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

被引:20
作者
Brauser, Annemarie [2 ,5 ]
Schroeder, Indra [1 ,3 ]
Gutsmann, Thomas [2 ]
Cosentino, Cristian [1 ]
Moroni, Anna [1 ]
Moroni, Anna [1 ]
Hansen, Ulf-Peter [4 ]
Winterhalter, Mathias [5 ]
机构
[1] Univ Milan, Dept Biol, I-20133 Milan, Italy
[2] Res Ctr Borstel, Ctr Med & Biosci, Div Biophys, D-23845 Borstel, Germany
[3] Tech Univ Darmstadt, D-64287 Darmstadt, Germany
[4] Univ Kiel, Dept Biol Struct, D-24098 Kiel, Germany
[5] Jacobs Univ Bremen, Sch Sci & Engn, D-28759 Bremen, Germany
关键词
OUTER-MEMBRANE PERMEABILITY; MAXIK SELECTIVITY FILTER; ESCHERICHIA-COLI PORIN; OPEN CHANNEL NOISE; K+ CHANNEL; CRYSTAL-STRUCTURES; MOLECULAR-BASIS; VOLTAGE; PERMEATION; MODELS;
D O I
10.1085/jgp.201210776
中图分类号
Q4 [生理学];
学科分类号
071003 ;
摘要
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 beta 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 similar to 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 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.
引用
收藏
页码:69 / 82
页数:14
相关论文
共 45 条
[1]   Fast and slow gating are inherent properties of the pore module of the K+ channel Kcv [J].
Abenavoli, Alessandra ;
DiFrancesco, Mattia Lorenzo ;
Schroeder, Indra ;
Epimashko, Svetlana ;
Gazzarrini, Sabrina ;
Hansen, Ulf Peter ;
Thiel, Gerhard ;
Moroni, Anna .
JOURNAL OF GENERAL PHYSIOLOGY, 2009, 134 (03) :219-229
[2]   Unimolecular study of the interaction between the outer membrane protein OmpF from E. coli and an analogue of the HP(2-20) antimicrobial peptide [J].
Apetrei, Aurelia ;
Asandei, Alina ;
Park, Yoonkyung ;
Hahm, Kyung-Soo ;
Winterhalter, Mathias ;
Luchian, Tudor .
JOURNAL OF BIOENERGETICS AND BIOMEMBRANES, 2010, 42 (02) :173-180
[3]   Voltage gating is a fundamental feature of porin and toxin β-barrel membrane channels [J].
Bainbridge, G ;
Gokce, I ;
Lakey, JH .
FEBS LETTERS, 1998, 431 (03) :305-308
[4]   Voltage-gating of Escherichia coli porin:: A cystine-scanning mutagenesis study of loop 3 [J].
Bainbridge, G ;
Mobasheri, H ;
Armstrong, GA ;
Lea, EJA ;
Lakey, JH .
JOURNAL OF MOLECULAR BIOLOGY, 1998, 275 (02) :171-176
[5]   Alteration of pore properties of Escherichia coli OmpF induced by mutation of key residues in anti-loop 3 region [J].
Bredin, J ;
Saint, N ;
Malléa, M ;
Dé, E ;
Molle, G ;
Pagès, JM ;
Simonet, V .
BIOCHEMICAL JOURNAL, 2002, 363 :521-528
[6]  
CACECI MS, 1984, BYTE, V9, P340
[7]   CRYSTAL-STRUCTURES EXPLAIN FUNCTIONAL-PROPERTIES OF 2 ESCHERICHIA-COLI PORINS [J].
COWAN, SW ;
SCHIRMER, T ;
RUMMEL, G ;
STEIERT, M ;
GHOSH, R ;
PAUPTIT, RA ;
JANSONIUS, JN ;
ROSENBUSCH, JP .
NATURE, 1992, 358 (6389) :727-733
[8]   Probing the orientation of reconstituted maltoporin channels at the single-protein level [J].
Danelon, C ;
Brando, T ;
Winterhalter, M .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2003, 278 (37) :35542-35551
[9]   Outer membrane permeability and antibiotic resistance [J].
Delcour, Anne H. .
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, 2009, 1794 (05) :808-816
[10]   FAST SINGLE-CHANNEL MEASUREMENTS RESOLVE THE BLOCKING EFFECT OF CS+ ON THE K+ CHANNEL [J].
DRABER, S ;
HANSEN, UP .
BIOPHYSICAL JOURNAL, 1994, 67 (01) :120-129