RESUMO
We have determined the solution structure of an alpha-toxin, CsE-V, isolated from the venom of the New World scorpion Centruroides sculpturatus Ewing (CsE). This toxin causes spontaneous rhythmic contractions in muscle. Unlike other New World toxins from CsE, this protein exhibits amino acid insertions and deletions at locations similar to Old World toxins and may thus represent a transition protein between the New World and Old World scorpion alpha-toxins. Sequence-specific assignments were made using 600 MHz 1H two-dimensional NMR data. NOESY, PH-COSY and amide-exchange data were used to deduce constraints for molecular modeling calculations. Distance geometry and dynamical simulated annealing calculations were performed to generate a family of 70 structures free of constraint violations. With respect to this family of structures, the energy-minimized average structure had root-mean-square deviations of 0.74 and 1.32 A for backbone and all atoms, respectively (excluding the C-terminal dipeptide, which is disordered). As with other scorpion toxins, the secondary structure of CsE-V consists of an alpha-helix, a three-strand anti-parallel beta-sheet, four beta-turns, and a hydrophobic patch that includes tyrosine residues in herringbone configuration. Unlike the CsE-v3 and -v1 proteins from C. sculpturatus, all of the proline residues were found to be in the trans configuration. The alpha-helix is slightly longer in CsE-V. The overall structure is more similar to the Old World alpha-toxin AaH-II from Androctonus australis Hector (r.m.s.d 1.59 A for backbone atoms of matching residues) than to the New World alpha-toxin CsE-v3 (r.m.s.d. 1.91 A). These structural data on CsE-V add further to our knowledge of the conformational repertoire exhibited by these sodium channel-binding neurotoxins.
Assuntos
Neurotoxinas/química , Venenos de Escorpião/química , Sequência de Aminoácidos , Animais , Simulação por Computador , Proteínas de Insetos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas de Répteis , Escorpiões , Homologia de Sequência de AminoácidosRESUMO
It is evident from the data reviewed that scorpion toxins can be distinguished on the basis of three properties: their effects on Na currents, their specific binding to excitable membranes, and the effects of depolarization and pH on binding and on effect. Additional work with other scorpion toxins is required to establish the degree of correlation between the three properties for each class of toxin. Further investigations with this family of homologous proteins will undoubtedly contribute not only to our understanding of the toxins themselves but also to our understanding of the structure and function of the Na channel.
Assuntos
Canais Iônicos/fisiologia , Venenos de Escorpião/farmacologia , Canais de Sódio , Sódio/metabolismo , Sequência de Aminoácidos , Animais , Canais Iônicos/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Modelos Moleculares , Conformação Proteica , Nós Neurofibrosos/fisiologia , Venenos de Escorpião/isolamento & purificaçãoRESUMO
From the venom of the American scorpion Centruroides sculpturatus Ewing we have isolated a minute peptide fraction (named CsEKerg1) which reversibly inhibits the current through ERG (ether-à-go-go-related gene) K(+) channels. Isolation was done by CM-cellulose column chromatography and reversed phase high-performance liquid chromatography. To test for an effect on ERG channels we used NG108-15 neuroblastomaxglioma hybrid cells voltage-clamped in the whole-cell mode. CsEKerg1 contains 43 amino acids and has a molecular weight of 4833. Its amino acid sequence is similar but not identical to that of ergtoxin, a peptide isolated recently from the venom of the Mexican scorpion Centruroides noxius [FASEB J. 13 (1999) 953]. Half inhibition of ERG current occurs at a peptide concentration of 1.12microg/ml.
Assuntos
Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/genética , Venenos de Escorpião/química , Venenos de Escorpião/farmacologia , Escorpiões/fisiologia , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Canais de Potássio Éter-A-Go-Go , Glioma , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Dados de Sequência Molecular , Neuroblastoma , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/isolamento & purificação , Ratos , Venenos de Escorpião/isolamento & purificação , Análise de Sequência de Proteína , Espectrometria de Massas por Ionização por Electrospray , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/fisiologiaRESUMO
Binding sites for cations were probed in the structures of protein neurotoxins from Centruroides sculpturatus by enhancement of terbium(III) fluorescence, detected by emission at 552 nm, when aromatic side-chains of the toxins were activated at 286 nm. Gadolinium, Gd(III), was used as a cation binding probe by observing its effects on nuclear magnetic resonance (NMR) spectra. Toxins CsE-v2 and v3, when bound to Tb(III), enhance luminescence of Tb(III) 20-fold whereas CsE-v1 enhances Tb(III) luminescence about 15-fold. Toxins CsE-I and V have no effect on the luminescence of Tb(III) implying that these latter two toxins have structures incompatible with efficient energy transfer from activated aromatic side-chains. Enhancement of fluorescence is pH dependent and is competitively inhibited by alkaline earth divalent cations and by other lanthanide(III) ions. Neodymium, Nd(III), with an ionic radius of 0.995 A is the most efficient of the lanthanide ions and the divalent cations in displacement of Tb(III) from the toxins. Relaxation enhancements of aromatic CH resonances by Gd(III) are apparent with tyrosines 4, 42, 38, 14-40 peak and tryptophan 47. Results from pH vs fluorescence studies suggest that carboxyl groups are involved in binding of Tb(III). Association constants (Ka) of the Tb(III)-CsE-v2 and v3 complexes are respectively 2.5 x 10(3) and 2.4 x 10(3) M-1 determined by fluorescence enhancement and 2.4 x 10(3) and 2.3 x 10(3) M-1 by equilibrium dialysis. Similarly Ka values for toxins CsE I and V are respectively 1.9 x 10(3) and 1.8 x 10(3) M-1 determined by equilibrium dialysis. Experimental evidence suggests that at least two Tb(III)s are bound per toxin molecule. The results from these studies are discussed in relation to the tertiary structure of toxin CsE-v3.
Assuntos
Metais Terras Raras , Neurotoxinas/metabolismo , Venenos de Escorpião/química , Sequência de Aminoácidos , Sítios de Ligação , Cátions , Diálise , Corantes Fluorescentes , Gadolínio , Concentração de Íons de Hidrogênio , Cinética , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Ligação Proteica , TérbioRESUMO
We report a preliminary high-resolution proton nuclear magnetic resonance characterization of the variant-3 toxin from the scorpion Centruroides sculpturatus Ewing (range Southwestern USA). This toxin assumes a well defined folded conformation in aqueous solutions at room temperature and undergoes reversible thermal denaturation. A number of amide hydrogens exhibit exchange life times varying from several minutes to several hours. A few tentative assignments of the low field aromatic CH resonances has been made on the basis of 2D-COSY and NOE experiments. The upfield shifts exhibited by Trp-47 suggest a unique microenvironment for this residue. The NMR data suggest that there is some degree of correlation between the solution structure of the variant-3 toxin and its crystallographic structure. Our studies provide a basis for a detailed elucidation of the structure-function relationships of these interesting scorpion toxins which bind to the sodium channels of excitable membranes and delay sodium current inactivation.
Assuntos
Venenos de Escorpião , Sequência de Aminoácidos , Animais , Cristalografia , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Neurotoxinas , Conformação Proteica , Desnaturação ProteicaAssuntos
Anafilaxia , Enterotoxinas , Staphylococcus , Animais , Antígenos , Feminino , Cobaias , Histamina/análise , Liberação de Histamina , Técnicas In Vitro , Pulmão/imunologia , Coelhos , Útero/imunologiaAssuntos
Antitoxinas/farmacologia , Proteínas/toxicidade , Escorpiões , Peçonhas/toxicidade , Aminoácidos/análise , Animais , Compostos Azo/farmacologia , Derivados de Benzeno/farmacologia , Cloromercurobenzoatos/farmacologia , Cisteamina/farmacologia , Cisteína/farmacologia , Ditiotreitol/farmacologia , Iodoacetatos/farmacologia , Iodobenzoatos/farmacologia , Éteres Metílicos/farmacologia , Camundongos , Reagentes de Sulfidrila/farmacologia , Ácidos Sulfônicos/farmacologia , Tioglicolatos/farmacologia , Toxinas Biológicas/análise , Ureia/farmacologiaAssuntos
Escorpiões , Serpentes , Toxinas Biológicas/toxicidade , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Galinhas , Dissulfetos/farmacologia , Ditiotreitol/uso terapêutico , Glutationa/uso terapêutico , Humanos , Membranas/efeitos dos fármacos , Camundongos , Junção Neuromuscular/efeitos dos fármacos , Peptídeos , Intoxicação/tratamento farmacológico , Intoxicação/mortalidade , Picadas de Escorpião/tratamento farmacológico , Mordeduras de Serpentes/tratamento farmacológico , Especificidade da Espécie , Transmissão Sináptica/efeitos dos fármacos , Toxinas Biológicas/análise , Peçonhas/análise , Peçonhas/toxicidadeRESUMO
The cellular electrical activity accompanying the very slow resting-tension oscillation (period 4-13 min) in chick skeletal muscle caused by scorpion neurotoxin, reduced [Ca2+]0 and Cd2+ was investigated. All three oscillogenic treatments were found to induce spontaneous, periodically recurring, electrical activation of muscle cells, including periodic bursting activity of individual cells. The period of the recurrent electrical activation approximated the period of the resting-tension oscillation. The data suggest that the three oscillogenic treatments act by way of a common mechanism, possibly a critical Ca2+ moiety, to effect the periodic electrical activation of the cells. Analysis of single cell and group discharge activity suggests that the cells are coupled by non-specific, electrotonic interactions, resulting in the integration of their individual periodic activities into a resting-tension oscillation of the whole muscle.
Assuntos
Cádmio/farmacologia , Cálcio/fisiologia , Músculos/fisiologia , Neurotoxinas/farmacologia , Venenos de Escorpião/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Galinhas , Eletrofisiologia , Técnicas In Vitro , Masculino , Contração Muscular/efeitos dos fármacos , Músculos/efeitos dos fármacosRESUMO
1. The specific binding to rat brain synaptosomes of a radiolabelled derivative of toxin II from the scorpion Centruroides suffusus suffusus could be prevented by toxins III and IV, but not by toxin V or variants 1-3, from the venom of Centruroides sculpturatus. 2. The specific binding of a similar derivative of toxin II from Androctonus australis Hector was not affected by any of the toxins from Centruroides sculpturatus. 3. There is biochemical evidence for only two distinct classes of Na channel receptors specific for known scorpion toxins.
Assuntos
Canais Iônicos/metabolismo , Receptores de Droga/metabolismo , Venenos de Escorpião/metabolismo , Sódio/metabolismo , Animais , Ligação Competitiva , Encéfalo/metabolismo , Classificação , Ratos , Sinaptossomos/metabolismoRESUMO
1. The effect of various toxin fractions isolated by Watt et al. (1978) from the venom of the scorpion Centruroides sculpturatus Ewing on the Na currents of the node of Ranvier has been studied with the voltage clamp method. 2. The toxin fractions were applied externally. The most potent fractions were toxins III, IV and V which were effective in concentrations of 0.33-3.33 microgram/ml. The effect of toxins III and IV was quite different from that of toxin V. 3. In toxin III or IV - treated nodes a strong depolarizing pulse was followed by a transient shift of the negative resistance branch of the INa (E) curve to more negative potentials. The amount of shift varied between -10 and -60 mV. A 500 ms depolarizing pulse of small amplitude produced a slowly developing Na inward current which slowly decayed after the end of the pulse. Inactivation was incomplete, even with 500 ms pulses to 0 mV. 4. The transient shift of the INa (E) curve was not seen in nodes treated with toxin V. This toxin merely caused slow and incomplete Na inactivation. The effect of toxin IV was not suppressed by a four times higher concentration of toxin V, suggesting that the two toxins act on different receptors. 5. Toxin I acted like toxin IV but was about 10 times less potent. The effect of high concentrations of variants 1, 2, 3, 5, 6 resembled tha of toxin V. 6. All effects observed with toxin III or IV were also seen with the whole venom (cf. Cahalan 1975).
Assuntos
Canais Iônicos/efeitos dos fármacos , Nós Neurofibrosos/efeitos dos fármacos , Venenos de Escorpião/farmacologia , Sódio/metabolismo , Toxinas Biológicas/farmacologia , Animais , Permeabilidade da Membrana Celular , Rana esculenta , Venenos de Escorpião/análise , Toxinas Biológicas/isolamento & purificaçãoRESUMO
(1) Gating currents were recorded from frog nodes of Ranvier treated either with toxins III or IV from the venom of the scorpion Centruroides sculpturatus or with the alkaloid toxin aconitine. (2) Toxins III or IV from Centruroides sculpturatus (which drastically reduce the sodium permeability PNa and slightly shift its voltage dependence in the depolarizing direction) caused a small depolarizing shift of the relation between charge (Qon) and membrane potential (E) without affecting the maximum charge Qon max. (3) On nodes treated with toxins III or IV from Centruroides sculpturatus, a depolarizing conditioning pulse (which transiently shifts the descending branch of the INa(E) curve by up to 60 mV in the hyperpolarizing direction) shifted the midpoint potential (Emid) of the Qon(E) curve by -17 mV and slightly increased the slope of the curve; it also decreased Qon max markedly but had little effect on Qon measured with small depolarizing pulses. By contrast, massive treatment with aconitine (which irreversibly shifts sodium activation in the hyperpolarizing direction) irreversibly shifted the midpoint potential of the Qon(E) curve from -28.5 to -69 mV and significantly increased Qon and Qoff measured with small depolarizing pulses; concomitantly, the voltage dependence of the on time constant of the charge movement [tau on(E)] was shifted by -44 mV. (4) The sodium current INa was exponential both in nodes treated with toxins III or IV of Centruroides sculpturatus and subjected to a depolarizing conditioning pulse and in aconitine-treated nodes; in the latter, INa started after a delay of 30-40 microseconds. The time constant of the sodium current. tau on Na, was larger than the time constant of the charge movement, tau on Q; the ratio tau on Q/tau on Na was 0.61 and 0.73 in the experiments with Centruroides sculpturatus toxins and aconitine, respectively. (5) The off time constant of the sodium current (tau off Na) was slightly increased in nodes treated with Centruroides sculpturatus toxins and subjected to a depolarizing conditioning pulse, whereas it was markedly increased in aconitine-treated nodes. With the former treatment, the off time constant of the charge movement (tau off Q) was unaffected but with aconitine treatment it was considerably increased although it remained smaller than tau off Na. Consequently, the ratio tau off Q/tau off Na (which is greater than or equal to 1 in untreated nodes) became smaller than one, reaching values as low as 0.58 and 0.44 in the experiments with Centruroides sculpturatus toxins and aconitine, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Aconitina/farmacologia , Aconitum/análogos & derivados , Canais Iônicos/fisiologia , Nós Neurofibrosos/fisiologia , Venenos de Escorpião/farmacologia , Animais , Batraquiotoxinas/farmacologia , Condutividade Elétrica , Potenciais da Membrana/efeitos dos fármacos , Rana esculenta , Nós Neurofibrosos/efeitos dos fármacos , Sódio/metabolismoRESUMO
In voltage clamped nodes of Ranvier inactivation of the sodium permeability is slowed by toxin V from the scorpion Centruroides sculpturatus, by sea anemone toxin ATX II or by internally applied KIO3. The slow decay of the Na inward current is markedly accelerated if the test pulse is preceded by a depolarizing conditioning pulse followed by a 10-500 ms pause. This phenomenon was studied in detail, using conditioning pulses of varying amplitude and up to 15 s duration. In nodes treated with toxin V a 20 ms conditioning pulse to positive potentials was sufficient to produce a clear acceleration of the decay of the Na current and a reduction of the inward current remaining at the end of a 50 ms test pulse, i.e. a weakening of the toxin effect. In nodes treated with ATX II or internal KIO3 longer conditioning pulses were required. A similar effect of conditioning pulses on the decaying phase of the Na current was also observed in untreated fibres. To study the phenomenon quantitatively we fitted the decaying phase of the inward Na current with the equation INa = A exp(-t/tau 1) + B exp(-t/tau 2) + C. The effect of depolarizing conditioning pulses could be described as an increase of A, a decrease of B and C and a reduction of the time constants tau 1 and tau 2. I50/Ipeak, the normalised inward current remaining at the end of a 50 ms test pulse, decreased exponentially with increasing duration of the conditioning pulse to a steady-state value. The time constant tau and the steady-state value depended on the potential during the conditioning pulse. For nodes treated with toxin V, tau was 0.24 s at 0 mV and 12 degrees C and half inhibition occurred at -42 mV. The time constant tau was larger for nodes treated with ATX II or internal KIO3. At positive potentials, I50 was reduced to 20% of the control value in toxin V-treated nodes, but only to 70% in KIO3-treated nodes. Recovery from the effect of the conditioning pulse was studied by varying the pause between conditioning pulse and test pulse; recovery was 66-100% complete after 500 ms. The results are interpreted by assuming that a depolarizing conditioning pulse (a) accelerates inactivation of the sodium permeability and (b) causes dissociation of the toxin-receptor complex or transition into an inactive state. The latter effect occurs in toxin V-treated fibres but not in those treated with ATX II or KIO3.
Assuntos
Canais Iônicos/efeitos dos fármacos , Compostos de Potássio , Nós Neurofibrosos/metabolismo , Venenos de Escorpião/farmacologia , Sódio/metabolismo , Animais , Cardiotônicos/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Venenos de Cnidários/farmacologia , Iodatos/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Neurotoxinas/farmacologia , Potássio/farmacologia , Rana esculenta , Nós Neurofibrosos/efeitos dos fármacos , Anêmonas-do-Mar , Tetrodotoxina/farmacologiaRESUMO
New toxins, VI and VII, were purified from the venom of the North American scorpion, Centruroides sculpturatus, and studied in the voltage-clamped frog node of Ranvier. These toxins reduced peak inward Na currents and caused a transient, depolarization-induced shift in the voltage dependence of Na activation. Their effects were indistinguishable from those of toxins I, III and IV, as previously described by Meves et al. (1982). Toxin VII, 0.2 microgram/ml, shifted the voltage dependence of steady-state inactivation (h infinity). In four fibers, the mean shift of the h infinity (E) curve was -17 mV, compared to a mean shift of -28 mV in the descending branch of the INa(E) curve. The h infinity (E) curve with toxin VII was monotonic and inactivation was incomplete at positive potentials. Decreasing the pH from 7.4 to 5.7 increased the shift in the voltage dependence of activation with toxin. The increase with toxin VI at pH 5.7 was reversible on returning to pH 7.4, but the increase with toxin VII was not. The lack of reversibility of the effect of pH with toxin VII was quantified in two ways: the mean value of INa (measured at -62 mV with a conditioning pulse) at pH 7.4 was 5.1 times larger after treatment than before treatment with 0.1 microgram/ml of toxin VII at pH 5.7; the average concentration of toxin VII required for a shift of -30 mV at pH 7.4 was decreased by a factor of 4.3, to 0.19 microgram/ml (26 nM), by pretreatment at pH 5.7.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Canais Iônicos/efeitos dos fármacos , Venenos de Escorpião/farmacologia , Sódio/metabolismo , Animais , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Fibras Nervosas Mielinizadas/efeitos dos fármacos , Rana esculenta , Nós Neurofibrosos/efeitos dos fármacosRESUMO
Recent progress in biochemical, structural and physiological studies has revealed several interesting properties of the toxins from the American scorpion, Centruroides sculpturatus. These toxins, together with similar toxins from other species of scorpions, comprise a unique family of homologous proteins with phylogenetically related structural differences. There is now evidence from both binding and electrophysiological studies that two distinct classes of toxins are present in the venom of C. sculpturatus. One class of toxins markedly slows inactivation of the sodium permeability but has no demonstrable effect on activation, whereas the second class induces a transient shift in the voltage-dependence of activation. Both groups make inactivation incomplete.
Assuntos
Neurotoxinas/isolamento & purificação , Venenos de Escorpião/isolamento & purificação , Sequência de Aminoácidos , Animais , Fenômenos Químicos , Química , Cromatografia , Eletrofisiologia , Canais Iônicos/efeitos dos fármacos , Conformação Proteica , Nós Neurofibrosos/efeitos dos fármacos , Nós Neurofibrosos/fisiopatologia , Sódio/metabolismoRESUMO
1. An oscillation in resting tension of the chick biventer cervicis muscle is induced by scorpion toxin (CsT-V), reduced [Ca]o and by Cd2+. Other chick muscles, e.g. semispinalis cervicis, anterior and posterior latissimus dorsi, also oscillate with the same treatments. The period of the oscillation in the biventer cervicis muscle is uniform, averaging 5-6 min. 2. The oscillation and the period are very sensitive to changes in temperature, [Ca]o, [K]o, ouabain and tetrodotoxin [TTX]. The oscillation is resistant to twitch blocking dosages of nicotinic antagonists. 3. The period of the oscillation varies inversely as the temperature (26-37 degrees C), log [K]o and [ouabain] and directly as the log [Ca]o and [TTX]. 4. These data support the inference that the oscillator is postjunctional and results from fluctuation in the electrochemical gradient of ions across the muscle cell membrane. The role, if any, of the Na+, K+ pump in the primary oscillogenic mechanism is not evident from our data.
Assuntos
Cádmio/farmacologia , Cálcio/farmacologia , Contração Muscular/efeitos dos fármacos , Músculos/fisiologia , Venenos de Escorpião/farmacologia , Animais , Bungarotoxinas/farmacologia , Galinhas , Masculino , Músculos/efeitos dos fármacos , Oscilometria , Tubocurarina/farmacologiaRESUMO
The solution structure of the CsE-v3 neurotoxin from the venom of the North American scorpion Centruroides sculpturatus Ewing (CsE) has been determined by a hybrid refinement procedure that employed distance geometry and dynamical simulated annealing. Distance constraints deduced from the nuclear Overhauser effect spectroscopy data and torsion angle constraints deduced from the vicinal coupling constant data were used in the refinement procedure. A family of simulated annealing structures that showed no constraint violations was generated. The energy-minimized average structure exhibited root-mean-square deviations of 0.121 nm for the backbone and 0.182 nm for all atoms, with respect to this family. These studies confirm the previously qualitative NMR findings about the secondary structural features, viz. the presence of a short alpha-helix composed of residues 23-31 and an antiparallel beta-sheet composed of the strands of residues 1-5, 45-50 and 36-42. A cluster of aromatic ring systems is located on one side of the protein. The solution and crystal structures have similar overall features, but show some minor differences.