Leviton I M, Fraimow H S, Carrasco N, Dougherty T J, Miller M H
Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, USA.
Antimicrob Agents Chemother. 1995 Feb;39(2):467-75. doi: 10.1128/AAC.39.2.467.
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.
在pH 6.6的KMES [K(+)-2-(N-吗啉代)乙磺酸]缓冲液中制备的大肠杆菌膜囊泡中测定了妥布霉素的摄取情况。摄取发生在用抗坏血酸和吩嗪硫酸甲酯供能的囊泡中，其中膜电位(Δψ)为-120 mV，但在用D-乳酸供能的囊泡中(Δψ = -95 mV)不发生摄取。向用D-乳酸供能的囊泡中添加尼日利亚菌素，尽管Δψ增加到-110 mV，也不会诱导妥布霉素摄取。然而，当分别通过添加尼日利亚菌素或缬氨霉素使Δψ增加或降低时，用抗坏血酸和吩嗪硫酸甲酯供能的囊泡中的摄取分别受到刺激或抑制，这证实了对全细胞的研究表明氨基糖苷类的摄取是由Δψ而非质子动力(ΔμH+)或ΔpH控制的。N-乙基马来酰亚胺阻止摄取，表明氨基糖苷类转运体是一种具有可及巯基的细胞质膜蛋白。摄取在囊泡和全细胞中均受控制这一观察结果表明，扩散是通过电压门控通道发生的。在用妥布霉素预加载的囊泡中，添加质子载体羰基氰化物间氯苯腙后没有发生外流。在敏感细胞中，氨基糖苷类本身会降低Δψ的幅度。我们提出了一种氨基糖苷类诱导杀伤的机制，其中氨基糖苷类本身通过降低Δψ的幅度来关闭电压门控通道。通道关闭导致在Δψ下降之前积累的氨基糖苷类被困住，这反过来又导致不可逆摄取和随后的杀菌作用。
