<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>

Quantifying the passage of the large peptide protamine (Ptm) across CymA, a passive channel for cyclodextrin uptake, is in the focus of this study. Using a reporter-pair based fluorescence membrane assay we detected the entry of Ptm into liposomes containing CymA. The kinetics of the Ptm entry was independent of its concentration suggesting that the permeation across CymA is the rate-limiting factor. Furthermore, we reconstituted single CymA channels into planar lipid bilayers and recorded the ion current fluctuations in the presence of Ptm. To this end, we were able to resolve the voltage-dependent entry of single Ptm peptide molecules into the channel. Extrapolation to zero voltage revealed about 1-2 events per second and long dwell times, in agreement with the liposome study. Applied-field and steered molecular dynamics simulations provided an atomistic view on the permeation. It can be concluded that a concentration gradient of 1 M Ptm leads to a translocation rate of about 1 molecule per second and per channel.

We report that the dynamics of antibiotic capture and transport across a voltage-biased OmpF nanopore is dominated by the electroosmotic flow rather than the electrophoretic force. By reconstituting an OmpF porin in an artificial lipid bilayer and applying an electric field across, we are able to elucidate the permeation of molecules, and their mechanism of transport. This field gives rise to an electrophoretic force acting directly on a charged substrate, but also indirectly via coupling to all other mobile ions causing an electroosmotic flow. The directionality and magnitude of this flow depends on the selectivity of the channel. Modifying the charge state of three different substrates (Norfloxacin, Ciprofloxacin, and Enoxacin) by varying the pH between 6 and 9, while the charge and selectivity of OmpF is conserved, allows us to work under conditions where EOF and electrophoretic forces add or oppose. This configuration allows us to identify and distinguish the contributions of the electroosmotic flow and the electrophoretic force on translocation. Statistical analysis of the resolvable dwell-times reveals rich kinetic details regarding the direction and the stochastic movement of antibiotics inside the nanopore. We quantitatively describe the electroosmotic velocity component experienced by the substrates, and their diffusion coefficients inside the porin with an estimate of the energy barrier experienced by the molecules, caused by the interaction with the channel wall, slowing down the permeation by several orders of magnitude.

<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>

Quantifying the passage of the large peptide protamine (Ptm) across CymA, a passive channel for cyclodextrin uptake, is in the focus of this study. Using a reporter-pair based fluorescence membrane assay we detected the entry of Ptm into liposomes containing CymA. The kinetics of the Ptm entry was independent of its concentration suggesting that the permeation across CymA is the rate-limiting factor. Furthermore, we reconstituted single CymA channels into planar lipid bilayers and recorded the ion current fluctuations in the presence of Ptm. To this end, we were able to resolve the voltage-dependent entry of single Ptm peptide molecules into the channel. Extrapolation to zero voltage revealed about 1-2 events per second and long dwell times, in agreement with the liposome study. Applied-field and steered molecular dynamics simulations provided an atomistic view on the permeation. It can be concluded that a concentration gradient of 1 M Ptm leads to a translocation rate of about 1 molecule per second and per channel.