The voltage-dependent activity of Escherichia coli porins in different planar bilayer reconstitutions (original) (raw)
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Channel-closing activity of porins from Escherichia coli in bilayer lipid membranes
Biochimica Et Biophysica Acta-biomembranes, 1986
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 t 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 sized conductance channesls 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 n 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.
European Biophysics Journal, 2002
The mechanism by which the membrane potential closes and opens voltage-dependent b-barrel membrane channels is not fully understood. OmpC porins form trimeric water-filled channels when incorporated into artificial bilayers, each monomer having a conductance of approximately 510 pS in 1 M KCl. These channels are relatively insensitive to membrane potential difference (pd) and close only when the pd exceeds ±250 mV. Another well-known trimer, OmpF, has a monomer conductance of approximately 780 pS in 1 M NaCl, is more sensitive to pd, and can be closed reversibly when a pd of more than ±150 mV is applied to the channel-containing membranes. With the aid of the 3D atomic structure of these channels determined by X-ray crystallography, and using site-directed mutagenesis, specific amino acids can be substituted in desired locations in the channel lumen. In this study we have used mutants 37C and 74C and attached fluorescence probes to them to monitor polarity changes in the channel lumen during gating. From the observed changes in polarity, we conclude that conformational changes occur in the channel which interrupt the electrolyte conducting pathway.
E.coli PhoE porin has an opposite voltage-dependence to the homologous OmpF
The EMBO Journal, 1998
We used patch clamp analysis to compare the electrophysiological behavior of two related porins from Escherichia coli, the anion-specific PhoE and the cationselective 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 cationselective 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.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1978
One of the major proteins of the outer membrane of Escherichia coli, the matrix protein (porin), has been isolated by detergent solubilisation. When the protein is added in concentrations of the order of 10 ng/cm 3 to the outer phases of a planar lipid bilayer membrane, the membrane conductance increases by many orders of magnitude. At lower protein concentrations the conductance increases in a stepwise fashion, the single conductance increment being about 2 nS (1 nS = 10-9 siemens = 10-9 ~-~) in 1 M KC1. The conductance pathway has an ohmic current vs. voltage character and a poor selectivity for chloride and the alkali ions. These findings are consistent with the assumption that the protein forms large aqueous channels in the membrane. From the average value of the single-channel conductance a channel diameter of about 0.9 nm is estimated. This channel size is consistent with the sugar permeability which has been reported for lipid vesicles reconstituted in the presence of the protein.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1989
Lipid bilayer experiments were pedonned with one OmpF-PhoE and several OmpC-PlmE hybrid porins of Escherichia co//K-12. All hybrid pores had approximately the same pore-forming activity, which indicated that the stnteture of the pores remained essentially unchanged by the genetic manipulation. This result was supported by sin,tie-channel experiments t~cause all pores had similar single-channel conductanees in potassium chloride. Measurements with other salts indicated a drastic change in the ionic selectivity when the fusion site in the ompC.phoE hybrid genes passed along the sequence of the porius from the N-terminal to the C-terminal end, Selectivity measurements using z~t membrane potentials showed that the selectivity suddenly changed from anion to cation selectivity when, relatively short portion from the N-terminal end of PhoE was replaced by the corresponding part of OmpC, The replacement of increasing portieus led to an increase in the cation selectivity until that of OmpC was reached. The change in the anion to cation selectivity is correlated with exchange of lysine-18 and serine-28 by aspmic acidL The anion saecttvlty of the phosphate starvation-inducible PhoE porin is closely related to the presence of several lysines spread along the primary sequence of the polypeptide chain.
Properties of chemically modified porin from Escherichia coli in lipid bilayer membranes
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1984
Purified porin OmpF from Escherichia coil 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.
Native and chemically modified porin channels fromSalmonella typhiTy2 in planar lipid bilayers
FEBS Letters, 1986
Native porins, from Salmonella typhi Ty2 outer membrane, and porins alkylated with pyridoxal phosphate (Pip) 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 KCI). 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 Pip dissociates porin channel trimers in a reversible fashion.
Ion selectivity of gram-negative bacterial porins
Journal of Bacteriology, 1985
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 cat...