Department of Cell Biology and Physiology, and Center for Investigation of Membrane Excitability Diseases, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Mol Pharm. 2013 Apr 1;10(4):1450-8. doi: 10.1021/mp400024d. Epub 2013 Mar 19.
Polyamines are ubiquitous organic cations implicated in many physiological processes. Because they are positively charged at physiological pH, carrier-mediated systems are necessary for effective membrane permeation, but the identity of specific polyamine transporter proteins in eukaryotic cells remains unclear. Polyspecific organic cation transporters (OCTs) interact with many natural and xenobiotic monovalent cations and have been reported to transport dicationic compounds, including the short polyamine putrescine. In this study, we used Xenopus oocytes expressing mammalian OCT1 (SLC22A1), OCT2 (SLC22A2), or OCT3 (SLC22A3) to assess binding and transport of longer-chain polyvalent polyamines. In OCT-expressing oocytes, [(3)H]MPP(+) uptake rates were 15- to 35-fold higher than in noninjected oocytes, whereas those for [(3)H]spermidine increased more modestly above the background, up to 3-fold. This reflected up to 20-fold lower affinity for spermidine than for MPP(+); thus, K(0.5) for MPP(+) was ~50 μM in OCT1, ~170 μM in OCT2, and ~60 μM in OCT3, whereas for spermidine, K(0.5) was ~1 mM in OCT1, OCT2, and OCT3. J(max) values for MPP(+) and spermidine were within the same range, suggesting that both compounds are transported at a similar turnover rate. To gain further insight into OCT substrate specificity, we screened a selection of structural polyamine analogues for effect on [(3)H]MPP(+) uptake. In general, blocking potency increased with overall hydrophobic character, which indicates that, as for monovalent cations, hydrophobicity is a major requirement for recognition in polyvalent OCT substrates and inhibitors. Our results demonstrate that the natural polyamines are low affinity, but relatively high turnover, substrates for OCTs. The identification of OCTs as polyamine transport systems may contribute to further understanding of the mechanisms involved in polyamine homeostasis and aid in the design of polyamine-like OCT-targeted drugs.
多胺是广泛存在的有机阳离子，参与许多生理过程。由于它们在生理 pH 值下带正电荷，因此需要载体介导的系统才能有效渗透细胞膜，但真核细胞中特定多胺转运蛋白的身份仍不清楚。多特异性有机阳离子转运体 (OCT) 与许多天然和外源性单价阳离子相互作用，并已被报道可转运二价化合物，包括短链多胺腐胺。在这项研究中，我们使用表达哺乳动物 OCT1（SLC22A1）、OCT2（SLC22A2）或 OCT3（SLC22A3）的非洲爪蟾卵母细胞来评估长链多价多胺的结合和转运。在表达 OCT 的卵母细胞中，[(3)H]MPP(+) 的摄取速率比未注射卵母细胞高 15-35 倍，而 [(3)H] 亚精胺的摄取速率则适度高于背景，最高可达 3 倍。这反映出亚精胺对 OCT 的亲和力低 20 倍左右，对 MPP(+) 的 K(0.5)在 OCT1 中约为 50 μM，在 OCT2 中约为 170 μM，在 OCT3 中约为 60 μM，而对于亚精胺，K(0.5)在 OCT1、OCT2 和 OCT3 中约为 1 mM。MPP(+) 和亚精胺的 J(max) 值在同一范围内，表明这两种化合物以相似的周转率进行转运。为了更深入地了解 OCT 底物特异性，我们筛选了一系列结构多胺类似物对 [(3)H]MPP(+)摄取的影响。一般来说，阻断效力随整体疏水性增加而增加，这表明，与单价阳离子一样，疏水性是多价 OCT 底物和抑制剂识别的主要要求。我们的结果表明，天然多胺是低亲和力但相对高周转率的 OCT 底物。将 OCT 鉴定为多胺转运系统可能有助于进一步了解多胺动态平衡涉及的机制，并有助于设计多胺样 OCT 靶向药物。
