in C. magus. A new 25-residue conopeptide, SO-3, was identified by us from the venom of C. striatus (Figure 1), a fish-eating snail inhabiting the South China Sea [6,7]. The amino acid sequence of SO-3 shows 72% identity to MVIIA, and 56% to another conopeptide derived from C. striatus, ω-conotoxin SVIB, which is a selective P/Q-type CaV channel blocker . Further bioactivity evaluation on the synthetic SO-3 showed that SO-3 is also a potent analgesic agent with lesser side effects than that of MVIIA [9–11]. Electrophysiological studies on voltage-sensitive ion channels indicated that SO-3 is an N-type CaV channel blocker and more selective towards N-type CaV channels than MVIIA .
2. Discovery and Synthesis of SO-3
The South China Sea is in a temperate zone and provides an optimal environment for Conus proliferation. To date, approximately 100 species of cone snails from this habitat have been identified, but C. striatus is the only available fish-eating snail near the coast. Conopeptide content in C. striatus venoms were examined by rapid amplification of 3′ cDNA ends of O-superfamily conopeptide cDNA [6,7]. Four new O-superfamily conopeptide sequences (SO-3~SO-6), and four previously biochemically characterized conopeptide sequences [ω-conotoxin SVIA, SVIA(m1), SVIA(m2) and SVIB] from C. striatus were identified. Conopeptide SO-3 is highly homologous to MVIIA from C. magus in sequence and has the same positive charges as MVIIA (see Table 1). SO-4~SO-6 have no homologous to suggest for their function [6,7].
The synthesis of the linear peptide SO-3 was performed using the 9-fluorenylmethoxycarbonyl (Fmoc) strategy [9,13–15]. Because SO-3 contains six cysteine residues maintained in three disulfide bridges, linear peptide SO-3 was subsequently folded in ammonium acetate buffer (pH 7.9) containing oxidized glutathione (GSSG) and glutathione (GSH) or cysteine at 4°C. Under these conditions, a 20% overall yield of oxidized peptide was obtained. After two steps of HPLC purification, the final product was greater than 98% purity. Using Matrix Assisted Laser Desorption/Ionization-Time Of Flight (MALDI-TOF) mass spectrometry, the molecular mass (single isotope) of SO-3 was 2560.1 Da, in excellent agreement with the calculated molecular mass of 2561.1 Da [9,13–15].
3. Biological Activity and Analgesic Effects of SO-3
Using a range of pain models, including the standard hot-plate, light radiation, and acetic acid stimulus in mice, and heat-elicited tail-flick latency and mechanical tail tests in rats, SO-3 demonstrated analgesic effects that were comparable to or slightly better than those observed for identical doses of MVIIA following either intracerebral or intrathecal injections [9–11]. The toxicity of SO-3 and MVIIA after intramuscular injection into red common goldfish (Carassius carassius) showed that no lethality was associated with SO-3 at doses as high as 8.5 μg/per goldfish. However, severe lethality was observed after MVIIA treatment at the same dose . In mice, when higher doses of SO-3 and MVIIA (>3.2 μg/kg) were intracerebrally delivered, some trembling or shaking activity was noted. Overall, high doses of SO-3 resulted in slightly lower adverse effects compared with MVIIA at the same doses as SO-3. The median lethal dose (LD50) of SO-3 was 13.5 mg/kg after intracerebral administration in mice, which is 18,000 times higher than the median effective dose (ED50 = 0.75 μg/kg; derived from the hot plate test). This difference suggests that SO-3 has a favorable safety index [9–11].
4. Ion Channel Target of SO-3
To ascertain the ion channel target of SO-3 is necessary and important for its further development as drug candidate. Conopeptides in the same superfamily share a characteristic arrangement of cysteine residues and a highly conserved signal sequence in their precursors [1–3]. Amino acid residue sequence analyses indicate that SO-3 belongs to O-superfamily conotoxins [6,7]. O-superfamily conotoxins mainly target voltage-sensitive ion channels. According to their different pharmacological targets, these O-superfamily conotoxins can be further divided into several functional families. For example, δ-conotoxins delay the inactivation of sodium channels, while μO-, κ-, ω- and χ-conotoxins block sodium, potassium, calcium and pacemaker channels, respectively [1–3]. In order to identify the ion channel target of SO-3, the characteristics of the effects of SO-3 on several voltage-sensitive ion channels were observed by using the whole-cell patch clamping techniques. Within the concentration of 100 μM, SO-3 had no effect on voltage-sensitive sodium currents, delayed rectifier potassium currents and transient outward potassium currents in primary cultured neonatal rat hippocampal neurons. However, similar to the selective N-type CaV channel blocker MVIIA, SO-3 blocks high voltage-activated (HVA) calcium currents in a concentration-dependent fashion. For both SO-3 and MVIIA, there was a plateau of inhibition at 1–3 μM. The calculated drug concentration to produce 50% channel inhibition (IC50) for SO-3 and MVIIA were 0.16 μM and 0.20 μM, respectively . These properties of SO-3 indicated that it is a novel CaV channel blocker and a member that belongs to a new ω-conotoxin family.
Based on different physiological and pharmacological properties, CaV channels have been categorized into several subtypes. At least four distinct types of HVA CaV channels, including the L-, N-, P/Q- and R-type channels, are expressed in cultured hippocampal neurons and are sensitive to different blockers [23–26]. We further studied whether specific types of calcium channels were inhibited by SO-3. As shown in Figure 2, four components of HVA calcium currents in hippocampal neurons were isolated by using the classical pharmacological protocol, only the N-type calcium currents were selectively blocked by 3 μM SO-3 . Considering the high expression of N-type CaV channels in dorsal root ganglia (DRG) neurons and the significance of N-type channels in DRG neurons for pain transduction , the effects of SO-3 on HVA calcium currents in DRG neurons were also observed and a similar conclusion was derived: 1 μM SO-3 selectively inhibited the pharmacologically isolated N-type calcium currents in DRG neurons (unpublished data).
In general, neurons co-express several types of CaV channels and the pharmacological isolation of CaV channel subtypes in neurons is not absolutely distinct. Recently, the single foreign ion channel gene expression was proved to giving a pure channel protein, which is very suitable for research on ion channel targeting. Thus, we further studied the selectivity of SO-3 on L-, N-, P/Q- or R-type calcium channels transiently expressed in Human Embryonic Kidney (HEK) 293 cells, respectively. SO-3 selectively blocked the expressed N-type calcium channel currents in a concentration-dependent manner, at 0.01–0.1 μM, and its effects on N-type currents were more obvious than the effects of MVIIA. A kinetic analysis of the SO-3 effects on the expressed N-type calcium channels showed that SO-3 blocked resting, open, and inactivated channels (unpublished data). The block effects of SO-3 and MVIIA on the expressed N-type calcium channels were both reversible and the recovery from block by SO-3 was slower than the recovery from block by MVIIA, which is consistent with the results in cultured hippocampal neurons . However, at higher concentrations (30 μM and 100 μM), SO-3 had inhibitory effects on non-N-type HVA currents recorded from both cultured hippocampal neurons  and HEK 293 cells expressing L-, P/Q- or R-type calcium channels (unpublished data), but its inhibitory effects on non-N-type HVA currents were less than those of MVIIA at the same higher concentrations. These results indicate that SO-3 is a new N-type CaV channel blocker and it possibly possesses more selectivity towards the N-type channels than MVIIA. However, how the pharmacological effect and clinical application will be influenced because of these differences between SO-3 and MVIIA should be studied.
5. Structure–activity Relationships of SO-3
The ω-, κ-, μO-, δ- and χ-conotoxins, which all belong to the O-superfamily conotoxins, contain the same cysteine framework C-C-CC-C-C, but ω-conotoxins have some structural characteristics different from κ-, μO-, δ- and χ-conotoxins. To date, more than ten ω-conotoxins have been identified from the venoms of fish-, worm- or mollusc-hunting Conus species. Almost all ω-conotoxins are Cterminally post-translationally modified and amidated (Table 1) [1–3]. Besides the conserved cysteine framework, there are three conserved residues Gly5, Tyr13, and Ser19 (numbering of MVIIA) throughout this set of peptides except ω-conotoxins SVIA and TxVII (L-type CaV channel blockers derived from C. textile; Table 1). It is known that the Tyr13 residue is important for binding to CaV channels [28–30]. Apart from these characteristics, all ω-conotoxins possess 4–6 positive charges (except ω-conotoxin TxVII) due to the high content of basic amino acid residues (Table 1), which are also known to play an important role in the blocking of CaV channels [29,31]. Among these three conotoxins derived from C. striatus, SVIA is the smallest ω-conotoxin without the Tyr13, Ser19 residues, and has relatively poor activity on most CaV channels found in mammalian systems ; whereas SO-3 has similar primary structural characteristics to SVIB and other derived ω-conotoxins [6,7], which may contribute to its selectivity on CaV channels (Table 1).
The CaV channels are complex proteins composed of four or five distinct subunits. The α1-subunit is the main subunit and it incorporates the conduction pore, the voltage sensor and gating apparatus, and the known sites of channel regulation by drugs and toxins. The α1-subunit is organized in four homologous domains (I-IV) with six transmembrane segments (S1–S6) in each domain. The S4 segment serves as the voltage sensor. The pore loop (P region) between transmembrane segments S5 and S6 in each domain determines ion conductance and selectivity. Although the auxiliary β-, α2δ- and γ-subunits modulate the properties of the channel complex, the pharmacological and electrophysiological diversity of CaV channels arises primarily from the existence of multiple α1- subunits. The P region of domain III (III P region) has been shown to be the main binding region for ω-conotoxins [32–35]. The model of the III P region was preliminarily simulated using the graphic molecular modeling program and the results showed that the interaction between the III P region and SO-3 was similar as the interaction between the III P region and MVIIA or other ω-conotoxins .
Most ω-conotoxins specifically target N- and/or P/Q-type CaV channels, expect ω-conotoxin TxVII, which selectively blocks the L-type subtype. TxVII contains high number of hydrophobic amino acid residues and possesses three negative charges (Table 1) [21,22]. Both ω-conotoxin SVIB and SO-3 were isolated from C. striatus. SVIB is selective for the P/Q-type CaV channels , whereas SO-3 selectively targets the N-type CaV channels . SO-3 has a 56% sequence identity to SVIB and a higher (72%) sequence identity to MVIIA. However, the identity in primary sequences is insufficient to define CaV channel selectivity. Sequence hypervariability has been observed between functionally homologous ω-conotoxins. ω-Conotoxins with high sequence identity may also have different selectivity (Table 1). Therefore, studies of secondary and tertiary structures of SO-3 and other ω- conotoxins might help to understand the subtype selectivity in blocking CaV channels. The molecular structure of ω-conotoxins is stable and this stability depends on three disulfide bridges and a short triple-stranded antiparallel β-sheet with four turns. In addition, the backbone conformation of ω- conotoxins was quite conserved [30,31,37,38]. The three-dimensional solution structure of SO-3 (Figure 3) was determined by 1H NMR and showed that it contains a short antiparallel β-sheet involving residues 6–9, 19–21, and 24–25, and the disulfide bridge pattern of SO-3 was identical to the cystine framework found in MVIIA and other ω-conotoxins [39,40]. These results indicate that the three-dimensional structure of SO-3 and MVIIA contributes to their selectively targeting the same Ntype CaV channels. Additionally, the previous structure-activity analysis of ω-conotoxins also indicate that loops 1 and 3 have little effect on selectivity, whereas loops 2 and 4 are critical determinants in controlling the selectivity of ω-conotoxins for N-, P/Q-, or R-type CaV channels [19,31,37,38]. Hence, the assumption that SO-3 and SVIB target different CaV channel subtypes due to the difference in their three-dimensional structure requires further studies.
Although both SO-3 and MVIIA are N-type CaV channel blockers, they demonstrate some different selectivity towards N-type CaV channels. Such a difference in function might be caused by the subtle difference in the tertiary structure. The most identified regions between SO-3 and MVIIA are the first three β-turns (residues 3–6, 9–12, 15–18) and the first strand of β-sheets (residues 6–8), indicating that these regions may be important for stabilizing the structures. The loop regions and the sheet from residues 19–25 have the lowest identity except for residue 24, indicating that these regions may be related to the difference in the function [39,40]. In addition, Tyr13 was found to be crucial for the calcium channel binding activity of ω-conotoxins, suggesting that minor structural changes in the region around Tyr13 may be responsible for the selectivity. Comparison of the tertiary structure indicated that for SO-3 and MVIIA, the region including Tyr13 is the most flexible region around the dihedral angles or the most disordered stretch of backbone [39,40]. This dispersion property suggests that the backbone conformation of ω-conotoxins blocking the N-type CaV channel is flexible.
Previously, some researchers have reported and discussed the reversibility of the blocking effects of several ω-conotoxins on the transiently expressed N-type calcium channels and these results were not consistent with each other. Taken together, the blocking effect of ω-conotoxin GVIA is poorly reversible, while that of both MVIIA and ω-conotoxin CVID are readily reversible [41–48]. Since ω- conotoxin GVIA dissociates very slowly from CaV channels, it may be difficult to control in a clinical setting, and is therefore not an ideal drug candidate. In our study, the block effects of SO-3 and MVIIA on CaV channels were both almost completely reversed, and the recovery from block by MVIIA was more rapid than the recovery from block by SO-3 . The recovery from block by ω-conotoxins is partly dependent on the divalent cation in extracellular solution , the holding potential [44,47], and the α2δ auxiliary subunit of CaV channels . Apart from these factors, the amino acid residue of ω- conotoxins at position 10 has a significant impact on the extent of the reversibility of these toxins . SO-3 and MVIIA, both with Arg at position 10 (Table 1), can reversibly block the CaV channels, whereas GVIA demonstrates different reversibility because of the different residue (Hydroxyproline) at position 10 (Table 1). The different extent of recovery from the block by SO-3 and MVIIA may due to the different sequences of the amino acid residues near this position or other unknown mechanisms.
6. Potential Therapeutic Implications of SO-3
N-type CaV channels are critical for pain transduction and modulation. Although they are located on pre-synaptic nerve terminals in both central and peripheral nervous systems, N-type channels are highly present at the pre-synaptic terminals of nociceptive neurons in dorsal horn of the spinal cord where they regulate the release of the key pro-nociceptive neurotransmitters such as glutamate, substance P, neurokinin A and/or calcitoningene-related peptide [33,49]. The crucial role of the N-type channels in nociception is also supported by the evidence that mice lacking the N-type channel gene have higher pain thresholds compared to wild-type mice [50–53]. It is reasonable to consider that Ntype CaV channel blockers have the therapeutic potential as a new class of analgesic agents [54,55]. However, based on the fact that N-type CaV channels are also located on numerous other synapses in non-pain pathways, including the pre-synaptic nerve terminals in sympathetic neurons, it is not surprising that N-type CaV channel blockers may result in adverse effects in analgesia [54,55]. These adverse effects include increased dizziness, blurred vision, nystagmus, sedation, anxiety, hallucinations, hypotension, etc. [4,5]. A similar pathological syndrome was observed in N-type channel α1-subunit knockout mice [56,57]. In contrast with opioids, which always give rise to dependence and tolerance, N-type CaV channel blockers do not seem to have these clinical limits and are thereby considered as the alternative for the alleviation of severe chronic pain states [54,55].
The side effects of the N-type CaV channel blockers in pain control may also arise from their activities at non-N-type CaV channels. Some non-N-type CaV channels are also involved in neurotransmitter release in most central synapses. P/Q-type CaV channels exist primarily in neuromuscular junction; selective P/Q-type CaV channel blockers (such as ω-conotoxin MVIIC and SVIB) are likely to be lethal at relatively low dose and are considered useless in pain treatment [58–60]. Consequently, ω-conotoxins, more selective for N-type CaV channels than P/Q- and other CaV channel types, can largely minimize the side effects in analgesic therapy. Recently, another ω-conotoxin (CVID, also known as AM336 and developed by Amrad Corporation) was reported that it is more selective towards the N-type CaV channels than MVIIA, and it may have more advantages relative to MVIIA for the alleviation of persistent pain states [61,62]. Our results mentioned previously also indicate that, SO-3, which shows a higher selectivity for N- vs. L-, P/Q- and R-type CaV channels in electrophysiological experiments, may have superior clinical utility in the management of severe chronic pain.
This work was supported by grants from National High-Tech Research and Development Program of China (2001AA624150) and National Natural Science Foundation of China (30572175). We thank Profs. Qiuyun Dai and Xiaowei Zhou (Beijing Institute of Biotechnology, Beijing, China) and Dr. Yongbin Yan (Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, China) for helpful comments on the manuscript.
- Olivera, B. M.; Cruzab, L. J. Conotoxins, in retrospect. Toxicon 2001, 39, 7–14. [Google Scholar]
- Terlau, H.; Olivera, B. M. Conus venoms: a rich source of novel ion channel-targeted peptides. Physiol. Rev 2004, 84, 41–68. [Google Scholar]
- Wang, C. Z.; Chi, C. W. Conus peptides-a rich pharmaceutical treasure. Acta Biochim. Biophys. Sin 2004, 36, 713–723. [Google Scholar]
- Staats, P. S.; Yearwood, T.; Charapata, S. G.; Presley, R. W.; Wallace, M. S.; Byas-Smith, M.; Fisher, R.; Bryce, D. A.; Mangieri, E. A.; Luther, R. R.; Mayo, M.; Mcguire, D.; Ellis, D. Intrathecal ziconotide in the treatment of refractory pain in patients with cancer or AIDS: a randomized controlled trial. JAMA 2004, 291, 63–70. [Google Scholar]
- Sharp, D. Novel pain relief via marine snails. Lancet 2005, 366, 439–440. [Google Scholar]
- Lu, B. S.; Yu, F.; Zhao, D.; Huang, P. T.; Huang, C. F. Conopeptides from Conus striatus and Conus textile by cDNA cloning. Peptides 1999, 20, 1139–1144. [Google Scholar]
- Lu, B. S.; Yu, F.; Wang, J. H.; Zhao, S. Q.; Zhao, D.; Dai, Q. Y.; Huang, P. T.; Huang, C. F. New O-superfamily conotoxins from Conus striatus inhabited near Chinese Hainan Island. Chin. Sci. Bull 2000, 45, 432–435. [Google Scholar]
- Ramilo, C. A.; Zafaralla, G. C.; Nadasdi, L.; Hammerland, L. G.; Yoshikami, D.; Gray, W. R.; Kristipati, R.; Ramachandran, J.; Miljanich, G.; Olivera, B. M. Novel α- and ω-conotoxins from Conus striatus venom. Biochemistry 1992, 31, 9919–9926. [Google Scholar]
- Dai, Q. Y.; Liu, F. Y.; Zhou, Y. R.; Lu, B. S.; Yu, F.; Huang, P. T. The synthesis of SO-3, a conopeptide with high analgesic activity derived from Conus striatus. J. Nat. Prod 2003, 66, 1276–1279. [Google Scholar]
- Liu, F. Y.; Dai, Q. Y.; Li, J. J.; Li, X. L.; Zhou, Y. R.; Huang, P. T. SO3-a new type of conotoxin with analgesic activity. Bull. Acad. Mil. Med. Sci 2001, 25, 174–176, , 179.. [Google Scholar]
- Liu, F. Y.; Dai, Q. Y.; Zhou, Y. R.; Li, X. L.; Huang, P. T. Studies on toxicity and drug dependence of conotoxin peptide SO3. Letters in Biotechnology 2002, 13, 191–193, , 213.. [Google Scholar]
- Wen, L.; Yang, S.; Qiao, H. F.; Liu, Z. W.; Zhou, W. X.; Zhang, Y. X.; Huang, P. T. SO-3, a new O-superfamily conopeptide derived from Conus striatus, selectively inhibits N-type calcium currents in cultured hippocampal neurons. Br. J. Pharmacol 2005, 145, 728–739. [Google Scholar]
- Zhou, Y. R.; Liu, F. Y.; Dai, Q. Y. Research on the relationship between refolding and bioactivity of the new type of conotoxin SO3. Letters in Biotechnology 2001, 12, 181–183. [Google Scholar]
- Zhou, Y. R.; Liu, F. Y.; Dai, Q. Y.; Huang, P. T. Optimization of synthetic and folding conditions of a novel ω-conotoxin peptide SO3. Letters in Biotechnology 2002, 13, 286–288. [Google Scholar]
- Zhou, Y. R.; Liu, F. Y.; Dai, Q. Y. Synthesis of ω-conotoxin and its derivatives. Journal of Chinese Biotechnology 2003, 23, 72–76. [Google Scholar]
- Olivera, B. M.; McIntosh, J. M.; Cruz, L. J.; Luque, F. A.; Gray, W. R. Purification and sequence of a presynaptic peptide toxin from Conus geographus venom. Biochemistry 1984, 23, 5087–5090. [Google Scholar]
- Olivera, B. M.; Cruz, L. J.; de Santos, V.; LeCheminant, G. W.; Griffin, D.; Zeikus, R.; McIntosh, J. M.; Galyean, R.; Varga, J.; Gray, W. R. Neuronal calcium channel antagonists. Discrimination between calcium channel subtypes using ω-conotoxin from Conus magus venom. Biochemistry 1987, 26, 2086–2090. [Google Scholar]
- Hillyard, D. R.; Monje, V. D.; Mintz, I. M.; Bean, B. P.; Nadasdi, L.; Ramachandran, J.; Miljanich, G.; Azimi-Zoonooz, A.; McIntosh, J. M.; Cruz, L. J.; Imperial, J. S.; Olivera, B. M. A new Conus peptide ligand for mammalian presynaptic Ca2+ channels. Neuron 1992, 9, 69–77. [Google Scholar]
- Lewis, R. J.; Nielsen, K. J.; Craik, D. J.; Loughnan, M. L.; Adams, D. A.; Sharpe, I. A.; Luchian, T.; Adams, D. J.; Bond, T.; Thomas, L.; Jones, A.; Matheson, J. L.; Drinkwater, R.; Andrews, P. R.; Alewood, P. F. Novel ω-conotoxins from Conus catus discriminate among neuronal calcium channel subtypes. J. Biol. Chem 2000, 275, 35335–35344. [Google Scholar]
- Miljanich, G. P.; Ramachandran, J. Antagonists of neuronal calcium channels: structure, function, and therapeutic implications. Annu. Rev. Pharmacol. Toxicol 1995, 35, 707–734. [Google Scholar]
- Fainzilber, M.; Lodder, J. C.; van der Schors, R. C.; Li, K. W.; Yu, Z.; Burlingame, A. L.; Geraerts, W. P.; Kits, K. S. A novel hydrophobic ω-conotoxin blocks molluscan dihydropyridinesensitive calcium channels. Biochemistry 1996, 35, 8748–8752. [Google Scholar]
- Sasaki, T.; Feng, Z. P.; Scott, R.; Grigoriev, N.; Syed, N. I.; Fainzilber, M.; Sato, K. Synthesis, bioactivity, and cloning of the L-type calcium channel blocker ω-conotoxin TxVII. Biochemistry 1999, 38, 12876–12884. [Google Scholar]
- Currie, K. P. M.; Fox, A. P. Comparison of N- and P/Q-type voltage-gated calcium channel current inhibition. J. Neurosci 1997, 17, 4570–4579. [Google Scholar]
- Nakashima, Y. M.; Todorovic, S. M.; Covey, D. F.; Lingle, C. J. The anesthetic steroid (+)-3α- hydroxy-5α-androstane-17β-carbonitrile blocks N-, Q-, and R-type, but not L- and P-type, high voltage-activated Ca2+ current in hippocampal and dorsal root ganglion neurons of the rat. Mol. Pharmacol 1998, 54, 559–568. [Google Scholar]
- Blalock, E. M.; Porter, N. M.; Landfield, P. W. Decreased G-protein-mediated regulation and shift in calcium channel types with age in hippocampal cultures. J. Neurosci 1999, 19, 8674–8684. [Google Scholar]
- Scamps, F.; Vigues, S.; Restituito, S.; Campo, B.; Roig, A.; Charnet, P.; Valmier, J. Sarcoendoplasmic ATPase blocker 2, 5-di (tert-butyl)-1, 4-benzohydroquinone inhibits N-, P-, and Qbut not T-, L-, or R-type calcium currents in central and peripheral neurons. Mol. Pharmacol 2000, 58, 18–26. [Google Scholar]
- Bell, T. J.; Thaler, C.; Castiglioni, A. J.; Helton, T. D.; Lipscombe, D. Cell-specific alternative splicing increases calcium channel current density in the pain pathway. Neuron 2004, 41, 127–138. [Google Scholar]
- Kim, J. I.; Takahashi, M.; Ohtake, A.; Wakamiya, A.; Sato, K. Tyr13 is essential for the activity of ω-conotoxin MVIIA and GVIA, specific N-type calcium channel blockers. Biochem. Biophys. Res. Commun 1995, 206, 449–454. [Google Scholar]
- Lew, M. J.; Flinn, J. P.; Pallaghy, P. K.; Murphy, R.; Whorlow, S. L.; Wright, C. E.; Norton, R. S.; Angus, J. A. Structure-function relationships of ω-conotoxin GVIA. Synthesis, structure, calcium channel binding, and functional assay of alanine-substituted analogs. J. Biol. Chem 1997, 272, 12014–12023. [Google Scholar]
- Nielsen, K. J.; Adams, D. A.; Alewood, P. F.; Lewis, R. J.; Thomas, L.; Schroeder, T.; Craik, D. J. Effects of chirality at Tyr13 on the structure-activity relationships of ω-conotoxins from conus magus. Biochemistry 1999, 38, 6741–6751. [Google Scholar]
- Nielsen, K. J.; Schroeder, T.; Lewis, R. Structure-activity relationships of ω-conotoxins at N-type voltage-sensitive calcium channels. J. Mol. Recognit 2000, 13, 55–70. [Google Scholar]
- Catterall, W. A. Structure and regulation of voltage-gated Ca2+ channels. Annu. Rev. Cell Dev. Biol. 2000, 16, 521–555. [Google Scholar]
- Jarvis, S. E.; Zamponi, G. W. Interactions between presynaptic Ca2+ channels, cytoplasmic messengers and proteins of the synaptic vesicle release complex. Trends Pharmacol. Sci 2001, 22, 519–525. [Google Scholar]
- Arikkath, J.; Campbell, K. P. Auxiliary subunits: essential components of the voltage-gated calcium channel complex. Curr. Opin. Neurobiol 2003, 13, 298–307. [Google Scholar]
- Catterall, W. A.; Striessnig, J.; Snutch, T. P.; Perez-Reyes, E. International Union of Pharmacology. XL. Compendium of voltage-gated ion channels: calcium channels. Pharmacol. Rev 2003, 55, 579–581. [Google Scholar]
- Yue, J. J.; Dai, Q. Y.; Wang, G. L.; Liang, L.; Yu, W. Y.; Huang, P. T. Computational simulation of interaction between calcium channel and conotoxin. Letters in Biotechnology 2002, 13, 281–285. [Google Scholar]
- Nielsen, K. J.; Thomas, L.; Lewis, R. J.; Alewood, P. F.; Craik, D. J. A consensus structure for ω- conotoxins with different selectivities for voltage-sensitive calcium channel subtypes: comparison of MVIIA, SVIB and SNX-202. J. Mol. Biol 1996, 263, 297–310. [Google Scholar]
- Nielsen, K. J.; Adams, D.; Thomas, L.; Bond, T.; Alewood, P. F.; Craik, D. J.; Lewis, R. J. Structure-activity relationships of ω-conotoxins MVIIA, MVIIC and 1, 4 loop splice hybrids at N and P/Q-type calcium channels. J. Mol. Biol 1999, 289, 1405–1421. [Google Scholar]
- Kohno, T.; Kim, J.I.; Kobayashi, K.; Kodera, Y.; Maeda, T.; Sato, K. Three-dimensional structure in solution of the calcium channel blocker ω-conotoxin MVIIA. Biochemistry 1995, 34, 10256–10265. [Google Scholar]
- Yan, Y. B.; Tu, G. Z.; Luo, X. C.; Dai, Q. Y.; Huang, P. T.; Zhang, R. Q. Three-dimensional solution structure of ω-conotoxin SO-3 determined by 1H NMR. Chin. Sci. Bull 2003, 48, 1097–1102. [Google Scholar]
- Williams, M. E.; Brust, P. F.; Feldman, D. H.; Patthi, S.; Simerson, S.; Maroufi, A.; McCue, A. F.; Velicelebi, G.; Ellis, S. B.; Harpold, M. M. Structure and functional expression of an ω- conotoxin-sensitive human N-type calcium channel. Science 1992, 257, 389–395. [Google Scholar]
- Kristipati, R.; Nadasdi, L.; Tarczy-Hornoch, K.; Lau, K.; Miljanich, G. P.; Ramachandran, J.; Bell, J. R. Characterization of the binding of ω-Conopeptides to different classes of non-L-type neuronal calcium channels. Mol. Cell Neurosc 1994, 5, 219–228. [Google Scholar]
- Lin, Z.; Haus, S.; Edgerton, J.; Lipscombe, D. Identification of functionally distinct isoforms of the N-type Ca2+ channel in rat sympathetic ganglia and brain. Neuron 1997, 18, 153–166. [Google Scholar]
- Stocker, J. W.; Nadasdi, L.; Aldrich, R. W.; Tsien, R. W. Preferential interaction of ω-conotoxins with inactivated N-type Ca2+ channels. J. Neurosci 1997, 17, 3002–3013. [Google Scholar]
- Feng, Z. P.; Hamid, J.; Doering, C.; Bosey, G. M.; Snutch, T. P.; Zamponi, G. W. Residue Gly1326 of the N-type calcium channel α1B subunit controls reversibility of ω-conotoxin GVIA and MVIIA block. J. Biol. Chem 2001, 276, 15728–15735. [Google Scholar]
- Liang, H.; Elmslie, K. S. Rapid and reversible block of N-type calcium channels (CaV 2.2) by ω- conotoxin GVIA in the absence of divalent cations. J. Neurosci 2002, 22, 8884–8890. [Google Scholar]
- Feng, Z. P.; Doering, C. J.; Winkfein, R. J.; Beedle, A. M.; Spafford, J. D.; Zamponi, G. W. Determinants of inhibition of transiently expressed voltage-gated calcium channels by ω- conotoxins GVIA and MVIIA. J. Biol. Chem 2003, 278, 20171–20178. [Google Scholar]
- Mould, J.; Yasuda, T.; Schroeder, C. I.; Beedle, A. M.; Doering, C. J.; Zamponi, G. W.; Adams, D. J.; Lewis, R. J. The α2δ auxiliary subunit reduces affinity of ω-conotoxins for recombinant N-type (Cav2.2) calcium channels. J. Biol. Chem 2004, 279, 34705–34714. [Google Scholar]
- Adams, D. J.; Smith, A. B.; Schroeder, C. I.; Yasuda, T.; Lewis, R. J. ω-Conotoxin CVID inhibits a pharmacologically distinct voltage-sensitive calcium channel associated with transmitter release from preganglionic nerve terminals. J. Biol. Chem 2003, 278, 4057–4062. [Google Scholar]
- Hatakeyama, S.; Wakamori, M.; Ino, M.; Miyamoto, N.; Takahashi, E.; Yoshinaga, T.; Sawada, K.; Imoto, K.; Tanaka, I.; Yoshizawa, T.; Nishizawa, Y.; Mori, Y.; Niidome, T.; Shoji, S. Differential nociceptive responses in mice lacking the α1B subunit of N-type Ca2+ channels. Neuroreport 2001, 12, 2423–2427. [Google Scholar]
- Kim, C.; Jun, K.; Lee, T.; Kim, S. S.; McEnery, M. W.; Chin, H.; Kim, H. L.; Park, J. M.; Kim, D. K.; Jung, S. J.; Kim, J.; Shin, H. S. Altered nociceptive response in mice deficient in the α1B subunit of the voltage-dependent calcium channel. Mol. Cell Neurosci 2001, 18, 235–245. [Google Scholar]
- Saegusa, H.; Kurihara, T.; Zong, S.; Kazuno, A.; Matsuda, Y.; Nonaka, T.; Han, W.; Toriyama, H.; Tanabe, T. Suppression of inflammatory and neuropathic pain symptoms in mice lacking the N-type Ca2+ channel. EMBO J 2001, 20, 2349–2356. [Google Scholar]
- Saegusa, H.; Matsuda, Y.; Tanabe, T. Effects of ablation of N- and R-type Ca2+ channels on pain transmission. Neurosci. Res 2002, 43, 1–7. [Google Scholar]
- Vanegasa, H.; Schaible, H. G. Effects of antagonists to high-threshold calcium channels upon spinal mechanisms of pain, hyperalgesia and allodynia. Pain 2000, 8, 9–18. [Google Scholar]
- Altier, C.; Zamponi, G. W. Targeting Ca2+ channels to treat pain: T-type versus N-type. Trends Pharmacol. Sci 2004, 25, 465–470. [Google Scholar]
- Ino, M.; Yoshinaga, T.; Wakamori, M.; Miyamoto, N.; Takahashi, E.; Sonoda, J.; Kagaya, T.; Oki, T.; Nagasu, T.; Nishizawa, Y.; Tanaka, I.; Imoto, K.; Aizawa, S.; Koch, S.; Schwartz, A.; Niidome, T.; Sawada, K.; Mori, Y. Functional disorders of the sympathetic nervous system in mice lacking the α1B subunit (CaV 2.2) of N-type calcium channels. Proc. Natl. Acad. Sci. USA 2001, 98, 5323–5328. [Google Scholar]
- Beuckmann, C. T.; Sinton, C. M.; Miyamoto, N.; Ino, M.; Yanagisawa, M. N-type calcium channel α1B subunit (CaV2.2) knock-out mice display hyperactivity and vigilance state differences. J. Neurosci 2003, 23, 6793–6797. [Google Scholar]
- Meir, A.; Ginsburg, S.; Butkevich, A.; Kachalsky, S. G.; Kaiserman, I.; Ahdut, R.; Demirgoren, S.; Rahamimoff, R. Ion channels in presynaptic nerve terminals and control of transmitter release. Physiol. Rev 1999, 79, 1019–1088. [Google Scholar]
- Bowersox, S. S.; Miljanich, G. P.; Sugiura, Y.; Li, C.; Nadasdi, L.; Hoffman, B. B.; Ramachandran, J.; Ko, C. P. Differential blockade of voltage-sensitive calcium channels at the mouse neuromuscular junction by novel ω-conopeptides and ω-agatoxin-IVA. J. Pharmacol. Exp. Ther 1995, 273, 248–256. [Google Scholar]
- Katz, E.; Protti, D. A.; Ferro, P. A.; Rosato, S.; Uchitel, O. D. Effects of Ca2+ channel blocker neurotoxins on transmitter release and presynaptic currents at the mouse neuromuscular junction. Br. J. Pharmacol 1997, 121, 1531–1540. [Google Scholar]
- Scott, D. A.; Wright, C. E.; Angus, J. A. Actions of intrathecal ω-conotoxins CVID, GVIA, MVIIA, and morphine in acute and neuropathic pain in the rat. Eur. J. Pharmacol 2002, 451, 279–286. [Google Scholar]
- Smith, M. T.; Cabot, P. J.; Ross, F. B.; Robertson, A. D.; Lewis, R. J. The novel N-type calcium channel blocker, AM336, produces potent dose-dependent antinociception after intrathecal dosing in rats and inhibits substance P release in rat spinal cord slices. Pain 2002, 96, 119–127. [Google Scholar]
Figure 1. Conus striatus inhabiting the South China Sea.
Figure 2. SO-3 selectively inhibiting the N-type calcium currents in cultured hippocampal neurons. Distinct CaV channel subtype blockers were applied to the cells as indicated by the horizontal bars to pharmacologically isolate the whole-cell HVA calcium currents. Additional application of SO-3 did not further inhibit the HVA currents after NCaV channel blocker MVIIA application, which indicated they inhibited the overlapping components of HVA currents (A). No overlapping component of HVA currents was inhibited by SO-3 and L-CaV blocker nimodipine (Nim) (B), or by SO-3 and P/Q-CaV channel blocker ω-agatoxin (Aga) (C). 3 μM SO-3 also had no effect on R-type currents, which were completely blocked by 0.4 mM Cd2+ (D). Currents were evoked by 150-ms depolarizing voltage step commands from −80 to 0 mV at 10 s intervals. Upper insets showed the current traces at each time point. Scale bars: 10 pA/pF, 50 ms. (Modified from Wen et al. ).
Figure 3. The three-dimensional structure of SO-3. The three-dimensional structure of SO-3 was determined by 1H NMR (PDB ID 1FYG). The figure shows the disulphide bridges (yellow balls and sticks) and the β-sheet region (cyan arrows). Photo was provided by Dr. Yongbin Yan from Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, China.
Table 1. Selected ω-conotoxins targeting different CaV channel subtypes.
|Peptide||Conus Specie||Sequence a||Subtype Targeted||Reference|
|ω-GVIA||C. geographus||CKSOGSSCSOTSYNCCR-SCNOYTKRCY* (4)||N-type|||
|ω-MVIIA||C. magus||CKGKGAKCSRLMYDCCTGSC--RSGKC* (5)||N-type|||
|ω-MVIIC||C. magus||CKGKGAPCRKTMYDCCSGSCGRR-GKC* (6)||P/Q/N-type|||
|ω-CVIA||C. catus||CKSTGASCRRTSYDCCTGSC--RSGRC* (4)||N-type|||
|ω-CVIB||C. catus||CKGKGASCRKTMYDCCRGSC--RSGRC* (6)||N/P/Q-type|||
|ω-CVIC||C. catus||CKGKGQSCSKLMYDCCTGSCSRR-GKC* (5)||N/P/Q-type|||
|ω-CVID||C. catus||CKSKGAKCSKLMYDCCSGSCSGTVGRC* (4)||N-type|||
|ω-TVIA||C. tulipa||CLSOGSSCSOTSYNCCR-SCNOYSRKCR* (4)||N-type|||
|ω-TxVII||C. textile||CKQADEPCDVFSLDCCTGIC---LGVCMW* (−3)||L-type||[21,22]|
|ω-SVIA||C. striatus||CRSSGSPCGVTS-ICC-GRC--YRGKCT* (4)||poor activity|||
|ω-SVIB||C. striatus||CKLKGQSCRKTSYDCCSGSCG-RSGKC* (5)||P/Q-type|||
|SO-3||C. striatus||CKAAGKPCSRIAYNCCTGSC--RSGKC* (5)||N-type|||
aAlmost all ω-conotoxins are C-terminally amidated (*), each net positive/negative charge is indicated in the braces after the sequence of the corresponding ω-conotoxin. The disulfide bridges motif of ω-conotoxins and its 4 loops (Δ) are also displayed above the sequences. Hydroxyproline residues are denoted by the letter O.
© 2006 by MDPI Reproduction is permitted for noncommercial purposes.