Neuronal calcium channel inhibitors. Synthesis of omega-conotoxin GVIA and effects on 45Ca uptake by synaptosomes

We previously described a 27-amino acid peptide neurotoxin from the venom of Conus geographus, omega-conotoxin GVIA, which inhibits neuronal voltage-activated calcium channels. In this paper we describe the total synthesis of omega-conotoxin GVIA and demonstrate that it efficiently blocks voltage-activated uptake of 45Ca by standard synaptosomal preparations from chick brain. Dihydropyridines do not block 45Ca uptake under these conditions. Thus, the omega-conotoxin-sensitive, but dihydropyridine-insensitive uptake of 45Ca2+ by chick brain synaptosomes serves as a functional assay for a Ca channel target of omega-conotoxin. The use of synthetic GVIA should rapidly accelerate our understanding of the molecular biology of Ca2+ channels and their role in neuronal function.     

Monoclonal antibodies against the presynaptic calcium channel antagonist omega-conotoxin GVI A from cone snail poison

Monoclonal antibodies have been prepared against omega-conotoxin GVI A, a peptide isolated from marine snails of the genus Conus (Conus geographus and Conus magus). This toxin is a blocker of select presynaptic Ca2+ channels in the central nervous system. Antigenic omega-conotoxin GVI A was synthesized as a covalent conjugate with bovine serum albumin and injected s.c. An ELISA assay combined with a competitive inhibition assay was used to select and characterize monoclonal antibodies able to recognize and bind the free toxin. Several of the antibodies were found to block omega-conotoxin GVI A inhibition of 45Ca transport into rat brain synaptosomes and to block omega-conotoxin GVI A binding to membranes from the same preparation. The antibodies recognize native, synthetic toxin, and are useful for analysis of toxin in biological fluids.     

Structural and dynamic characterization of omega-conotoxin MVIIA: the binding loop exhibits slow conformational exchange

omega-Conotoxin MVIIA is a 25-residue, disulfide-bridged polypeptide from the venom of the sea snail Conus magus that binds to neuronal N-type calcium channels. It forms a compact folded structure, presenting a loop between Cys8 and Cys15 that contains a set of residues critical for its binding. The loop does not have a unique defined structure, nor is it intrinsically flexible. Broadening of a subset of resonances in the NMR spectrum at low temperature, anomalous temperature dependence of the chemical shifts of some resonances, and exchange contributions to J(0) from (13)C relaxation measurements reveal that conformational exchange affects the residues in this loop. The effects of this exchange on the calculated structure of omega-conotoxin MVIIA are discussed. The exchange appears to be associated with a change in the conformation of the disulfide bridge Cys8-Cys20. The implications for the use of the omega-conotoxins as a scaffold for carrying other functions is discussed.     

Omega-conotoxin CVID inhibits a pharmacologically distinct voltage-sensitive calcium channel associated with transmitter release from preganglionic nerve terminals

Neurotransmitter release from preganglionic parasympathetic neurons is resistant to inhibition by selective antagonists of L-, N-, P/Q-, R-, and T-type calcium channels. In this study, the effects of different omega-conotoxins from genus Conus were investigated on current flow-through cloned voltage-sensitive calcium channels expressed in Xenopus oocytes and nerve-evoked transmitter release from the intact preganglionic cholinergic nerves innervating the rat submandibular ganglia. Our results indicate that omega-conotoxin CVID from Conus catus inhibits a pharmacologically distinct voltage-sensitive calcium channel involved in neurotransmitter release, whereas omega-conotoxin MVIIA had no effect. omega-Conotoxin CVID and MVIIA inhibited depolarization-activated Ba(2+) currents recorded from oocytes expressing N-type but not L- or R-type calcium channels. High affinity inhibition of the CVID-sensitive calcium channel was enhanced when position 10 of the omega-conotoxin was occupied by the smaller residue lysine as found in CVID instead of an arginine as found in MVIIA. Given that relatively small differences in the sequence of the N-type calcium channel alpha(1B) subunit can influence omega-conotoxin access (Feng, Z. P., Hamid, J., Doering, C., Bosey, G. M., Snutch, T. P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 15728-15735), it is likely that the calcium channel in preganglionic nerve terminals targeted by CVID is a N-type (Ca(v)2.2) calcium channel variant.     

Novel alpha- and omega-conotoxins from Conus striatus venom.

Three neurotoxic peptides from the venom of Conus striatus have been purified, biochemically characterized, and chemically synthesized. One of these, an acetylcholine receptor blocker designated alpha-conotoxin SII, has the sequence GCCCNPACGPNYGCGTSCS. In contrast to all other alpha-conotoxins, SII has three disulfide bonds (instead of two), has no net positive charge, and has a free C-terminus. The other two paralytic peptides are Ca channel-targeted omega-conotoxins, SVIA and SVIB. omega-SVIA is the smallest natural omega-conotoxin so far characterized and has the sequence CRSSGSPCGVTSICCGRCYRGKCT-NH2. Although omega-conotoxin SVIA is a potent paralytic toxic in lower vertebrate species, it was much less effective in mammals. The third toxin, omega-conotoxin SVIB, has the sequence CKLKGQSCRKTSYDCCSGSCGRSGKC-NH2. This peptide has a different pharmacological specificity from other omega-conotoxins previously purified from Conus venoms; only omega-conotoxin SVIB has proven to be lethal to mice upon ic injection. Binding competition experiments with rat brain synaptosomal membranes indicate that the high-affinity binding site for omega-conotoxin SVIB is distinct from the high-affinity omega-conotoxin GVIA or MVIIA site.     

Novel omega-conotoxins from Conus catus discriminate among neuronal calcium channel subtypes

omega-Conotoxins selective for N-type calcium channels are useful in the management of severe pain. In an attempt to expand the therapeutic potential of this class, four new omega-conotoxins (CVIA-D) have been discovered in the venom of the piscivorous cone snail, Conus catus, using assay-guided fractionation and gene cloning. Compared with other omega-conotoxins, CVID has a novel loop 4 sequence and the highest selectivity for N-type over P/Q-type calcium channels in radioligand binding assays. CVIA-D also inhibited contractions of electrically stimulated rat vas deferens. In electrophysiological studies, omega-conotoxins CVID and MVIIA had similar potencies to inhibit current through central (alpha(1B-d)) and peripheral (alpha(1B-b)) splice variants of the rat N-type calcium channels when coexpressed with rat beta(3) in Xenopus oocytes. However, the potency of CVID and MVIIA increased when alpha(1B-d) and alpha(1B-b) were expressed in the absence of rat beta(3), an effect most pronounced for CVID at alpha(1B-d) (up to 540-fold) and least pronounced for MVIIA at alpha(1B-d) (3-fold). The novel selectivity of CVID may have therapeutic implications. (1)H NMR studies reveal that CVID possesses a combination of unique structural features, including two hydrogen bonds that stabilize loop 2 and place loop 2 proximal to loop 4, creating a globular surface that is rigid and well defined.     

Structure-activity relationships of omega-conotoxins MVIIA, MVIIC and 14 loop splice hybrids at N and P/Q-type calcium channels.

The omega-conotoxins are a set of structurally related, four-loop, six cysteine containing peptides, that have a range of selectivities for different subtypes of the voltage-sensitive calcium channel (VSCC). To investigate the basis of the selectivity displayed by these peptides, we have studied the binding affinities of two naturally occurring omega-conotoxins, MVIIA and MVIIC and a series of 14 MVIIA/MVIIC loop hybrids using radioligand binding assays for N and P/Q-type Ca2+channels in rat brain tissue. A selectivity profile was developed from the ratio of relative potencies at N-type VSCCs (using [125I]GVIA radioligand binding assays) and P/Q-type VSCCs (using [125I]MVIIC radioligand binding assays). In these peptides, loops 2 and 4 make the greatest contribution to VSCC subtype selectivity, while the effects of loops 1 and 3 are negligible. Peptides with homogenous combinations of loop 2 and 4 display clear selectivity preferences, while those with heterogeneous combinations of loops 2 and 4 are less discriminatory. 1H NMR spectroscopy revealed that the global folds of MVIIA, MVIIC and the 14 loop hybrid peptides were similar; however, several differences in local structure were identified. Based on the binding data and the 3D structures of MVIIA, GVIA and MVIIC, we have developed a preliminary pharmacophore based on the omega-conotoxin residues most likely to interact with the N-type VSCC.     

Determinants of inhibition of transiently expressed voltage-gated calcium channels by omega-conotoxins GVIA and MVIIA

The Conus magus peptide toxin omega-conotoxin MVIIA is considered an irreversible, specific blocker of N-type calcium channels, and is now in clinical trials as an intrathecal analgesic. Here, we have examined the action of MVIIA on mutant and wild type calcium channels transiently expressed in tsA-201 cells. Although we have shown previously that mutations in a putative external EF-hand motif in the domain IIIS5-H5 region alters block by both omega-conotoxin GVIA and MVIIA (Feng, Z. P., Hamid, J., Doering, C., Bosey, G. M., Snutch, T. P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 15728-15735), the introduction of five point mutations known to affect GVIA blocking (and located downstream of the EF-hand) affected MVIIA block to a smaller degree compared with GVIA. These data suggest that despite some overlap, MVIIA and GVIA block does not share identical channel structural determinants. At higher concentrations (approximately 3 microm), MVIIA reversibly blocked L-, P/Q-, and R-type, but not T-type channels, indicating that the overall architecture of the MVIIA site is conserved in all types of high voltage-activated calcium channels. A kinetic analysis of the MVIIA effects on the N-type channel showed that MVIIA blocked resting, open, and inactivated channels. Although the development of MVIIA block did not appear to be voltage-, nor frequency-dependent, the degree of recovery from block strongly depended on the potential applied during washout. Interestingly, the degree of washout was highly variable and appeared to weakly depend on the holding potential applied during toxin application. We propose a model in which N-type calcium channels can form both reversible and irreversible complexes with MVIIA.     

Structure-activity relationships of omega-conotoxins at N-type voltage-sensitive calcium channels

Due to their selectivity towards voltage-sensitive calcium channels (VSCCs) omega-conotoxins are being exploited as a new class of therapeutics in pain management and may also have potential application in ischaemic brain injury. Here, the structure-activity relationships (SARs) of several omega-conotoxins including GVIA, MVIIA, CVID and MVIIC are explored. In addition, the three-dimensional structures of these omega-conotoxins and some structurally related peptides that form the cysteine knot are compared, and the effects of the solution environment on structure discussed. The diversity of binding and functional assays used to measure omega-conotoxin potencies at the N-type VSCC warranted a re-evaluation of the relationship between these assays. With one exception, [A22]-GVIA, this analysis revealed a linear correlation between functional (peripheral N-type VSCCs) and radioligand binding assays (central N-type VSCCs) for the omega-conotoxins and analogues that were tested over three studies. The binding and functional results of several studies are compared in an attempt to identify and distinguish those residues that are important in omega-conotoxin function as opposed to those that form part of the structural scaffold. Further to determining what omega-conotoxin residues are important for VSCC binding, the range of possible interactions between the ligand and channel are considered and the factors that influence the selectivity of MVIIA, GVIA and CVID towards N-type VSCCs examined.     

The alpha2delta auxiliary subunit reduces affinity of omega-conotoxins for recombinant N-type (Cav2.2) calcium channels

The omega-conotoxins from fish-hunting cone snails are potent inhibitors of voltage-gated calcium channels. The omega-conotoxins MVIIA and CVID are selective N-type calcium channel inhibitors with potential in the treatment of chronic pain. The beta and alpha(2)delta-1 auxiliary subunits influence the expression and characteristics of the alpha(1B) subunit of N-type channels and are differentially regulated in disease states, including pain. In this study, we examined the influence of these auxiliary subunits on the ability of the omega-conotoxins GVIA, MVIIA, CVID and analogues to inhibit peripheral and central forms of the rat N-type channels. Although the beta3 subunit had little influence on the on- and off-rates of omega-conotoxins, coexpression of alpha(2)delta with alpha(1B) significantly reduced on-rates and equilibrium inhibition at both the central and peripheral isoforms of the N-type channels. The alpha(2)delta also enhanced the selectivity of MVIIA, but not CVID, for the central isoform. Similar but less pronounced trends were also observed for N-type channels expressed in human embryonic kidney cells. The influence of alpha(2)delta was not affected by oocyte deglycosylation. The extent of recovery from the omega-conotoxin block was least for GVIA, intermediate for MVIIA, and almost complete for CVID. Application of a hyperpolarizing holding potential (-120 mV) did not significantly enhance the extent of CVID recovery. Interestingly, [R10K]MVIIA and [O10K]GVIA had greater recovery from the block, whereas [K10R]CVID had reduced recovery from the block, indicating that position 10 had an important influence on the extent of omega-conotoxin reversibility. Recovery from CVID block was reduced in the presence of alpha(2)delta in human embryonic kidney cells and in oocytes expressing alpha(1B-b). These results may have implications for the antinociceptive properties of omega-conotoxins, given that the alpha(2)delta subunit is up-regulated in certain pain states.     

A new omega-conotoxin that targets N-type voltage-sensitive calcium channels with unusual specificity.

A new specific voltage-sensitive calcium channel (VSCC) blocker has been isolated from the venom of the fish-hunting cone snail Conus consors. This peptide, named omega-Ctx CNVIIA, consists of 27 amino acid residues folded by 3 disulfide bridges. Interestingly, loop 4, which is supposed to be crucial for selectivity, shows an unusual sequence (SSSKGR). The synthesis of the linear peptide was performed using the Fmoc strategy, and the correct folding was achieved in the presence of guanidinium chloride, potassium buffer, and reduced/oxidized glutathione at 4 degrees C for 3 days. Both synthetic and native toxin caused an intense shaking activity, characteristic of omega-conotoxins targeting N-type VSCC when injected intracerebroventricularly to mice. Binding studies on rat brain synaptosomes revealed that the radioiodinated omega-Ctx CNVIIA specifically and reversibly binds to high-affinity sites with a K(d) of 36.3 pM. Its binding is competitive with omega-Ctx MVIIA at low concentration (K(i) = 2 pM). Moreover, omega-Ctx CNVIIA exhibits a clear selectivity for N-type VSCCs versus P/Q-type VSCCs targeted respectively by radioiodinated omega-Ctx GVIA and omega-Ctx MVIIC. Although omega-Ctx CNVIIA clearly blocked N-type Ca(2+) current in chromaffin cells, this toxin did not inhibit acetylcholine release evoked by nerve stimuli at the frog neuromuscular junction, in marked contrast to omega-Ctx GVIA. omega-Ctx CNVIIA thus represents a new selective tool for blocking N-type VSCC that displays a unique pharmacological profile and highlights the diversity of voltage-sensitive Ca(2+) channels in the animal kingdom.     

Identification of the receptor for omega-conotoxin in brain. Probable components of the calcium channel

Recently omega-conotoxin GVIA was shown to specifically block neuronal and other calcium channels. In this work, an azidonitrobenzoyl derivative of mono-[125I]iodo-omega-conotoxin GVIA was used to identify the components of its receptor site in synaptic plasma membrane by photoaffinity labeling. Components of Mr approximately equal to 310,000, approximately equal to 230,000, and 34,000 were specifically photolabeled. The characteristics of photolabeling of these three components were consistent with those of the specific binding of omega-conotoxin GVIA to synaptic plasma membrane with respect to the effects of metal ions, conventional calcium antagonists, and an agonist (1,4-dihydropyridines, verapamil, and diltiazem, etc.), omega-conotoxins GVIIA and GVIIB. Furthermore, the distribution of these three components in subcellular fractions from rat brain as estimated by photolabeling was in good agreement with that of the specific binding of omega-conotoxin GVIA to its receptor. These findings indicate that the components of Mr approximately equal to 310,000, approximately equal to 240,000, and 34,000 are the receptor for omega-conotoxin GVIA and suggest that these components are constituents of the voltage-sensitive calcium channel in brain. No specific photolabeling was observed in the plasma membrane of human erythrocytes, probably indicating the absence of the receptor for omega-conotoxin GVIA in the membrane.     

Function and solution structure of Huwentoxin-X, a specific blocker of N-type calcium channels, from the Chinese bird spider Ornithoctonus huwena

Huwentoxin-X (HWTX-X) is a novel peptide toxin, purified from the venom of the spider Ornithoctonus huwena. It comprises 28 amino acid residues including six cysteine residues as disulfide bridges linked in the pattern of I-IV, II-V, and III-VI. Its cDNA, determined by rapid amplification of 3' and 5' cDNA ends, encodes a 65-residue prepropeptide. HWTX-X shares low sequence homology with omega-conotoxins GVIA and MVIIA, two well known blockers of N-type Ca2+ channels. Nevertheless, whole cell studies indicate that it can block N-type Ca2+ channels in rat dorsal root ganglion cells (IC50 40 nm) and the blockage by HWTX-X is completely reversible. The rank order of specificity for N-type Ca2+ channels is GVIA approximately HWTX-X > MVIIA. In contrast to GVIA and MVIIA, HWTX-X had no detectable effect on the twitch response of rat vas deferens to low frequency electrical stimulation, indicating that HWTX-X has different selectivity for isoforms of N-type Ca2+ channels, compared with GVIA or MVIIA. A comparison of the structures of HWTX-X and GVIA reveals that they not only adopt a common structural motif (inhibitor cystine knot), but also have a similar functional motif, a binding surface formed by the critical residue Tyr, and several basic residues. However, the dissimilarities of their binding surfaces provide some insights into their different selectivities for isoforms of N-type Ca2+ channels.     

A new Conus peptide ligand for mammalian presynaptic Ca2+ channels.

Voltage-sensitive Ca2+ channels that control neurotransmitter release are blocked by omega-conotoxin (omega-CgTx) GVIA from the marine snail Conus geographus, the most widely used inhibitor of neurotransmitter release. However, many mammalian synapses are omega-CgTx-GVIA insensitive. We describe a new Conus peptide, omega-CgTx-MVIIC, that is an effective inhibitor of omega-CgTx-GVIA-resistant synaptic transmission. Ca2+ channel targets that are inhibited by omega-CgTx-MVIIC but not by omega-CgTx-GVIA include those mediating depolarization-induced 45Ca2+ uptake in rat synaptosome preparations, "P" currents in cerebellar Purkinje cells, and a subset of omega-CgTx-GVIA-resistant currents in CA1 hippocampal pyramidal cells. The characterization of omega-CgTx-MVIIC by a combination of molecular genetics and chemical synthesis defines a general approach for obtaining ligands with novel receptor subtype specificity from Conus.     

Synthesis, bioactivity, and cloning of the L-type calcium channel blocker omega-conotoxin TxVII.

omega-Conotoxin TxVII is the first conotoxin reported to block L-type currents. In contrast to other omega-conotoxins, its sequence is characterized by net negative charge and high hydrophobicity, although it retains the omega-conotoxin cysteine framework. In order to obtain structural information and to supply material for further characterization of its biological function, we synthesized TxVII and determined its disulfide bond pairings. Because a linear precursor with free SH groups showed a strong tendency to aggregate and to polymerize, we examined many different conditions for air oxidation and concluded that a mixture of cationic buffer and hydrophobic solvent was the most effective for the folding of TxVII. Synthetic TxVII was shown to suppress the slowly inactivating voltage-dependent calcium current in cultured Lymnaea RPeD1 neurons and furthermore to suppress synaptic transmission between these neurons and their follower cells. In contrast, TxVII did not block calcium flux through L-type channels in PC12 cells, suggesting a phyletic or subtype specificity in this channel family. Disulfide bond pairings of TxVII and its isomers were determined by enzymatic fragmentation in combination with chemical synthesis, thus revealing that TxVII has the same disulfide bond pattern as other omega-conotoxins. Furthermore, the CD spectrum of TxVII is similar to those of omega-conotoxins MVIIA and MVIIC. The precursor sequence of TxVII was determined by cDNA cloning and shown to be closest to that of delta-conotoxin TxVIA, a sodium channel inactivation inhibitor. Thus TxVII conserves the structural fold of other omega-conotoxins, and the TxVIA/TxVII branch of this family reveals the versatility of its structural scaffold, allowing evolution of structurally related peptides to target different channels.     

mu-conotoxin GIIIA, a peptide ligand for muscle sodium channels: chemical synthesis, radiolabeling, and receptor characterization

The peptide conotoxin GIIIA from Conus geographus L. venom, which specifically blocks sodium channels in muscle, has been synthesized by a solid-phase method. The three disulfide bridges were formed by air oxidation. After HPLC purification, the synthetic product was shown to be identical with the native conotoxin GIIIA from Conus geographus. A high specific activity, 125I derivative of mu-conotoxin was prepared and used for binding assays to the Na channel from Electrophorus electric organ. Specific binding could be abolished by competition with tetrodotoxin. The radiolabeled toxin was specifically cross-linked to the Na channel. These studies demonstrate that mu-conotoxin GIIIA can be used to define the guanidinium toxin binding site and will be a useful ligand for understanding functionally important differences between Na channel subtypes.     

Calcium channel antagonists. Omega-conotoxin defines a new high affinity site

The omega-conotoxins, a class of Ca2+ channel antagonists from fish-hunting marine snails, have recently been described (Olivera, B. M., McIntosh, J. M., Zeikus, R., Gray, W. R., Varga, J., Rivier, J., de Santos, V., and Cruz, L. J. (1985) Science, 230, 1338-1343). One of these peptide neurotoxins, omega-conotoxin GVIA, was radiolabeled with iodine, and the 125I-labeled toxin was shown to bind specifically to high affinity sites on chick brain synaptosomes. The toxin-receptor complex was extremely stable; addition of an excess of unlabeled toxin did not cause significant displacement of the labeled toxin after 2 h. Binding competition data suggest that omega-conotoxin defines a new high affinity receptor site affecting voltage-activated Ca2+ channels, distinct from both the verapamil and dihydropyridine target sites.     

Purification and sequence of a presynaptic peptide toxin from Conus geographus venom

A novel toxin, omega-conotoxin (omega-CgTX), from the venom of the fish-eating marine mollusc Conus geographus has been purified and biochemically characterized. Recently, this omega-conotoxin has been shown to inhibit the voltage-activated entry of Ca2+, thus providing a potentially powerful probe for exploring the vertebrate presynaptic terminal [Kerr, L. M., & Yoshikami, D. (1984) Nature (London) 308, 282-284]. The toxin is a basic 27 amino acid peptide amide with three disulfide bridges. An unusual feature is a remarkable preponderance of hydroxylated amino acids. The sequence of omega-CgTx GVIA is Cys-Lys-Ser- Hyp-Gly5-Ser-Ser-Cys-Ser-Hyp10-Thr-Ser-Tyr-Asn-Cys15-C ys-Arg-Ser- Cys-Asn20-Hyp-Tyr-Thr-Lys-Arg25-Cys-Tyr-NH2.     

Combinatorial synthesis of omega-conotoxin MVIIC analogues and their binding with N- and P/Q-type calcium channels

Omega-conotoxin MVIIC (MVIIC) blocks P/Q-type calcium channels with high affinity and N-type calcium channels with low affinity, while the highly homologous omega-conotoxin MVIIA blocks only N-type calcium channels. We wished to obtain MVIIC analogues more selective for P/Q-type calcium channels than MVIIC to elucidate structural differences among the channels, which discriminate the omega-conotoxins. To prepare a number of MVIIC analogues efficiently, we developed a combinatorial method which includes a random air oxidation step. Forty-seven analogues were prepared in six runs and some of them exhibited higher selectivity for P/Q-type calcium channels than MVIIC in binding assays.