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

植物多种K^转运蛋白的功能研究

基本信息

批准号：
11660082
负责人：
UOZUMI Nobuyuki
金额：
$ 2.43万
依托单位：
NAGOYA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11660082/
关键词：
K^+channel transporter Arabidopsis thaliana topology Na^+Escherichia coli AtHKT1 膜蛋白質 KAT1 糖鎖修飾 Arabidopsis

项目摘要

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.

1.拟南芥HKT 1同源cDNA（AtHKT 1）及其在异源系统中离子转运模式的表征。AtHKT 1介导了光滑异种卵母细胞的内向Na^+电流、酿酒酵母的Na^+摄取和大肠杆菌的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HEK 293转染子以及免疫荧光检测的独立实验表明，AtHKT 1含有8个跨膜片段。我们的模型比其他小组提出的模型更准确。3.野生型AtHKT 1具有N-糖基化位点。一种工程化的非糖基化蛋白质变体在非洲爪蟾卵母细胞中介导Na^+电流，而不像野生型蛋白质那样丧失单价阳离子选择性，这表明糖基化对于AtHKT 1在质膜中的表达或AtHKT1.4的Na^+转运活性都不是必需的。尽管野生型植物超极化激活K^+通道KAT 1对外部Na^+不敏感，但突变型通道T256 Q和T256 E的表观解离常数却被Na^+显著抑制。在极端超极化时，T256 E的阻断作用明显减轻。孔螺旋内256位的突变使选择性过滤器重排，使Na^+渗透到孔中。当脱落酸处理蚕豆保卫细胞的提取物加入到拟南芥KAT 1内向整流钾通道的羧基端时，多肽的磷酸化被抑制.在大肠杆菌基因表达系统中，利用PhoA融合技术，确定了K^+转运蛋白AtKUP 1的拓扑结构。我们在AtKUP 1中鉴定了7个跨膜片段。