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1.
Biochem Biophys Res Commun ; 506(4): 826-832, 2018 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-30389137

RESUMO

Voltage-gated sodium channels play important roles in human physiology. However, their complexity hinders the understanding of their physiology and pathology at atomic level. We took advantage of the structural reports of similar channels obtained by cryo-EM (EeNav1.4, and NavPaS), and constructed models of human Nav1.4 channels at closed and open states. The open-state model is very similar to the recently published cryo-EM structure of hNav1.4. The comparison of both models shows shifts of the voltage sensors (VS) of DIII and DIV. The activated position of VS-DII in the closed model was demonstrated by Ts1 docking, thereby confirming the requirement that VS-DI, VS-DII and VS-DIII must be activated for the channel to open. The interactions observed with VS-DIII suggest a stepwise, yet fast, transition from resting to activated state. These models provide structural insights on the closed-open transition of the channel.


Assuntos
Ativação do Canal Iônico , Modelos Biológicos , Músculo Esquelético/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.4/química , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Humanos , Simulação de Acoplamento Molecular
2.
Life Sci ; 192: 144-150, 2018 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-29183797

RESUMO

AIMS: Ca2+ and cAMP are important intracellular modulators. In order to generate intracellular signals with various amplitudes, as well as different temporal and spatial properties, a tightly and precise control of these modulators in intracellular compartments is necessary. The aim of this study was to evaluate the effects of elevated and sustained cAMP levels on voltage-dependent Ca2+ currents and proliferation in pituitary tumor GH3 cells. MAIN METHODS: Effect of long-term exposure to forskolin and dibutyryl-cyclic AMP (dbcAMP) on Ca2+ current density and cell proliferation rate were determined by using the whole-cell patch-clamp technique and real time cell monitoring system. The cAMP levels were assayed, after exposing transfected GH3 cells with the EPAC-1 cAMP sensor to forskolin and dbcAMP, by FRET analysis. KEY FINDINGS: Sustained forskolin treatment (24 and 48h) induced a significant increase in total Ca2+ current density in GH3 cells. Accordingly, dibutyryl-cAMP incubation (dbcAMP) also elicited increase in Ca2+ current density. However, the maximum effect of dbcAMP occurred only after 72h incubation, whereas forskolin showed maximal effect at 48h. FRET-experiments confirmed that the time-course to elevate intracellular cAMP was distinct between forskolin and dbcAMP. Mibefradil inhibited the fast inactivating current component selectively, indicating the recruitment of T-type Ca2+ channels. A significant increase on cell proliferation rate, which could be related to the elevated and sustained intracellular levels of cAMP was observed. SIGNIFICANCE: We conclude that maintaining high levels of intracellular cAMP will cause an increase in Ca2+ current density and this phenomenon impacts proliferation rate in GH3 cells.


Assuntos
Canais de Cálcio/metabolismo , AMP Cíclico/metabolismo , Animais , Bucladesina/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo T/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colforsina/farmacologia , Mibefradil/farmacologia , Técnicas de Patch-Clamp , Neoplasias Hipofisárias/metabolismo , Ratos , Vasodilatadores/farmacologia
3.
Toxicon ; 119: 152-8, 2016 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-27263806

RESUMO

The genes of the five disulfide-bonded peptide toxins 1 and 2 (named Oxytoxins or Oxotoxins) from the spider Oxyopes lineatus were cloned into the expression vector pQE30 containing a 6His-tag and a Factor Xa proteolytic cleavage region. These two recombinant vectors were transfected into Escherichia coli BL21 cells and expressed under induction with isopropyl thiogalactoside (IPTG). The product of each gene was named HisrOxyTx1 or HisrOxyTx2, and the protein expression was ca 14 and 6 mg/L of culture medium, respectively. Either recombinant toxin HisrOxyTx1 or HisrOxyTx2 were found exclusively in inclusion bodies, which were solubilized using a chaotropic agent, and then, purified using affinity chromatography and reverse-phase HPLC (RP-HPLC). The HisrOxyTx1 and HisrOxyTx2 products, obtained from the affinity chromatographic step, showed several peptide fractions having the same molecular mass of 9913.1 and 8030.1 Da, respectively, indicating that both HisrOxyTx1 and HisrOxyTx2 were oxidized forming several distinct disulfide bridge arrangements. The isoforms of both HisrOxyTx1 and HisrOxyTx2 after DTT reduction eluted from the column as a single protein component of 9923 and 8040 Da, respectively. In vitro folding of either HisrOxyTx1 or HisrOxyTx2 yielded single oxidized components, which were cleaved independently by the proteolytic enzyme Factor Xa to give the recombinant peptides rOxyTx1 and rOxyTx2. The experimental molecular masses of rOxyTx1 and rOxyTx2 were 8059.0 and 6176.4 Da, respectively, which agree with their expected theoretical masses. The recombinant peptides rOxyTx1 and rOxyTx2 showed lower but comparable toxicity to the native toxins when injected into lepidopteran larvae; furthermore, rOxyTx1 was able to inhibit calcium ion currents on dorsal unpaired median (DUM) neurons from Periplaneta americana.


Assuntos
Dissulfetos/química , Inseticidas/química , Peptídeos/química , Aranhas/química , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Masculino , Camundongos
4.
Toxicon ; 104: 73-82, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26220799

RESUMO

A potent insecticidal toxin, ß/δ-PrIT1, molecular mass of 5598.86 [M+H](+), was characterized from Phoneutria reidyi spider venom. Its partial amino acid sequence showed high similarity with insecticidal spider toxins from the genus Phoneutria. ß/δ-PrIT1 was very toxic (LD50 = 4 nmol/g) to flies (Musca domestica), but not to mice (Mus musculus). Kinetic studies showed that (125)I-ß/δ-PrIT1 binds to two distinct sites in insect sodium channels, with close affinity (Kd1 = 34.7 pM and Kd2 = 35.1 pM). Its association is rather fast (t1/2(1) = 1.4 min, t1/2(2) = 8.5 min) and its dissociation is a slower process (t1/2(1) = 5.4 min, t1/2(2) = 32.8 min). On rat brain synaptosomes ß/δ-PrIT1 partially competed (∼30%) with the beta-toxin (125)I-CssIV, but did not compete with the alpha-toxin of reference (125)I-AaII, nor with the beta-toxin (125)I-TsVII. On cockroach nerve cord synaptosomes, ß/δ-PrIT1 did not compete with the anti-insect toxin (125)I-LqqIT1, but it competed (IC50 = 80 pM) with the "alpha-like" toxin (125)I-BomIV. In cockroach neurons, ß/δ-PrIT1 inhibited the inactivation of Nav-channels and it shifted the sodium channel activation to hyperpolarizing potentials. These results indicate two different binding sites for ß/δ-PrIT1, leading to two different pharmacological responses. ß/δ-PrIT1 is one of the most toxic spider toxins to insects without apparent toxicity to mammals, and provide new model for the development of insecticides.


Assuntos
Inseticidas/farmacologia , Venenos de Aranha/farmacologia , Aranhas/química , Sinaptossomos/metabolismo , Animais , Sítios de Ligação , Brasil , Baratas/citologia , Baratas/efeitos dos fármacos , Dípteros/efeitos dos fármacos , Feminino , Inseticidas/química , Masculino , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Ratos Wistar , Canais de Sódio/metabolismo , Venenos de Aranha/química
5.
Biochimie ; 94(12): 2756-63, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22968173

RESUMO

Phoneutria nigriventer toxin Tx1 (PnTx1, also referred to in the literature as Tx1) exerts inhibitory effect on neuronal (Na(V)1.2) sodium channels in a way dependent on the holding potential, and competes with µ-conotoxins but not with tetrodotoxin for their binding sites. In the present study we investigated the electrophysiological properties of the recombinant toxin (rPnTx1), which has the complete amino acid sequence of the natural toxin with 3 additional residues: AM on the N-terminal and G on the C-terminal. At the concentration of 1.5 µM, the recombinant toxin inhibits Na(+) currents of dorsal root ganglia neurons (38.4 ± 6.1% inhibition at -80 mV holding potential) and tetrodotoxin-resistant Na(+) currents (26.2 ± 4.9% at the same holding potential). At -50 mV holding potential the inhibition of the total current reached 71.3 ± 2.3% with 1.5 µM rPnTx1. The selectivity of rPnTx1 was investigated on ten different isoforms of voltage-gated sodium channels expressed in Xenopus oocytes. The order of potency for rPnTx1 was: rNa(V)1.2 > rNa(V)1.7 ≈ rNa(V)1.4 ≥ rNa(V)1.3 > mNa(V)1.6 ≥ hNa(V)1.8. No effect was seen on hNa(V)1.5 and on the arthropods isoforms (DmNa(V)1, BGNa(V)1.1a and VdNa(V)1). The IC(50) for Na(V)1.2 was 33.7 ± 2.9 nM with a maximum inhibition of 83.3 ± 1.9%. The toxin did not alter the voltage-dependence of channel gating and was effective on Na(V)1.2 channels devoid of inactivation. It was ineffective on neuronal calcium channels. We conclude that rPnTx1 has a promising selectivity, and that it may be a valuable model to achieve pharmacological activities of interest for the treatment of channelopathies and neuropathic pain.


Assuntos
Neuropeptídeos/farmacologia , Proteínas Recombinantes/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio Disparados por Voltagem/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Ligação Competitiva , Células Cultivadas , Conotoxinas/metabolismo , Conotoxinas/farmacologia , Relação Dose-Resposta a Droga , Feminino , Gânglios Espinais/citologia , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Masculino , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/fisiologia , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Oócitos/fisiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacologia , Ratos , Ratos Wistar , Proteínas Recombinantes/metabolismo , Venenos de Aranha/metabolismo , Venenos de Aranha/farmacologia , Tetrodotoxina/metabolismo , Tetrodotoxina/farmacologia , Xenopus laevis
6.
Biochemistry ; 48(14): 3078-88, 2009 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-19231838

RESUMO

In this work, Phoneutria nigriventer toxins PnTx2-5 and PnTx2-6 were shown to markedly delay the fast inactivation kinetics of neuronal-type sodium channels. Furthermore, our data show that they have significant differences in their interaction with the channel. PnTx2-6 has an affinity 6 times higher than that of PnTx2-5, and its effects are not reversible within 10-15 min of washing. PnTx2-6 partially (59%) competes with the scorpion alpha-toxin AaHII, but not with the scorpion beta-toxin CssIV, thus suggesting a mode of action similar to that of site 3 toxins. However, PnTx2-6 is not removed by strong depolarizing pulses, as in the known site 3 toxins. We have also established the correct PnTx2-5 amino acid sequence and confirmed the sequence of PnTx2-6, in both cases establishing that the cysteines are in their oxidized form. A structural model of each toxin is proposed. They show structures with poor alpha-helix content. The model is supported by experimental and theoretical tests. A likely binding region on PnTx2-5 and PnTx2-6 is proposed on the basis of their different affinities and sequence differences.


Assuntos
Peptídeos/farmacologia , Canais de Sódio/efeitos dos fármacos , Venenos de Aranha/farmacologia , Cinética , Modelos Moleculares , Neuropeptídeos/química , Neuropeptídeos/farmacologia , Peptídeos/química , Ligação Proteica , Conformação Proteica , Venenos de Escorpião , Canais de Sódio/metabolismo , Venenos de Aranha/química , Relação Estrutura-Atividade
7.
J Gen Physiol ; 132(2): 251-63, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18663133

RESUMO

Alpha-scorpion toxins bind in a voltage-dependent way to site 3 of the sodium channels, which is partially formed by the loop connecting S3 and S4 segments of domain IV, slowing down fast inactivation. We have used Ts3, an alpha-scorpion toxin from the Brazilian scorpion Tityus serrulatus, to analyze the effects of this family of toxins on the muscle sodium channels expressed in Xenopus oocytes. In the presence of Ts3 the total gating charge was reduced by 30% compared with control conditions. Ts3 accelerated the gating current kinetics, decreasing the contribution of the slow component to the ON gating current decay, indicating that S4-DIV was specifically inhibited by the toxin. In addition, Ts3 accelerated and decreased the fraction of charge in the slow component of the OFF gating current decay, which reflects an acceleration in the recovery from the fast inactivation. Site-specific fluorescence measurements indicate that Ts3 binding to the voltage-gated sodium channel eliminates one of the components of the fluorescent signal from S4-DIV. We also measured the fluorescent signals produced by the movement of the first three voltage sensors to test whether the bound Ts3 affects the movement of the other voltage sensors. While the fluorescence-voltage (F-V) relationship of domain II was only slightly affected and the F-V of domain III remained unaffected in the presence of Ts3, the toxin significantly shifted the F-V of domain I to more positive potentials, which agrees with previous studies showing a strong coupling between domains I and IV. These results are consistent with the proposed model, in which Ts3 specifically impairs the fraction of the movement of the S4-DIV that allows fast inactivation to occur at normal rates.


Assuntos
Venenos de Escorpião/farmacologia , Animais , Regulação da Expressão Gênica , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Oócitos , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/genética , Canais de Sódio/metabolismo , Xenopus
8.
J Gen Physiol ; 130(3): 257-68, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17698594

RESUMO

Several naturally occurring polypeptide neurotoxins target specific sites on the voltage-gated sodium channels. Of these, the gating modifier toxins alter the behavior of the sodium channels by stabilizing transient intermediate states in the channel gating pathway. Here we have used an integrated approach that combines electrophysiological and spectroscopic measurements to determine the structural rearrangements modified by the beta-scorpion toxin Ts1. Our data indicate that toxin binding to the channel is restricted to a single binding site on domain II voltage sensor. Analysis of Cole-Moore shifts suggests that the number of closed states in the activation sequence prior to channel opening is reduced in the presence of toxin. Measurements of charge-voltage relationships show that a fraction of the gating charge is immobilized in Ts1-modified channels. Interestingly, the charge-voltage relationship also shows an additional component at hyperpolarized potentials. Site-specific fluorescence measurements indicate that in presence of the toxin the voltage sensor of domain II remains trapped in the activated state. Furthermore, the binding of the toxin potentiates the activation of the other three voltage sensors of the sodium channel to more hyperpolarized potentials. These findings reveal how the binding of beta-scorpion toxin modifies channel function and provides insight into early gating transitions of sodium channels.


Assuntos
Proteínas de Insetos/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Proteínas Musculares/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Neurotoxinas/farmacologia , Venenos de Escorpião/farmacologia , Canais de Sódio/efeitos dos fármacos , Regulação Alostérica , Animais , Sítios de Ligação , Feminino , Cinética , Potenciais da Membrana/efeitos dos fármacos , Modelos Biológicos , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Oócitos , Técnicas de Patch-Clamp , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Ratos , Canais de Sódio/química , Canais de Sódio/metabolismo , Espectrometria de Fluorescência , Xenopus
9.
Neuropharmacology ; 52(8): 1650-62, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17517422

RESUMO

The inhibitor cystine-knot motif identified in the structure of CSTX-1 from Cupiennius salei venom suggests that this toxin may act as a blocker of ion channels. Whole-cell patch-clamp experiments performed on cockroach neurons revealed that CSTX-1 produced a slow voltage-independent block of both mid/low- (M-LVA) and high-voltage-activated (HVA) insect Ca(v) channels. Since C. salei venom affects both insect as well as rodent species, we investigated whether Ca(v) channel currents of rat neurons are also inhibited by CSTX-1. CSTX-1 blocked rat neuronal L-type, but no other types of HVA Ca(v) channels, and failed to modulate LVA Ca(v) channel currents. Using neuroendocrine GH3 and GH4 cells, CSTX-1 produced a rapid voltage-independent block of L-type Ca(v) channel currents. The concentration-response curve was biphasic in GH4 neurons and the subnanomolar IC(50) values were at least 1000-fold lower than in GH3 cells. L-type Ca(v) channel currents of skeletal muscle myoballs and other voltage-gated ion currents of rat neurons, such as I(Na(v)) or I(K(v)) were not affected by CSTX-1. The high potency and selectivity of CSTX-1 for a subset of L-type channels in mammalian neurons may enable the toxin to be used as a molecular tool for the investigation of this family of Ca(v) channels.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/fisiologia , Neurônios/efeitos dos fármacos , Venenos de Aranha/química , Venenos de Aranha/farmacologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Baratas/citologia , Relação Dose-Resposta a Droga , Estimulação Elétrica/métodos , Gânglios Sensitivos/citologia , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Nitrendipino/farmacologia , Técnicas de Patch-Clamp , Ratos
10.
Cell Biochem Biophys ; 44(3): 424-30, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16679529

RESUMO

Recently, we proposed a quantitative model to explain the molecular mechanism of action of the Tityus serrulatus Ts3 alpha-toxin on sodium channels. In this model, the toxin acts as a stop that prevents the segment S4 of domain IV from reaching its outermost position, thus impairing the normal fast inactivation without affecting activation. In the present work, we analyze the predictions of the proposed model with regard to the voltage-dependent transitions to and from inactivation. Our results show that the recovery from inactivation was significantly faster in Ts3-bound channels and that there was no significant voltage dependence. The transition to inactivated state from open state in Ts3-modified channels presented a small but significant voltage dependence, which may derive from an intrinsic voltage dependence of inactivation or by a short movement of IVS4 in the presence of bound Ts3. We also studied the thermodynamic parameters of the voltage-dependent displacement of Ts3 from its binding site. We have observed that the activation energy to remove the toxin is 27 kJ/mol, part of which derives from the imposed depolarizing potential and the movement of an equivalent electrical charge of 0.54 e(0). These results support the proposed model.


Assuntos
Ativação do Canal Iônico/fisiologia , Neurotoxinas/farmacologia , Venenos de Escorpião/farmacologia , Canais de Sódio/fisiologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Estimulação Elétrica/métodos , Eletrofisiologia , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Mutagênese Sítio-Dirigida , Neurotoxinas/metabolismo , Venenos de Escorpião/isolamento & purificação , Venenos de Escorpião/metabolismo , Bloqueadores dos Canais de Sódio/antagonistas & inibidores , Canais de Sódio/efeitos dos fármacos , Termodinâmica , Canais de Ânion Dependentes de Voltagem
11.
Br J Pharmacol ; 142(7): 1115-22, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15249424

RESUMO

The voltage-dependent displacement of the scorpion Tityus serrulatus alpha-toxin Ts3 was investigated in native sodium channels of GH3 cells by examining the removal of its effects in toxin-free solution. Toxin at saturating concentration was pulsed (approximately 1 s) directly onto the cell, thus causing an eight-fold increase of the slow component (taus = 6 ms) of fast inactivation, and a three-fold increase of the time constant of its fast component. At 0 mV, maximal conductance was achieved in cells before and after treatment with Ts3, and no displacement of the toxin could be detected. Toxin displacement occurred if stronger depolarising pulses (> 100 mV) were applied. The rate of displacement depended on the amplitude and duration of the pulses, and was not related with outward Na+ flux. We propose a model in which activation does not require complete movement of segment S4 of domain IV (IVS4) and that a more extensive movement of this segment is needed for normal fast inactivation. A kinetic model is presented that can account for the typical effects of site 3 toxins.


Assuntos
Neurotoxinas/farmacologia , Venenos de Escorpião/farmacologia , Canais de Sódio/fisiologia , Animais , Ativação do Canal Iônico , Cinética , Neurotoxinas/metabolismo , Técnicas de Patch-Clamp , Ratos , Venenos de Escorpião/metabolismo , Fatores de Tempo , Células Tumorais Cultivadas
12.
FEBS Lett ; 523(1-3): 219-23, 2002 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-12123835

RESUMO

A cDNA with 403 nucleotides encoding the precursor of the toxin PnTx2-6 was cloned and sequenced. Subsequent analysis revealed that the precursor begins with a signal peptide and a glutamate-rich propeptide. The succeeding peptide confirmed the reported sequence of PnTx2-6. The purified toxin exerted complex effects on Na(+) current of frog skeletal muscle. There was a marked decrease of the inactivation kinetics, and a shift to hyperpolarizing potentials of both the Na(+) conductance and the steady-state inactivation voltage dependences, along with a reduction of the current amplitude. The concentration dependence of the modified current suggests a K(D) of 0.8 microM for the toxin-channel complex.


Assuntos
Músculo Esquelético/efeitos dos fármacos , Neuropeptídeos/farmacologia , Canais de Sódio/fisiologia , Aranhas/química , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/análise , Condutividade Elétrica , Eletrofisiologia , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/fisiologia , Cinética , Dados de Sequência Molecular , Músculo Esquelético/fisiologia , Neuropeptídeos/genética , Peptídeos/genética , Peptídeos/farmacologia , Rana catesbeiana , Canais de Sódio/efeitos dos fármacos , Venenos de Aranha/genética , Venenos de Aranha/farmacologia , Aranhas/genética
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