RESUMEN
BACKGROUND: Almost all Tityus characterized toxins are from subgenera Atreus and Tityus, there are only a few data about toxins produced by Archaeotityus, an ancient group in Tityus genus. METHODS: Tityus (Archaeotityus) mattogrossensis crude venom was fractionated by high performance liquid chromatography, the major fractions were tested in a frog sciatic nerve single sucrose-gap technique. Two fractions (Tm1 and Tm2) were isolated, partially sequenced by MALDI-TOF/MS and electrophysiological assayed on HEK293 Nav 1.3, HEK293 Nav 1.6, DUM and DRG cells. RESULTS: The sucrose-gap technique showed neurotoxicity in four fractions. One fraction caused a delay of action potential repolarization and other three caused a reduction in amplitude. An electrophysiological assay showed that Tm1 is active on HEK293 Nav 1.3, HEK293 Nav 1.6, DUM and DRG cells, and Tm2 on HEK293 Nav 1.3 and DRG cells, but not in HEK293 Nav 1.6. In addition, Tm1 and Tm2 did promote a shift to more negative potentials strongly suggesting that both are α-NaScTx. CONCLUSION: Although Tityus (Archaeotityus) mattogrossensis is considered an ancient group in Tityus genus, the primary structure of Tm1 and Tm2 is more related to Tityus subgenus. The patch clamp electrophysiological tests suggest that Tm1 and Tm2 are NaScTx, and also promoted no shift to more negative potentials, strongly suggesting that both are α-NaScTx. This paper aimed to explore and characterize for the first time toxins from the ancient scorpion Tityus (Archaeotityus) mattogrossensis.
RESUMEN
Background: Almost all Tityus characterized toxins are from subgenera Atreus and Tityus, there are only a few data about toxins produced by Archaeotityus, an ancient group in Tityus genus. Methods: Tityus (Archaeotityus) mattogrossensis crude venom was fractionated by high performance liquid chromatography, the major fractions were tested in a frog sciatic nerve single sucrose-gap technique. Two fractions (Tm1 and Tm2) were isolated, partially sequenced by MALDI-TOF/MS and electrophysiological assayed on HEK293 Nav 1.3, HEK293 Nav 1.6, DUM and DRG cells. Results: The sucrose-gap technique showed neurotoxicity in four fractions. One fraction caused a delay of action potential repolarization and other three caused a reduction in amplitude. An electrophysiological assay showed that Tm1 is active on HEK293 Nav 1.3, HEK293 Nav 1.6, DUM and DRG cells, and Tm2 on HEK293 Nav 1.3 and DRG cells, but not in HEK293 Nav 1.6. In addition, Tm1 and Tm2 did promote a shift to more negative potentials strongly suggesting that both are α-NaScTx. Conclusion: Although Tityus (Archaeotityus) mattogrossensis is considered an ancient group in Tityus genus, the primary structure of Tm1 and Tm2 is more related to Tityus subgenus. The patch clamp electrophysiological tests suggest that Tm1 and Tm2 are NaScTx, and also promoted no shift to more negative potentials, strongly suggesting that both are α-NaScTx. This paper aimed to explore and characterize for the first time toxins from the ancient scorpion Tityus (Archaeotityus) mattogrossensis.(AU)
Asunto(s)
Animales , Venenos de Escorpión/aislamiento & purificación , Venenos de Escorpión/clasificación , Cromatografía Liquida/métodos , Sacarosa/análisis , Fenómenos Electrofisiológicos/fisiologíaRESUMEN
Abstract Background: Almost all Tityus characterized toxins are from subgenera Atreus and Tityus, there are only a few data about toxins produced by Archaeotityus, an ancient group in Tityus genus. Methods: Tityus (Archaeotityus) mattogrossensis crude venom was fractionated by high performance liquid chromatography, the major fractions were tested in a frog sciatic nerve single sucrose-gap technique. Two fractions (Tm1 and Tm2) were isolated, partially sequenced by MALDI-TOF/MS and electrophysiological assayed on HEK293 Nav 1.3, HEK293 Nav 1.6, DUM and DRG cells. Results: The sucrose-gap technique showed neurotoxicity in four fractions. One fraction caused a delay of action potential repolarization and other three caused a reduction in amplitude. An electrophysiological assay showed that Tm1 is active on HEK293 Nav 1.3, HEK293 Nav 1.6, DUM and DRG cells, and Tm2 on HEK293 Nav 1.3 and DRG cells, but not in HEK293 Nav 1.6. In addition, Tm1 and Tm2 did promote a shift to more negative potentials strongly suggesting that both are -NaScTx. Conclusion: Although Tityus (Archaeotityus) mattogrossensis is considered an ancient group in Tityus genus, the primary structure of Tm1 and Tm2 is more related to Tityus subgenus. The patch clamp electrophysiological tests suggest that Tm1 and Tm2 are NaScTx, and also promoted no shift to more negative potentials, strongly suggesting that both are -NaScTx. This paper aimed to explore and characterize for the first time toxins from the ancient scorpion Tityus (Archaeotityus) mattogrossensis.
RESUMEN
Sea anemones produce a wide variety of biologically active compounds, such as the proteinaceous neurotoxins and cytolysins. Herein we report a new peptide, purified to homogeneity from the neurotoxic fraction of B. caissarum venom, by using gel filtration followed by rp-HPLC, naming it as BcIV. BcIV is a 41 amino acid peptide (molecular mass of 4669 amu) possessing 6 cysteines covalently linked by three disulfide bonds. This toxin has 45 and 48% of identity when compared to APETx1 and APETx2 from Anthopleura elegantissima, respectively, and 42% of identity with Am-II and BDS-I and-II obtained from Antheopsis maculata and Anemonia sulcata, respectively. This neurotoxin presents only a weak-paralyzing action (minimal Lethal Dose close to 2000 microg/kg) in swimming crabs Callinectes danae. This appears to be a different effect to that caused by the type 1 sea anemone toxin BcIII that is lethal to the same animals at lower doses (LD50=219 microg/kg). Circular dichroism spectra of BcIII and BcIV show a high content of beta-strand secondary structure in both peptides, very similar to type 1 sodium channel toxins from various sea anemones, and to APETx1 and APETx2 from A. elegantissima, a HERG channel modulator and an ASIC3 inhibitor, respectively. Interestingly, BcIII and BcIV have similar effects on the action potential of the crab leg nerves, suggesting the same target in this tissue. As BcIII was previously reported as a Na+ channel effector and BcIV is inactive over Na+ currents of mammalian GH3 cells, we propose a species-specific action for this new molecule. A molecular model of BcIV was constructed using the structure of the APETx1 as template and putative key residues are discussed.
Asunto(s)
Venenos de Cnidarios/química , Toxinas Marinas/química , Toxinas Marinas/toxicidad , Neurotoxinas/química , Neurotoxinas/toxicidad , Canales de Sodio/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Bioensayo , Braquiuros/efectos de los fármacos , Dicroismo Circular , Venenos de Cnidarios/toxicidad , Electrofisiología , Dosificación Letal Mediana , Toxinas Marinas/aislamiento & purificación , Modelos Moleculares , Datos de Secuencia Molecular , Neurotoxinas/aislamiento & purificación , Parálisis/inducido químicamente , Péptidos/química , Péptidos/aislamiento & purificación , Péptidos/toxicidad , Anémonas de Mar/metabolismoRESUMEN
A number of neurotoxins from venoms of invertebrates and plants are ligands for voltage-gated Na+ channels and are useful tools for studying Na+ channel function and structure. Using whole-cell recordings from vagal afferent nodose neurons, we studied neurotoxins that target Na+ channels. We asked whether Ts3 (an alpha-scorpion toxin) and/or veratridine (a lipid-soluble toxin), could modify the TTX-resistant Na+ current generated by vagal afferent nodose neurons. Nodose TTX-resistant current was not affected by Ts3, whereas Ts3 slowed inactivation of the current generated by TTX-sensitive current component. We found that veratridine inhibited the TTX-resistant Na+ currents on rat nodose neurons. Interestingly, veratridine-modified Na+ channels developed a persistent current that accounted for the large tail current observed. We propose that veratridine modifies TTX-resistant Na+ channels through a mechanism distinct from its actions on other voltage-gated Na+ channels.
Asunto(s)
Neuronas Aferentes/metabolismo , Neurotoxinas/toxicidad , Venenos de Escorpión/toxicidad , Canales de Sodio/efectos de los fármacos , Sodio/metabolismo , Veratridina/toxicidad , Animales , Transporte Biológico Activo , Transporte Iónico/efectos de los fármacos , Masculino , Neurotoxinas/metabolismo , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Venenos de Escorpión/metabolismo , Canales de Sodio/metabolismo , Tetrodotoxina , Nervio Vago/metabolismo , Veratridina/metabolismoRESUMEN
1. The aim of this work was to study the effects of N-salicyloyltryptamine (STP), a novel anticonvulsant agent, on voltage-gated ion channels in GH3 cells. 2. In this study, we show that STP at 17 microM inhibited up to 59.2+/-10.4% of the Ito and 73.1+/-8.56% of the IKD K+ currents in GH3 cells. Moreover, the inhibitory activity of the drug STP on K+ currents was dose-dependent (IC50=34.6+/-8.14 microM for Ito) and partially reversible after washing off. 3. Repeated stimulation at 1 Hz (STP at 17 microM) led to the total disappearance of Ito current, and an enhancement of IKD. 4. In the cell-attached configuration, application of STP to the bath increased the open probability of large-conductance Ca2+-activated K+ channels. 5. STP at 17 microM inhibited the L-type Ca2+ current by 54.9+/-7.50% without any significant changes in the voltage dependence. 6. STP at 170 microM inhibited the TTX-sensitive Na+ current by 22.1+/-2.41%. At a lower concentration (17 microM), no effect on INa was observed. 7. The pharmacological profile described here might contribute to the neuroprotective effect exerted by this compound in experimental 'in vivo' models.
Asunto(s)
Anticonvulsivantes/farmacología , Canales de Calcio Tipo L/efectos de los fármacos , Canales de Potasio/efectos de los fármacos , Salicilatos/farmacología , Canales de Sodio/efectos de los fármacos , Triptaminas/farmacología , Animales , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Técnicas de Placa-Clamp , Neoplasias Hipofisarias/patología , RatasRESUMEN
At the neuromuscular junction, several endogenous substances have been shown to act presynaptically to modify transmitter release. Here we show that angiotensin 1-7, a vasoactive peptide of the renin-angiotensin system, increased quantal content in a dose-dependent manner, with a maximal increase of 78% at 250 nM. At the same dose, angiotensin 1-7 increased paired pulse facilitation by 70%. This is the first report of angiotensin 1-7 altering a cholinergic synapse.