Role of membrane potential and hydrogen bonding in the mechanism of translocation of guanidinium-rich peptides into cells
Role of membrane potential and hydrogen bonding in the mechanism of translocation of guanidinium-rich peptides into cells
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DOI：
10.1021/ja0482536
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发表时间：
2004-08-11
影响因子：
15
通讯作者：
Wender, PA
中科院分区：
文献类型：
--
作者：
Rothbard, JB;Jessop, TC;Wender, PA
本文所述的结果支持关于富含胍的转运蛋白如何附着于小货物（MW约为100）的机制假说。<3000）可以迁移穿过细胞的脂质膜并直接进入胞质溶胶。发现精氨酸低聚物几乎完全分配到水-辛醇双层的水层中。然而，当在月桂酸钠（一种代表性的带负电荷的膜成分）存在下进行相同的分配实验时，精氨酸低聚物几乎完全（>95%）分配到辛醇层中。相比之下，鸟氨酸低聚物在添加和不添加月桂酸钠的情况下几乎完全分配到水层中。在月桂酸钠存在下，富胍和富铵低聚物之间的不同分配与前者形成双齿氢键合离子对的能力一致。单-和二甲基化的精氨酸低聚物，像鸟氨酸只能有效地形成单齿氢键，制备和发现表现出差的细胞摄取。离子对形成将曾经的水溶性试剂转化为脂溶性试剂，从而减少富含胍的转运蛋白通过脂质双层的能量损失。富含胍的转运蛋白的摄取是一个能量依赖性过程，这种对细胞ATP的需求现在通过在存在降低膜电位的试剂的情况下抑制富含胍的转运蛋白摄取而合理化。具体地，在具有高钾离子浓度的缓冲液中孵育细胞或用短杆菌肽A预处理细胞使Fl-aca-arg 8-CONH 2的细胞摄取减少> 90%。此外，用缬氨霉素使细胞超极化的倒数实验使摄取增加>1.5倍。总之，我们提出，水溶性的，带正电荷的胍头基的转运形成双齿氢键与H-键受体功能的细胞表面上。所得的离子对复合物分区进入脂质双层，并以与膜电位相关的速率迁移。复合物在膜的内叶上解离，转运蛋白进入胞质溶胶。这一假设并不排除其他机制的吸收，包括内吞作用，这可能是占主导地位的大型货物。
The results described herein support a mechanistic hypothesis for how guanidine-rich transporters attached to small cargos (MW ca. <3000) can migrate across the lipid membrane of a cell and directly enter the cytosol. Arginine oligomers are found to partition almost completely into the aqueous layer of a water−octanol bilayer. However, when the same partitioning experiment is conducted in the presence of sodium laurate, a representative negatively charged membrane constituent, the arginine oligomer partitions almost completely (>95%) into the octanol layer. In contrast, ornithine oligomers partition almost exclusively into the water layer with and without added sodium laurate. The different partitioning between guanidinium-rich and ammonium-rich oligomers in the presence of sodium laurate is consistent with the ability of the former to form a bidentate hydrogen bonded ion pair. Mono- and dimethylated arginine oligomers, which like ornithine can only efficiently form monodentate hydrogen bonds, were prepared and found to exhibit poor cellular uptake. Ion pair formation converts a once water-soluble agent to a lipid-soluble agent, thereby reducing the energetic penalty for passage of guanidine-rich transporters through the lipid bilayer. Uptake of guanidine-rich transporters is known to be an energy-dependent process, and this requirement for cellular ATP is now rationalized by the inhibition of guanidine-rich transporter uptake in the presence of agents that reduce the membrane potential. Specifically, incubation of cells in buffers with high potassium ion concentrations or pretreatment of cells with gramicidin A reduces the cellular uptake of Fl-aca-arg8-CONH2by >90%. Furthermore, the reciprocal experiment of hyperpolarizing the cell with valinomycin increased uptake by >1.5 times. In summary, we propose that the water-soluble, positively charged guanidinium headgroups of the transporter form bidentate hydrogen bonds with H-bond acceptor functionality on the cell surface. The resultant ion pair complexes partition into the lipid bilayer and migrate across at a rate related to the membrane potential. The complex dissociates on the inner leaf of the membrane, and the transporter enters the cytosol. This hypothesis does not preclude uptake by other mechanisms, including endocytosis, which is likely to dominate with large cargos.