Study on function of the diverse K^+ transporters from plants

1. 拟南芥HKT1同源cDNA （AtHKT1）及其在异源系统中离子转运模式的研究。AtHKT1介导异种laevis卵母细胞内Na^+电流、酿酒酵母Na^+摄取和大肠杆菌K^+摄取。利用大肠杆菌表达系统和PhoA报告酶融合体、真核无细胞系统中的糖基化反应和HEK293转染物以及免疫荧光检测进行的独立实验表明，AtHKT1含有8个跨膜片段。我们的模型是一个比其他小组提出的模型更精确的拓扑结构。野生型AtHKT1具有n -糖基化位点。一种工程化的非糖基化蛋白变体介导了laevis卵母细胞中的Na^+电流，而没有像野生型蛋白那样失去一价阳离子的选择性，这表明糖基化对于AtHKT1在质膜中的表达和AtHKT1.4的Na^+易位活性都不是必需的。虽然野生型植物超极化激活K^+通道KAT1对外部Na^+不敏感，但互耦通道T256Q和T256E明显受到Na^+的抑制，其表观解离常数明显降低。在极端超极化时，T256E的阻滞明显缓解。孔螺旋内256位的突变重新排列了选择性过滤器，允许Na^+渗透到孔中。当将脱落酸处理蚕豆保护细胞的提取物加入拟南芥KAT1向内校正K^+通道的羧基端时，观察到肽的磷酸化。利用PhoA融合法测定了大肠杆菌基因表达系统中K^+转运体AtKUP1的拓扑结构。我们在AtKUP1中鉴定了7个跨膜片段。

1. A cDNA homologous to HKT1 from Arabidopsis (AtHKT1) and the characterization of its mode of ion transport in heterologous systems. The AtHKT1 mediates inward Na^+ currents in Xenopous laevis oocytes and Na^+ uptake in Saccharomyces cerevisiae, and K^+ uptake in Escherichia coli.2. Independent experiments using as E.coli-expression system and PhoA reporter enzyme fusions, glycosylation reactions in a eukaryotic cell free system and HEK293 transfectants and immunofluorescence detection showed that AtHKT1 contains eight transmembrane-spanning segments. Our model is a more accurate topology than the model proposed by other groups.3. The wild type AtHKT1 possesses the N-glycosylation site. An engineered unglycosylated protein variant mediated Na^+ currents in Xenopous laevis oocytes without loss of monovalent cation selectivity just as the wild type protein, indicating that glycosylation is not essential for either the expression of AtHKT1 in the plasma membrane of the Na^+ translocation activity of AtHKT1.4. Although the wild-type plant hyperpolarization-activating K^+ channel, KAT1, was insensitive to external Na^+, the mutanl channels, T256Q and T256E, were significantly depressed by Na^+ with their apparent dissociation constants. At the extreme hyperpolarization the blocking was relieved significantly in the T256E.The mutation at position 256 within the pore helix rearranged the selectivity filter and allow Na^+ to penetrate into the pore.5. When extracts from abscisic acid-treating Vicia fava guard cell were added into the carboxyl-terminus of an inward-rectifying K^+ channel from Arabidopsis KAT1, phosphorylation of the peptides were observed.6. The topology of K^+ transporter, AtKUP1, were determined by PhoA fusion approach in E.coli gene expression system. We identified seven transmembrane segments in the AtKUP1.