RESUMO
Nonstructural protein 2B of foot-and-mouth disease (FMD) virus (FMDV) is comprised of a small, hydrophobic, 154-amino-acid protein. Structure-function analyses demonstrated that FMDV 2B is an ion channel-forming protein. Infrared spectroscopy measurements using partially overlapping peptides that spanned regions between amino acids 28 and 147 demonstrated the adoption of helical conformations in two putative transmembrane regions between residues 60 and 78 and between residues 119 and 147 and a third transmembrane region between residues 79 and 106, adopting a mainly extended structure. Using synthetic peptides, ion channel activity measurements in planar lipid bilayers and imaging of single giant unilamellar vesicles (GUVs) revealed the existence of two sequences endowed with membrane-porating activity: one spanning FMDV 2B residues 55 to 82 and the other spanning the C-terminal region of 2B from residues 99 to 147. Mapping the latter sequence identified residues 119 to 147 as being responsible for the activity. Experiments to assess the degree of insertion of the synthetic peptides in bilayers and the inclination angle adopted by each peptide regarding the membrane plane normal confirm that residues 55 to 82 and 119 to 147 of 2B actively insert as transmembrane helices. Using reverse genetics, a panel of 13 FMD recombinant mutant viruses was designed, which harbored nonconservative as well as alanine substitutions in critical amino acid residues in the area between amino acid residues 28 and 147. Alterations to any of these structures interfered with pore channel activity and the capacity of the protein to permeabilize the endoplasmic reticulum (ER) to calcium and were lethal for virus replication. Thus, FMDV 2B emerges as the first member of the viroporin family containing two distinct pore domains.IMPORTANCE FMDV nonstructural protein 2B is able to insert itself into cellular membranes to form a pore. This pore allows the passage of ions and small molecules through the membrane. In this study, we were able to show that both current and small molecules are able to pass though the pore made by 2B. We also discovered for the first time a virus with a pore-forming protein that contains two independent functional pores. By making mutations in our infectious clone of FMDV, we determined that mutations in either pore resulted in nonviable virus. This suggests that both pore-forming functions are independently required during FMDV infection.
Assuntos
Permeabilidade da Membrana Celular , Vírus da Febre Aftosa/metabolismo , Febre Aftosa/metabolismo , Bicamadas Lipídicas/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Sequência de Aminoácidos , Animais , Células Cultivadas , Cricetinae , Febre Aftosa/genética , Febre Aftosa/virologia , Vírus da Febre Aftosa/genética , Transporte de Íons , Mutação , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios Proteicos , Homologia de Sequência , Proteínas não Estruturais Virais/genéticaRESUMO
We have computed the pK(a)'s of the ionizable residues of a protein ion channel, the Staphylococcus aureus toxin alpha-hemolysin, by using two types of input structures, namely the crystal structure of the heptameric alpha-hemolysin and a set of over four hundred snapshots from a 4.38 ns Molecular Dynamics simulation of the protein inserted in a phospholipid planar bilayer. The comparison of the dynamic picture provided by the Molecular Simulation with the static one based on the X-ray crystal structure of the protein embedded in a lipid membrane allows analyzing the influence of the fluctuations in the protein structure on its ionization properties. We find that the use of the dynamic structure provides interesting information about the sensitivity of the computed pK(a) of a given residue to small changes in the local structure. The calculated pK(a) are consistent with previous indirect estimations obtained from single-channel conductance and selectivity measurements.
Assuntos
Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Fenômenos Químicos , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Eletricidade Estática , Membrana Celular/química , Membrana Celular/metabolismo , Gráficos por Computador , Cristalografia por Raios X , Simulação de Dinâmica Molecular , Conformação Proteica , Staphylococcus aureus , Fatores de TempoRESUMO
Recently developed nanometer-sized synthetic pores display several properties so far believed to be distinctive features of a large variety of biological wide ion channels. Thus conductance in the pS-nS range, pH-dependent ion selectivity, fluctuations of current between open and closed states, flux inhibition caused by protons or divalent cations, current rectification, and the ability to perform selective macromolecule sizing and counting are found in synthetic and biological channels alike. Despite other differences such as pore size and geometry, the similarities open a new field for exploring specific technological applications via the chemical modification of synthetic pores with biological molecules. This article reviews some of the basic concepts and theories relevant to ion transport in nanopores with titratable charges stressing the analogies between synthetic pores and biological ion channels. The ultimate goal is to show that continuum theories may account for the essential features of these systems. A simple electrodiffusion model and its comparison with experimental results are chosen as a case study.
Assuntos
Canais Iônicos , Transporte de Íons , Modelos Biológicos , Canais Iônicos/química , Canais Iônicos/metabolismo , Membranas Artificiais , Nanoestruturas , Porinas/química , Porinas/metabolismoRESUMO
We have considered the access resistance (AR) of a single conducting channel placed in a membrane bathed by an electrolyte. The classical expression for AR is due to Hall, who modeled the electrolyte as an ohmic conducting homogeneous medium. This approach is discussed in the present paper and it is shown that it is not valid in all cases, but depends on the ion concentration in solution and the ratio between solution and channel resistivities. To get a new expression for AR, we have combined the use of one-dimensional Nernst-Planck and Poisson (NPP) equations for the mouth of the channel and three-dimensional NPP equations for the outside solution. The influence of ion gradients and the channel itself on AR tums out to be considerable in diluted solutions (and also in the case of small channels in any solution). This influence is weaker in concentrated solutions, for which AR is well described by Hall's expression.
Assuntos
Canais Iônicos/fisiologia , Modelos Biológicos , Membrana Celular , Impedância Elétrica , Eletrólitos , Transporte de Íons , Concentração OsmolarRESUMO
One of the contributions to the surface potential in charged phospholipid monolayers at air-water interfaces is the double layer potential. In this note several misconceptions found in the literature concerning the relationship between the double layer potential and the degree of dissociation of the lipid polar headgroups are critically analyzed. The deviations of the double layer potential measurements from the Gouy-Chapman theory observed by several authors are explained by taking into account the dependence of the degree of dissociation with concentration, area per lipid molecule and pH.
Assuntos
Lipídeos de Membrana/química , Fosfolipídeos/química , Ar , Eletrólitos/química , Concentração de Íons de Hidrogênio , Íons , Modelos Químicos , Ácidos Esteáricos/química , TemperaturaRESUMO
Synthetic nanopores with fixed charges exhibit ionic equilibrium and transport properties that resemble those displayed by biological ion channels. We present an electrodiffusion model based on the Nernst-Planck flux equations, which allows for a qualitative description of the steady state ionic transport through a nanopore when the membrane fixed charges and all mobile carriers (including the water ions) are properly taken into account. In particular, we study the current-voltage curve, the electrical conductance, the reversal potential (a measure of the nanopore ionic selectivity), as well as the flux inhibition by protons and divalent cations in the nanopore. The model clearly shows how the changes in the ionization state of the fixed charges with pH and salt concentration dictate the electrical properties of the nanopore. The agreement between the model predictions and previous experimental data allows us to identify which are the main characteristics that permit a simple description of this complex system.
Assuntos
Íons , Transporte Biológico , Fenômenos Biofísicos , Biofísica , Cátions , Difusão , Relação Dose-Resposta a Droga , Condutividade Elétrica , Concentração de Íons de Hidrogênio , Modelos Químicos , Modelos Estatísticos , Prótons , Sais/farmacologiaRESUMO
The binding of a polar macromolecule to a large ion channel is studied theoretically, paying special attention to the influence of external conditions (applied voltage and ion strength of solution). The molecule behavior in bound state is considered as random thermal fluctuations within a limited fraction of its phase space. The mean duration of molecule binding (residence time tau r) is represented as the mean first passage time to reach the boundary of that restricted domain. By invoking the adiabatic approximation we reduce the problem to one dimension with the angle between macromolecule dipole and channel axes being the key variable of the problem. The model accounts for experimental measurements of tau r for the antibiotic Ampicillin within the bacterial porin OmpF of Escherichia coli. By assuming that the electrical interaction between Ampicillin dipole and OmpF ionizable groups affects the fluctuations, we find that the biased residence time-voltage dependence observed in experiments is the result of the strong transversal electric field in OmpF constriction with a tilt approximately 30 degrees aside the cis side.
Assuntos
Ampicilina/química , Ampicilina/metabolismo , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/fisiologia , Modelos Biológicos , Porinas/química , Porinas/metabolismo , Sítios de Ligação , Simulação por Computador , Eletroquímica/métodos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Cinética , Modelos Químicos , Ligação Proteica , Eletricidade EstáticaRESUMO
The ion selectivity of a channel can be quantified in several ways by using different experimental protocols. A wide, mesoscopic channel, the OmpF porin of the outer membrane of E. coli, serves as a case study for comparing and analysing several measures of the channel cation-anion permeability in chlorides of alkali metals (LiCl, NaCl, KCl, CsCl). We show how different insights can be gained and integrated to rationalize the global image of channel selectivity. To this end, reversal potential, channel conductance and bi-ionic potential (two different salts with a common anion on each side of the channel but with the same concentration) experiments are discussed in light of an electrodiffusion model based on the Poisson-Nernst-Planck formalism. Measurements and calculations based on the atomic crystal structure of the channel show that each protocol displays a particular balance between the different sources of selectivity.
Assuntos
Ativação do Canal Iônico , Modelos Químicos , Modelos Moleculares , Porinas/química , Porinas/ultraestrutura , Simulação por Computador , Sensibilidade e EspecificidadeRESUMO
The membrane surface charge modifies the conductance of ion channels by changing the electric potential and redistributing the ionic composition in their vicinity. We have studied the effects of lipid charge on the conductance of a multi-state channel formed in planar lipid bilayers by the peptide antibiotic alamethicin. The channel conductance was measured in two lipids: in a neutral dioleoylphosphatidylethanolamine (DOPE) and a negatively charged dioleoylphosphatidylserine (DOPS). The charge state of DOPS was manipulated by the pH of the membrane-bathing solution. We find that at high salt concentrations (e.g., 2 M NaCl) the effect of the lipid charge is below the accuracy of our measurements. However, when the salt concentration in the membrane-bathing solution is decreased, the surface charge manifests itself as an increase in the conductance of the first two channel levels that correspond to the smallest conductive alamethicin aggregates. Our analysis shows that both the salt and pH dependence of the surface charge effect can be rationalized within the nonlinear Poisson-Boltzmann approach. Given channel conductance in neutral lipids, we use different procedures to account for the surface charge (e.g., introduce averaging over the channel aperture and take into account Na+ adsorption to DOPS heads), but only one adjustable parameter: an effective distance from the nearest lipid charge to the channel mouth center. We show that this distance varies by 0.3-0.4 nm upon channel transition from the minimal conducting aggregate (level L0) to the next larger one (level L1). This conclusion is in accord with a simple geometrical model of alamethicin aggregation.
Assuntos
Alameticina/química , Canais Iônicos/química , Antibacterianos/química , Fenômenos Biofísicos , Biofísica , Condutividade Elétrica , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Ionóforos/química , Bicamadas Lipídicas/química , Modelos Químicos , Fosfatidiletanolaminas/química , Fosfatidilserinas/química , Cloreto de Sódio , Eletricidade EstáticaRESUMO
We manipulate lipid bilayer surface charge and gauge its influence on gramicidin A channel conductance by two strategies: titration of the lipid charge through bulk solution pH and dilution of a charged lipid by neutral. Using diphytanoyl phosphatidylserine (PS) bilayers with CsCl aqueous solutions, we show that the effects of lipid charge titration on channel conductance are masked 1) by conductance saturation with Cs+ ions in the neutral pH range and 2) by increased proton concentration when the bathing solution pH is less than 3. A smeared charge model permits us to separate different contributions to the channel conductance and to introduce a new method for "bilayer pKa" determination. We use the Gouy-Chapman expression for the charged surface potential to obtain equilibria of protons and cations with lipid charges. To calculate cation concentration at the channel mouth, we compare different models for the ion distribution, exact and linearized forms of the planar Poisson-Boltzmann equation, as well as the construction of a "Gibbs dividing surface" between salt bath and charged membrane. All approximations yield the intrinsic pKain of PS lipid in 0.1 M CsCl to be in the range 2.5-3.0. By diluting PS surface charge at a fixed pH with admixed neutral diphytanoyl phosphatidylcholine (PC), we obtain a conductance decrease in magnitude greater than expected from the electrostatic model. This observation is in accord with the different conductance saturation values for PS and PC lipids reported earlier (, Biochim. Biophys. Acta. 552:369-378) and verified in the present work for solvent-free membranes. In addition to electrostatic effects of surface charge, gramicidin A channel conductance is also influenced by lipid-dependent structural factors.