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Modulation of enrofloxacin binding in OmpF by Mg(2+) as revealed by the analysis of fast flickering single-porin current

One major determinant of the efficacy of antibiotics on Gram-negative bacteria is the passage through the outer membrane. During transport of the fluoroquinolone enrofloxacin through the trimeric outer membrane protein OmpF of Escherichia coli, the antibiotic interacts with two binding sites within...

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Autores principales: Brauser, Annemarie, Schroeder, Indra, Gutsmann, Thomas, Cosentino, Cristian, Moroni, Anna, Hansen, Ulf-Peter, Winterhalter, Mathias
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Rockefeller University Press 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3382719/
https://www.ncbi.nlm.nih.gov/pubmed/22689827
http://dx.doi.org/10.1085/jgp.201210776
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author Brauser, Annemarie
Schroeder, Indra
Gutsmann, Thomas
Cosentino, Cristian
Moroni, Anna
Hansen, Ulf-Peter
Winterhalter, Mathias
author_facet Brauser, Annemarie
Schroeder, Indra
Gutsmann, Thomas
Cosentino, Cristian
Moroni, Anna
Hansen, Ulf-Peter
Winterhalter, Mathias
author_sort Brauser, Annemarie
collection PubMed
description One major determinant of the efficacy of antibiotics on Gram-negative bacteria is the passage through the outer membrane. During transport of the fluoroquinolone enrofloxacin through the trimeric outer membrane protein OmpF of Escherichia coli, the antibiotic interacts with two binding sites within the pore, thus partially blocking the ionic current. The modulation of one affinity site by Mg(2+) reveals further details of binding sites and binding kinetics. At positive membrane potentials, the slow blocking events induced by enrofloxacin in Mg(2+)-free media are converted to flickery sojourns at the highest apparent current level (all three pores flickering). This indicates weaker binding in the presence of Mg(2+). Analysis of the resulting amplitude histograms with β distributions revealed the rate constants of blocking (k(OB)) and unblocking (k(BO)) in the range of 1,000 to 120,000 s(−1). As expected for a bimolecular reaction, k(OB) was proportional to blocker concentration and k(BO) independent of it. k(OB) was approximately three times lower for enrofloxacin coming from the cis side than from the trans side. The block was not complete, leading to a residual conductivity of the blocked state being ∼25% of that of the open state. Interpretation of the results has led to the following model: fast flickering as caused by interaction of Mg(2+) and enrofloxacin is related to the binding site at the trans side, whereas the cis site mediates slow blocking events which are also found without Mg(2+). The difference in the accessibility of the binding sites also explains the dependency of k(OB) on the side of enrofloxacin addition and yields a means of determining the most plausible orientation of OmpF in the bilayer. The voltage dependence suggests that the dipole of the antibiotic has to be adequately oriented to facilitate binding.
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spelling pubmed-33827192013-01-01 Modulation of enrofloxacin binding in OmpF by Mg(2+) as revealed by the analysis of fast flickering single-porin current Brauser, Annemarie Schroeder, Indra Gutsmann, Thomas Cosentino, Cristian Moroni, Anna Hansen, Ulf-Peter Winterhalter, Mathias J Gen Physiol Article One major determinant of the efficacy of antibiotics on Gram-negative bacteria is the passage through the outer membrane. During transport of the fluoroquinolone enrofloxacin through the trimeric outer membrane protein OmpF of Escherichia coli, the antibiotic interacts with two binding sites within the pore, thus partially blocking the ionic current. The modulation of one affinity site by Mg(2+) reveals further details of binding sites and binding kinetics. At positive membrane potentials, the slow blocking events induced by enrofloxacin in Mg(2+)-free media are converted to flickery sojourns at the highest apparent current level (all three pores flickering). This indicates weaker binding in the presence of Mg(2+). Analysis of the resulting amplitude histograms with β distributions revealed the rate constants of blocking (k(OB)) and unblocking (k(BO)) in the range of 1,000 to 120,000 s(−1). As expected for a bimolecular reaction, k(OB) was proportional to blocker concentration and k(BO) independent of it. k(OB) was approximately three times lower for enrofloxacin coming from the cis side than from the trans side. The block was not complete, leading to a residual conductivity of the blocked state being ∼25% of that of the open state. Interpretation of the results has led to the following model: fast flickering as caused by interaction of Mg(2+) and enrofloxacin is related to the binding site at the trans side, whereas the cis site mediates slow blocking events which are also found without Mg(2+). The difference in the accessibility of the binding sites also explains the dependency of k(OB) on the side of enrofloxacin addition and yields a means of determining the most plausible orientation of OmpF in the bilayer. The voltage dependence suggests that the dipole of the antibiotic has to be adequately oriented to facilitate binding. The Rockefeller University Press 2012-07 /pmc/articles/PMC3382719/ /pubmed/22689827 http://dx.doi.org/10.1085/jgp.201210776 Text en © 2012 Brauser et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
spellingShingle Article
Brauser, Annemarie
Schroeder, Indra
Gutsmann, Thomas
Cosentino, Cristian
Moroni, Anna
Hansen, Ulf-Peter
Winterhalter, Mathias
Modulation of enrofloxacin binding in OmpF by Mg(2+) as revealed by the analysis of fast flickering single-porin current
title Modulation of enrofloxacin binding in OmpF by Mg(2+) as revealed by the analysis of fast flickering single-porin current
title_full Modulation of enrofloxacin binding in OmpF by Mg(2+) as revealed by the analysis of fast flickering single-porin current
title_fullStr Modulation of enrofloxacin binding in OmpF by Mg(2+) as revealed by the analysis of fast flickering single-porin current
title_full_unstemmed Modulation of enrofloxacin binding in OmpF by Mg(2+) as revealed by the analysis of fast flickering single-porin current
title_short Modulation of enrofloxacin binding in OmpF by Mg(2+) as revealed by the analysis of fast flickering single-porin current
title_sort modulation of enrofloxacin binding in ompf by mg(2+) as revealed by the analysis of fast flickering single-porin current
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3382719/
https://www.ncbi.nlm.nih.gov/pubmed/22689827
http://dx.doi.org/10.1085/jgp.201210776
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