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

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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.