Electrophysiological Characteristics of the PhoE Porin Channel from Escherichia coli. Implications for the Possible Existence of a Superfamily of Ion Channels

Berrier, Catherine; Besnard, Madeleine; Ghazi, Alexandre

doi:10.1007/s002329900193

Cited by 26 publications

(32 citation statements)

References 31 publications

(40 reference statements)

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“…Interestingly, in the case of PL/PL membranes the voltage required for closure of PhoE is Յ125 mV in both directions, which is in the same order of magnitude as the proposed transmembrane voltage across the IM. Similar values have also been found in patch-clamp experiments on giant vesicles made from azolectin (33). Therefore, as was previously suggested (34), if trimers were missorted to the IM, pores would be closed to prevent a short circuit destroying the electrochemical potential across the IM.…”

Section: Figsupporting

confidence: 86%

Pore Formation and Function of Phosphoporin PhoE of Escherichia coli Are Determined by the Core Sugar Moiety of Lipopolysaccharide

Hagge

Cock

Gutsmann

et al. 2002

Journal of Biological Chemistry

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The lipid matrix of the outer membrane of Gram-negative bacteria is an asymmetric bilayer composed of a phospholipid inner leaflet and a lipopolysaccharide outer leaflet. Incorporated into this lipid matrix are, among other macromolecules, the porins, which have a sieve-like function for the transport or exclusion of hydrophilic substances. It is known that a reduced amount of porins is found in the outer membrane of rough mutants as compared with wild-type bacteria. This observation was discussed to be caused by a reduced number of insertion sites in the former. We performed electrical measurements on reconstituted planar bilayers composed of lipopolysaccharide on one side and a phospholipid mixture on the other side using lipopolysaccharide from various rough mutant strains of Salmonella enterica serovar Minnesota. We found that pore formation by PhoE trimers that were added to the phospholipid side of the bilayers increased with the increasing length of the lipopolysaccharide core sugar moiety. These results allow us to conclude that the length of the sugar moiety of lipopolysaccharide is the parameter governing pore formation and that no particular insertion sites are required. Furthermore, we found that the voltage gating of the porin channels is strongly dependent on the composition of the lipid matrix.The cell envelope of Gram-negative bacteria consists of the cytoplasmic membrane, the peptidoglycan layer, and an additional barrier, the outer membrane (OM), 1 (1) which is strictly asymmetric with respect to its lipid composition. Whereas the inner membrane (IM) is composed on both sides of phospholipids, the OM consists of a phospholipid inner leaflet and a lipopolysaccharide (LPS) outer leaflet. The LPS consists of an oligo-or polysaccharide portion covalently linked to a lipid component termed lipid A, which anchors the molecule in the membrane (2). In wild-type strains, the polysaccharide portion consists of an O-specific chain and the core oligosaccharide. Rough mutant strains do not express the O-specific chain, but retain core oligosaccharides of varying length. The LPS of various rough mutants are characterized by chemotypes in a sequence of decreasing length of the core sugar as Ra (complete core), Rb, Rc, Rd, and Re. Deep-rough LPS (Re LPS) represents the minimal structure of LPS consisting of only lipid A and two 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) monosaccharides (3) (Fig. 1).The OM protects the cell from harmful agents like antibiotics and toxins and against changes in osmotic pressure. Transmembrane proteins, the porins, allowing the uptake and disposal of small hydrophilic compounds such as nutrients and waste products (4), are assembled in the OM. In Escherichia coli OmpF and OmpC represent the general diffusion pores. The phosphoporin PhoE is synthesized when cells are grown under phosphate limitation (5). PhoE has a molecular weight of M r 36,822 and an exclusion size of M r ϳ600 and is weakly anion selective (6). The crystal structure of PhoE has been solved (7). The channel-formin...

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Section: Figsupporting

confidence: 86%

Pore Formation and Function of Phosphoporin PhoE of Escherichia coli Are Determined by the Core Sugar Moiety of Lipopolysaccharide

Hagge

Cock

Gutsmann

et al. 2002

Journal of Biological Chemistry

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“…Our results confirm the requirement of both PorA and PorH to form a proper ion channel (8). They show in addition that the PorAPorH channel shares the characteristic fingerprints of channels such as bacterial porin (34) and also mitochondrial porin (35): closure at positive and negative potential with fast and slow kinetics, asymmetric voltage dependence, and multiple conductance states. Similar porin channels can be found in related families of bacteria, C. diphtheria, C. efficiens, and N. farcinia (8,36,37).…”

Section: Purification Of C Glutamicum and Cf-expressed Proteins-supporting

confidence: 84%

Functional Expression of the PorAH Channel from Corynebacterium glutamicum in Cell-free Expression Systems

Rath

Demange

Saurel

et al. 2011

Journal of Biological Chemistry

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PorA and PorH are two small membrane proteins from the outer membrane of Corynebacterium glutamicum, which have been shown to form heteromeric ion channels and to be posttranslationally modified by mycolic acids. Any structural details of the channel could not be analyzed so far due to tremendous difficulties in the production of sufficient amounts of protein samples. Cell-free (CF) expression is a new and remarkably successful strategy for the production of membrane proteins for which toxicity, membrane targeting, and degradation are key issues. In addition, reaction conditions can easily be modified to modulate the quality of synthesized protein samples. We developed an efficient CF expression strategy to produce the channel subunits devoid of post-translational modifications.15 N-labeled PorA and PorH samples were furthermore characterized by NMR and gave well resolved spectra, opening the way for structural studies. The comparison of ion channel activities of CF-expressed proteins with channels isolated from C. glutamicum gave clear insights on the influence of the mycolic acid modification of the two subunits on their functional properties.

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“…Two types of channel activity were detected. The first, observed very infrequently and only with some preparations, displayed the characteristics of porin channels, as described (24). These channels had a high conductance, were maximally open at 0 mV, and closed at positive and negative potential with an asymmetric voltage dependence.…”

Section: Resultsmentioning

confidence: 83%

Secretin PulD: Association with pilot PulS, structure, and ion-conducting channel formation

Nouwen

Ranson²,

Saibil³

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

178

234

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The outer membrane protein PulD (secretin) of Klebsiella oxytoca is required for transport of pullulanase across this membrane. We have purified a multimeric PulD complex from an Escherichia coli strain expressing all the proteins involved in pullulanase secretion. The outer membrane-anchored lipoprotein PulS was found to copurify with PulD. The molar ratio of the two proteins is close to 1:1, and the size of the complex is Ϸ1 MDa. Scanning transmission electron and cryo-electron microscopy analyses showed that the purified complex is a cylindrical structure having a central cavity of Ϸ7.6 nm and peripheral radial spokes. Fusion of proteoliposomes containing the purified complex with a planar lipid bilayer resulted in the appearance of small, voltage-activated, ion-conducting channels. We conclude that the central cavity seen in the electron microscope is part of a large gated channel and propose that the observed current f luctuations correspond to voltage-induced, relatively minor displacements of domains in the purified complex rather than to a complete opening of the secretin channel.

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Electrophysiological Characteristics of the PhoE Porin Channel from Escherichia coli. Implications for the Possible Existence of a Superfamily of Ion Channels

Cited by 26 publications

References 31 publications

Pore Formation and Function of Phosphoporin PhoE of Escherichia coli Are Determined by the Core Sugar Moiety of Lipopolysaccharide

Pore Formation and Function of Phosphoporin PhoE of Escherichia coli Are Determined by the Core Sugar Moiety of Lipopolysaccharide

Functional Expression of the PorAH Channel from Corynebacterium glutamicum in Cell-free Expression Systems

Secretin PulD: Association with pilot PulS, structure, and ion-conducting channel formation

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