The Vibrio cholerae porins OmpU and OmpT have distinct channel properties

Numerous environmental signals regulate the production of virulence factors and the composition of the outer membrane of Vibrio cholerae. In particular, bile promotes the ToxR-dependent expression of the porin OmpU. Strains expressing solely OmpU are more resistant to bile, are better able to colonize the intestine, and produce more cholera toxin than strains expressing solely the OmpT porin. To gain some understanding in the physiological relevance and the molecular mechanism underlying these porin-dependent phenotypes, we have undertaken a thorough electrophysiological characterization of the channel properties of the two porins. Purified OmpU or OmpT was reconstituted in liposomes suitable for patch clamp and in planar lipid bilayers. The high resolution of the patch clamp technique allowed us to analyze in detail the behavior of single OmpU and OmpT channels. Both channels exhibit closing transitions to various conductance states. OmpT is a much more dynamic channel than OmpU, displaying frequent and prolonged closures, even at low transmembrane potentials. With a critical voltage for closure V(c) of approximately +/-90 mV, OmpT is much more voltage-sensitive than OmpU (with a V(c) of approximately +/-160 mV), a feature that is also readily apparent in the voltage dependence of closing probability observed in patch clamp in the +/-100 mV range. OmpT has low ionic selectivity (P(K)/P(Cl) = approximately 4), whereas OmpU is more cation-selective (P(K)/P(Cl) = approximately 14). The distinct functional properties of the two porins are likely to play an integrated role with environmental regulation of their expression. For example, the higher selectivity of OmpU for cations provides a possible explanation for the protective role played by this porin in a bile-containing environment, because this type of selectivity would restrict the flux of anionic bile salts through the outer membrane and thus would reduce the exposure of the cytoplasmic membrane to this natural detergent.     

Size and Dynamics of the Vibrio cholerae Porins OmpU and OmpT Probed by Polymer Exclusion

The trimeric OmpU and OmpT porins form large, triple-barrel hydrophilic channels in the outer membrane of the pathogen Vibrio cholerae. They have distinct pore properties, such as conductance, block by deoxycholic acid, and sensitivity to acidic pH. Their three-dimensional structures are unknown, but they share significant sequence homologies. To gain insight into the molecular basis for the distinct functional properties of these two similar porins, we carried out polymer exclusion experiments using planar lipid bilayer and patch-clamp electrophysiology. By studying the partitioning of polyethylene glycols (PEGs) of different molecular weights into each porin, we determined an effective radius of 0.55 nm and 0.43 nm for OmpU and OmpT respectively, and found an increased OmpU effective radius at acidic pH. PEGs or high buffer ionic strength promotes the appearance of single step closures in OmpU similar to the acidic-pH induced closures we documented previously. In addition, these closing events can be triggered by nonpenetrating PEGs applied asymmetrically. We believe our results support a model whereby acidic pH, high ionic strength, or exposure to PEGs stabilizes a less conductive state that corresponds to the appearance of an additional resistive element on one side of the OmpU protein and common to the three monomers.     

Deoxycholic acid blocks vibrio cholerae OmpT but not OmpU porin

OmpT and OmpU are general diffusion porins of the human intestinal pathogen Vibrio cholerae. The sole presence of OmpT in the outer membrane sensitizes cells to the bile component deoxycholic acid, and the repression of OmpT in the intestine may play an important role in the adaptation of cells to the host environment. Here we report a novel important functional difference between the two porins, namely the sensitivity to deoxycholic acid. Single channel recordings show that submicellar concentrations of sodium deoxycholate induce time-resolved blocking events of OmpT but are devoid of any effect on OmpU. The effects are dose-, voltage-, and pH-dependent. They are elicited by deoxycholate applied to either side of the membrane, with some asymmetry in the sensitivity. The voltage dependence remains even when deoxycholate is applied symmetrically, indicating that it is intrinsic to the binding site. The pH dependence suggests that the active form is the neutral deoxycholic acid and not the negatively charged species. The results are interpreted as deoxycholic acid acting as an open-channel blocker, which may relate to deoxycholic acid permeation.     

Altered pore properties and kinetic changes in mutants of the Vibrio cholerae porin OmpU

The electrophysiological technique of patch-clamp was used to characterize the pore properties of site-directed mutants in the Vibrio cholerae general diffusion porin OmpU. Changes in conductance and selectivity were observed, thus confirming the predicted pore location of these residues, based on homology with the Escherichia coli porins OmpF and OmpC. Some mutants acquire a weak selectivity for cations, which mirrors the properties of the homologous, deoxycholic acid sensitive, OmpT porin of V. cholerae. However, the mutants remain insensitive to deoxycholic acid, like wildtype OmpU. This result suggests that channel selectivity is not an important determinant in the sensitivity to this drug, and is in agreement with our finding that the neutral deoxycholic acid, and not deoxycholate, is the actual active form in channel block. Modifications in the kinetics of spontaneous closures were also noted, and are similar to those found for the E. coli channels. In addition, mutants at the D116 residue on the L3 loop display marked transitions to sub-conductance states. The results reported here are compared to a phenotypical characterization of the mutants in terms of permeability to maltodextrins and beta-lactam antibiotic sensitivity. No strict correlations are observed, suggesting that distinct, but somewhat overlapping, molecular determinants control electrophysiological properties and substrate permeability.     

Porins of Vibrio cholerae: purification and characterization of OmpU.

Three outer membrane proteins with molecular masses of 40, 38, and 27 kDa of the hypertoxinogenic strain 569B of Vibrio cholerae have been purified to homogeneity. The synthesis of all the three proteins is regulated by the osmolarity of the growth medium. The pore-forming ability of the 40-kDa protein, OmpT, and the 38-kDa protein, OmpU, has been demonstrated by using liposomes, in which these proteins were embedded. The 27-kDa protein, OmpX, though osmoregulated, is not a porin. OmpU constitutes 30% of the total outer membrane protein when grown in the presence of 1.0% NaCl in the growth medium and 60% in the absence of NaCl. OmpU is an acidic protein and is a homotrimer of 38-kDa monomeric units. Its secondary structure contains predominantly a beta-sheet, and three to four Ca2+ ions are associated with each monomeric unit. Removal of Ca2+ irreversibly disrupts the structure and pore-forming ability of the protein. The pore size of OmpU is 1.6 nm, and the specific activity of the OmpU channel is two- to threefold higher than that of Escherichia coli porin OmpF, synthesis of which resembles that of OmpU with respect to the osmolarity of the growth medium. The pore size of OmpT, which is analogous to OmpC of E. coli, is smaller than that of OmpU. Southern blot hybridization of V. cholerae genomic DNA digested with several restriction endonucleases with nick-translated E. coli ompF as the probe revealed no nucleotide sequence homology between the ompU and ompF genes. OmpU is also not antigenically related to OmpF. Anti-OmpF antiserum, however, cross-reacted with the 45-kDa V. cholerae outer membrane protein, OmpS, the synthesis of which is regulated by the presence of maltose in the growth medium. OmpU hemagglutinated with rabbit and human blood. This toxR-regulated protein is one of the possible virulence determinants in V. cholerae (V. L. Miller and J. J. Mekalanos, J. Bacteriol. 170:2575-2583, 1988).     

Altered pore properties and kinetic changes in mutants of the Vibrio cholerae Porin OmpU

The electrophysiological technique of patch-clamp was used to characterize the pore properties of site-directed mutants in the Vibrio cholerae general diffusion porin OmpU. Changes in conductance and selectivity were observed, thus confirming the predicted pore location of these residues, based on homology with the Escherichia coli porins OmpF and OmpC. Some mutants acquire a weak selectivity for cations, which mirrors the properties of the homologous, deoxycholic acid sensitive, OmpT porin of V. cholerae. However, the mutants remain insensitive to deoxycholic acid, like wildtype OmpU. This result suggests that channel selectivity is not an important determinant in the sensitivity to this drug, and is in agreement with our finding that the neutral deoxycholic acid, and not deoxycholate, is the actual active form in channel block. Modifications in the kinetics of spontaneous closures were also noted, and are similar to those found for the E. coli channels. In addition, mutants at the D116 residue on the L3 loop display marked transitions to sub-conductance states. The results reported here are compared to a phenotypical characterization of the mutants in terms of permeability to maltodextrins and β-lactam antibiotic sensitivity. No strict correlations are observed, suggesting that distinct, but somewhat overlapping, molecular determinants control electrophysiological properties and substrate permeability.     

Refolding and functional assembly of the Vibrio cholerae porin OmpU recombinantly expressed in the cytoplasm of Escherichia coli

OmpU is one of the major outer membrane porins of Vibrio cholerae. OmpU has been biochemically characterized previously for its 'porin'-property. However, previous studies have used the OmpU protein extracted from the bacterial outer membrane envelope fractions. Such method of isolation imposes limitations on the availability of the protein reagent, and also enhances the possibility of the OmpU preparation being contaminated with lipid molecules of bacterial outer membrane origin, especially lipopolysaccharides (LPS). Here we report a strategy of purifying the V. cholerae OmpU protein recombinantly overexpressed in heterologous protein expression system in Escherichia coli, without its being incorporated into the bacterial membrane fraction. In our strategy, the majority of the protein was expressed as insoluble inclusion body in the E. coli cytoplasm, the protein was dissolved by denaturation in 8M urea, refolded, and purified to homogeneity in presence of detergent. Our strategy allowed isolation of the recombinant OmpU protein with significantly enhanced yield as compared to that of the wild type protein extracted from the V. cholerae membrane fraction. The recombinant V. cholerae OmpU protein generated in our study displayed functional channel-forming property in the synthetic liposome membrane, thus confirming its 'porin'-property. To the best of our knowledge, this is the first report showing an efficient refolding and functional assembly of the V. cholerae OmpU porin recombinantly expressed as inclusion body in the cytoplasm of a heterologous host E. coli.     

Homology models of the Yersinia pseudotuberculosis and Yersinia pestis general porins and comparative analysis of their functional and antigenic regions

The amino acid sequences of the Yersinia pseudotuberculosis porin (YPS) and Y. pestis porin (YPT) have recently deduced but their three-dimensional structures were not known. These sequences were analyzed using the servers 3D-PSSM and PredPort. The YPS and YPT porins were shown to have a high degree of identity (above 50%) in primary and secondary structures. The three-dimensional models of the Yersinia pseudotuberculosis porin (YPS) and Y. pestis porin (YPT) were obtained using the homology modeling approach, SWISS-MODEL Protein Modeling Server and 3-D structure of PhoE porin from E. coli as template. The superposition of the Calpha-atoms of the monomers of the Yersinia porins and PhoE porin gave a root mean square deviations of 0.47 A and 0.43 A for YPS and YPT respectively. Yersinia porins were found to be very similar in their three-dimensional structure to other non-specific enterobacterial porins, having the same features of overall fold and disposition of loop L3. The intrinsic structures of the monomer pores of YPS and YPT were investigated and their conductances were predicted with the program HOLE. The good correspondence between the theoretical and experimental magnitudes of YPS conductance was found. The Yersinia porins were determined to be unusual in containing the substitution, Glu replaced by Val, in a highly conserved pentapeptide (Pro-Glu-Phe-Gly-Gly-Asp), located in the loop L3 tip that disturbs the functionally important cluster of the acidic amino acids in the constriction site. Comparative analysis of structural organization of YPS and E. coli OmpF porin in the regions involved in subunit association and pore lumen was performed. The YPS porin functional properties were predicted. The differences between these porins in polar interactions playing a significant role in stabilization of the porin trimers were found and discussed in term of the variations in trimer stability. The Yersinia porins were shown to have the highest degree of the structural similarity. The differences between the porins were observed in their external loops. Their loops L6 and loops L8 showed 71.4 and 52.9% of sequence identity, respectively. The arrangement of charged residues clustered in the channel external vestibule of these porins was found to be also different suggesting the possible differences in their functional properties. The surface exposed regions of Yersinia porins involved in their potential sequential antigenic determinants were compared. The structural basis of their cross reactivity and antigenic differences is discussed.     

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).     

Plasticity of Escherichia coli porin channels. Dependence of their conductance on strain and lipid environment.

The conductance properties of three members of the porin family which form channels across the outer membrane of Gram-negative bacteria were compared. With their endogenous lipopolysaccharide (LPS) bound, the closely related porins F and C from Escherichia coli reveal significantly different conductance steps and closing potentials, with values of 0.82 nS (nanosiemens) and 89 mV for F-type channels, and 0.49 nS and 158 mV for C-type pores (1 M NaCl), respectively. On the basis of their closing potentials, the two channel types can be distinguished unequivocally. If reconstituted in asolectin and extraneous LPS, porin C forms F-type in addition to C-type channels. Substitution of asolectin by mitochondrial lipids yields the native C-type pores only. Both channel types can be induced to assume the mutually other channel configuration by variation of ionic strength. A multiplicity of channel subtypes is observed by variation of the pH of the medium. The three channels within a trimer are, however, consistently of the same type. Since structural studies have revealed a single channel per monomer, the several conductance steps observed are likely to reflect distinct configurations of the same channel. Best channel recoveries were observed if endogenous LPS remained associated to porin during purification. Significant yields could nevertheless be obtained also if LPS was removed from porin and replaced with various precursors or chemically synthesized analogues. As function requires the presence of glycolipids, yet crystallization is perturbed by heterodisperse endogenous LPS, the smallest monodisperse analogues yielding good channel recovery were determined. The minimal synthetic moiety is a monoglucosaminetetraacyl compound. The characteristics of porin B from E. coli BE are shown to be indistinguishable from those of porin F. The conductance properties of this porin, refolded from random coil configuration, are indistinguishable from those exhibited by native protein. The formation of channels is thus encoded by the sequence of the mature polypeptide alone.     

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.     

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.     

Effects of conjugated and unconjugated bile acids on the activity of the Vibrio cholerae porin OmpT

During infection, the enteric pathogen Vibrio cholerae encounters a bile-containing environment. Previous studies have shown that bile and/or bile acids exert several effects on the virulence and physiology of the bacterial cells. These observations have led to the suggestion that bile acids may play a signaling role in infection. We have previously reported that the bile component deoxycholic acid blocks the general diffusion porin OmpT in a dose-dependent manner, presumably as it transits through the pore. V. cholerae colonizes the distal jejunum and ileum, where a mixture of various conjugated and unconjugated bile acids are found. In this work, we have used patch clamp electrophysiology to investigate the effects of six bile acids on OmpT. Two bile acids (deoxycholic and chenodeoxycholic acids) were found to block OmpT at physiological concentrations below 1 mM, while glycodeoxycholic acid was mildly effective and cholic, lithocholic and taurodeoxycholic acids were ineffective in this range. The block was also voltage-dependent. These observations suggest the presence of a specific binding site inside the OmpT pore. Since deconjugation is due to the activity of the endogenous flora, the preferential uptake of some unconjugated bile acids by OmpT may signal the presence of a hospitable environment. The results are also discussed in terms of the possible molecular interactions between the penetrating bile acid molecule and the channel wall.     

1/f-Noise of Open Bacterial Porin Channels

General diffusion pores and specific porin channels from outer membranes of gram-negative bacteria were reconstituted into lipid bilayer membranes. The current noise of the channels was investigated for the different porins in the open state and in the ligand-induced closed state using fast Fourier transformation. The open channel noise exhibited 1/f-noise for frequencies up to 200 Hz. The 1/f-noise was investigated using the Hooge formula (Hooge, Phys. Lett. 29A: 139–140 (1969)), and the Hooge parameter α was calculated for all bacterial porins used in this study. The 1/f-noise was in part caused by slow inactivation and activation of porin channels. However, when care was taken that during the noise measurement no opening or closing of porin channels occurred, the Hooge Parameter α was a meaningful number for a given channel. A linear relationship was observed between α and the single-channel conductance, g, of the different porins. This linear relation between single-channel conductance and the Hooge parameter α could be qualitatively explained by assuming that the passing of an ion through a bacterial porin channel is—to a certain extent—influenced by nonlinear effects between channel wall and passing ion. Received: 8 May 1996/Revised: 27 January 1997     

Native and chemically modified porin channels from Salmonella typhi Ty2 in planar lipid bilayers

Native porins, from Salmonella typhi Ty2 outer membrane, and porins alkylated with pyridoxal phosphate (Plp) were studied in planar lipid bilayers. The conductance of bilayers exposed to native or chemically modified porins increases in discrete jumps. Conductance histograms for native porins displayed two major peaks at 1.7 and 6.7 nS (in 0.5 M KCl). On the other hand, Plp-treated porins exhibited a single major peak at 1 nS. The relation between bilayer conductance and native porin concentration was linear. However, this relation became logarithmic in the presence of modified porins. The results support the notion that alkaline reduction of S. typhi Ty2 porins with Plp dissociates porin channel trimers in a reversible fashion.     

Rectification properties and pH-dependent selectivity of meningococcal class 1 porin

We studied the current rectification properties and selectivity of class 1 porin (PorA) from Neisseria meningitidis (strain H44/76 Δ3Δ4) reconstituted in planar lipid membranes varying salt concentrations and pH. PorA channel shows voltage gating with a characteristic time remarkably longer than other porins. Its current-voltage asymmetry, evaluated as the current rectification ratio, changes nonmonotonically with salt concentration. Interestingly, it reaches its maximum value at physiological concentration. Porin selectivity, quantified by reversal potential measurements, is also significantly asymmetric. Depending on the direction of the salt gradient, the channel becomes more or less selective (10:1 vs. 5:1 Na⁺/Cl⁻). Besides, the reversal potential measurements suggest that porin inserts directionally following the concentration gradient. Measurements over a wide range of pH show that although PorA is strongly cation selective at pH >6, its selectivity gradually changes to anionic in an acidic medium (pH < 4). We show that a continuum electrodiffusion model quantitatively accounts for conductance and reversal potential measurements at positive and negative applied voltages.     

Molecular and functional characterisation of the Serratia marcescens outer membrane protein Omp1

Serratia marcescens outer membrane contains three different general diffusion porins: Omp1, Omp2 and Omp3. Omp1 was cloned and sequenced and it shows a great homology to the family of outer membrane porins that comprises the general porins of enteric bacteria. The gene for Omp1 was transferred into an expression plasmid and was expressed in Escherichia coli UH302 (E. coli UH302 pOM100), a porin deficient strain. Its expression confers a higher susceptibility towards different antibiotics to this strain. Omp1 was purified to homogeneity from outer membrane of E. coli UH302 pOM100. Reconstitution of the purified protein into black lipid bilayers demonstrated that it is a channel-forming component with a single-channel conductance of approximately 2 nS in 1 M KCl similar to that of other porins from enteric bacteria. Omp1 is slightly cation-selective. Its homology to already crystallised members of the family of enteric porins whose three-dimensional-structures are known and allowed the design of a topology model for Omp1. The charge distribution within a porin monomer is similar as in other general diffusion pores. The positively charged amino acids localised at the beta-strands opposite the external loop L3, which restrict the pore diameter in the porin monomer.