Electroosmosis Dominates Electrophoresis of Antibiotic Transport Across the Outer Membrane Porin F

Abstract

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 it, 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 in which electroosmotic flow 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, which slows down the permeation by several orders of magnitude.

Figure 1

Charge states of molecules at different pH. The net charge and the location of charged regions are depicted in the figure. The molecules have a net positive charge at pH 6, are zwitterionic at pH 7, and are negatively charged at pH 9. To see this figure in color, go online.

Figure 2

(A) A single channel current recording from reconstituted OmpF porin at both ±100 mV (blank) is shown. (B) Given is a single channel current recording of OmpF porin showing real-time detection of CFX at ±100 mV with a concentration of 0.25 mM in 1 M KCl at pH 6 cut out of a section of the trace that shows clear and distinct blockages. (C and D) Shown is a schematic of the direction of the electric field and the EOF at +100 and −100 mV, respectively. To see this figure in color, go online.

Figure 3

(A) A single channel current recording from reconstituted OmpF porin at both ±100 mV (blank) is shown. (B) Given is a single channel current recording of OmpF porin showing real-time detection of CFX at ±100 mV with a concentration of 0.25 mM in 1 M KCl at pH 9 cut out of a section of the trace that shows clear and distinct blockages. (C and D) Shown is a schematic of the direction of the electric field and the EOF at +100 and −100 mV, respectively. To see this figure in color, go online.

Figure 4

Event rates and dwell times of molecule translocation through OmpF in the presence of 0.25 mM NFX, CFX, and ENX 1 M KCl with 10 mM MES at pH 6 and 1 M KCl with 10 mM CAPS at pH 9. Event rates were determined by counting the total number of events and dividing by the total observation time. Mean dwell times are obtained by fitting dwell time data to single exponentials assuming a two-state Markov process over at least 1000 events; for details, see Supporting Materials and Methods. The error bars depict the standard deviation of the mean over at least three sets of independent experimental measurements. To see this figure in color, go online.

Figure 5

Determination of v_EOF and the free-energy term. Dwell time ratios are fitted to Eq. 4, as explained in the text. The expected linear behavior is apparent at |V_m| ≥ 50 mV. The data at |V_m| = 25 mV are excluded from the fit. The deviation at |V_m| = 25 mV is discussed at length within the text. The error bars depict the standard deviation of the mean over at least three sets of independent experimental measurements. To see this figure in color, go online.

Figure 6

Sketch of a simple free-energy landscape for OmpF. We choose a symmetric free-energy profile that linearly increases toward the constriction zone of the porin, where the total free-energy change experienced by the molecule is ΔF = 12kT − 20kT. To see this figure in color, go online.

Figure 7

Testing the validity of the EOF model at pH 7. The solid line depicts the expected behavior of the molecules at pH 7 (Eq. 5) calculated from the parameters obtained from Eq. 4. The error bars depict the standard deviation of the mean over at least three sets of independent experimental measurements. To see this figure in color, go online.