Ionic partition and transport in multi-ionic channels: a molecular dynamics simulation study of the OmpF bacterial porin

We performed all-atom molecular dynamics simulations studying the partition of ions and the ionic current through the bacterial porin OmpF and two selected mutants. The study is motivated by new, interesting experimental findings concerning their selectivity and conductance behavior at neutral pH. The mutations considered here are designed to study the effect of removal of negative charges present in the constriction zone of the wild-type OmpF channel (which contains, on one side, a cluster with three positive residues, and on the other side, two negatively charged residues). Our results show that these mutations induce an exclusion of cations from the constriction zone of the channel, substantially reducing the flow of cations. In fact, the partition of ions inside the mutant channels is strongly inhomogeneous, with regions containing an excess of cations and regions containing an excess of anions. Interestingly, the overall number of cations inside the channel is larger than the number of anions, this excess being different for each protein channel. We found that the differences in ionic charge inside these channels are justified by the differences in electric charge between the wild-type OmpF and the mutants, following an electroneutral balance.     

Ion permeation and selectivity of OmpF porin: a theoretical study based on molecular dynamics,Brownian dynamics,and continuum electrodiffusion theory

Three different theoretical approaches are used and compared to refine our understanding of ion permeation through the channel formed by OmpF porin from Escherichia coli. Those approaches are all-atom molecular dynamics (MD) in which ions, solvent, and lipids are represented explicitly, Brownian dynamics (BD) in which ions are represented explicitly, while solvent and lipids are represented as featureless dielectrics, and Poisson-Nernst-Planck (PNP) electrodiffusion theory in which both solvent and local ion concentrations are represented as a continuum. First, the ability of the different theoretical approaches in reproducing the equilibrium average ion density distribution in OmpF porin bathed by a 1M KCl symmetric salt solution is examined. Under those conditions the PNP theory is equivalent to the non-linear Poisson-Boltzmann (PB) theory. Analysis shows that all the three approaches are able to capture the important electrostatic interactions between ions and the charge distribution of the channel that govern ion permeation and selectivity in OmpF. The K(+) and Cl(-) density distributions obtained from the three approaches are very consistent with one another, which suggests that a treatment on the basis of a rigid protein and continuum dielectric solvent is valid in the case of OmpF. Interestingly, both BD and continuum electrostatics reproduce the distinct left-handed twisted ion pathways for K(+) and Cl(-) extending over the length of the pore which were observed previously in MD. Equilibrium BD simulations in the grand canonical ensemble indicate that the channel is very attractive for cations, particularly at low salt concentration. On an average there is 1.55 K(+) inside the pore in 10mM KCl. Remarkably, there is still 0.17 K(+) on average inside the pore even at a concentration as low as 1microM KCl. Secondly, non-equilibrium ion flow through OmpF is calculated using BD and PNP and compared with experimental data. The channel conductance in 0.2M and 1M KCl calculated using BD is in excellent accord with the experimental data. The calculations reproduce the experimentally well-known conductance-concentration relation and also reveal an asymmetry in the channel conductance (a larger conductance is observed under a positive transmembrane potential). Calculations of the channel conductance for three mutants (R168A, R132A, and K16A) in 1M KCl suggest that the asymmetry in the channel conductance arises mostly from the permanent charge distribution of the channel rather than the shape of the pore itself. Lastly, the calculated reversal potential in a tenfold salt gradient (0.1:1M KCl) is 27.4(+/-1.3)mV (BD) and 22.1(+/-0.6)mV (PNP), in excellent accord with the experimental value of 24.3mV. Although most of the results from PNP are qualitatively reasonable, the calculated channel conductance is about 50% higher than that calculated from BD probably because of a lack of some dynamical ion-ion correlations.     

Distinct sensitivities of OmpF and PhoE porins to charged modulators

The inhibition of the anion-selective PhoE porin by ATP and of the cation-selective OmpF porin by polyamines has been previously documented. In the present study, we have extended the comparison of the inhibitor-porin pairs by investigating the effect of anions (ATP and aspartate) and positively charged polyamines (spermine and cadaverine) on both OmpF and PhoE with the patch-clamp technique, and by comparing directly the gating kinetics of the channels modulated by their respective substrates. The novel findings reported here are (1) that the activity of PhoE is completely unaffected by polyamines, and (2) that the kinetic changes induced by ATP on PhoE or polyamines on OmpF suggest different mechanisms of inhibition. ATP induces a high degree of flickering in the PhoE-mediated current and appears to behave as a blocker of ion flow during its presumed transport through PhoE. Polyamines modulate the kinetics of openings and closings of OmpF, in addition to promoting a blocker-like flickering activity. The strong correlation between sensitivity to inhibitors and ion selectivity suggests that some common molecular determinants are involved in these two properties and is in agreement with the hypothesis that polyamines bind inside the pore of cationic porins.     

Translocation and interactions of L-arabinose in OmpF porin: A molecular dynamics study

The passage of a natural substrate, L-arabinose (L-ARA) through Escherichia coli porin embedded in an artificial bilayer, is studied by equilibrium molecular dynamics simulations. We investigate the early stage of translocation process of L-ARA from intra-cellular to extra-cellular side (Int-to-Ext) across the bilayer. The average trajectory path over all L-ARA molecules along with quantum-mechanical configuration-optimizations at PM3 level predict the existence of at least three trapping zones. The common feature within all these zones is that L-ARA remains perpendicular to the channel axis. It is remarkable how the orientation and translational-rotational motion of L-ARA molecule play a role in its transport through OmpF channel. These simulations are important for better understanding of permeation process in OmpF channel. They also provide an insight into the chiral recognition of translocation process in protein nanochannels from substrate and protein prospects and help interpret experiments on permeation process of small dipolar molecules across biological membranes.     

Porin channels in Escherichia coli: studies with liposomes reconstituted from purified proteins.

Rates of diffusion of uncharged and charged solute molecules through porin channels were determined by using liposomes reconstituted from egg phosphatidylcholine and purified Escherichia coli porins OmpF (protein 1a), OmpC (protein 1b), and PhoE (protein E). All three porin proteins appeared to produce channels of similar size, although the OmpF channel appeared to be 7 to 9% larger than the OmpC and PhoE channels in an equivalent radius. Hydrophobicity of the solute retarded the penetration through all three channels in a similar manner. The presence of one negative charge on the solute resulted in about a threefold reduction in penetration rates through OmpF and OmpC channels, whereas it produced two- to tenfold acceleration of diffusion through the PhoE channel. The addition of the second negatively charged group to the solutes decreased the diffusion rates through OmpF and OmpC channels further, whereas diffusion through the PhoE channel was not affected much. These results suggest that PhoE specializes in the uptake of negatively charged solutes. At the present level of resolution, no sign of true solute specificity was found in OmpF and OmpC channels; peptides, for example, diffused through both of these channels at rates expected from their molecular size, hydrophobicity, and charge. However, the OmpF porin channel allowed influx of more solute molecules per unit time than did the equivalent weight of the OmpC porin when the flux was driven by a concentration gradient of the same size. This apparent difference in "efficiency" became more pronounced with larger solutes, and it is likely to be the consequence of the difference in the sizes of OmpF and OmpC channels.     

Thermal Motion of DNA in an MspA Pore

We report on an experiment and calculations that determine the thermal motion of a voltage-clamped single-stranded DNA-NeutrAvidin complex in a Mycobacterium smegmatis porin A nanopore. The electric force and diffusion constant of DNA inside a Mycobacterium smegmatis porin A pore were determined to evaluate the thermal position fluctuations of DNA. We show that an out-of-equilibrium state returns to equilibrium so quickly that experiments usually measure a weighted average over the equilibrium position distribution. Averaging over the equilibrium position distribution is consistent with results of state-of-the-art nanopore sequencing experiments. It is shown how a reduction in thermal position fluctuations can be achieved by increasing the electrophoretic force used in nanopore sequencing devices.     

Brownian dynamics simulation of ion flow through porin channels

Bacterial porins, which allow the passage of solutes across the outer bacterial membrane, are structurally well characterized. They therefore lend themselves to detailed studies of the determinants of ion flow through transmembraneous channels. In a comparative study, we have performed Brownian dynamics simulations to obtain statistically significant transfer efficiencies for cations and anions through matrix porin OmpF, osmoporin OmpK36, phosphoporin PhoE and two OmpF charge mutants.The simulations show that the electrostatic potential at the highly charged channel constriction serves to enhance ion permeability of either cations or anions, dependent on the type of porin. At the same time translocation of counterions is not severely impeded. At the constriction, cations and anions follow distinct trajectories, due to the segregation of basic and acidic protein residues.Simulated ion selectivity and relative conductance agree well with experimental values, and are dependent crucially on the charge constellation at the pore constriction. The experimentally observed decrease in ion selectivity and single channel conductance with increasing ionic strength is well reproduced and can be attributed to electrostatic shielding of the pore lining.     

Antibiotic translocation through membrane channels: temperature-dependent ion current fluctuation for catching the fast events

Temperature-dependent facilitated permeation of antibiotics through membrane channels was investigated. Here we reconstituted single OmpF trimers from the outer membrane of Escherichia coli (E. coli) into a planar lipid bilayer. The penetration of ampicillin through OmpF causes fluctuation in the ion current, and analysis of the fluctuations at different temperatures allows us to determine the mode of permeation. The residence time of the drug inside the channel decays strongly with temperature, reaching the resolution limit of the instrument at 30°C. The number of events increases exponentially with temperature up to 30°C and then gradually decreases as temperature increases. At room temperature, we observe about 25 events per second per monomer of the trimeric channel and an extrapolation to 37°C gives roughly 50 events. The activation energy for ampicillin translocation through OmpF is estimated to be around 13 kT. Temperature-dependent study gives new insights into the faster translocation of small substrates through biological nanopores.     

Crystal structure of Omp32, the anion-selective porin from Comamonas acidovorans, in complex with a periplasmic peptide at 2.1 A resolution

BACKGROUND: Porins provide diffusion channels for salts and small organic molecules in the outer membrane of bacteria. In OmpF from Escherichia coli and related porins, an electrostatic field across the channel and a potential, originating from a surplus of negative charges, create moderate cation selectivity. Here, we investigate the strongly anion-selective porin Omp32 from Comamonas acidovorans, which is closely homologous to the porins of pathogenic Bordetella and Neisseria species. RESULTS: The crystal structure of Omp32 was determined to a resolution of 2.1 A using single isomorphous replacement with anomalous scattering (SIRAS). The porin consists of a 16-stranded beta barrel with eight external loops and seven periplasmic turns. Loops 3 and 8, together with a protrusion located within beta-strand 2, narrow the cross-section of the pore considerably. Arginine residues create a charge filter in the constriction zone and a positive surface potential at the external and periplasmic faces. One sulfate ion was bound to Arg38 in the channel constriction zone. A peptide of 5.8 kDa appeared bound to Omp32 in a 1:1 stoichiometry on the periplasmic side close to the symmetry axis of the trimer. Eight amino acids of this peptide could be identified, revealing specific interactions with beta-strand 1 of the porin. CONCLUSIONS: The Omp32 structure explains the strong anion selectivity of this porin. Selectivity is conferred by a positive potential, which is not attenuated by negative charges inside the channel, and by an extremely narrow constriction zone. Moreover, Omp32 represents the anchor molecule for a peptide which is homologous to proteins that link the outer membrane to the cell wall peptidoglycan.     

Permeation properties of an engineered bacterial OmpF porin containing the EEEE-locus of Ca2+ channels

The selectivity filter of the bacterial porin OmpF carries a small net charge close to -1 e and is therefore only slightly cation-selective. Calcium channels, on the other hand, contain four negatively charged glutamates, the EEEE-locus, and are among the most selective cation channels known. We aimed to turn the essentially nonselective OmpF into a Ca2+-selective channel. To that end, two additional glutamates (R42E and R132E) were introduced in the OmpF constriction zone that already contains D113 and E117. Mutant OmpF containing this DEEE-locus has a high Ca2+ over Cl- selectivity and a Na+ current with a strongly increased sensitivity to 1 mM Ca2+. The charge/space competition model, initially applied to the L-type Ca2+ channel, identifies the fixed charge and filter volume as key determinants of ion selectivity, with the precise atomic arrangement having only second-order effects. By implication, the reproduction of fixed charge and filter volume should transform two channels into channels of similar selectivity, even if the two belong to entirely different ion channel families, as is the case for OmpF and the L-type Ca2+ channel. The results presented here fit quite well in the framework of charge/space competition theory.     

Properties of chemically modified porin from Escherichia coli in lipid bilayer membranes

Purified porin OmpF from Escherichia coli outer membrane was chemically modified by acetylation and succinylation of amino groups and by amidation of the carboxyl groups. Native and chemically modified porins were incorporated into lipid bilayer membranes and the permeability properties of the pores were studied. Acetylation and succinylation of the porin trimers had almost no influence on the single channel conductance in the presence of small cations and anions and the cation selectivity remained essentially unchanged as compared with the native porin. Amidation had also only little influence on the single channel conductance and changed the pore conductance at maximum by less than 50%, whereas the cation selectivity of the porin is completely lost after amidation. The results suggest that the structure of the porin pore remains essentially unchanged after chemical modification of the pores and that their cation selectivity is caused by an excess of negatively charged groups inside the pore and/or on the surface of the protein. Furthermore, it seems very unlikely that the pore contains any positively charged group at neutral pH.     

Residue ionization and ion transport through OmpF channels

Single trimeric channels of the general bacterial porin, OmpF, were reconstituted into planar lipid membranes and their conductance, selectivity, and open-channel noise were studied over a wide range of proton concentrations. From pH 1 to pH 12, channel transport properties displayed three characteristic regimes. First, in acidic solutions, channel conductance is a strong function of pH; it increases by approximately threefold as the proton concentration decreases from pH 1 to pH 5. This rise in conductance is accompanied by a sharp increase in cation transport number and by pronounced open-channel low-frequency current noise with a peak at ~pH 2.5. Random stepwise transients with amplitudes at ~1/5 of the monomer conductance are major contributors to this noise. Second, over the middle range (pH 5 ÷ pH 9), channel conductance and selectivity stay virtually constant; open channel noise is at its minimum. Third, over the basic range (pH 9 ÷ pH 12), channel conductance and cation selectivity start to grow again with an onset of a higher frequency open-channel noise. We attribute these effects to the reversible protonation of channel residues whose pH-dependent charge influences transport by direct interactions with ions passing through the channel.     

Role of charged residues at the OmpF porin channel constriction probed by mutagenesis and simulation

The channel constriction of OmpF porin, a pore protein in the bacterial outer membrane, is highly charged due to the presence of three arginines (R42, R82, and R132) and two acidic residues (D113 and E117). The influence of these charges on ion conductance, ion selectivity, and voltage gating has been studied with mutants D113N/E117Q, R42A/R82A/R132A/D113N/E117Q, and V18K/G131K, which were designed to remove or add protein charge at the channel constriction. The crystal structures revealed no or only local changes compared to wild-type OmpF, thus allowing a comparative study. The single-channel conductance of the isosteric D113N/E117Q variant was found to be 2-fold reduced, and that of the pentuple mutant was 70% of the wild-type value, despite a considerably larger pore cross section. Ion selectivity was drastically altered by the mutations with cation/anion permeability ratios ranging from 1 to 12. Ion flow through these and eight other mutants, which have been characterized previously, was simulated by Brownian dynamics based on the detailed crystal structures. The calculated ion selectivity and relative channel conductance values agree well with the experimental data. This demonstrates that ion translocation through porin is mainly governed by pore geometry and charge, the two factors that are properly represented in the simulations.     

Molecular dynamics simulation of water permeation through the alpha-hemolysin channel

The alpha-hemolysin (AHL) nanochannel is a non-selective channel that allows for uncontrolled transport of small molecules across membranes leading to cell death. Although it is a bacterial toxin, it has promising applications, ranging from drug delivery systems to nano-sensing devices. This study focuses on the transport of water molecules through an AHL nanochannel using molecular dynamics (MD) simulations. Our results show that AHL can quickly transport water across membranes. The first-passage time approach was used to estimate the diffusion coefficient and the mean exit time. To study the energetics of transport, the potential of mean force (PMF) of a water molecule along the AHL nanochannel was calculated. The results show that the energy barriers of water permeation across a nanopore are always positive along the channel and the values are close to thermal energy (k_BT). These findings suggest that the observed quick permeation of water is due to small energy barriers and a hydrophobic inner channel surface resulting in smaller friction. We speculate that these physical mechanisms are important in how AHL causes cell death.     

Chemical modification of the bacterial porin OmpF: gain of selectivity by volume reduction

OmpF is an essentially nonselective porin isolated from the outer membrane of Escherichia coli. Here we report on the manipulation of the ion selectivity of OmpF by chemical modification with MTS reagents (MTSET, MTSEA, and MTSES) and the (rather bulky) tripeptide glutathione, all cysteine specific. When recorded in a gradient of 0.1//1 M CaCl2 or 0.1//1 M NaCl, pH 7.4 solutions, measured reversal potentials of the most cation-selective modified mutants were (virtually) identical to the Nernst potential of Ca2+ or Na+. Compared to this full cation selectivity, the anion-selective modified mutants performed somewhat less but nevertheless showed high anion selectivity. We conclude that a low permanent charge in combination with a narrow pore can render the same selectivity as a highly charged but wider pore. These results favor the view that both the electrostatic potential arising form the fixed charge in the pore and the space available at the selectivity filter contribute to the charge selection (i.e., cation versus anion selectivity) of a biological ion channel.     

Increased salt concentration promotes competitive block of OmpF channel by protons

Porins are channel-forming proteins that are located in the outer membranes (OM) of Gram-negative bacteria and allow the influx of hydrophilic nutrients and the extrusion of waste products. The fine regulation of the ion transport through these wide channels could play an important role in the survival of the bacteria in acidic media. We investigate here the mechanism responsible for the pH sensitivity of the trimeric porin OmpF, of Escherichia coli. Planar lipid bilayer electrophysiology and site-directed mutagenesis were used to study the effect of pH on the ion conductive properties of the OmpF channel in its fully open, "nongated" conformation. At low pH we observe a large drop in the OmpF open channel conductance that is accompanied by a substantial increase of the current noise. These channel features are strongly dependent on the salt concentration and we propose that they are originated by competitive binding of cations and protons occurring in the narrow central constriction of the channel. This subtle mechanism reveals to be capital for the channel function because it not only drives the channel sensitivity to pH but is also indispensable for the particularly efficient permeation mechanism of the channel at physiological conditions (~neutral pH).     

E.coli PhoE porin has an opposite voltage-dependence to the homologous OmpF.

We used patch clamp analysis to compare the electrophysiological behavior of two related porins from Escherichia coli, the anion-specific PhoE and the cation-selective OmpF. Outer membrane fractions were obtained from strains expressing just one of these porin types, and the channels were reconstituted into liposomes without prior purification. We show that the orientation of the reconstituted channels is not random and is the same for both PhoE and OmpF. Like cation-selective porins, PhoE shows fast and slow gating to closed levels of various amplitudes, testifying that the channels visit multiple functional states and behave as cooperative entities. The voltage-dependence of PhoE closure is asymmetric, but strikingly, occurs at voltages of inverse polarity from those promoting closures of OmpC and OmpF. Both slow kinetics and inverse voltage-dependence are removed when 70 amino acids from the N-terminal of OmpF are introduced into the homologous region of PhoE. This novel observation regarding the voltage-dependence of the two channel types, along with published results on PhoE and OmpF mutants, allows us to propose a molecular mechanism for voltage sensing and sensor charge movements in bacterial porins. It also offers new cues on the possible physiological relevance in bacteria of this common form of channel modulation.     

Transport at the nanoscale: temperature dependence of ion conductance

Temperature dependent ion conductance in nanopores is measured in a wide range of electrolyte concentrations and compared with molecular modeling. Single outer membrane protein F (OmpF) channels from E. coli are reconstituted into planar lipid bilayers. In qualitative agreement with the experimental data, applied-field molecular dynamics unraveled atomistic details of the ion transport. Comparing the temperature dependence of the channel conductance with that of the bulk conductivity in the range from 0 to 90°C revealed that at low salt concentrations the transport is mainly driven along the pore surface. Increasing the salt concentration saturates the surface charge transport and induces ion transport in the center of the nanopore. The confinement of the nanopore then favors the formation of ion pairs. Stepping up the temperature reduces the life time of the ion pairs and increases the channel conductance more than expected from the bulk behavior.     

High Salt Concentrations Increase Permeability through OmpC Channels of Escherichia coli

OmpF and OmpC porin channels are responsible for the passage of small hydrophilic solutes across the outer membrane of Escherichia coli. Although these channels are two of the most extensively studied porin channels, what had yet remained elusive was the reason why OmpC shows markedly lower permeability than OmpF, despite having little difference in its channel size. The OmpC channel, however, is known to contain a larger number of ionizable residues than the OmpF channel. In this study, we examined the channel property of OmpF and OmpC using the intact cell of E. coli, and we found that the permeability of several β-lactams and lactose through OmpC became increased to the level comparable with OmpF with up to 0.3 m salt that may increase the Debye-Hückel shielding or with 2% ethanol or 0.3 m urea that may perturb the short range ordering of water molecules. Replacing 10 pore-lining residues that show different ionization behavior between OmpC and OmpF led to substantial conversion of channel property with respect to their permeability and response to external salt concentration. We thus propose that the overall configuration of ionizable residues in the channel that may orient water molecules and the electrostatic profile of the channel play a decisive role in defining the channel property of the OmpC porin rather than its channel size.