Modulation of enrofloxacin binding in OmpF by Mg2+ as revealed by the analysis of fast flickering single-porin current

Abstract

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 Mg2+ reveals further details of binding sites and binding kinetics. At positive membrane potentials, the slow blocking events induced by enrofloxacin in Mg2+-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 Mg2+. Analysis of the resulting amplitude histograms with β distributions revealed the rate constants of blocking (kOB) and unblocking (kBO) in the range of 1,000 to 120,000 s−1. As expected for a bimolecular reaction, kOB was proportional to blocker concentration and kBO independent of it. kOB 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 Mg2+ 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 Mg2+. The difference in the accessibility of the binding sites also explains the dependency of kOB 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.

Orientation of the OmpF Porin in Planar Lipid Bilayers

“…[6,7] The lumen of E. coli OmpF is also exploited for the surfaced isplay of lipoproteins, [8] protein export, [9] and the import of antibacterial colicins. [14] Investigationsi nto OmpF transport in planarl ipid bilayers (PLB) by single-channel recording have revealed molecular details about ion permeation, [15,16] antibiotic binding, [17][18][19][20][21] and colicin interactions. The b strands are connected by eight short b-hairpin turns (T1-T8) at the periplasmic end of each subunit and eight long, irregular loops (L1-L8)t hat protrude from the bilayer surface.…”

“…[22][23][24][25] However,t hese studies have been conducted withoutk nowing the orientation of OmpF in the bilayer.I nt he convention used in this paper,O mpF is added to the cis compartment of the PLB apparatus, and can insert into the bilayer with either the periplasmic turns or extracellular loops first. [21] Maltoporin, an E. coli sugar transporter with as imilar structure to OmpF,h as also been proposed to insert with ap reference for the periplasmic side first. [15] However,i th as not been established which asymmetry corresponds to which OmpF orientation.…”

How to resolve microsecond current fluctuations in single ion channels: The power of beta distributions

“…78,94,116 The best fit was obtained when blocking was assumed to switch between I min D 0.3 I true and I true . 80 This demonstrates that with sufficient data quality the lower level of the block (or channel closing) can be estimated from the fit in the same way as the true open-channel current.…”

“…11A). 80 The open probability of the 3 individual pores is nearly 100% as can be seen from the infrequent closing events (arrows in Fig. 11A, B).…”

“…For example, this is the case for the antibiotic enrofloxacin, which is blocking the E. coli porin OmpF in the presence of millimolar concentration of Mg 2C (Fig. 2D, arrow labeled "b" in bottom trace and 80 ). In blocking experiments, the full current amplitude is usually known from control experiments (Fig.…”