RESUMO
Conorfamides (CNFs) are toxins initially characterized from the venom duct of the venomous marine snail Conus spurius from the Gulf of Mexico; at their C-termini, these toxins are amidated and have high sequence similarity with the molluskan cardioexcitatory tetrapeptide Phe-Met-Arg-Phe-NH2 (FMRFamide or FMRFa) and other FMRFa-related peptides (FaRPs) found in the five molluskan classes, and in other invertebrate and vertebrate phyla. These peptides were the first FaRPs found to be present in any venom, and they are biologically active in mice, limpets, and/or freshwater snails. However, the molecular targets of the known CNFs (CNF-Sr1 and CNF-Sr2 from C. spurius, and CNF-Vc1 from C. victoriae) remain unidentified. Very recently, three FaRPs from C. textile have been found to potentiate the currents of acid-sensing ion channels. In this work, we characterized a novel conorfamide, CNF-Sr3 (ATSGPMGWLPVFYRF-NH2), comprised of 15 amino acid residues, and with a specific blocking activity for the Shaker subtype of the voltage-gated potassium channels, without significant effect on the Shab, Shaw, Shal and Eag channels. This peptide is the third type of disulfide-free conotoxins that has been discovered to target K+ channels.
Assuntos
Caramujo Conus/química , Venenos de Moluscos/química , Neuropeptídeos/farmacologia , Peptídeos/farmacologia , Superfamília Shaker de Canais de Potássio/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Baculoviridae , Células HEK293 , Humanos , Venenos de Moluscos/síntese química , Venenos de Moluscos/farmacologia , Neuropeptídeos/síntese química , Neuropeptídeos/química , Peptídeos/química , Bloqueadores dos Canais de Potássio/química , Bloqueadores dos Canais de Potássio/farmacologia , Células Sf9/virologiaRESUMO
The transient receptor potential (TRP) superfamily is subdivided into several subfamilies on the basis of sequence similarity, which is highly heterogeneous but shows a molecular architecture that resembles the one present in members of the Kv channel superfamily. Because of this diversity, they produce a large variety of channels with different gating and permeability properties. Elucidation of these particular features necessarily requires comparative studies based on structural and functional data. The present study aims to compilate, analyze, and determine, in a coherent way, the relationship between intrinsic side-chain flexibility and the allosteric coupling in members of the TRPV, TRPM, and TRPC families. Based on the recently determined structures of TRPV1 and TRPV2, we have generated protein models for single subunits of TRPV5, TRPM8, and TRPC5 channels. With these models, we focused our attention on the apparently crucial role of the GP dipeptide at the center of the S4-S5 linker and discussed its role in the interaction with the TRP domain, specifically with the highly-conserved Trp during this coupling. Our analysis suggests an important role of the S4-S5L flexibility in the thermosensitivity, where heat-activated channels possess rigid S4-S5 linkers, whereas cold-activated channels have flexible ones. Finally, we also present evidence of the key interaction between the conserved Trp residue of the TRP box and of several residues in the S4-S5L, importantly the central Pro. Proteins 2017; 85:630-646. © 2016 Wiley Periodicals, Inc.
Assuntos
Dipeptídeos/química , Canais de Cátion TRPC/química , Canais de Cátion TRPM/química , Canais de Cátion TRPV/química , Triptofano/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Glicina/química , Ativação do Canal Iônico , Cinética , Camundongos , Modelos Moleculares , Prolina/química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , TermodinâmicaRESUMO
In this work, we studied the characteristics of recovery from desensitization of the light-elicited current of crayfish. Applying a two-flash protocol, we found that the first flash triggers a current that activates with a noticeable latency, reaches a peak value, and thereafter decays along a single exponential time course. In comparison with the first-elicited current, the current elicited by the second flash not only presents an expected smaller peak current, depending on the time between flashes, but it also displays a different latency and decay time constant. Recovery of the first flash values of these current parameters depends on the circadian time at which the experiments are conducted, and on the presence of pigment-dispersing hormone. Our data also suggest the existence of distinctive desensitized states, whose induction depends on circadian time and the presence of pigment-dispersing hormone.
Assuntos
Astacoidea/fisiologia , Ritmo Circadiano , Hormônios de Invertebrado/metabolismo , Células Fotorreceptoras de Invertebrados/fisiologia , Algoritmos , Animais , Aquicultura , Astacoidea/crescimento & desenvolvimento , Fenômenos Eletrofisiológicos , Olho , Técnicas In Vitro/veterinária , Cinética , Muda , Tempo de ReaçãoRESUMO
Opisthacanthus cayaporum belongs to the Liochelidae family, and the scorpions from this genus occur in southern Africa, Central America and South America and, therefore, can be considered a true Gondwana heritage. In this communication, the isolation, primary structure characterization, and Kâº-channel blocking activity of new peptide from this scorpion venom are reported. OcyKTx2 is a 34 amino acid long peptide with four disulfide bridges and molecular mass of 3807 Da. Electrophysiological assays conducted with pure OcyKTx2 showed that this toxin reversibly blocks Shaker B Kâº-channels with a Kd of 82 nM, and presents an even better affinity toward hKv1.3, blocking it with a Kd of â¼18 nM. OcyKTx2 shares high sequence identity with peptides belonging to subfamily 6 of α-KTxs that clustered very closely in the phylogenetic tree included here. Sequence comparison, chain length and number of disulfide bridges analysis classify OcyKTx2 into subfamily 6 of the α-KTx scorpion toxins (systematic name, α-KTx6.17).
Assuntos
Canal de Potássio Kv1.3/antagonistas & inibidores , Peptídeos/metabolismo , Venenos de Escorpião/metabolismo , Superfamília Shaker de Canais de Potássio/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Células Cultivadas , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos/química , Peptídeos/isolamento & purificação , Bloqueadores dos Canais de Potássio/química , Bloqueadores dos Canais de Potássio/isolamento & purificação , Bloqueadores dos Canais de Potássio/metabolismo , Ligação Proteica , Venenos de Escorpião/química , Venenos de Escorpião/isolamento & purificação , Escorpiões/metabolismo , Alinhamento de Sequência , Análise de Sequência de ProteínaRESUMO
Quinidine is a commonly used antiarrhythmic agent and a tool to study ion channels. Here it is reported that quinidine equilibrates within seconds across the Sf9 plasma membrane, blocking the open pore of Shab channels from the intracellular side of the membrane in a voltage-dependent manner with 1:1 stoichiometry. On binding to the channels, quinidine interacts with pore K(+) ions in a mutually destabilizing manner. As a result, when the channels are blocked by quinidine with the cell bathed in an external medium lacking K(+), the Shab conductance G(K) collapses irreversibly, despite the presence of a physiological [K(+)] in the intracellular solution. The quinidine-promoted collapse of Shab G(K) resembles the collapse of Shaker G(K) observed with 0 K(+) solutions on both sides of the membrane: thus the extent of G(K) drop depends on the number of activating pulses applied in the presence of quinidine, but is independent of the pulse duration. Taken together the observations indicate that, as in Shaker, the quinidine-promoted collapse of Shab G(K) occurs during deactivation of the channels, at the end of each activating pulse, with a probability of 0.1 per pulse at 80 mV. It appears that when Shab channels are open, the pore conformation able to conduct is stable in the absence of K(+), but on deactivation this conformation collapses irreversibly.
Assuntos
Permeabilidade da Membrana Celular/fisiologia , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/fisiologia , Potássio/metabolismo , Quinidina/administração & dosagem , Quinidina/farmacocinética , Canais de Potássio Shab/fisiologia , Animais , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Condutividade Elétrica , Potenciais da Membrana/efeitos dos fármacos , Canais de Potássio Shab/efeitos dos fármacos , SpodopteraRESUMO
In this communication we reported the study of a cation channel present in the cytoplasmic membrane of the nitrogen fixing bacterium Rhizobium etli. Inner-membrane (IM) vesicles were purified and fused into planar lipid bilayers (PLBs), under voltage clamp conditions. We have found that fusion of IM-enriched vesicle fractions with these model membranes leads, mainly (>30% of 46 experiments), to the reconstitution of high-conductance channels. Following this strategy, the activity of a channel with main open conductance of 198 pS, in symmetrical 100 mM KCl, was recorded. The single-channel conductance increase to 653 pS in the presence of a 5:1 (cis to trans) gradient of KCl. The channel exhibits voltage dependency and a weak selectivity for cations showing a permeability ratios of P (Rb)/P (K) = 0.96, P (Na)/P (K) = 0.07, and a conductance ratio of gamma(Rb)/gamma(K) = 1.1. The channel here characterized represents a previously undescribed Rhizobium channel although its precise role in rhizobial physiology remains yet to be determined.
Assuntos
Proteínas de Bactérias/metabolismo , Canais Iônicos/metabolismo , Rhizobium etli/fisiologia , Cátions/metabolismo , Membrana Celular/metabolismo , Bicamadas Lipídicas/metabolismo , Rhizobium etli/metabolismo , Microbiologia do SoloRESUMO
Shab channels are fairly stable with K(+) present on only one side of the membrane. However, on exposure to 0 K(+) solutions on both sides of the membrane, the Shab K(+) conductance (G(K)) irreversibly drops while the channels are maintained undisturbed at the holding potential. Herein it is reported that the drop of G(K) follows first-order kinetics, with a voltage-dependent decay rate r. Hyperpolarized potentials drastically inhibit the drop of G(K). The G(K) drop at negative potentials cannot be explained by a shift in the voltage dependence of activation. At depolarized potentials, where the channels undergo a slow inactivation process, G(K) drops in 0 K(+) with rates slower than those predicted based on the behavior of r at negative potentials, endowing the r-V(m) relationship with a maximum. Regardless of voltage, r is very small compared with the rate of ion permeation. Observations support the hypothesized presence of a stabilizing K(+) site (or sites) located either within the pore itself or in its external vestibule, at an inactivation-sensitive location. It is argued that part of the G(K) stabilization achieved at hyperpolarized potentials could be the result of a conformational change in the pore itself.
Assuntos
Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/fisiologia , Potássio/metabolismo , Canais de Potássio Shab/fisiologia , Spodoptera/metabolismo , Animais , Linhagem Celular , Condutividade Elétrica , Concentração de Íons de HidrogênioRESUMO
A novel toxin was identified, purified and characterized from the venom of the Mexican scorpion Hadrurus gertschi (abbreviated HgeTx1). It has a molecular mass of 3950 atomic mass units (a.m.u.) and contains 36 amino acids with four disulfide bridges established between Cys1-Cys5, Cys2-Cys6, Cys3-Cys7 and Cys4-Cys8. It blocks reversibly the Shaker B K(+)-channels with a Kd of 52nM. HgeTx1 shares 60%, 45% and 40% sequence identity, respectively, with Heterometrus spinnifer toxin1 (HsTX1), Scorpio maurus K(+)-toxin (maurotoxin) and Pandinus imperator toxin1 (Pi1), all four-disulfide bridged toxins. It is 57-58% identical with the other scorpion K(+)-channel toxins that contain only three disulfide bridges. Sequence comparison, chain length and number of disulfide bridges analysis classify HgeTx1 into subfamily 6 of the alpha-KTx scorpion toxins (systematic name: alpha-KTx 6.14).
Assuntos
Bloqueadores dos Canais de Potássio/química , Venenos de Escorpião/química , Superfamília Shaker de Canais de Potássio/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Células Cultivadas , Insetos/citologia , Dados de Sequência Molecular , Bloqueadores dos Canais de Potássio/isolamento & purificação , Venenos de Escorpião/isolamento & purificação , Escorpiões/química , Alinhamento de SequênciaRESUMO
The Venezuelan scorpion Tityus discrepans is known to cause human fatalities. We describe the first complete proteomic analysis of its venom. By HPLC 58 different fractions were obtained and 205 different components were identified by MS analysis. Components having molecular masses from 272 to 57 908 amu were found. Forty homogeneous components had their N-terminal amino acid sequence determined by Edman degradation, from which two new peptides named TdK2 and TdK3 (meaning T. discrepans (Td) K(+) channel toxins 2 and 3) were fully characterized. The first contains 34 amino acid residues with a molecular mass of 3451 amu, and the second has 36 amino acids with 3832 amu. Both peptides are tightly bound by three disulfide bridges. TdK2 was shown to block reversibly the Shaker B K(+)-channel expressed heterologously in Sf9 cells. The systematic number assigned to TdK2 is alpha-KTx-18.2 and that of TdK3 is alpha-KTx-18.3. Comparative analysis of the amino acid sequences found suggests that this venom contains peptides highly similar to those that block K(+) channels, as well as those that modify the gating mechanisms of Na(+) channels, found in other scorpions. Additionally, peptides similar to defensins were also identified.
Assuntos
Proteoma/análise , Venenos de Escorpião/química , Venenos de Escorpião/toxicidade , Escorpiões/química , Toxinas Biológicas/química , Sequência de Aminoácidos , Animais , Técnicas de Cultura de Células , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Eletrofisiologia , Hidrólise , Peso Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Mapeamento de Peptídeos , Venenos de Escorpião/isolamento & purificação , Escorpiões/genética , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Spodoptera/citologia , Tripsina/farmacologiaRESUMO
The Colombian scorpion Tityus pachyurus is toxic to humans and is capable of producing fatal accidents, but nothing is known about its venom components. This communication reports the separation of at least 57 fractions from the venom by high performance liquid chromatography. From these, at least 104 distinct molecular weight compounds were identified by mass spectrometry analysis. The complete amino acid sequences of three peptides were determined and the partial sequences of three others were also identified. Electrophysiological experiments conducted with ion-channels expressed heterologously on Sf9 cells showed the presence of a potent Shaker B K(+)-channel blocker. This peptide (trivial name Tpa1) contains 23 amino acid residues closely packed by three disulfide bridges with a molecular mass of 2,457 atomic mass units. It is the third member of the sub-family 13, for which the systematic name is proposed to be alpha-KTx13.3. The mice assay showed clearly the presence of toxic peptides to mammals. One of them named Tpa2, containing 65 amino acid residues with molecular mass of 7,522.5 atomic mass units, is stabilized by four disulfide bridges. It was shown to modify the Na(+)-currents of F-11 and TE671 cells in culture, similar to the beta scorpion toxins. These results demonstrate the presence of toxic peptides in the venom of T. pachyurus and confirm that accidents with this species of scorpion should be considered an important human hazard in Colombia.
Assuntos
Canais de Potássio/efeitos dos fármacos , Venenos de Escorpião/química , Venenos de Escorpião/toxicidade , Canais de Sódio/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Humanos , Técnicas In Vitro , Dose Letal Mediana , Camundongos , Dados de Sequência Molecular , Bloqueadores dos Canais de Potássio/química , Bloqueadores dos Canais de Potássio/isolamento & purificação , Bloqueadores dos Canais de Potássio/toxicidade , Proteômica , Venenos de Escorpião/genética , Escorpiões/química , Escorpiões/genética , Escorpiões/patogenicidade , Homologia de Sequência de Aminoácidos , Superfamília Shaker de Canais de Potássio/antagonistas & inibidores , Bloqueadores dos Canais de Sódio/química , Bloqueadores dos Canais de Sódio/isolamento & purificação , Bloqueadores dos Canais de Sódio/toxicidade , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
A previously undescribed plasma membrane cation channel from Phaseolus vulgaris bean roots was studied after its incorporation into planar lipid bilayers. The channel allows the passage of monovalent cations excluding the flux of both anions (Cl-) and divalent cations (Ca2+). The channel presents a high ( approximately 213 pS) conductance in (300 mM Kcis+)/ (150 mMKtrans+) conditions. The probability of opening (Po) is low at all the tested voltages, but it increases significantly at trans-negative potentials. Permeability ratios (Pcation/PK+) under bi-ionic conditions follow the sequence: K+ (1.0)>NH4+ (0.86)>Na+ (0.78). Under the same conditions, the conductance ratios (gamma cation/gamma K+) follow the sequence: NH4+ (1.1) > or = K+ (1.0)>Na+ (0.80). The low probability of opening exhibited by the channel upon its incorporation into a lipid bilayer makes it a candidate to regulation by (and therefore participation in) cellular signalling networks.
Assuntos
Cálcio/química , Canais Iônicos/química , Bicamadas Lipídicas/química , Phaseolus/metabolismo , Raízes de Plantas/metabolismo , Potássio/química , Sódio/química , Cátions , Condutividade Elétrica , Ativação do Canal Iônico , Potenciais da MembranaRESUMO
The venom of the scorpion Tityus costatus contains peptides toxic to humans but scarce information on their structure and function is available. Here, we report the separation of 50 different components by high performance liquid chromatography and the identification of approximately 90 distinct components by mass spectrometry analysis, with molecular weights varying from 413 to 45482 atomic mass units. Four peptides were fully sequenced: (i) a butantoxin-like peptide that blocks Shaker K+ channel; (ii) an insect toxin-like peptide; (iii) a scorpine-like peptide, and a short heptapeptide of unknown function. Fifteen peptides were directly sequenced at the N-terminal region, among which are components toxic to mice. A cDNA library was constructed and 13 clones were isolated and sequenced. Some of these peptides and genes are similar to other known scorpion toxins. Based on these results, stings by scorpions of the species Tityus costatus should be taken with caution by medical doctors.
Assuntos
Venenos de Escorpião/química , Escorpiões/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Brasil , Linhagem Celular , Camundongos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/isolamento & purificação , Canais de Potássio/efeitos dos fármacos , Venenos de Escorpião/genética , Venenos de Escorpião/toxicidade , Escorpiões/genética , Especificidade da EspécieRESUMO
Ion channels are key participants in physiological processes of plant cells. Here, we report the first characterization of a high conductance, Cl(-)-permeable channel, present in enriched fractions of plasma membranes of bean root cells. The Cl(-) channel was incorporated into planar lipid bilayers and its activity was recorded under voltage clamp conditions. The channel is voltage-dependent, excludes the passage of cations (K(+), Na(+), and Ca(2+)), and is inhibited by micromolar concentrations of Zn(2+). The Cl(-) conductance here characterized represents a previously undescribed channel of plant cells.
Assuntos
Canais de Cloreto/metabolismo , Bicamadas Lipídicas/metabolismo , Phaseolus/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Membrana Celular/metabolismo , Ativação do Canal Iônico , Técnicas de Patch-Clamp , Phaseolus/anatomia & histologia , Zinco/metabolismoRESUMO
A peptide was isolated from the venom of the scorpion Tityus trivittatus. It is an isoform of the toxin TsTX-IV earlier described [Toxicon 37 (1999) 651] and identical to butantoxin [Arch. Biochem. Biophys. 379 (2000) 18], both isolated from the Brazilian scorpion Tityus serrulatus. This newly characterized peptide contains 40 amino acid residues with a molecular mass of [M+H(+)] 4507.0, cross-linked by four disulfide bridges, made between the cysteine pairs: Cys2-Cys5, Cys10-Cys31, Cys16-Cys36 and Cys20-Cys38. It blocks in a completely reversible manner the Shaker B K(+)-channels, with a K(d) around 660nM. It belongs to the sub-family 12 and it is now being classified as alpha-KTx 12.2.
Assuntos
Dissulfetos/química , Neurotoxinas/química , Bloqueadores dos Canais de Potássio/química , Venenos de Escorpião/química , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Dados de Sequência Molecular , Neurotoxinas/farmacologia , Peptídeos , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/metabolismo , Isoformas de Proteínas , Venenos de Escorpião/farmacologia , Superfamília Shaker de Canais de PotássioRESUMO
We describe an interaction between external Ca(2+) ions and Shaker K channels that is important in the gating of the channels. The interaction was first detected as a partial block of inward K(+) current in elevated Ca(2+), beginning near -40 mV and becoming stronger at more negative voltage. Surprisingly, the time course of the block can be resolved as a rapid decay of inward current magnitude following a repolarizing step. The rapid decay of current is shown to be the result of channel block by using a two-pulse procedure that monitors the time course of gate closing. As a result of block, the decay of the tail current after repolarization is two to three times faster than gate closing. With physiological values for voltage and calcium concentration, block is readily detectable from tail time course, implying that it occurs as a normal concomitant of gate closing in Shaker. The slight voltage dependence of block from -60 to -100 mV suggests that Ca(2+) is bound (with low affinity) near the outer mouth of the channel. Elevated calcium quickens the inward gating current recorded as Shaker channels are closing; this current approximately doubles in amplitude and has a faster time course and quicker rising phase. When combined, the results suggest that calcium accelerates the first step in closing of the channel gate, perhaps by changing the channel's ion-occupancy state.
Assuntos
Cálcio/farmacologia , Canais de Potássio/fisiologia , Animais , Ativação do Canal Iônico/fisiologia , Cinética , Potenciais da Membrana/efeitos dos fármacos , Canais de Potássio/efeitos dos fármacos , Superfamília Shaker de Canais de Potássio , Spodoptera , TransfecçãoRESUMO
Two novel toxic peptides (Tc30 and Tc32) were isolated and characterized from the venom of the Brazilian scorpion Tityus cambridgei. The first have 37 and the second 35 amino acid residues, with molecular masses of 3,871.8 and 3,521.5, respectively. Both contain three disulfide bridges but share only 27% identity. They are relatively potent inhibitors of K(+)-currents in human T lymphocytes with K(d) values of 10 nM for Tc32 and 16 nM for Tc30, but they are less potent or quite poor blockers of Shaker B K(+)-channels, with respective K(d) values of 74 nM and 4.7 microM. Tc30 has a lysine in position 27 and a tyrosine at position 36 identical to those of charybdotoxin. These two positions conform the dyad considered essential for activity. On the contrary, Tc32 has a serine in the position equivalent to lysine 27 of charybdotoxin and does not contain any aromatic amino acid. Due to its unique primary sequence and to its distinctive preference for K(+)-channels of T lymphocytes, it was classified as the first example of a new subfamily of K(+)-channel-specific peptides (alpha-KT x 18.1). Tc30 is a member of the Tityus toxin II-9 subfamily and was given the number alpha-KT x 4.4.