Preliminary Study On Potassium Channel MIRK From Melon Corresponding Of Plant Salt Tolerance

In our previous work, a K~+ channel gene MIRK(Melon Inward Rectifying K~+ Channel,Gene Bank accession number:DQ116940)was cloned from muskmelon"Chunli"(Cucumis melon L.). The full length cDNA of MIRK was 2506bp, encoding a 701 amino acids (aa) polypeptide, meanwhile belonging to the Shaker channel group 2 (KAT-like subfamily) according to phylogenetic tree. It mainly expressed in leaves and fruits, and could further improve the salt tolerance of plant after expressed in Arabidopsis thaliana.MIRK subcellular localization was investigated by RT-PCR analyses first. The result revealed that MIRK was preferentially expressed in guard cells and in veins. This was exactly in line with the model of KAT1 subfamily which expressed mainly in guard cells.As MIRK transformation could increase salt tolerance in Arabidopsis, the next aim of this study is to understand the relationship between MIRK potassium channel genes and external Na~+. Therefore, MIRK and its relationship with external sodium was further assessed by expression in a S. cerevisiae mutant strain (W?6) defective for K~+ uptake. The results indicated that Wâ–³6, expressing MIRK grew well in the presence of 1 mmol/L K+. In the presence of 1 mmol/L K~+ and 100 mmol/L Na~+, the positive effect of MIRK expression on cell growth was strongly inhibited by the addition of Na~+, while that of KAT1 expression was not inhibited. Thus, taken as a whole, the data provided physiological evidence, in growing cells, both that MIRK can mediate K~+ uptake and that this capacity was suspressed by Na~+.Additionally, MIRK was transferred into Xenopus oocytes and used two-electrode voltage clamp technique to validate the results of yeast experiment. The results shown a blockage of MIRK currents by external Na~+, the inhibitory effect seemed to be dependent on the Na~+/K~+ external concentration ratio but not voltage dependent. Moreover, patch-clamp experiments were performed on guard cell protoplasts of melon cv Chunli in order to assess the contribution of MIRK-like channels to K~+ uptake in this cell type. Na~+ blockage phenomenon was found remaining, however the sensitivity to Na~+ was lower in guard cells than in MIRK-expressing oocytes. This suggested that MIRK-like channels co-exist in guard cells with other Shaker channels insensitive to Na~+.At last, the research was proceeded further by performing site-directed mutagenesis experiment after amino acid align and ultimately obtained three mutants of MIRK. We furthermore tranfered the mutants to Xenopus ooocytes and used double electrode voltage clamp techniques as a tool to search for molecular base of blockage site. The results implied that the depressing effect was not determined by single amino acid but might involve multiple amino acids like 232-234 serine, lysine and glutamine and 241 asparagine residues.