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. 1995 Feb;39(2):467–475. doi: 10.1128/aac.39.2.467

Tobramycin uptake in Escherichia coli membrane vesicles.

I M Leviton 1, H S Fraimow 1, N Carrasco 1, T J Dougherty 1, M H Miller 1
PMCID: PMC162562  PMID: 7726517

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 (delta psi) was -120 mV, but not in vesicles energized with D-lactate (delta psi = -95 mV). The addition of nigericin to vesicles energized with D-lactate did not induce tobramycin uptake despite an increase in delta psi to -110 mV. However, when delta psi 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 delta psi rather than by proton motive force (delta microH+) or delta pH. 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 delta psi. We propose a mechanism of aminoglycoside-induced killing in which aminoglycosides themselves close the voltage-gated channel by decreasing the magnitude of delta psi. Channel closure causes aminoglycosides accumulated prior to the fall in delta psi to be trapped, which in turn causes irreversible uptake and subsequent bactericidal effects.

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Selected References

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