Poulin R, Zhao C, Verma S, Charest-Gaudreault R, Audette M
Laboratory of Molecular Endocrinology, Laval University Medical Research Centre, 2705 Laurier Blvd., Ste.Foy, Quebec, Canada G1V4G2.
Biochem J. 1998 Mar 15;330 ( Pt 3)(Pt 3):1283-91. doi: 10.1042/bj3301283.
The mechanism of mammalian polyamine transport is poorly understood. We have investigated the role of plasma-membrane potential (DeltaPsipm) in putrescine and spermidine uptake in ZR-75-1 human breast cancer cells. The rate of [3H]putrescine and [3H]spermidine uptake was inversely correlated to extracellular [K+] ([K+]o) and to DeltaPsipm, as determined by the accumulation of [3H]tetraphenylphosphonium bromide (TPP). Inward transport was unaffected by a selective decrease in mitochondrial potential (DeltaPsimit) induced by valinomycin at low [K+]o, but was reduced by approximately 60% by the rheogenic protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP), which rapidly (<=15 min) collapsed both DeltaPsipm and DeltaPsimit. Plasma-membrane depolarization by high [K+]o or CCCP did not enhance putrescine efflux in cells pre-loaded with [3H]putrescine, suggesting that decreased uptake caused by these agents did not result from a higher excretion rate. On the other hand, the electroneutral K+/H+ exchanger nigericin (10 microM) co-operatively depressed -3H-TPP, [3H]putrescine and [3H]spermidine uptake in the presence of ouabain. Suppression of putrescine uptake by nigericin+ouabain was Na+-dependent, suggesting that plasma-membrane repolarization by the electrogenic Na+ pump was required upon acidification induced by nigericin, due to the activation of the Na+/H+ antiporter. The sole addition of 5-N, N-hexamethylene amiloride, a potent inhibitor of the Na+/H+ antiporter, strongly inhibited putrescine uptake in a competitive fashion -Ki 4.0+/-0.9 (S.D.) microM-, while being a weaker antagonist of spermidine uptake. The potency of a series of amiloride analogues to inhibit putrescine uptake was clearly different from that of the Na+/H+ antiporter, and resembled that noted for Na+ co-transport proteins. These data demonstrate that putrescine and spermidine influx is mainly unidirectional and strictly depends on DeltaPsipm, but not DeltaPsimit. This report also provides first evidence for a high-affinity amiloride-binding site on the putrescine carrier, which provides new insight into the biochemical properties of this transporter.
哺乳动物多胺转运的机制目前还知之甚少。我们研究了质膜电位（ΔΨpm）在ZR-75-1人乳腺癌细胞摄取腐胺和亚精胺过程中的作用。[3H]腐胺和[3H]亚精胺的摄取速率与细胞外[K+]（[K+]o）以及由[3H]四苯基溴化膦（TPP）积累所测定的ΔΨpm呈负相关。在低[K+]o条件下，缬氨霉素诱导的线粒体电位（ΔΨmit）选择性降低对内向转运没有影响，但质子载体羰基氰化物间氯苯腙（CCCP）可使ΔΨpm和ΔΨmit迅速（<=15分钟）消失，从而使摄取减少约60%。高[K+]o或CCCP引起的质膜去极化并未增强预先加载[3H]腐胺的细胞中腐胺的外流，这表明这些试剂导致的摄取减少并非由于排泄率升高。另一方面，在哇巴因存在的情况下，电中性的K+/H+交换剂尼日利亚菌素（10 microM）协同抑制[3H]-TPP、[3H]腐胺和[3H]亚精胺的摄取。尼日利亚菌素+哇巴因对腐胺摄取的抑制作用是Na+依赖性的，这表明由于Na+/H+反向转运体的激活，尼日利亚菌素诱导酸化后，电生性Na+泵使质膜复极化是必需的。单独添加5-N，N-六亚甲基阿米洛利（一种有效的Na+/H+反向转运体抑制剂）以竞争性方式强烈抑制腐胺摄取 -Ki为4.0±0.9（标准差） microM-，而对亚精胺摄取的拮抗作用较弱。一系列阿米洛利类似物抑制腐胺摄取的效力与Na+/H+反向转运体明显不同，与Na+共转运蛋白的情况相似。这些数据表明，腐胺和亚精胺的内流主要是单向的，并且严格依赖于ΔΨpm，而不依赖于ΔΨmit。本报告还首次提供了腐胺载体上存在高亲和力阿米洛利结合位点的证据，这为该转运体的生化特性提供了新的见解。
