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1.
Channels (Austin) ; 15(1): 648-665, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34658293

RESUMO

K+ ions exert a structural effect that brings stability to K+ selective pores. Thus, upon bathing Shab channels in 0 K+ solutions the ion conductance, GK, irreversibly collapses. Related to this, studies with isolated KcsA channels have suggested that there is a transition [K+] around which the pore takes one of two conformations, either the low (non-conducting) or high K+ (conducting) crystal structures. We examined this premise by looking at the K+-dependency of GK stability of Shab channels within the cell membrane environment. We found that: K+ effect on GK stability is highly asymmetrical, and that as internal K+ is replaced by Na+ GK drops in a way that suggests a transition internal [K+]. Additionally, we found that external permeant ions inhibit GK drop with a potency that differs from the global selectivity-sequence of K+ pores; the non-permeant TEA inhibited GK drop in a K+-dependent manner. Upon lowering internal [K+] we observed an influx of Na+ at negative potentials. Na+ influx was halted by physiological external [K+], which also restored GK stability. Hyperpolarized potentials afforded GK stability but, as expected, do not restore GK selectivity. For completeness, Na+ interaction with Shab was also assessed at depolarized potentials by looking at Na block followed by permeation (pore unblock) at positive potentials, in solutions approaching the 0 K+ limit. The stabilizing effect of negative potentials along with the non-parallel variation of Na+ permeability and conductance-stability herein reported, show that pore stability and selectivity, although related, are not strictly coupled.


Assuntos
Potássio , Sódio , Membrana Celular , Concentração de Íons de Hidrogênio , Íons
2.
J Vis Exp ; (153)2019 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-31762449

RESUMO

A method to study desensitization and recovery of crayfish photoreceptors is presented. We performed intracellular electrical recordings of photoreceptor cells in isolated eyestalks using the discontinuous single electrode-switched voltage-clamp configuration. First, with a razor blade we made an opening in the dorsal cornea to get access to the retina. Thereafter, we inserted a glass electrode through the opening, and penetrated a cell as reported by the recording of a negative potential. Membrane potential was clamped at the photoreceptor's resting potential and a light-pulse was applied to activate currents. Finally, the two light-flash protocol was employed to measure current desensitization and recovery. The first light-flash triggers, after a lag period, the transduction ionic current, which after reaching a peak amplitude decays towards a desensitized state; the second flash, applied at varying time intervals, assesses the state of the light-activated conductance. To characterize the light-elicited current, three parameters were measured: 1) latency (the time elapsed between light flash delivery and the moment in which current achieves 10% of its maximum value); 2) peak current; and 3) desensitization time constant (exponential time constant of the current decay phase). All parameters are affected by the first pulse. To quantify recovery from desensitization, the ratio p2/p1 was employed versus time between pulses. p1 is the peak current evoked by the first light-pulse, and p2 is the peak current evoked by the second pulse. These data were fitted to a sum of exponential functions. Finally, these measurements were carried out as function of circadian time.


Assuntos
Astacoidea , Luz , Células Fotorreceptoras/efeitos da radiação , Animais , Transporte de Íons/efeitos da radiação , Potenciais da Membrana/efeitos da radiação , Células Fotorreceptoras/citologia , Células Fotorreceptoras/metabolismo
3.
Toxicon ; 138: 53-58, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28774677

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/virologia
4.
Proteins ; 85(4): 630-646, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28066924

RESUMO

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 , Termodinâmica
5.
J Cell Physiol ; 232(8): 2019-2032, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27255432

RESUMO

Several reports credit mibefradil with tumor suppressing properties arising from its known inhibition of Ca2+ currents. Given that mibefradil (Mb) is also known to inhibit K+ channels, we decided to study the interaction between this organic compound and the tumor-related Kv10.1 channel. Here we report that Mb modulates the gating of Kv10.1. Mb induces an apparent inactivation from both open and early closed states where the channels dwell at hyperpolarized potentials. Additionally, Mb accelerates the kinetics of current activation, in a manner that depends on initial conditions. Our observations suggest that Mb binds to the voltage sensor domain of Kv10.1 channels, thereby modifying the gating of the channels in a way that in some, but not all, aspects opposes to the gating effects exerted by divalent cations. J. Cell. Physiol. 232: 2019-2032, 2017. © 2016 Wiley Periodicals, Inc.


Assuntos
Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Ativação do Canal Iônico/efeitos dos fármacos , Mibefradil/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Sítios de Ligação , Canais de Potássio Éter-A-Go-Go/genética , Canais de Potássio Éter-A-Go-Go/metabolismo , Células HEK293 , Humanos , Cinética , Potenciais da Membrana , Mibefradil/metabolismo , Modelos Biológicos , Bloqueadores dos Canais de Potássio/metabolismo , Ligação Proteica , Transfecção
6.
Artigo em Inglês | MEDLINE | ID: mdl-27783925

RESUMO

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 Reação
7.
PLoS One ; 10(3): e0120431, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25798591

RESUMO

Potassium channels allow the selective flux of K⁺ excluding the smaller, and more abundant in the extracellular solution, Na⁺ ions. Here we show that Shab is a typical K⁺ channel that excludes Na⁺ under bi-ionic, Na(o)/K(i) or Na(o)/Rb(i), conditions. However, when internal K⁺ is replaced by Cs⁺ (Na(o)/Cs(i)), stable inward Na⁺ and outward Cs⁺ currents are observed. These currents show that Shab selectivity is not accounted for by protein structural elements alone, as implicit in the snug-fit model of selectivity. Additionally, here we report the block of Shab channels by external Ca²âº ions, and compare the effect that internal K⁺ replacement exerts on both Ca²âº and TEA block. Our observations indicate that Ca²âº blocks the channels at a site located near the external TEA binding site, and that this pore region changes conformation under conditions that allow Na⁺ permeation. In contrast, the latter ion conditions do not significantly affect the binding of quinidine to the pore central cavity. Based on our observations and the structural information derived from the NaK bacterial channel, we hypothesize that Ca²âº is probably coordinated by main chain carbonyls of the pore's first K⁺-binding site.


Assuntos
Cálcio/farmacologia , Potássio/metabolismo , Canais de Potássio Shab/antagonistas & inibidores , Animais , Sítios de Ligação/efeitos dos fármacos , Césio/metabolismo , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Permeabilidade/efeitos dos fármacos , Porosidade , Bloqueadores dos Canais de Potássio , Quinidina/farmacologia , Células Sf9 , Canais de Potássio Shab/química , Canais de Potássio Shab/metabolismo , Sódio/metabolismo , Spodoptera
8.
Pflugers Arch ; 467(10): 2069-79, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25398373

RESUMO

Protein mobility is important to achieve protein function. Intrinsic flexibility associated with motion underlies this important issue and the analysis of side chain flexibility gives insights to understand it. In this work, the S5-P-S6 pore modulus (PM) of members of Kv and HCN channels was examined by a combination of sequence alignment, residue composition analysis, and intrinsic side chain flexibility. The PM sequences were organized as a database that was used to reveal and correlate the functional diversity of each analyzed family. Specifically, we focused our attention on the crucial role of the S4-S5 linker and its well-described interaction with the S6 T during the electromechanical coupling. Our analysis suggests the presence of a Gly-hinge in the middle of the S4-S5 linkers. This apparent Gly-hinge links a flexible N-terminal segment with a rigid C-terminal one, although in Kv7 channels, the latter segment is even more flexible. Instead, HCN channels exhibit a putative Thr-hinge and is rich in aromatic residues, in consequence, their linker is more rigid. Concerning S6, we confirm the presence of the two flexible kinks previously described and we provide the complete segmental flexibility profiles for the different families. Our results are discussed in terms of the relation between residue composition, conservation, and local conformational flexibility. This provides important insights to understand and differentiate the characteristic gating properties of these channels as well as their implications in cell physiology.


Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/química , Ativação do Canal Iônico , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sequência Conservada , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Potenciais da Membrana , Dados de Sequência Molecular , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Estrutura Terciária de Proteína
9.
Peptides ; 46: 40-6, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23684923

RESUMO

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 K⁺-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 K⁺-channels with a Kd of 82 nM, and presents an even better affinity toward hKv1.3, blocking it with a Kd of ∼18 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 Proteína
10.
Channels (Austin) ; 7(3): 225-8, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23584197

RESUMO

We have recently examined slow inactivation of Shab channels. Here we extend our characterization of Shab slow inactivation by presenting the properties of recovery from inactivation. The observations support our proposal that Shab reaches the same inactivated state either from open or closed states and suggest that closed and open state inactivation share the same mechanism. Regarding the latter, we also show that external K (+) and TEA slow down recovery from inactivation in agreement with the hypothesis that the mechanism of Shab inactivation qualitatively differs from C-type inactivation.


Assuntos
Cálcio/metabolismo , Óxido Nítrico/metabolismo , Células de Purkinje/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais
11.
Channels (Austin) ; 7(2): 97-108, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23419584

RESUMO

Herein, we report the first characterization of Shab slow inactivation. Open Shab channels inactivate within seconds, with two voltage-independent time constants. Additionally, Shab presents significant closed-state inactivation. We found that with short depolarizing pulses, shorter than the slowest inactivation time constant, the resulting inactivation curve has a marked U-shape, but as pulse duration increases, approaching steady-state conditions, the U-shape vanishes, and the resulting inactivation curves converge to the classical Boltzmann h∞ curve. Regarding the mechanism of inactivation, we found that external K (+) and TEA facilitate both open- and closed-state inactivation, while the cavity blocker quinidine hinders inactivation. These results together with our previous observations regarding the K (+) -dependent stability of the K (+) conductance, suggest the novel hypothesis that inactivation of Shab channels, and possibly that of other Kv channels whose inactivation is facilitated by K (+) , does not involve a significant narrowing of the extracellular entry of the pore. Instead, we hypothesize that there is only a rearrangement of a more internal segment of the pore that affects the central cavity and halts K (+) conduction.


Assuntos
Proteínas de Drosophila/fisiologia , Ativação do Canal Iônico , Potássio/farmacologia , Canais de Potássio Shab/fisiologia , Animais , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/química , Drosophila melanogaster/química , Drosophila melanogaster/fisiologia , Potássio/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Estrutura Terciária de Proteína , Quinidina/farmacologia , Células Sf9 , Canais de Potássio Shab/antagonistas & inibidores , Canais de Potássio Shab/química , Spodoptera , Tetraetilamônio/farmacologia
12.
Channels (Austin) ; 5(1): 56-64, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21084865

RESUMO

Celecoxib is a drug designed to selectively inhibit COX-2, an inflammation-inducible cyclooxygenase isoform, over the constitutively expressed COX-1 isoform. In addition to this selective inhibition it is now known that celecoxib exerts a variety of effects on several types of ion channels, thus producing secondary physiological effects. In this work we demonstrate that at therapeutically relevant concentrations celecoxib interacts with Shab K(+) channels specifically promoting a fast inactivation gating (without blocking the pore or significantly affecting other gating processes). At least two celecoxib molecules bind to each channel promoting a fast inactivation that develops from both open and closed states. Channel inactivation in turn causes a reduction of the size of I(K). Taken together, our observations show that in addition to its intended therapeutic target celecoxib is a useful tool to further study the mechanism of Shab channel inactivation.


Assuntos
Inibidores de Ciclo-Oxigenase 2/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Potássio/metabolismo , Pirazóis/farmacologia , Canais de Potássio Shab/efeitos dos fármacos , Sulfonamidas/farmacologia , Animais , Baculoviridae/genética , Celecoxib , Linhagem Celular , Inibidores de Ciclo-Oxigenase 2/metabolismo , Cinética , Potenciais da Membrana , Ligação Proteica , Pirazóis/metabolismo , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Canais de Potássio Shab/genética , Canais de Potássio Shab/metabolismo , Sulfonamidas/metabolismo , Transfecção
13.
Artigo em Inglês | MEDLINE | ID: mdl-20713169

RESUMO

Visual photoreceptors are structures involved in the expression and synchronization of crayfish circadian rhythm of sensitivity to light (electroretinogram, ERG). Considering the relevant role of Pigment dispersing hormone (PDH) in the invertebrate circadian system organization, we study the effect of this substance on the electrical activity of crayfish visual photoreceptors during the 24-h cycle. The study demonstrates that: (1) PDH affects the electrical response to light of crayfish visual photoreceptor cells in a circadian time-dependent manner. (2) The kinetics of the light-elicited current of crayfish visual photoreceptor cells, as well as the ionic permeability underlying the electrical response to light vary over the 24-h cycle. (3) PDH modifies the kinetics and ionic permeability underlying the light-elicited current of crayfish visual photoreceptor cells in a circadian time-dependent manner.


Assuntos
Astacoidea/efeitos dos fármacos , Astacoidea/fisiologia , Ritmo Circadiano/efeitos dos fármacos , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Peptídeos/farmacologia , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/metabolismo , Animais , Astacoidea/efeitos da radiação , Ritmo Circadiano/efeitos da radiação , Cinética , Luz , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/efeitos da radiação , Células Fotorreceptoras de Vertebrados/efeitos da radiação , Fatores de Tempo
14.
J Physiol ; 588(Pt 15): 2691-706, 2010 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-20547671

RESUMO

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 , Spodoptera
15.
Arch Microbiol ; 192(7): 595-602, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20502878

RESUMO

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 Solo
16.
J Membr Biol ; 234(1): 13-27, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20177670

RESUMO

Rhizobium etli is equipped with several systems to handle both hyper- and hypo-osmotic stress. For adaptation to hypo-osmotic stress, R. etli possesses a single gene with clear homology to MscS, four MscS-like channels and one ortholog of MscL (ReMscL, identity approximately 44% compared to Escherichia coli MscL). We subcloned and expressed the ReMscL channel ortholog from R. etli in E. coli to examine its activity by patch clamp in giant spheroplasts and characterized it at the single-channel level. We obtained evidence that ReMscL prevents the lysis of E. coli null mutant log-phase cells upon a rapid, osmotic downshock and identified a slight pH dependence for ReMscL activation. Here, we describe the facilitation of ReMscL activation by arachidonic acid (AA) and a reversible inhibitory effect of Gd(3+). The results obtained in these experiments suggest a stabilizing effect of micromolar AA and traces of Gd(3+) ions in the partially expanded conformation of the protein. Finally, we discuss a possible correlation between the number of gene paralogs for MS channels and the habitats of several microorganisms. Taken together, our data show that ReMscL may play an important role in free-living rhizobacteria during hypo-osmotic shock in the rhizosphere.


Assuntos
Proteínas de Bactérias/genética , Canais Iônicos/genética , Mecanotransdução Celular , Rhizobium etli/genética , Sequência de Aminoácidos , Ácido Araquidônico/farmacologia , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Gadolínio/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Canais Iônicos/metabolismo , Dados de Sequência Molecular , Pressão Osmótica , Técnicas de Patch-Clamp , Rhizobium etli/metabolismo , Alinhamento de Sequência
17.
Eur Biophys J ; 38(7): 1013-27, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19424690

RESUMO

Mechanosensitive (MS) channels play a major role in protecting bacterial cells against hypo-osmotic shock. To understand their function, it is important to identify the conserved motifs using sequence analysis methods. In this study, the sequence conservation was investigated by an in silico analysis to generate sequence logos. We have identified new conserved motifs in the domains TM1, TM2 and the cytoplasmic helix from 231 homologs of MS channel of large conductance (MscL). In addition, we have identified new motifs for the TM3 and the cytoplasmic carboxy-terminal domain from 309 homologs of MS channel of small conductance (MscS). We found that the conservation in MscL homologs is high for TM1 and TM2 in the three domains of life. The conservation in MscS homologs is high only for TM3 in Bacteria and Archaea.


Assuntos
Proteínas Arqueais/química , Proteínas de Bactérias/química , Sequência Conservada , Canais Iônicos/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência Consenso , Citoplasma , Evolução Molecular , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
18.
Biophys J ; 93(12): 4197-208, 2007 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-17704149

RESUMO

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 Hidrogênio
19.
Toxicon ; 48(8): 1046-53, 2006 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-17030052

RESUMO

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 Sequência
20.
Proteomics ; 6(12): 3718-27, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16705749

RESUMO

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/farmacologia
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