Polarity-dependent voltage-gated porin channels from Escherichia coli in lipid bilayer membranes.

A porin preparation from Escherichia coli 0111:B4 consisting of Omp F and Omp C (with Omp F in excess) was purified by salt extraction procedures and investigated in bilayer lipid membranes formed according to the Montal-Mueller technique. The porin preparation was added to the KCl electrolyte compartment of the Montal-Mueller cell which was connected to the voltage source. As the porin incorporated into the membrane, asymmetric, voltage-gated ion channels were formed. Transmembrane voltages greater than +50 mV (measured with respect to the side of porin addition) caused channel closing, while negative voltages, on the other hand, had no effect on channel behaviour but did increase the rate of porin incorporation at higher voltages. With porin added to both compartments voltage gating no longer occurred. Single-channel conductances corresponded to effective pore diameters of 1.5 nm for opening events and 1.18 nm for channel closing events. The number of charges involved in gating was approximately 2.     

Permeability properties of chemically modified porin trimers from Escherichia coli B

The pore-forming protein of the outer membrane of Escherichia coli, porin, was chemically modified with acetic anhydride, succinic anhydride, and glycinamide. Extensive modification of amino groups of the functional porin trimers caused reduced diffusion rates of the negatively charged solutes such as p-nitrophenyl phosphate and AMP, but did not reduce significantly the diffusion of positively charged molecules carbobenzoxy-glycyl-prolyl-arginine-p-nitranilide and tosyl-glycyl-prolyl-arginine-p-nitranilide. Modification of carboxyl groups of trimers caused decreased diffusion rates of the positively charged solutes more significantly than the diffusion rates of negatively charged solutes. The results suggest that the ionic interactions play an important role for the diffusion of charged solutes through the porin pore. The diffusion of p-nitrophenyl alpha-D-glucoside, an uncharged solute, ws not influenced significantly by modification of either amino or carboxyl groups. This observation suggests that modifications only occurred in areas outside of the narrowest portion of the pore or, alternatively, that amino and carboxyl groups are exclusively located at noncylindrical area of the pore. The structural integrity of the acetylated and the succinylated trimers seemed well preserved. On the other hand, modification of carboxyl groups decreased the thermal stability of trimers and extensive modifications caused the dissociation of trimers into monomers at 37 degrees C.     

Channel-closing activity of porins from Escherichia coli in bilayer lipid membranes

The opening and closing of the ompF porin from Escherichia coli JF 701 was investigated by reconstituting the purified protein into planar bilayer membranes. The electrical conductance changes across the membranes at constant potential were used to analyze the size and aggregate nature of the porin channel complexes and the relative number of opening and closing events. We found that, when measured at pH 5.5, the channel conductance diminished and the number of closing events increased when the voltage was greater than 100 mV. The results suggest that the number of smaller sized conductance channels increases above this potential. There was also an increase in the smaller subunits and in the closing events when the pH was lowered to 3.5, and these changes were further enhanced by increasing the voltage. We propose that both lowering the pH and elevating the potential across the membrane stabilize the porin in a conformation in which the subunits are less tightly associated and the subunits open in a non-cooperative manner. These same conditions also appear to stabilize the closed state of the pore.     

Properties of the porin of Haemophilus influenzae type b in planar lipid bilayer membranes

The major outer membrane protein (40 kDa) of the bacterium Haemophilus influenzae type b is a porin which forms transmembrane permeability channels. It has an exclusion limit for oligosaccharides of about 1.4 kDa. When this protein was added to the aqueous phase which was bathing a planar lipid bilayer, it caused the conductance of the membrane to increase by several orders of magnitude. At low protein concentrations (2-10 pM), the conductance of the membrane increased in a stepwise fashion with an average single-channel conductance of 1.1 nS in 1 M KCl. Single-channel experiments were performed with a variety of different salts. The conductance of single channels was proportional to the specific conductance of the aqueous solution which was bathing the membrane. Current through the pores was proportional to the applied voltage, indicating that these pores are not voltage-controlled. The 40 kDa porin was very slightly cation-selective: the pores were about 1.6-times more permeable to potassium ions than to chloride ions. These properties of the 40 kDa porin are those of large water-filled channels and are characteristic of most bacterial porins. The single-channel conductance of the porin is, however, much smaller than might be expected from its exclusion limit. A model is proposed which could explain the differences in apparent pore size.     

Pore formation by LamB of Escherichia coli in lipid bilayer membranes.

Lipid bilayer experiments were performed in the presence of different Escherichia coli LamB preparations. These LamB preparations formed two types of pores in the membranes. Large pores, which had a single-channel conductance of 2.7 nS and comprised about 1 to 6% of the total pores, were presumably contaminants which might have been induced together with LamB. LamB itself formed small pores with a single-channel conductance of 160 pS in 1 M KCl. These pores could be completely blocked by the addition of maltose and maltodextrins. Titration of the pore conductance with maltotriose suggested that there was a binding site inside the pores with a Ks of 2.5 X 10(-4) M for maltotriose. On the basis of our data we concluded that the structure of the LamB channels is quite different from the structures of the channels of general diffusion porins, such as OmpF and OmpC.     

Pore formation by the mitochondrial porin of rat brain in lipid bilayer membranes

The porin of the outer membrane of rat-brain mitochondria was isolated and purified. The protein showed a single band of apparent Mr 35,500 on dodecyl sulfate-containing polyacrylamide gels. The incorporation of rat-brain porin into artificial lipid bilayer membranes showed that it is able to form pores with an average single-channel conductance of 400 pS in 0.1 M KCI. The pores were found to be voltage-dependent and switched to substrates at higher transmembrane potentials. The voltage-dependence of the rat brain pore was considerably smaller than that of the other known eukaryotic porins. The possible role of the rat-brain porin in the regulation of transport process across the outer mitochondrial membrane is discussed.     

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.     

Ionic selectivity of bilayer lipid membranes modified by triforine

It is found that bilayer lipid membranes acquire little cationic selectivity in the presence of systemic fungicide triforine at physiological pH, and besides potassium selectivity exceeds the sodium one. A decrease of pH to 3.5 leads to substitution of cationic selectivity by the anionic one. It is suggested that selectivity of the membranes modified by triforine is determined both by charge and dipole moments of the fungicide molecule.     

Properties of lipid bilayer membranes made from lipids containing phytanic acid

Besides the preparation of phytanic acid (3,7,11,15-tetramethylhexadecylic acid) according to the Dumas-Stass reaction, the synthesis of four different lipids containing phytanic acid residues is described. Diphytanoyl phophatidylcholine was systhesized beginning from glycerylphosphorylcholine, whereas the other lipids, diphytanoyl phosphatidylethanolamine, diphytanoyl phosphatidylserine and monophytanoyl glyceride were prepared by total synthesis.Some properties of lipid bilayer membranes made from the lipids containing phytanic acid were investigated. The specific capacity of these membranes was measured. Its value of approximately 400 nF cm^?2 was found to be similar to the value of membranes from lipids with unbranched fatty acid residues. Charge pulse experiments were performed using dipicrylamine as a molecular probe of membrane structure. The results were discussed on the basis of a higher viscosity of the membranes from lipids containing phytanic acid residues compared with unbranched fatty acid residues.     

Trigonal crystals of porin from Escherichia coli

Trigonal crystals of the integral membrane protein porin from Escherichia coli have been grown and characterized. They belong to space group P321 with unit cell constants a = b = LL8.4, c = 52.7 A, alpha = beta = 90 degrees, gamma = 120 degrees. The crystals grow as well-defined hexagonal prisms to a size of 0.25 mm in all dimensions, and diffract to 2.7 A. The molecular symmetry coincides with 3-fold crystallographic symmetry, giving two trimers per unit cell (1 monomer/asymmetric unit). This corresponds to VM = 2.9 A3/Da. Native X-ray data to 3.0 A resolution have been collected on a FAST area detector and a search for heavy atom derivatives is underway.     

Paradoxical lipid dependence of pores formed by the Escherichia coli alpha-hemolysin in planar phospholipid bilayer membranes

alpha-Hemolysin (HlyA) is an extracellular protein toxin (117 kDa) secreted by Escherichia coli that targets the plasma membranes of eukaryotic cells. We studied the interaction of this toxin with membranes using planar phospholipid bilayers. For all lipid mixtures tested, addition of nanomolar concentrations of toxin resulted in an increase of membrane conductance and a decrease in membrane stability. HlyA decreased membrane lifetime up to three orders of magnitude in a voltage-dependent manner. Using a theory for lipidic pore formation, we analyzed these data to quantify how HlyA diminished the line tension of the membrane (i.e., the energy required to form the edge of a new pore). However, in contrast to the expectation that adding the positive curvature agent lysophosphatidylcholine would synergistically lower line tension, its addition significantly stabilized HlyA-treated membranes. HlyA also appeared to thicken bilayers to which it was added. We discuss these results in terms of models for proteolipidic pores.     

Supported bilayer lipid membranes modified with a phosphate ionophore

This article reports the electrical responses of a phosphate ionophore, the cyclic polyamine 3-decyl-1,5,8-triazacyclodecane-2,4-dione (N₃-cyclic amine) incorporated into metal supported bilayer lipid membranes (s-BLM). Teflon coated silver wire was used as a support. In a potentiometric mode, the ionophore had a response that was linearly related to the logarithm of HPO₄²⁻ concentration and was also dependant on pH. Selectivity coefficients for other anions compared to HPO₄²⁻ ions, determined by the separate solution method, fell within the range 1.73 × 10⁻⁴ to 6.38 × 10⁻².     

A porin activity of purified lambda-receptor protein from Escherichia coli in reconstituted vesicle membranes.

The receptor protein for bacteriophage λ was purified to homogeneity from a mutant strain of $\text{Escherichia coli}$ K-12 producing reduced amounts of porin. In the reconstituted vesicle membranes the λ-receptor formed permeability channels that allowed the diffusion of maltose, lactose, sucrose, raffinose, amino acids, and nucleosides, but essentially not of stachyose. The permeability channels made of λ-receptor thus had a relatively low specificity for solute molecules. The active form of the protein seemed to be an oligomer of λ-receptor proteins.     

Outer membrane protein K of Escherichia coli: purification and pore-forming properties in lipid bilayer membranes.

Protein K, a recently described outer membrane protein correlated with encapsulation in Escherichia coli (Paakkanen et al., J. Bacteriol. 139:835-841, 1979), has been purified to apparent homogeneity. Purification was based upon the noncovalent association of protein K with peptidoglycan, and the purified protein was shown to form sodium dodecyl sulfate-resistant oligomers on polyacrylamide gels. Incorporation of small amounts (10(-10) to 10(-11) M) of purified protein K into artificial lipid bilayers resulted in an increase, by many orders of magnitude, in membrane conductance. The increased conductance resulted from the formation of large, water-filled, ion-permeable channels exhibiting single-channel conductance in 1.0 M KCl of 1.83 nS. The membrane conductance showed a linear relationship between current and applied voltage and was not voltage induced or regulated. The channel was permeable to large organic ions (e.g., Tris+ Cl-) and, based upon a pore length of 7.5 nm, a minimum channel diameter of 1.2 nm was estimated; these properties resemble values for other enteric porins. The possible biological role of the pores produced by protein K is discussed.     

Charge-pulse relaxation studies with lipid bilayer membranes modified by alamethicin

Charge-pulse relaxation studies with the alamethicin-lipid membrane system reveal a triphasic decay of membrane voltage. At short times (resolution time 2 microseconds), where a voltage decay due to the orientation of alamethicin dipoles from the interface into the membranes interior ("gating current") could possibly be expected, only a slow decrease with a time constant determined by the bare membrane conductance occurs. After approximately 1 ms (depending on the experimental conditions) the formation of alamethicin pores starts, leading to an increase in the voltage decay rate. When the characteristic voltage Vcpc is approached, pores close and after passing Vcpc the voltage decreases slowly again according to the bare membrane conductance. Vcpc is determined as a function of the initially applied voltage Vo, alamethicin and KCl concentration. Since the membrane voltage decreases continuously, the system does not reach the equilibrium states obtained at constant voltages. Taking the presented experimental results into account the estimate of the electrical potential at the functional membrane of photosynthesis induced by a saturating single turnover flash of deltaphio approximately 105-135 mV (Zickler, Witt and Boheim (1976) FEBS Lett. 66, 142-148) is changed to deltaphio approximately 200 mV.     

Photoelectrospectrometry of bilayer lipid membranes

Three different bilayer lipid membrane systems were studied under visible and ultraviolet illumination. The first system consisted of a bilayer lipid membrane formed with a mixture of phospholipids and cholesterol, to one side of which purple membrane fragments from Halobacterium halobium were added. The second system consisted of a membrane formed from spinach chloroplast extract. When either of these membrane systems was illuminated with ultraviolet and visible radiation, photopotentials were observed and photoelectric action spectra were recorded (the technique is termed photoelectrospectrometry). Each spectrum had a definite structure which was characteristic of each of the modified membranes. The third system studied consisted of an otherwise photoinactive membrane formed with a mixture of phospholipids and cholesterol, to one side of which chymotrypsin was added. When the membrane was illuminated with visible light no photoresponse was observed. On the other hand, a photopotential which increased with incubation time was observed when the membrane was illuminated with ultraviolet light. Since, in our systems, the photoresponses have been observed to be due to certain species incorporated into the membrane, it appears that photoelectrospectrometry is a useful tool for studying lipid-protein interactions, constituent organization and energy transfer in membranes.     

Lipopolysaccharide-free Escherichia coli OmpF and Pseudomonas aeruginosa protein P porins are functionally active in lipid bilayer membranes.

Escherichia coli porin OmpF and Pseudomonas aeruginosa porin protein P were eluted from sodium dodecyl sulfate-polyacrylamide gels. The resultant porin preparations were found to be devoid of detectable lipopolysaccharide (LPS) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining for LPS, direct enzyme-linked immunosorbent assays with LPS-specific monoclonal antibodies, and 2-keto-3-deoxyoctulosonic acid assays. The average conductances, ionic selectivities and incorporation rates of the electroeluted porins were identical to those of their conventionally purified counterparts. These data suggest that LPS is not required per se for porin function.