Noise analysis of ion current through the open and the sugar-induced closed state of the LamB channel of Escherichia coli outer membrane: evaluation of the sugar binding kinetics to the channel interior

Nekolla, S.; Andersen, Christian; Benz, Roland

doi:10.1016/s0006-3495(94)80929-4

Cited by 92 publications

(146 citation statements)

References 46 publications

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“…The frequency of closing events is sugar concentration dependent and is related to the k on rate of sugar binding, while the time the channel is in the closed state reflects the k off rate. The power spectrum of the time course of the ion current was fitted with a Lorentzian, yielding k on and k off rates (11,15). In symmetric-sugar conditions (sugar added to both compartments), these rate constants reflect access from and release to both sides of the channel.…”

Section: Methodsmentioning

confidence: 99%

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Sugar Transport through Maltoporin of Escherichia coli : Role of the Greasy Slide

Gelder¹,

Dumas²,

Bartoldus

et al. 2002

J Bacteriol

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The lining of the maltodextrin-specific maltoporin (LamB) channel exhibits a string of aromatic residues, the greasy slide, part of which has been shown previously by crystallography to be involved in substrate binding. To probe the functional role of the greasy slide, alanine scanning mutagenesis has been performed on the six greasy slide residues and Y118 at the channel constriction. The mutants were characterized by an in vivo uptake assay and sugar-induced-current-noise analysis. Crystallographic analysis of the W74A mutant showed no perturbation of the structure. All mutants showed considerably decreased maltose uptake rates in vivo (<10% of the wild-type value), indicating the functional importance of the investigated residues. Substitutions at the channel center revealed appreciably increased (up to 100-fold) in vitro half-saturation concentrations for maltotriose and maltohexaose binding to the channel. Sugar association rates, however, were significantly affected also by the mutations at either end of the slide (W74A, W358A, and F227A), an effect which became most apparent upon nonsymmetrical sugar addition. The kinetic data are discussed on the basis of an asymmetric one-site two-barrier model, which suggests that, at low substrate concentrations, as are found under physiological conditions, only the heights of the extracellular and periplasmic barriers, which are reduced by the presence of the greasy slide, determine the efficiency of this facilitated diffusion channel.Solute uptake into cells of gram-negative bacteria requires the crossing of two membranes, the outer and the inner membranes, which are separated by the periplasmic space (16). The outer membrane can be considered a defense wall protecting the cell against harmful compounds such as toxins and bile salts. Since a complete seal would impede the import of essential nutrients into the cell, several pore-forming proteins (porins) are located in the outer membrane (19). Transport of solutes through these water-filled channels most commonly proceeds by simple diffusion. For specific porins, diffusional transport is aided by the presence of a binding site.Maltoporin (LamB) specifically facilitates the translocation of malto-oligosaccharides across the outer membrane barrier. Its crystal structure (20) is trimeric, with each monomer consisting of an 18-stranded ␤-barrel enclosing a channel. Long loops protrude into the extracellular space, and short turns face the periplasm. Three loops fold into the ␤-barrel, with loop L3 constricting the channel about halfway through. In the channel lining, there are six contiguous aromatic residues, which form an elongated apolar stripe, the greasy slide, extending from the vestibule through the channel constriction to the periplasmic outlet (Fig. 1). The first of these residues, W74, is provided by L2 of a neighboring subunit. The next three residues, Y41, Y6, and W420, are situated in the middle of the channel. They are followed by W358 and F227, which reside in the wide periplasmic exit hall. A comparable ...

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Section: Methodsmentioning

confidence: 99%

“…Association and dissociation rates were derived from sugar-induced current-noise analysis (2,15). The role of the residues of the polar tracks has been investigated in a separate study (8).…”

mentioning

confidence: 99%

Sugar Transport through Maltoporin of Escherichia coli : Role of the Greasy Slide

Gelder¹,

Dumas²,

Bartoldus

et al. 2002

J Bacteriol

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“…Inspection of the porin structure suggests that the permeation of molecules through the channel is driven by molecular interactions with the surface rather than by free diffusion 68,69 . The first evidence for facilitated diffusion through a binding site was found in maltoporin, a maltose-specific channel from the outer membrane in E. coli [70][71][72][73] . a closer look at the OmpF structure revealed that it possesses a possible affinity site for ampicillin molecules (FIG.…”

Section: Physico-chemical Basis Of Porin Transportmentioning

confidence: 99%

“…The average through maltoporin (lamb) led to the development of an asymmetric single-binding-site model [70][71][72][73] The symbols P and aP indicate a channel state that is unoccupied or occupied by a bound antibiotic molecule, respectively; a and a′′ indicate free extracellular and periplasmic substrate, respectively.…”

Section: Box 3 | Electrophysiology Studiesmentioning

confidence: 99%

The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria

2008

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“…Derivation of the Individual Rate Constants of Sugar Binding-The binding of carbohydrates to the maltoporin channels was assumed for a long time to be symmetric (9,12), so that once a sugar molecule is bound inside the pore it would have equal probability to translocate or to exit to the same side. Only recent measurements performed on individual maltoporin trimers demonstrated that the association rate is 3-5-fold different depending on the sugar entrance side (18,19).…”

Section: For Details)mentioning

confidence: 99%

Probing the Orientation of Reconstituted Maltoporin Channels at the Single-protein Level

Danelon

Brando

Winterhalter

2003

Journal of Biological Chemistry

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Recently we have shown that maltoporin channels reconstituted into black lipid membranes have pronounced asymmetric properties in both ion conduction and sugar binding. This asymmetry revealed also that maltoporin insertion is directional. However, the orientation in the lipid bilayer remained an open question. To elucidate the orientation, we performed point mutations at each side of the channel and analyzed the ion current fluctuation caused by an asymmetric maltohexaose addition. In a second series we used a chemically modified maltohexaose sugar molecule with inhibited entry possibility from the periplasmic side. In contrast to the natural outer cell wall of bacteria, we found that the maltoporin inserts in artificial lipid bilayer in such a way that the long extracellular loops are exposed to the same side of the membrane than protein addition. Based on this orientation, the directional properties of sugar binding were correlated to physiological conditions. We found that nature has optimized maltoporin channels by lowering the activation barriers at each extremity of the pore to trap sugar molecules from the external medium and eject them most efficiently to the periplasmic side.

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Noise analysis of ion current through the open and the sugar-induced closed state of the LamB channel of Escherichia coli outer membrane: evaluation of the sugar binding kinetics to the channel interior

Cited by 92 publications

References 46 publications

Sugar Transport through Maltoporin of Escherichia coli : Role of the Greasy Slide

Sugar Transport through Maltoporin of Escherichia coli : Role of the Greasy Slide

The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria

Probing the Orientation of Reconstituted Maltoporin Channels at the Single-protein Level

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