Tobramycin uptake in Escherichia coli membrane vesicles

I. M. Leviton, H. S. Fraimow, N. Carrasco, T. J. Dougherty, M. H. Miller

Research output: Contribution to journalArticlepeer-review

14 Scopus citations

Abstract

The uptake of tobramycin was measured in Escherichia coli membrane vesicles prepared in KMES [K+-2(N-morpholino)ethanesulfonic acid] buffer at pH 6.6. Uptake occurred in vesicles energized with ascorbic acid and phenazine methosulfate, in which the electrical potential (ΔΨ) was -120 mV, but not in vesicles energized with D-lactate (ΔΨ = -95 mV). The addition of nigericin to vesicles energized with D-lactate did not induce tobramycin uptake despite an increase in ΔΨ to -110 mV. However, when ΔΨ was increased or decreased by the addition of nigericin or valinomycin, respectively, uptake in vesicles energized with ascorbic acid and phenazine methosulfate was stimulated or inhibited, respectively, confirming studies with whole cells showing that uptake of aminoglycosides is gated by ΔΨ rather than by proton motive force (Δμ(K+)) or ΔpH. N-ethylmaleimide prevented uptake, suggesting that the aminoglycoside transporter is a cytoplasmic membrane protein with accessible sulfhydryl groups. The observation that uptake is gated in vesicles as well as in whole cells suggested that diffusion occurs through a voltage-gated channel. In vesicles preloaded with tobramycin, no efflux occurred after the addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone. In susceptible cells, aminoglycosides themselves decreased the magnitude of ΔΨ. We propose a mechanism of aminoglycoside-induced killing in which aminoglycosides themselves close the voltage-gated channel by decreasing the magnitude of ΔΨ. Channel closure causes aminoglycosides accumulated prior to the fall in ΔΨ to be trapped, which in turn causes irreversible uptake and subsequent bactericidal effects.

Original languageEnglish (US)
Pages (from-to)467-475
Number of pages9
JournalAntimicrobial agents and chemotherapy
Volume39
Issue number2
DOIs
StatePublished - 1995
Externally publishedYes

ASJC Scopus subject areas

  • Pharmacology
  • Pharmacology (medical)
  • Infectious Diseases

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