RESUMEN
The neurotoxins of venomous scorpion act on ion channels. Whether these neurotoxins are retained in processed Buthus martensii Karsch scorpions used in traditional Chinese medicine materials is unknown. Comprehensive mass spectrometry-based proteomic characterization of functionally active toxins in the processed medicinal scorpion material revealed 22 full-length and 44 truncated thermostable potassium channel-modulatory toxins that preserved six conserved cysteine residues capable of forming the three disulfide bonds necessary for toxicity. Additionally, a broad spectrum of degraded toxin fragments was found, indicating their relative thermal instability which enabled toxicity reduction. Furthermore, the suppression of interleukin-2 (IL-2) production in Jurkat cells and the reduced delayed-type hypersensitivity (DTH) response demonstrated that the extracts have immunoregulatory activity both in vitro and in vivo. Our work describes the first "map" of functionally active scorpion toxins in processed scorpion medicinal material, which is helpful to unveil the pharmaceutical basis of the processed scorpion medicinal material in traditional Chinese medicine. BIOLOGICAL SIGNIFICANCE: Scorpions have been used as medicinal materials in China for more than one thousand years. This is an example of the well-known "Combat poison with poison" strategy common to traditional Chinese medicine. In the past 30â¯years, extensive investigations of Chinese scorpions have indicated that the neurotoxins in the scorpion venom are the main toxic components and they target various ion channels in cell membranes. However, whether these neurotoxins are retained in processed Buthus martensii Karsch scorpions used for traditional Chinese medicine remains unknown. Our study described the thermal stability and instability of potassium channel-modulatory neurotoxins in processed scorpions and helps to understand the pharmaceutical basis underling the strategy of "combat poison with poison to cure diseases".
Asunto(s)
Medicina Tradicional China , Neurotoxinas/análisis , Bloqueadores de los Canales de Potasio/análisis , Proteoma/análisis , Venenos de Escorpión/análisis , Animales , Estabilidad de Medicamentos , Femenino , Células HEK293 , Humanos , Células Jurkat , Neurotoxinas/metabolismo , Péptidos/análisis , Péptidos/metabolismo , Preparaciones Farmacéuticas/análisis , Preparaciones Farmacéuticas/química , Preparaciones Farmacéuticas/metabolismo , Bloqueadores de los Canales de Potasio/aislamiento & purificación , Bloqueadores de los Canales de Potasio/metabolismo , Canales de Potasio/metabolismo , Estabilidad Proteica , Proteoma/metabolismo , Proteómica/métodos , Ratas , Ratas Endogámicas Lew , Venenos de Escorpión/química , Venenos de Escorpión/metabolismo , Escorpiones/química , Escorpiones/metabolismo , TemperaturaRESUMEN
Venom gland transcriptomic and proteomic analyses have improved our knowledge on the diversity of the heterogeneous components present in scorpion venoms. However, most of these studies have focused on species from the family Buthidae. To gain insights into the molecular diversity of the venom components of scorpions belonging to the family Superstitioniidae, one of the neglected scorpion families, we performed a transcriptomic and proteomic analyses for the species Superstitionia donensis. The total mRNA extracted from the venom glands of two specimens was subjected to massive sequencing by the Illumina protocol, and a total of 219,073 transcripts were generated. We annotated 135 transcripts putatively coding for peptides with identity to known venom components available from different protein databases. Fresh venom collected by electrostimulation was analyzed by LC-MS/MS allowing the identification of 26 distinct components with sequences matching counterparts from the transcriptomic analysis. In addition, the phylogenetic affinities of the found putative calcins, scorpines, La1-like peptides and potassium channel κ toxins were analyzed. The first three components are often reported as ubiquitous in the venom of different families of scorpions. Our results suggest that, at least calcins and scorpines, could be used as molecular markers in phylogenetic studies of scorpion venoms.
Asunto(s)
Proteínas de Artrópodos , Venenos de Escorpión , Animales , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/metabolismo , Glándulas Exocrinas/metabolismo , Perfilación de la Expresión Génica , Filogenia , Proteómica , Venenos de Escorpión/genética , Venenos de Escorpión/metabolismo , EscorpionesRESUMEN
Human voltage-gated potassium channel Kv1.3 is an important pharmacological target for the treatment of autoimmune and metabolic diseases. Increasing clinical demands stipulate an active search for efficient and selective Kv1.3 blockers. Here we present a new, reliable, and easy-to-use analytical system designed to seek for and study Kv1.3 ligands that bind to the extracellular vestibule of the K(+)-conducting pore. It is based on Escherichia coli spheroplasts with the hybrid protein KcsA-Kv1.3 embedded into the membrane, fluorescently labeled Kv1.3 blocker agitoxin-2, and confocal laser scanning microscopy as a detection method. This system is a powerful alternative to radioligand and patch-clamp techniques. It enables one to search for Kv1.3 ligands both among individual compounds and in complex mixtures, as well as to characterize their affinity to Kv1.3 channel using the "mix and read" mode. To demonstrate the potential of the system, we performed characterization of several known Kv1.3 ligands, tested nine spider venoms for the presence of Kv1.3 ligands, and conducted guided purification of a channel blocker from scorpion venom.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Escherichia coli/genética , Canal de Potasio Kv1.3/química , Microscopía Confocal/métodos , Animales , Escherichia coli/química , Escherichia coli/metabolismo , Expresión Génica , Humanos , Canal de Potasio Kv1.3/genética , Canal de Potasio Kv1.3/metabolismo , Ligandos , Canales de Potasio con Entrada de Voltaje/química , Canales de Potasio con Entrada de Voltaje/genética , Canales de Potasio con Entrada de Voltaje/metabolismo , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Venenos de Escorpión/química , Venenos de Escorpión/genética , Venenos de Escorpión/metabolismo , Escorpiones , Esferoplastos/química , Esferoplastos/genética , Esferoplastos/metabolismo , Venenos de Araña/química , ArañasRESUMEN
Sublethally irradiated mice were administered with scorpion venom peptides (SVP) or with PBS in the saline control group, 3 days before and 7 consecutive days after irradiation. Hematopoietic recovery was assessed by bone marrow (BM) cell proliferation index (PI) and colony forming unit-granulocyte/macrophage (CFU-GM), spleen weight index (SI) and thymus weight index (TI), colony-forming unit-spleen (CFU-S) and peripheral leukocyte counts. In addition, IL-1alpha and SCF levels in BM, IL-6 and GM-CSF levels in serum were determined. In SVP treated groups, PI was improved dramatically versus control mice on day 22 after irradiation. The number of CFU-GM colonies in all SVP treated groups was higher than the control groups. The difference of the number of CFU-GM colonies between SVPV group (0.2 mg/kg) and the control was significant on day 5 and 10 after irradiation (p < 0.05). SVPIV (0.2 mg/kg) could activate the CFU-S formation on day 10 after irradiation. SI was in peak value on day 15 after irradiation in all groups and the SI value of SVPV treated group was higher than control group (p < 0.05). Our results suggest that SVP may be valuable natural peptides that relieve myelosuppression caused by radiation. The effect of SVP accelerating the hematopoietic recovery was potentially through a mechanism of stimulating the release of cytokines.
Asunto(s)
Células Madre Hematopoyéticas/efectos de los fármacos , Mielopoyesis/efectos de los fármacos , Péptidos/farmacología , Venenos de Escorpión/metabolismo , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/efectos de la radiación , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Factor Estimulante de Colonias de Granulocitos y Macrófagos/sangre , Células Progenitoras de Granulocitos y Macrófagos/efectos de los fármacos , Células Progenitoras de Granulocitos y Macrófagos/efectos de la radiación , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de la radiación , Interleucina-1alfa/sangre , Interleucina-6/sangre , Recuento de Leucocitos , Masculino , Ratones , Ratones Congénicos , Ratones Endogámicos BALB C , Mielopoyesis/efectos de la radiación , Tamaño de los Órganos/efectos de los fármacos , Tamaño de los Órganos/efectos de la radiación , Péptidos/metabolismo , Bazo/efectos de los fármacos , Bazo/patología , Bazo/efectos de la radiación , Factor de Células Madre/sangre , Timo/efectos de los fármacos , Timo/patología , Timo/efectos de la radiación , Factores de Tiempo , Irradiación Corporal TotalRESUMEN
The pace of resistance against antibiotics almost exceeds that of the development of new drugs. As many bacteria have become resistant to conventional antibiotics, new drugs or drug resources are badly needed to combat antibiotic-resistant pathogens, like methicillin-resistant Staphylococcus aureus (MRSA). Antimicrobial peptides, rich sources existing in nature, are able to effectively kill multidrug-resistant pathogens. Here, imcroporin, a new antimicrobial peptide, was screened and isolated from the cDNA library of the venomous gland of Isometrus maculates. The MIC of imcroporin against MRSA was 50 microg/ml, 8-fold lower than that of cefotaxime and 40-fold lower than that of penicillin. Imcroporin killed bacteria rapidly in vitro, inhibited bacterial growth, and cured infected mice. These results revealed that imcroporin could be considered a potential anti-infective drug or lead compound, especially for treating antibiotic-resistant pathogens.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/uso terapéutico , Venenos de Escorpión/farmacología , Venenos de Escorpión/uso terapéutico , Escorpiones/metabolismo , Secuencia de Aminoácidos , Animales , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Péptidos Catiónicos Antimicrobianos/metabolismo , Cefotaxima/farmacología , Cefotaxima/uso terapéutico , Femenino , Hemólisis/efectos de los fármacos , Humanos , Masculino , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Ratones , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Penicilinas/farmacología , Penicilinas/uso terapéutico , Venenos de Escorpión/metabolismo , Escorpiones/química , Homología de Secuencia de Aminoácido , Infecciones Estafilocócicas/tratamiento farmacológico , Vancomicina/farmacología , Vancomicina/uso terapéuticoRESUMEN
Recently, a solid-state NMR study revealed that scorpion toxin binding leads to conformational changes in the selectivity filter of potassium channels. The exact nature of the conformational changes, however, remained elusive. We carried out all-atom molecular dynamics simulations that enabled us to cover the complete pathway of toxin approach and binding, and we validated our simulation results by using solid-state NMR data and electrophysiological measurements. Our structural model revealed a mechanism of cooperative toxin-induced conformational changes that accounts both for the signal changes observed in solid-state NMR and for the tight interaction between KcsA-Kv1.3 and Kaliotoxin. We show that this mechanism is structurally and functionally closely related to recovery from C-type inactivation. Furthermore, our simulations indicate heterogeneity in the binding modes of Kaliotoxin, which might serve to enhance its affinity for KcsA-Kv1.3 further by entropic stabilization.
Asunto(s)
Canal de Potasio Kv1.3/metabolismo , Canales de Potasio con Entrada de Voltaje , Venenos de Escorpión/metabolismo , Animales , Simulación por Computador , Electrofisiología , Femenino , Canal de Potasio Kv1.3/química , Canal de Potasio Kv1.3/genética , Microinyecciones , Modelos Moleculares , Conformación Molecular , Mutación , Resonancia Magnética Nuclear Biomolecular , Oocitos/metabolismo , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/química , Bloqueadores de los Canales de Potasio/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , Estructura Secundaria de Proteína , ARN Complementario/administración & dosificación , Venenos de Escorpión/química , Escorpiones , Electricidad Estática , XenopusRESUMEN
Scorpion venom represents a tremendous hitherto unexplored resource for understanding ion channels. BmK86 is a novel K+ -channel toxin gene isolated from a cDNA library of Mesobuthus martensii Karsch, which encodes a signal peptide of 22 amino acid residues and a mature toxin of 35 residues with three disulfide bridges. The genomic sequence of BmK86 consists of two exons disrupted by an intron of 72 bp. Comparison with the other scorpion toxins BmK86 shows low sequence similarity. The GST-BmK86 fusion protein was successfully expressed in Escherichia coli. The fusion protein was cleaved by enterokinase and the recombinant BmK86 was purified by HPLC. Using whole-cell patch-clamp recording, the recombinant BmK86 was found to inhibit the potassium current of mKv1.3 channel expressed in COS7 cells. These results indicated that BmK86 belongs to a representative member of a novel subfamily of alpha-KTxs. The systematic number assigned to BmK86 is alpha-KTx26.1.
Asunto(s)
Bloqueadores de los Canales de Potasio/metabolismo , Venenos de Escorpión/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Clonación Molecular , ADN Complementario , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Bloqueadores de los Canales de Potasio/química , Bloqueadores de los Canales de Potasio/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Venoms from scorpions contain extremely rich bioactive peptides that often carry diverse functions and are presumably needed to achieve synergistic effects for rapidly immobilizing prey and defending themselves. BotLVP1 is a unique heterodimer protein recently found in the scorpion Buthus occitanus tunetanus venom that is structurally related to scorpion toxins affecting sodium channels (NaScTxs) but exhibits adipocyte lipolysis activity. We have isolated and identified two cDNA clones encoding subunits alpha and beta of a BotLVP1-like peptide (named BmLVP1) from the Chinese scorpion Buthus martensii venom gland and determined the first complete gene structure of this subfamily. These results highlight a genetic link between these lipolysis activating peptides and NaScTxs. Comparison of cDNA and genomic sequences combined with protein structural and functional analysis provides evidence supporting the existence of RNA editing mechanism in scorpion venom glands, which could mediate functional switch of BmLVP1 gene, from adipocyte lipolysis to neurotoxicity, by altering the wrapper disulfide bridge (WDB) pattern of the peptides.
Asunto(s)
Disulfuros/metabolismo , Lipólisis , Péptidos/metabolismo , Venenos de Escorpión/metabolismo , Escorpiones/metabolismo , Adenina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario/genética , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Péptidos/genética , Estructura Terciaria de Proteína , Edición de ARN/genética , Venenos de Escorpión/química , Venenos de Escorpión/genética , Escorpiones/química , Escorpiones/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Canales de Sodio/genética , Canales de Sodio/metabolismo , Homología Estructural de Proteína , Transcripción Genética/genéticaRESUMEN
In a recent note to Nature, R. MacKinnon has raised the possibility that potassium channel gating modifiers are able to partition in the phospholipid bilayer of neuronal membranes and that by increasing their partial concentration adjacent to their receptor, they affect channel function with apparent high affinity (Lee and MacKinnon (2004) Nature 430, 232-235). This suggestion was adopted by Smith et al. (Smith, J. J., Alphy, S., Seibert, A. L., and Blumenthal, K. M. (2005) J. Biol. Chem. 280, 11127-11133), who analyzed the partitioning of sodium channel modifiers in liposomes. They found that certain modifiers were able to partition in these artificial membranes, and on this basis, they have extrapolated that scorpion beta-toxins interact with their channel receptor in a similar mechanism as that proposed by MacKinnon. Since this hypothesis has actually raised a new conception, we examined it in binding assays using a number of pharmacologically distinct scorpion beta-toxins and insect and mammalian neuronal membrane preparations, as well as by analyzing the rate by which the toxin effect on gating of Drosophila DmNa(v)1 and rat brain rNa(v)1.2a develops. We show that in general, scorpion beta-toxins do not partition in neuronal membranes and that in the case in which a depressant beta-toxin partitions in insect neuronal membranes, this partitioning is unrelated to its interaction with the receptor site and the effect on the gating properties of the sodium channel. These results negate the hypothesis that the high affinity of beta-toxins for sodium channels is gained by their ability to partition in the phospholipid bilayer and clearly indicate that the receptor site for scorpion beta-toxins is accessible to the extracellular solvent.
Asunto(s)
Membrana Celular/metabolismo , Membrana Dobles de Lípidos/química , Neuronas/metabolismo , Fosfolípidos/química , Venenos de Escorpión/metabolismo , Canales de Sodio/química , Animales , Encéfalo/metabolismo , Drosophila , Fosfolípidos/metabolismo , Unión Proteica , ARN Complementario/metabolismo , Ratas , Canales de Sodio/metabolismo , Solventes/químicaRESUMEN
By using single wavelength anomalous diffraction phasing based on the anomalous signal from copper atoms, the crystal structure of atratoxin was determined at the resolution of 1.5 A and was refined to an ultrahigh resolution of 0.87 A. The ultrahigh resolution electron density maps allowed the modeling of 38 amino acid residues in alternate conformations and the location of 322 of 870 possible hydrogen atoms. To get accurate information at the atomic level, atratoxin-b (an analog of atratoxin with reduced toxicity) was also refined to an atomic resolution of 0.92 A. By the sequence and structural comparison of these two atratoxins, Arg(33) and Arg(36) were identified to be critical to their varied toxicity. The effect of copper ions on the distribution of hydrogen atoms in atratoxin was discussed, and the interactions between copper ions and protein residues were analyzed based on a statistical method, revealing a novel pentahedral copper-binding motif.
Asunto(s)
Neurotoxinas/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Anisotropía , Secuencia de Bases , Clonación Molecular , Cobre/química , Cristalografía por Rayos X , ADN Complementario/metabolismo , Bases de Datos como Asunto , Venenos Elapídicos , Electrones , Hidrógeno/química , Proteínas de Insectos , Iones , Modelos Moleculares , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Unión Proteica , Conformación Proteica , Venenos de Escorpión/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
We have studied the interaction between the SK2 channel and different scorpion toxins in order to find similarity and differences to other K+ channels. Beside apamin, ScTX is a high affinity blocker of the SK2 channel, whereas CTX is unable to block current through SK2. In order to prove that the ScTX affinity can be explained by the character of the different residues in the outer pore of the SK channels we introduced point mutations that render SK2 K+ channel SK1 and SK3 like. Directed by the results of the toxin receptor on the ShakerK+ channel, we changed single amino acids of the SK2 K+ channel that should render it sensitive to other peptide toxins like CTX a blocker of the IK channel, or KTX a blocker of the voltage-dependent channel Kv1.1 and Kv1.3. Amino acids V342G, S344E, and G384D of SK2 were changed to amino acids known from ShakerK+ channel to improve Shaker K+ channel CTX sensitivity. Interestingly SK2 V342G became CTX sensitive with a Kd of 19 nM and was also KTX sensitive Kd=97 nM. SK2 S344E (KdCTX = 105 nM,KdKTX = 144 nM) and G348D (KdCTX = 31 nM,Kd KTX = 89 nM) became also CTX and KTX sensitive with a lower affinity. The mutant channels SK V342G, SK2 S344E and SK2 G348D showed reduced ScTX sensitivity (Kd = 6 nM,Kd = 48 nM, and Kd = 12 nM). Because the exchange of a single residue could create a new high affinity binding site for CTX and KTX we concluded that the outer vestibule around position V342, S344, and G348 of the SK2 K+ channel pore is very similar to those of voltage-gated K+ channels such as the Shaker K+ channel, Kv1.1 and Kv1.3 channels and also to the prokaryotic KcsA channel. From mutant cycle analysis of KTX position H34 and SK2 position V342G, S344E, and G348D we could deduce that KTX binds in a similar way to SK2 channel mutant pore than to the Kv1.1 pore. We have studied the interaction between the SK2 channel and different scorpion toxins in order to find similarity and differences to other K+ channels. Beside apamin, ScTX is a high affinity blocker of the SK2 channel, whereas CTX is unable to block current through SK2. In order to prove that the ScTX affinity can be explained by the character of the different residues in the outer pore of the SK channels we introduced point mutations that render SK2 K+ channel SK1 and SK3 like. Directed by the results of the toxin receptor on the ShakerK+ channel, we changed single amino acids of the SK2 K+ channel that should render it sensitive to other peptide toxins like CTX a blocker of the IK channel, or KTX a blocker of the voltage-dependent channel Kv1.1 and Kv1.3. Amino acids V342G, S344E, and G384D of SK2 were changed to amino acids known from ShakerK+ channel to improve Shaker K+ channel CTX sensitivity. Interestingly SK2 V342G became CTX sensitive with a Kd of 19 nM and was also KTX sensitive Kd = 97 nM. SK2 S344E (KdCTX = 105 nM,KdKTX = 144 nM) and G348D (KdCTX = 31 nM,Kd KTX = 89 nM) became also CTX and KTX sensitive with a lower affinity. The mutant channels SK V342G, SK2 S344E and SK2 G348D showed reduced ScTX sensitivity (Kd = 6 nM,Kd = 48 nM, and Kd = 12 nM). Because the exchange of a single residue could create a new high affinity binding site for CTX and KTX we concluded that the outer vestibule around position V342, S344, and G348 of the SK2 K+ channel pore is very similar to those of voltage-gated K+ channels such as the Shaker K+ channel, Kv1.1 and Kv1.3 channels and also to the prokaryotic KcsA channel. From mutant cycle analysis of KTX position H34 and SK2 position V342G, S344E, and G348D we could deduce that KTX binds in a similar way to SK2 channel mutant pore than to the Kv1.1 pore.
Asunto(s)
Caribdotoxina/metabolismo , Canales de Potasio Calcio-Activados , Canales de Potasio/metabolismo , Venenos de Escorpión/metabolismo , Escorpiones/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Apamina/genética , Apamina/metabolismo , Sitios de Unión/fisiología , Calcio/metabolismo , Células Cultivadas , Caribdotoxina/genética , ADN Complementario/genética , Electrofisiología , Vectores Genéticos , Humanos , Concentración de Iones de Hidrógeno , Canales de Potasio de Conductancia Intermedia Activados por el Calcio , Potenciales de la Membrana/fisiología , Datos de Secuencia Molecular , Canales de Potasio/genética , Canales de Potasio/fisiología , Unión Proteica/fisiología , Venenos de Escorpión/genética , Alineación de Secuencia , Canales de Potasio de Pequeña Conductancia Activados por el Calcio , TransfecciónRESUMEN
Scorpion alpha-K(+) channel toxins are a large family of polypeptides with a similar structure but diverse pharmacological activities. Despite many structural and functional data available at present, little progress has been made in understanding the toxin's molecular basis responsible for the functional diversification. In this paper, we report the first complete cDNA sequences of toxins belonging to subfamily 6 and identify five new members, called alpha-KTx 6.6-6.10. By analyzing the rates of mutations that occurred in the corresponding cDNAs, we suggest that accelerated evolution in toxin-coding regions may be associated with the functional diversification of this subfamily. To pinpoint sites probably involved in the functional diversity of alpha-KTx family, we analyzed this family of sequences using the evolutionary trace method. This analysis highlighted one channel-binding surface common for all the members. This surface is composed of one conserved lysine residue at position 29 assisted by other residues at positions 10, 26, 27, 32, 34, and 36. Of them, the positions 29, 32, and 34 have been reported to be the most major determinants of channel specificity. Interestingly, another contrary surface was also observed at a higher evolutionary time cut-off value, which may be involved in the binding of ERG (ether-a-go-go-related gene) channel-specific toxins. The good match between the trace residues and the functional epitopes of the toxins suggested that the evolutionary trace results reported here can be applied to predict channel-binding sites of the toxins. Because, the side-chain variation in the trace positions is strongly linked with the functional alteration and channel-binding surface transfer of alpha-KTx family, we conclude that our findings should also be important for the rational design of new toxins targeting a given potassium channel with high selectivity.
Asunto(s)
Evolución Molecular , Canales de Potasio/metabolismo , Venenos de Escorpión/química , Venenos de Escorpión/metabolismo , Escorpiones , Toxinas Biológicas/química , Toxinas Biológicas/genética , Animales , Secuencia de Bases , Sitios de Unión , Clonación Molecular , ADN Complementario/genética , Bases de Datos Genéticas , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Canales de Potasio/química , Unión Proteica , Venenos de Escorpión/genética , Venenos de Escorpión/farmacología , Escorpiones/química , Escorpiones/genética , Alineación de Secuencia , Relación Estructura-Actividad , Especificidad por Sustrato , Toxinas Biológicas/clasificación , Toxinas Biológicas/farmacologíaRESUMEN
The goals of this study are to investigate the mechanism and site of action whereby a human ether-a-go-go-related gene (HERG)-specific scorpion peptide toxin, ErgTx, suppresses HERG current. We apply cysteine-scanning mutagenesis to the S5-P and P-S6 linkers of HERG and examine the resulting changes in ErgTx potency. Data are compared with the characteristics of charybdotoxin (ChTx, or its analogs) binding to the Shaker channel. ErgTx binds to the outer vestibule of HERG but may not physically occlude the pore. In contrast to ChTx. Shaker interaction, elevating [K](o) (from 2 to 98 mm) does not affect ErgTx potency, and through-solution electrostatic forces only play a minor role in influencing ErgTx.HERG interaction. Cysteine mutations of three positions in S5-P linker (Trp-585, Gly-590, and Ile-593) and 1 position in P-S6 linker (Pro-632) induce profound changes in ErgTx binding (DeltaDeltaG > 2 kcal/mol). We propose that the long S5-P linker of the HERG channel forms an amphipathic alpha-helix that, together with the P-S6 linker, forms a hydrophobic ErgTx binding site. This study paves the way for future mutant cycle analysis of interacting residues in the ErgTx.HERG complex, which, in conjunction with NMR determination of the ErgTx solution structure, will yield information about the topology of HERG's outer vestibule.
Asunto(s)
Bloqueadores de los Canales de Calcio/química , Proteínas de Transporte de Catión , Proteínas de Unión al ADN , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/química , Venenos de Escorpión/química , Transactivadores , Secuencia de Aminoácidos , Sitios de Unión , Bloqueadores de los Canales de Calcio/metabolismo , Caribdotoxina/química , Cisteína/química , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Canal de Potasio ERG1 , Electrofisiología , Canales de Potasio Éter-A-Go-Go , Humanos , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Oocitos/metabolismo , Potasio , Canales de Potasio/metabolismo , Unión Proteica , Venenos de Escorpión/metabolismo , Homología de Secuencia de Aminoácido , Canales de Potasio de la Superfamilia Shaker , Tetraetilamonio/farmacología , Factores de Tiempo , Regulador Transcripcional ERGRESUMEN
A new neurotoxic component named BmK abT was purified from the venom of Chinese scorpion Buthus martensi Karsch. The molecular weight of BmK abT was determined to be 7212 Da on a mass spectrum. The minimum lethal dose of BmK abT was tested to be about 1.5 microg per mouse by intracerebroventricular injection, and the dose induced significant paralysis effect on cockroach was about 5 microg by i.p. injection. The partial amino acid sequence indicated that it was a distinctive polypeptide in the scorpion neurotoxin family. Thereafter, the whole amino acid sequence of mature BmK abT was deduced from cDNA sequence by 5'- and 3'-rapid amplification of cDNA ends. Finally, it was defined to be composed of 63 residues with amidation at the C-terminal residue. By sequence comparison, BmK abT was found to be most similar to Ts VII, a beta-toxin from the New World scorpion. The patch-clamp recording on DRG neurons, unexpectedly, showed this toxin could prolong the action potential and increase the amplitude of the peak Na+ currents, which are the typical characters of alpha-toxin. These results suggested that BmK abT was a new toxic component found in the Old World scorpion species structurally similar to beta-toxins, but functionally similar to alpha-toxins.
Asunto(s)
Venenos de Escorpión/metabolismo , Escorpiones/química , Potenciales de Acción/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Cucarachas , ADN Complementario/metabolismo , Electrofisiología , Ganglios Espinales/efectos de los fármacos , Espectrometría de Masas , Ratones , Datos de Secuencia Molecular , Canales de Potasio/química , Canales de Potasio/efectos de los fármacos , ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Canales de Sodio/química , Canales de Sodio/efectos de los fármacos , Venenos de Araña/química , Venenos de Araña/toxicidadRESUMEN
Maurotoxin is a 34-residue toxin isolated from the venom of the Tunisian chactoid scorpion Scorpio maurus palmatus and contains four disulfide bridges that are normally found in long-chain toxins of 60-70 amino acid residues, which affect voltage-gated sodium channels. However, despite the unconventional disulfide-bridge pattern of maurotoxin, the conformation of this toxin remains similar to that of other toxins acting on potassium channels. Here, we analyzed the effects of synthetic maurotoxin on voltage-gated Shaker potassium channels (ShB) expressed in Xenopus oocytes. Maurotoxin produces a strong, but reversible, inhibition of the ShB K+ current with an IC50 of 2 nM. Increasing concentrations of the toxin induce a progressively higher block at saturating concentrations. At nonsaturating concentrations of the toxin (5-20 nM), the channel block appears slightly more pronounced at threshold potentials suggesting that the toxin may have a higher affinity for the closed state of the channel. At the single channel level, the toxin does not modify the unitary current amplitude, but decreases ensemble currents by increasing the number of depolarizing epochs that failed to elicit any opening. A point mutation of Lys23 to alanine in maurotoxin produces a 1000-fold reduction in the IC50 of block by the toxin suggesting the importance of this charged residue for the interaction with the channel. Maurotoxin does not affect K+ currents carried by Kir2.3 channels in oocytes or Na+ currents carried by the alphaIIa channel expressed in CHO cells.
Asunto(s)
Bloqueadores de los Canales de Potasio , Canales de Potasio/metabolismo , Venenos de Escorpión/farmacología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Disulfuros , Relación Dosis-Respuesta a Droga , Expresión Génica , Microinyecciones , Datos de Secuencia Molecular , Oocitos , Técnicas de Placa-Clamp , Conformación Proteica , ARN Complementario/metabolismo , Venenos de Escorpión/química , Venenos de Escorpión/metabolismo , Canales de Potasio de la Superfamilia Shaker , Canales de Sodio/metabolismo , Xenopus laevisRESUMEN
Five full-length cDNAs encoding the precursors of two 'short chain' scorpion non-toxic peptides active on Ca(2+)-activated K(+) channels (BmP02 and BmP03) and two novel putative long chain K(+) channel-blocking peptides (named BmTXKbeta and BmTXKbeta2) were first isolated from the venom gland cDNA library of the Chinese scorpion Buthus martensii Karsch (BmK). BmTXKbeta2 showed a high similarity with AaTXKbeta, while BmTXKbeta was completely different in the deduced primary structure from the long chain and short chain scorpion toxins already characterized. Thus, BmTXKbeta expands the scorpion long chain K(+) channel-blocking peptide family. Although little sequence similarity exists between the above two short and two long peptides, they are similar at the positions of six cysteines, suggesting that they should all share a similar scaffold composed of an alpha-helix and a three-stranded beta-sheet.
Asunto(s)
Péptidos/genética , Péptidos/metabolismo , Bloqueadores de los Canales de Potasio , Precursores de Proteínas/genética , Venenos de Escorpión/genética , Venenos de Escorpión/metabolismo , Regiones no Traducidas 3' , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/análisis , Regulación de la Expresión Génica , Datos de Secuencia Molecular , Péptidos/farmacología , Reacción en Cadena de la Polimerasa , Precursores de Proteínas/metabolismo , Procesamiento Postranscripcional del ARN , Venenos de Escorpión/química , Venenos de Escorpión/farmacología , Análisis de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Potassium-channel-blocking scorpion toxins (alpha-K-toxins) have been shown to be valuable tools for the study of potassium channels. Here we report two toxins, cobatoxin 1 and 2, of 32 amino acids, containing three disulphide bridges, that were isolated from the venom of the Mexican scorpion Centruroides noxius. Their primary sequences show less than 40% identity to other alpha-K-toxins. It is therefore proposed that they belong to subfamily 9. The cDNA of cobatoxin 1 encodes a putative signal peptide, a putative short propeptide, the mature peptide and two amino acids that are processed to leave cobatoxin 1 amidated at the C-terminus. In rat brain synaptosomal membranes cobatoxin 1 and cobatoxin 2 bind to a common binding site of alpha-K-toxins with Ki values of 109 pM and 87 pM, respectively. Moreover, they block the Shaker and Kv1.1 K+ channels with moderate affinities, with Kd values of around 0.7 microM and 4.1 microM (Shaker) and 0.5 microM and 1.0 microM (Kv1.1), respectively. A three-dimensional model of cobatoxin 1 was generated and used to interpret the obtained functional data on a structural basis.
Asunto(s)
Bloqueadores de los Canales de Potasio , Venenos de Escorpión/química , Venenos de Escorpión/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/metabolismo , Clonación Molecular , ADN Complementario , Radioisótopos de Yodo , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Ratas , Venenos de Escorpión/genética , Venenos de Escorpión/metabolismo , Homología de Secuencia de Aminoácido , Sinaptosomas/metabolismoRESUMEN
An immortal, cloned cell line (RCMH), obtained from human skeletal muscle was established in our laboratory and shown to express muscle specific proteins. We measured ligand binding to ion channels, ion currents using whole cell patch clamp and intracellular calcium both in cells grown in complete media and in cells grown for 4-40 days in media supplemented with hormones and nutrients (differentiating media). Markers for differentiated muscle, such as the muscle isoform of creatine kinase and the cytoskeletal proteins alpha-actinin, alpha-sarcomeric actin, myosin and titin were present in early stages. Receptors for gamma toxin from Tityus serrulatus scorpion venom, a specific modulator for voltage dependent sodium channels, were present (0.9-1.0 pmol mg-1 protein) during stage 1 (0-6 days in culture with differentiating media) and increased by 50% in stage 3 (more than 10 days in differentiating media). High and low affinity dihydropyridine receptors present in stage 1 convert into a single type of high affinity receptors in stage 3. Both intracellular calcium release and InsP3 receptors were evident in stage 1 but ryanodine receptors were expressed only in stage 3. RCMH cells showed no voltage sensitive currents in stage 1. Between 7 and 10 days in differentiating media (stage 2), an outward potassium current was observed. Small inward currents appeared only in stage 3; we identified both tetrodotoxin sensitive and tetrodotoxin resistant sodium currents as well as calcium currents. This pattern is consistent with the expression of voltage dependent calcium release before appearance of both the action potential and ryanodine receptors.
Asunto(s)
Canales Iónicos/biosíntesis , Músculo Esquelético/citología , Músculo Esquelético/fisiología , Biomarcadores , Calcio/metabolismo , Diferenciación Celular , Línea Celular Transformada , Membrana Celular/fisiología , Creatina Quinasa/metabolismo , Proteínas del Citoesqueleto/análisis , Humanos , Canales Iónicos/fisiología , Isoenzimas , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Proteínas Musculares/análisis , Técnicas de Placa-Clamp , Venenos de Escorpión/metabolismo , Canales de Sodio/biosíntesis , Tetrodotoxina/farmacologíaRESUMEN
Two novel peptides were purified from the venom of the scorpion Pandinus imperator, and were named Pi2 and Pi3. Their complete primary structures were determined and their blocking effects on Shaker B K+ channels were studied. Both peptides contain 35 amino acids residues, compacted by three disulfide bridges, and reversibly block the Shaker B K+ channels. They have only one amino acid changed in their sequence, at position 7 (a proline for a glutamic acid). Whereas peptide Pi2, containing the Pro7, binds the Shaker B K+ channels with a Kd of 8.2 nm, peptide Pi3 containing the Glu7 residue has a much lower affinity of 140 nm. Both peptides are capable of displacing the binding of 125I-noxiustoxin to brain synaptosome membranes. Since these two novel peptides are about 50% identical to noxiustoxin, the present results support previous data published by our group showing that the amino-terminal region of noxiustoxin, and also the amino-terminal sequence of the newly purified homologues: Pi2, and Pi3, are important for the recognition of potassium channels.
Asunto(s)
Canales de Potasio/metabolismo , Venenos de Escorpión/aislamiento & purificación , Escorpiones/química , Secuencia de Aminoácidos , Animales , Unión Competitiva , Línea Celular , ADN Complementario/genética , Vectores Genéticos/genética , Cinética , Datos de Secuencia Molecular , Nucleopoliedrovirus/genética , Canales de Potasio/efectos de los fármacos , Ratas , Venenos de Escorpión/química , Venenos de Escorpión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Canales de Potasio de la Superfamilia Shaker , Spodoptera/citología , Relación Estructura-ActividadRESUMEN
A new ligand (Ts kappa) of the apamin binding site on rat brain synaptosomes (K0.5 = 300 pM) was purified and characterized from the venom of Tityus serrulatus. It is a polypeptide toxin of 35 amino acid residues, with three disulfide bridges. Its cDNA was amplified from a venom gland cDNA library and the nucleotide sequence determined. A model of Ts kappa was constructed by amino acid replacement using charybdotoxin structure as determined by 1H nuclear magnetic resonance as starting model.