Kanamycin Uptake into E. Coli Is Facilitated by OmpF and OmpC Porin Channels Located in the Outer Membrane

Abstract

<p>Despite decades of therapeutic application of aminoglycosides, it is still a matter of debate if porins contribute to the translocation of the antibiotics across the bacterial outer membrane. Here, we quantified the uptake of kanamycin across the major porin channels OmpF and OmpC present in the outer membrane of <i>E. coli.</i> Our analysis revealed that, despite its relatively large size, about 10 - 20 kanamycin molecules per second permeate through OmpF and OmpC under a 10 mM concentration gradient, whereas OmpN does not allow the passage. Molecular simulations elucidate the uptake mechanism of kanamycin through these porins. Whole-cell studies with a decisive set of <i>E. coli</i> porin mutants provide evidence that translocation of kanamycin <i>via</i> porins is relevant for antibiotic potency.</p>

Uptake of aminoglycosides through outer membrane porins in Escherichia coli

“…Moreover, antibiotic ions have much lower conductances and thus the single channel conductance values for LamB are only estimates ( Table 1 ). Further the single channel conductance of OmpF, OmpC, ChiP, LamB were also measured for 1M KCl ( Table S3 ) which are in agreement with previous measurements 6,26,32,33 . For this we prepared lipid membranes in the respective electrolyte solution and added the respective protein on one side, usually the ground connected side.…”

“…Our data indicates that kanamycin, amikacin and gentamicin can all translocate through ChiP of E. coli. The permeation of kanamycin through OmpF and OmpC was already previously characterized 26 and revisited here.…”

“…The polycations displace divalent cations cross-bridging neighboring LPS molecules by competitive binding, which results in destabilization and permeabilization of the outer membrane 25 . Our recent study 26 demonstrated the contribution and relevance of the major outer membrane porins OmpF and OmpC in the molecular uptake of kanamycin, one of the most abundantly used aminoglycosides, into E. coli, showing a new diffusion route of these aminoglycosides through outer membrane porins.…”

“…A small volume from the dilution was added to the cis side of the chamber containing 2.5 ml of 1M KCl for single channel measurements. For reversal potential measurements the gradient was created with the pure antibiotic solution 26,36 . Throughout the experiments the pH was maintained at 7.0 ± 0.5.…”

“…Planar lipid bilayer and electrical recording: Single and multichannel measurements. Planar lipid bilayers conferring to Montal and Mueller were formed as published 26,34 . Briefly, an aperture in a Teflon septum with a diameter of 100-120 μm was pre-painted with hexadecane dissolved in n-hexane at 1-5% (v/v) and the cuvette compartments were dried for 10-15 min, in-order to eliminate the solvent.…”