RESUMO
The first conotoxin affecting the voltage-gated potassium channels of the EAG family was identified and characterized from the venom of the vermivorous species Conus spurius from the Gulf of Mexico. This conopeptide, initially named Cs68 and later designated κO-SrVIA, is extremely hydrophobic and comprises 31 amino acid residues, including six Cysteines in the framework VI/VII, and a free C-terminus. It inhibits the currents mediated by two human EAG subtypes, Kv10.1 (IC50 = 1.88 ± 1.08 µM) and Kv11.1 (IC50 = 2.44 ± 1.06 µM), and also the human subtype Kv1.6 (IC50 = 3.6 ± 1.04 µM). Despite its clear effects on potassium channels, it shares a high sequence identity with δ-like-AtVIA and δ-TsVIA. Also, κO-SrVIA is the third conopeptide from the venom of C. spurius with effects on potassium channels, and the seventh conotoxin that blocks Kv1.6 channels.
Assuntos
Conotoxinas , Caramujo Conus , Canais de Potássio Éter-A-Go-Go , Animais , Humanos , Sequência de Aminoácidos , Conotoxinas/farmacologia , Conotoxinas/química , Caramujo Conus/química , Canais de Potássio Éter-A-Go-Go/efeitos dos fármacos , Canais de Potássio Éter-A-Go-Go/metabolismo , Canais de Potássio Éter-A-Go-Go/toxicidade , Peptídeos/químicaRESUMO
Background: Conotoxins exhibit great potential as neuropharmacology tools and therapeutic candidates due to their high affinity and specificity for ion channels, neurotransmitter receptors or transporters. The traditional methods to discover new conotoxins are peptide purification from the crude venom or gene amplification from the venom duct. Methods: In this study, a novel O1 superfamily conotoxin Tx6.7 was directly cloned from the genomic DNA of Conus textile using primers corresponding to the conserved intronic sequence and 3' UTR elements. The mature peptide of Tx6.7 (DCHERWDW CPASLLGVIYCCEGLICFIAFCI) was synthesized by solid-phase chemical synthesis and confirmed by mass spectrometry. Results: Patch clamp experiments on rat DRG neurons showed that Tx6.7 inhibited peak calcium currents by 59.29 ± 2.34% and peak potassium currents by 22.33 ± 7.81%. In addition, patch clamp on the ion channel subtypes showed that 10 µM Tx6.7 inhibited 56.61 ± 3.20% of the hCaV1.2 currents, 24.67 ± 0.91% of the hCaV2.2 currents and 7.30 ± 3.38% of the hNaV1.8 currents. Tx6.7 had no significant toxicity to ND7/23 cells and increased the pain threshold from 0.5 to 4 hours in the mouse hot plate assay. Conclusion: Our results suggested that direct cloning of conotoxin sequences from the genomic DNA of cone snails would be an alternative approach to obtaining novel conotoxins. Tx6.7 could be used as a probe tool for ion channel research or a therapeutic candidate for novel drug development.(AU)
Assuntos
Animais , Cálcio/isolamento & purificação , Conotoxinas/genética , Caramujo Conus/químicaRESUMO
Conotoxin sr5a had previously been identified in the vermivorous cone snail Conus spurius. This conotoxin is a highly hydrophobic peptide, with the sequence IINWCCLIFYQCC, which has a cysteine pattern "CC-CC" belonging to the T-1 superfamily. It is well known that this superfamily binds to molecular targets such as calcium channels, G protein-coupled receptors (GPCR), and neuronal nicotinic acetylcholine receptors (nAChR) and exerts an effect mainly in the central nervous system. However, its effects on other molecular targets are not yet defined, suggesting the potential of newly relevant molecular interactions. To find and demonstrate a potential molecular target for conotoxin sr5a electrophysiological assays were performed on three subtypes of voltage-activated sodium channels (NaV1.5, NaV1.6, and NaV1.7) expressed in HEK-293 cells with three different concentrations of sr5a(200, 400, and 600 nM). 200 nM sr5a blocked currents mediated by NaV1.5 by 33%, NaV1.6 by 14%, and NaV1.7 by 7%. The current-voltage (I-V) relationships revealed that conotoxin sr5a exhibits a preferential activity on the NaV1.5 subtype; the activation of NaV1.5 conductance was not modified by the blocking effect of sr5a, but sr5a affected the voltage-dependence of inactivation of channels. Since peptide sr5a showed a specific activity for a sodium channel subtype, we can assign a pharmacological family and rename it as conotoxin µ-SrVA.
Assuntos
Conotoxinas , Caramujo Conus , Receptores Nicotínicos , Animais , Humanos , Sequência de Aminoácidos , Canais de Cálcio/metabolismo , Conotoxinas/química , Caramujo Conus/química , Cisteína/metabolismo , Células HEK293 , Peptídeos/metabolismo , Receptores Nicotínicos/metabolismo , Caramujos/metabolismoRESUMO
We isolated a new dimeric conotoxin with inhibitory activity against neuronal nicotinic acetylcholine receptors. Edman degradation and transcriptomic studies indicate a homodimeric conotoxin composed by two chains of 47 amino acid in length. It has the cysteine framework XX and 10 disulfide bonds. According to conotoxin nomenclature, it has been named as αD-FrXXA. The αD-FrXXA conotoxin inhibited the ACh-induced response on nAChR with a IC50 of 125 nM on hα7, 282 nM on hα3ß2, 607 nM on α4ß2, 351 nM on mouse adult muscle, and 447 nM on mouse fetal muscle. This is first toxin characterized from C. fergusoni and, at the same time, the second αD-conotoxin characterized from a species of the Eastern Pacific.
Assuntos
Conotoxinas , Caramujo Conus , Receptores Nicotínicos , Sequência de Aminoácidos , Animais , Conotoxinas/química , Caramujo Conus/química , Camundongos , Antagonistas Nicotínicos/metabolismo , Antagonistas Nicotínicos/farmacologia , Receptores Nicotínicos/metabolismo , Caramujos/metabolismoRESUMO
The venoms of Conus snails contain neuroactive peptides named conotoxins (CTXs). Some CTXs are nicotinic acetylcholine receptor (nAChRs) antagonists. nAChRs modulate the release of neurotransmitters and are implicated in several pathophysiologies. One venom peptide from Conus archon, a vermivorous species from the Mexican Pacific, was purified by RP-HPLC and its activity on human α7, α3ß2, and α7ß2 nAChRs was assessed by the two-electrode voltage clamp technique. At 36.3 µM the purified peptide (F27-1, renamed tentatively ArchIIIA) slowly reversibly inhibited the ACh-induced response of the hα7 subtype by 44.52 ± 5.83%, while it had low or no significant effect on the response of the hα3ß2 and hα7ß2 subtypes; the EC50 of the inhibiting effect was 45.7 µM on the hα7 subtype. This peptide has 15 amino acid residues and a monoisotopic mass of 1654.6 Da (CCSALCSRYHCLPCC), with three disulfide bridges and a free C-terminus. This sequence with a CC-C-C-CC arrangement (framework III) belongs to the M superfamily of conotoxins, corresponding to the mini-M´s (M-1-M-3) conotoxins; due to its size and inter-Cys spacings it is an M-2 conotoxin. This toxin is a novel mini-M conotoxin affecting ligand-gated ion channels, like the maxi-M CTX ψ-conotoxins and α-MIIIJ conotoxin (nAChRs blockers). This peptide seems to be homologous to the reg3b conotoxin (from Conus regius) with an identity of 93.3%, differing only in the third residue in the sequence, serine for threonine, both uncharged polar residues. We obtained, in silico, a probable 3D structure, which is consistent with its effect on neuronal subtypes.
Assuntos
Conotoxinas , Caramujo Conus , Antagonistas Nicotínicos , Receptores Nicotínicos , Animais , Conotoxinas/química , Conotoxinas/farmacologia , Caramujo Conus/química , Humanos , Antagonistas Nicotínicos/química , Antagonistas Nicotínicos/farmacologia , Peptídeos/metabolismo , Receptores Nicotínicos/metabolismoRESUMO
Cone snails are marine gastropod mollusks with one of the most powerful venoms in nature. The toxins, named conotoxins, must act quickly on the cone snails´ prey due to the fact that snails are extremely slow, reducing their hunting capability. Therefore, the characteristics of conotoxins have become the object of investigation, and as a result medicines have been developed or are in the trialing process. Conotoxins interact with transmembrane proteins, showing specificity and potency. They target ion channels and ionotropic receptors with greater regularity, and when interaction occurs, there is immediate physiological decompensation. In this review we aimed to evaluate the structural features of conotoxins and the relationship with their target types.
Assuntos
Conotoxinas/química , Caramujo Conus/química , Caramujo Conus/metabolismo , Animais , Conotoxinas/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Peçonhas/química , Peçonhas/metabolismoRESUMO
Conorfamides (CNFs) are toxins initially characterized from the venom duct of the venomous marine snail Conus spurius from the Gulf of Mexico; at their C-termini, these toxins are amidated and have high sequence similarity with the molluskan cardioexcitatory tetrapeptide Phe-Met-Arg-Phe-NH2 (FMRFamide or FMRFa) and other FMRFa-related peptides (FaRPs) found in the five molluskan classes, and in other invertebrate and vertebrate phyla. These peptides were the first FaRPs found to be present in any venom, and they are biologically active in mice, limpets, and/or freshwater snails. However, the molecular targets of the known CNFs (CNF-Sr1 and CNF-Sr2 from C. spurius, and CNF-Vc1 from C. victoriae) remain unidentified. Very recently, three FaRPs from C. textile have been found to potentiate the currents of acid-sensing ion channels. In this work, we characterized a novel conorfamide, CNF-Sr3 (ATSGPMGWLPVFYRF-NH2), comprised of 15 amino acid residues, and with a specific blocking activity for the Shaker subtype of the voltage-gated potassium channels, without significant effect on the Shab, Shaw, Shal and Eag channels. This peptide is the third type of disulfide-free conotoxins that has been discovered to target K+ channels.
Assuntos
Caramujo Conus/química , Venenos de Moluscos/química , Neuropeptídeos/farmacologia , Peptídeos/farmacologia , Superfamília Shaker de Canais de Potássio/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Baculoviridae , Células HEK293 , Humanos , Venenos de Moluscos/síntese química , Venenos de Moluscos/farmacologia , Neuropeptídeos/síntese química , Neuropeptídeos/química , Peptídeos/química , Bloqueadores dos Canais de Potássio/química , Bloqueadores dos Canais de Potássio/farmacologia , Células Sf9/virologiaRESUMO
Conus marine snails (â¼500 species) are tropical predators that use venoms mainly to capture prey and defend themselves from predators. The principal components of these venoms are peptides that are known as "conotoxins" and generally comprise 7-40 amino acid residues, including 0-5 disulfide bridges and distinct posttranslational modifications. The most common molecular targets of conotoxins are voltage- and ligand-gated ion channels, G protein-coupled receptors, and neurotransmitter transporters, to which they bind, typically, with high affinity and specificity. Due to these properties, several conotoxins have become molecular probes, medicines, and leads for drug design. Conotoxins have been classified into genetic superfamilies based on the signal sequence of their precursors, and into pharmacological families according to their molecular targets. The objective of this work was to identify and analyze partial cDNAs encoding conotoxin precursors belonging to the A superfamily from Conus brunneus, Conus nux, and Conus princeps. These are vermivorous species of the Mexican Pacific coast from which only one A-conotoxin, and few O- and I2-conotoxins have been reported. Employing RT-PCR, we identified 30 distinct precursors that contain 13 different predicted mature toxins. With the exception of two groups of four highly similar peptides, these toxins are diverse at both the sequence and the physicochemical levels, and they belong to the 4/3, 4/4, 4/5, 4/6, and 4/7 structural subfamilies. These toxins are predicted to target diverse nicotinic acetylcholine receptor (nAChR) subtypes: nx1d, muscle; pi1a-pi1d, α3ß2, α7, and/or α9α10; br1a, muscle, α3ß4, and/or α4ß2; and nx1a-nx1c/pi1g and pi1h, α3ß2, α3ß4, α9ß10, and/or α7.
Assuntos
Conotoxinas/química , Caramujo Conus/química , Precursores de Proteínas/química , Sequência de Aminoácidos , Animais , México , Dados de Sequência Molecular , Oceano Pacífico , Processamento de Proteína Pós-Traducional , Homologia de Sequência de AminoácidosRESUMO
A novel peptide, RsXXIVA, was isolated from the venom duct of Conus regularis, a worm-hunting species collected in the Sea of Cortez, México. Its primary structure was determined by mass spectrometry and confirmed by automated Edman degradation. This conotoxin contains 40 amino acids and exhibits a novel arrangement of eight cysteine residues (C-C-C-C-CC-CC). Surprisingly, two loops of the novel peptide are highly identical to the amino acids sequence of ω-MVIIA. The total length and disulfide pairing of both peptides are quite different, although the two most important residues for the described function of ω-MVIIA (Lys2 and Tyr13) are also present in the peptide reported here. Electrophysiological analysis using superior cervical ganglion (SCG) neurons indicates that RsXXIVA inhibits CaV2.2 channel current in a dose-dependent manner with an EC50 of 2.8 µM, whose effect is partially reversed after washing. Furthermore, RsXXIVA was tested in hot-plate assays to measure the potential anti-nociceptive effect to an acute thermal stimulus, showing an analgesic effect in acute thermal pain at 30 and 45 min post-injection. Also, the toxin shows an anti-nociceptive effect in a formalin chronic pain test. However, the low affinity for CaV2.2 suggests that the primary target of the peptide could be different from that of ω-MVIIA.
Assuntos
Analgésicos/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Conotoxinas/farmacologia , Caramujo Conus/química , Dor Aguda/tratamento farmacológico , Sequência de Aminoácidos , Analgésicos/química , Analgésicos/isolamento & purificação , Animais , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/isolamento & purificação , Canais de Cálcio Tipo N/efeitos dos fármacos , Canais de Cálcio Tipo N/metabolismo , Dor Crônica/tratamento farmacológico , Conotoxinas/química , Conotoxinas/isolamento & purificação , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Masculino , Espectrometria de Massas , México , Camundongos , Camundongos Endogâmicos ICR , Peptídeos/química , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Ratos , Ratos Wistar , Gânglio Cervical Superior/efeitos dos fármacos , Gânglio Cervical Superior/metabolismo , Fatores de TempoRESUMO
As part of continuing studies of the venom components present in Conus austini (syn.: Conus cancellatus), a vermivorous cone snail collected in the western Gulf of Mexico, Mexico, two major peptides, as14a and as14b, were purified and characterized. Their amino acid sequences were determined by automatic Edman sequencing after reduction and alkylation. Their molecular masses, established by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, confirmed the chemical analyses and indicated that as14a and as14b have free C-termini. Each peptide contains 4-Cys residues arranged in a pattern (C-C-C-C, framework 14). The primary structure of as14a is GGVGRCIYNCMNSGGGLNFIQCKTMCY (experimental monoisotopic mass 2883.92Da; calculated monoisotopic mass 2884.20Da), whereas that of as14b is RWDVDQCIYYCLNGVVGYSYTECQTMCT (experimental monoisotopic mass 3308.63Da; calculated monoisotopic mass 3308.34Da). Both purified peptides elicited scratching and grooming activity in mice, and as14b also caused body and rear limb extension and tail curling immediately upon injection. The high sequence similarity of peptide as14a with peptide vil14a from the vermivorous C. villepinii suggests that the former might block K+ channels.