Outer-membrane protein PhoE from Escherichia coli forms anion-selective pores in lipid-bilayer membranes

Porin PhoE of the outer membrane of Escherichia coli was isolated and purified. Reconstitution experiments with lipid bilayer membranes showed that this protein formed pores which had a single channel conductance of 210 pS at 0.1 M KCl. The PhoE pores were obviously not voltage-controlled or regulated. In contrast to pores formed by the OmpF porin from E. coli the PhoE channel was found to be anion-selective at neutral pH. Chloride is about three to ten times more permeable through the pore than alkali ions. On the basis of the observed pH dependence of the permeability ratio of anions and cations, this anionic selectivity is explained by the assumption that the PhoE pore contains an excess of fixed positive charges.     

Modification of the conductance,selectivity and concentration-dependent saturation of Pseudomonas aeruginosa protein P channels by chemical acetylation

Protein P, an anion-specific channel-forming protein from the outer membrane of Pseudomonas aeruginosa was chemically modified by acetylation and syccinylation of its accessible amino groups. The chemically modified protein retained its ability to form oligomers on sodium dodecyl sulfate polyacrylamide gels, whereas only the acetylated protein formed channels in reconstitution experiments with lipid bilayers. Acetylated protein P demonstrated a substantially reduced mean single channel conductance (25 pS at 1 M KCl) compared to the native protein P channels (250 pS at 1 M KCl) when reconstituted into black lipid bilayer membranes. The homogeneous size distribution of single-channel conductances suggested that all of the protein P molecules had been acetylated. Zero-current potential measurements demonstrated that the acetylated protein P channel was only weakly selective for anions and allowed the permeation of cations, in contrast to the native protein P channels, which were more than 100-fold selective for anions over cations. The dependence of conductance on salt concentration was changed upon acetylation, in that acetylated protein P demonstrated a linear concentration-conductance relationship, whereas native protein P channels became saturated at high salt concentrations. These data strongly suggested that the basis of anion selectivity for native protein P channels is fixed amino groups. In agreement with this, we could demonstrate a 2.5-fold decrease in single-channel conductance between pH 7 and pH 9, between which pH values the ?-amino groups of amino acids would start to become deprotonated. Two alternative schemes for the topography of the protein P channel and localization of the fixed amino groups are presented and discussed.     

Ion selectivity of gram-negative bacterial porins.

Twelve different porins from the gram-negative bacteria Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, and Yersinia pestis were reconstituted into lipid bilayer membranes. Most of the porins, except outer membrane protein P, formed large, water-filled, ion-permeable channels with a single-channel conductance between 1.5 and 6 nS in 1 M KCl. The ions used for probing the pore structure had the same relative mobilities while moving through the porin pore as they did while moving in free solution. Thus the single-channel conductances of the individual porins could be used to estimate the effective channel diameters of these porins, yielding values ranging from 1.0 to 2.0 nm. Zero-current potential measurements in the presence of salt gradients across lipid bilayer membranes containing individual porins gave results that were consistent with the conclusions drawn from the single-channel experiments. For all porins except protein P, the channels exhibited a greater cation selectivity for less mobile anions and a greater anion selectivity for less mobile cations, which again indicated that the ions were moving inside the pores in a fashion similar to their movement in the aqueous phase. Three porins, PhoE and NmpC of E. coli and protein P of P. aeruginosa, formed anion-selective pores. PhoE and NmpC were only weakly anion selective, and their selectivity was dependent on the mobility of the ions. In contrast, cations were unable to enter the selectivity filter of the protein P channel. This resulted in a high anion selectivity for all salts tested in this study. The other porins examined, including all of the known constitutive porins of the four gram-negative bacteria studied, were cation selective with a 3- to 40-fold preference for K+ ions over Cl- ions.     

Modification of positional distribution of fatty acids in phosphatidylinositol of rabbit neutrophils stimulated with formylmethionyl-leucyl-phenylalanine

Protein P, an anion-specific channel-forming protein from the outer membrane of Pseudomonas aeruginosa was chemically modified by acetylation and syccinylation of its accessible amino groups. The chemically modified protein retained its ability to form oligomers on sodium dodecyl sulfate polyacrylamide gels, whereas only the acetylated protein formed channels in reconstitution experiments with lipid bilayers. Acetylated protein P demonstrated a substantially reduced mean single channel conductance (25 pS at 1 M KCl) compared to the native protein P channels (250 pS at 1 M KCl) when reconstituted into black lipid bilayer membranes. The homogeneous size distribution of single-channel conductances suggested that all of the protein P molecules had been acetylated. Zero-current potential measurements demonstrated that the acetylated protein P channel was only weakly selective for anions and allowed the permeation of cations, in contrast to the native protein P channels, which were more than 100-fold selective for anions over cations. The dependence of conductance on salt concentration was changed upon acetylation, in that acetylated protein P demonstrated a linear concentration-conductance relationship, whereas native protein P channels became saturated at high salt concentrations. These data strongly suggested that the basis of anion selectivity for native protein P channels is fixed amino groups. In agreement with this, we could demonstrate a 2.5-fold decrease in single-channel conductance between pH 7 and pH 9, between which pH values the epsilon-amino groups of amino acids would start to become deprotonated. Two alternative schemes for the topography of the protein P channel and localization of the fixed amino groups are presented and discussed.     

Role of lysines in ion selectivity of bacterial outer membrane porins

The epsilon-amino groups of available lysine residues of the OmpC, OmpF and PhoE porin proteins of Escherichia coli and of the protein P porin of Pseudomonas aeruginosa, were modified by the bulky reagent trinitrobenzenesulphonic acid. Approximately 78% of the lysines of the anion-selective protein P and PhoE porins were modified whereas only 40-50% of the lysines of the cation selective OmpF and OmpC porins were altered. After modification, the three E. coli porins had very similar high selectivities for cations over anions, in contrast to the native porins which varied 86-fold in ion selectivity. Despite the large size of the trinitrophenyl group attached to modified lysines (i.e., a disc of approx. 0.86 nm diameter X 0.36 nm high) relative to the reported size of the constrictions of the E. coli porins (1.0-1.2 nm diameter), only the anion-selective PhoE porin was substantially blocked after trinitrophenylation. The protein P porin channel was relatively unaffected by trinitrophenylation, in contrast to previous data showing dramatic effects of acetylation of lysines on protein P conductance and selectivity. This favoured a model in which the critical lysines involved in anion binding by protein P were present in a constriction of the channel that was too small for trinitrobenzenesulphonic acid to enter. Overall, the data suggest that both the number and relative position of charged lysines are major determinants of ion selectivity.     

Identification of two general diffusion channels in the outer membrane of pea mitochondria.

Reconstitution experiments were performed on lipid bilayer membranes in the presence of detergent solubilized mitochondrial membranes of pea seedlings (Pisum sativum). The addition of the detergent-solubilized material to the membranes resulted in a strong increase of the membrane conductance. To identify the proteins responsible for membrane activity the detergent extracts were applied to a hydroxyapatite (HTP) column and the fractions were tested for channel formation. The eluate of the column contained a protein which migrated as a single band with an apparent molecular mass of 30 kDa on SDS-PAGE. This channel was identified as the porin of pea mitochondria since it formed voltage-dependent channels with single-channel conductances of 1.5 and 3.7 nS in 1 M KCl and an estimated effective diameter of about 1.7 nm. Further elution of the column with KCl containing solutions yielded fractions which resulted in the formation of transient channels in lipid bilayer membranes. These channels had a single-channel conductance of 2.2 nS in 1 M KCl and had also the characteristics of general diffusion pores with an estimated effective diameter of 1.2 nm. Zero-current membrane potential measurements suggested that pea porin was anion-selective in the open state. The selectivity of the second channel was investigated by the measurement of the reversal potential. It was also slightly anion-selective. Its possible role in the metabolism of mitochondria is discussed.     

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.     

Ion selectivity reversal and induction of voltage-gating by site-directed mutations in the Paracoccus denitrificans porin

The porin from Paracoccus denitrificans, a slightly anion specific outer membrane pore protein, was expressed in Escherichia coli, isolated from inclusion bodies, and refolded in the presence of urea and detergents. The purified recombinant protein was reconstituted into black lipid bilayer membranes and showed no difference in its functional properties in comparison to the native porin isolated from P.denitrificans membranes. To investigate the molecular basis of its ion selectivity and voltage-gating, a series of site-directed mutants was constructed, comprising acidic residues located on the third extracellular loop (L3), which forms the constriction zone of the channel, and basic residues along the opposing barrel wall. Measurements using zero-current membrane potentials indicated that the selectivity changed drastically from a slight anion to a distinct cation selectivity with the exchange of residues R29 and R31 by glutamate, whereas replacements on the L3 loop went largely unaffected. However, when assaying the voltage-dependent closure of channels, only mutations located on the L3 loop showed an effect, in contrast to the voltage-independent recombinant and native Paracoccus porin.     

Bordetella pertussis major outer membrane porin protein forms small, anion-selective channels in lipid bilayer membranes.

The major outer membrane protein of molecular weight 40,000 (the 40K protein) of a virulent isolate of Bordetella pertussis was purified to apparent homogeneity. The purified protein formed an oligomer band (of apparent molecular weight 90,000) on sodium dodecyl sulfate-polyacrylamide gels after solubilization at low temperatures. The porin function of this protein was characterized by the black lipid bilayer method. The 40K protein formed channels smaller than all other constitutive major outer membrane porins studied to date. The average single-channel conductance in 1 M KCl was 0.56 nS. This was less than a third of the conductance previously observed for Escherichia coli porins. Zero-current potential measurements made of the porin to determine its ion selectivity revealed the porin to be more than 100-fold selective for anions over cations. The single-channel conductance was measured as a function of salt concentration. The data could be fitted to a Lineweaver-Burk plot suggesting an anion binding site with a Kd of 1.17 M Cl- and a maximum possible conductance through the channel of 1.28 nS.     

One single lysine residue is responsible for the special interaction between polyphosphate and the outer membrane porin PhoE of Escherichia coli

Site-directed mutagenesis was performed with the phosphate starvation-inducible outer membrane porin PhoE of Escherichia coli K-12 to study the molecular basis of its anion selectivity. Lysines 18, 29, 64, and 125 were replaced by glutamic acids, and the properties of the mutant porins were investigated in in vivo and in vitro experiments. Lipid bilayer experiments showed that all these mutations had no influence on the pore structure because PhoE and the mutants had the same single channel conductance in KCl solution. Selectivity measurements revealed that the mutations changed the ionic selectivity of PhoE, but the change was dependent on the location of the lysine. Replacement of Lys18 and Lys29 by glutamic acid had a relatively small influence. The effect of the Lys64 substitution was somewhat larger, and the effect of the replacement of Lys125 resulted in the most drastic change in selectivity and in the loss of the interaction of PhoE with polyphosphate, whereas the replacement of the other lysines had no effect on the polyphosphate interaction behavior. The results are consistent with the assumption that the charge spot in PhoE consists of only 1 lysine per monomer, located in position 125 of the primary sequence and probably close to the pore interior.     

Porin from Thiobacillus versutus

The porin of Thiobacillus versutus IFO 14567 was isolated by extraction of cell-envelopes with sodium dodecyl sulfate. It exhibited strong porin-activity after reconstitution into artificial lipid bilayer membranes. The diameter of the pore was determined as 1.6 nm, with a weak selectivity for cations being observed. The porin migrated as a single band (Mr 35 kDa) on SDS-polyacrylamide gel-electrophoresis after heating (100 degrees C, 5 min). The porin oligomer was not sensitive towards EDTA. Analytical ultracentrifugation studies demonstrated the native oligomer to be a trimer.     

Characterisation of channels induced in planar bilayer membranes by detergent solubilised Escherichia coli porins

Purified OmpF, OmpC, NmpC, PhoE and Lc (Protein 2) porins from the Escherichia coli outer membrane were incorporated into planar phospholipid bilayer membranes and the permeability properties of the pores studied. Triton X-100 solubilised porin samples showed large and reproducible increases in membrane conductivity composed of discreet single-channel events. The magnitude of the cation selectivity found for the porins was in the order OmpC greater than OmpF greater than NmpC = Lc; PhoE was anion selective. For the cation selective porins the cation/anion permeability ratios in a variety of solutes ranged from 6 to 35. Further information on the internal structure of the porins was obtained by examination of the single-channel conductance and this was used to interpret macroscopic observations and to estimate single-channel diameters. The same porins solubilised in SDS exhibited slight conductance increase with no observable single-channel activity. Use of on-line microcomputer techniques confirmed the ohmic current vs. voltage behaviour for all the single porin channels examined.     

Pore forming activity of the major outer membrane protein of Rhodobacter capsulatus in lipid bilayer membranes

Porin of the outer membrane of Rhodobacter capsulatus St. Louis (ATCC 23782) was isolated and reconstituted into lipid bilayer membranes. The porin was obtained either by the sodium dodecyl sulfate treatment of cell envelopes (SDS-porin) or by saline extraction of whole cells (NaCl-porin). Nanomolar concentrations of both porin preparations resulted in a strong conductance increase of the lipid bilayer membranes by many orders of magnitude. At small protein concentrations the conductance increased in a stepwise fashion, the average single channel conductance being about 0.35 nS in 0.1 M KCl for SDS-porin and NaCl-porin as well. The single channel conductance was a linear function of the specific conductance of the aqueous phase. The results were consistent with the assumption that the porin formed large water-filled transmembrane channels in the membrane. From the average value of the single channel conductance in 0.1 M KCl an effective channel diameter of about 1.5 nm was estimated for both types of porins.Abbreviations EDTA ethylenediamine tetraacetic acid - SDS sodium dodecyl sulfate     

Ion selectivity of colicin E1: Modulation by pH and membrane composition

Colicin E1 is a plasmid-encoded bacteriocidal protein which, though water soluble when secreted by its host bacterium, spontaneously interacts with planar lipid bilayers to form voltage-gated ion channels. In asolectin bilayers, the preference for anions over cations exhibited by these channels at low pH can be reversed by raising the pH on either side of the membrane. When incorporated into membranes composed of either of the two zwitterionic lipids, bacterial phosphatidylethanolamine and diphytanoyl phosphatidylcholine, colicin E1 channels were nearly ideally anion selective in the limit of low pH and moderately cation selective at the high pH limit. In phosphatidylcholine membranes, however, the response of these channels to changes in pH exhibited a pattern of behavior peculiar to this lipid. If the side of the membrane on which the protein had been introduced (the cis side) was exposed to pH 4.0, all the channels in the bilayer, whether opened or closed, became refractory to further changes in pH. This irreversibility has been interpreted as evidence that the selectivity of colicin E1 is under the control of a pH-sensitive conformational change. Protonation of groups on the cis side of the membrane appear to be essential to the conversion to the anion-selective state. These groups are rendered kinetically inaccessible to the aqueous phase when the transition takes place in phosphatidylcholine membranes.     

The cell wall porin of the gram-positive bacterium Nocardia asteroides forms cation-selective channels that exhibit asymmetric voltage dependence

Detergent-solubilized cell wall extracts of the gram-positive, strictly aerobic bacterium Nocardia asteroides contain channel-forming activity as judged from reconstitution experiments using lipid bilayer membranes. The cell wall porin was identified as a protein with an apparent molecular mass of about 84 kDa based on SDS-PAGE. The porin was purified to homogeneity using preparative SDS-PAGE. The 84-kDa protein was no longer observed after heating in SDS buffer. The presumed dissociation products were not observed on SDS-polyacrylamide gels. The cell wall porin increased the specific conductance of artificial lipid bilayer membranes from phosphatidylcholine/phosphatidylserine mixtures by the formation of cation-selective channels, which had an average single-channel conductance of 3.0 nS in 1 M KCl. The single-channel conductance was only moderately dependent on the bulk aqueous KCl concentration, which indicated negative point charge effects on the channel properties. The analysis of the concentration dependence of the single-channel conductance using the effect of negative charges on channel conductance suggested that the diameter of the cell wall channel is about 1.4 nm. Asymmetric addition of the cell wall porin to lipid bilayer membranes resulted in an asymmetric voltage dependence. The cell wall channel switched into substates, when the cis side of the membrane, the side of the addition of the protein, had negative polarity. Positive potentials at the cis side had no influence on the conductance of the cell wall channel. Received: 23 September 1998 / Accepted: 9 December 1998     

Pores from mitochondrial outer membranes of yeast and a porin-deficient yeast mutant: A comparison

Reconstitution experiments were performed on lipid bilayer membranes in the presence of purified mitochondrial porin from yeast and of detergent-solubilized mitochondrial outer membranes of a porin-free yeast mutant. The addition of the porin resulted in a strong increase of the membrane conductance, which was caused by the formation of ion-permeable channels in the membranes. Yeast porin has a single-channel conductance of 4.2 nS in 1 M KCl. In the open state it behaves as a general diffusion pore with an effective diameter of 1.7 nm and possesses properties similar to other mitochondrial porins. Surprisingly, the membrane conductance also increased in the presence of detergent extracts of the mitochondrial outer membrane of the mutant. Single-channel recordings of lipid bilayer membranes in the presence of small concentration of the mutant membranes suggested that this membrane also contained a pore. The reconstituted pores had a single-channel conductance of 2.0 nS in 1 M KCl and the characteristics of general diffusion pores with an estimated effective diameter of 1.2 nm. This means that the pores present in the mitochondrial outer membranes of the yeast mutant have a much smaller effective diameter than normal mitochondrial porins. Zero-current membrane potential measurements suggested that the second mitochondrial porin is slightly cation-selective, while yeast porin is slightly anion-selective in the open state but highly cation-selective in the closed state. The possible role of these pores in the metabolism of mitochondria is discussed.     

Interaction of Clostridium perfringens iota-toxin with lipid bilayer membranes. Demonstration of channel formation by the activated binding component Ib and channel block by the enzyme component Ia.

The interaction between model lipid membranes and the binding component (Ib) of the ADP-ribosylating iota-toxin of Clostridium perfringens was studied in detail. Ib had to be activated by trypsin to result in channel formation in artificial lipid bilayers. The channels formed readily by Ib had a small single-channel conductance of about 85 picosiemens in 1 m KCl. Channel function was blocked in single-channel and multichannel experiments by the enzymatic component Ia in a pH-dependent manner. The strong Ia-mediated channel block of Ib occurred only when the pH was at least lowered to pH 5.6. The single-channel conductance showed a linear dependence on the bulk aqueous KCl concentration, which indicated that the channel properties were more general than specific. Zero current membrane potential measurements suggested the Ib channel has an approximately 6-fold higher permeability for potassium ions than for chloride. The selectivity ratio changed for salts composed of cations and anions of different mobility in the aqueous phase, again suggesting that Ib formed a water-filled general diffusion pore. Asymmetric addition of activated Ib to lipid bilayer membranes resulted in an asymmetric voltage dependence, indicating its full orientation within the membrane. Titration experiments with chloroquine and different tetraalkylammonium ions suggested that the Ib channel was blocked by these compounds but had only a weak affinity to them. In vivo measurements using Vero cells demonstrate that chloroquine and related molecules also did not efficiently block intoxication of the cells by iota-toxin. The possible role of Ib in the translocation of iota-toxin across the target cell membrane is discussed.     

Outer membrane porin protein of Campylobacter jejuni

Abstract Protein e, a 43-kDa protein from the outer membrane of Campylobacter jejuni UA580, was purified and reconstituted into lipid bilayer membranes. It was shown to form small channels with a single channel conductance of 8.82 nS in 1M KCl. Zero current potential measurements demonstrated that the channel was approx. 10-fold selective for K⁺ over Cl⁻ ions. A porin with a similar single channel conductance was observed in fractions from the outer membrane of Campylobacter fetus UA60.     

Salmonella typhimurium contains an anion-selective outer membrane porin induced by phosphate starvation.

A mutant of Salmonella typhimurium was selected that is constitutive for the pho regulon. It exhibited constitutive glycerol-3-phosphate transport activity and synthesized a new outer membrane porin. Upon measurement of porin activity in black lipid films, it exhibited anion selectivity. It therefore appears analogous to the Escherichia coli PhoE porin.     

Evidence for the Presence of a Porin in the Membrane of Glyoxysomes of Castor Bean

Glyoxysomes of endosperm tissue of castor bean (Ricinus communis L.) seedlings were solubilized in a detergent and added to a lipid bilayer. Conductivity measurements revealed that the glyoxysomal preparation contained a porin-like channel. Using an electrophysiological method, which we established for semiquantitative determination of porin activity, we were able to demonstrate that glyoxysomal membranes purified by sucrose density gradient centrifugation contain an integral membrane protein with porin activity. The porin of glyoxysomes was shown to have a relatively small single-channel conductance of about 330 picosiemens in 1 M KCl and to be strongly anion selective. Thus, the glyoxysomal porin differs from the other previously characterized porins in the outer membrane of mitochondria or plastids, but is similar to the porin of spinach (Spinacia oleracea L.) leaf peroxisomes. Our results suggest that, in analogy to the porin of leaf peroxisomes, the glyoxysomal porin facilitates the passage of small metabolites, such as succinate, citrate, malate, and aspartate, through the membrane.