Molecular analysis of ion transporters in plant and cyanobacteria involved in response to high osmolality

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1. We have identified the localization of Arabidopsis Na transporter AtHKT1 in plant cells. The specificity of the anti-AtHKT1 antibodies was confirmed. Immunoelectron microscopy using the antibodies demonstrate that AtHKT1 is targeted to the plasma membrane in xylem parenchyma cells in leaves.2. AtHKT1 disruption alleles caused large increases in the Na content of the xylem sap and conversely reduced the Na content of the phloem sap. The athkt1 mutant alleles had a smaller and inverse influence on the K content compared with the Na content to the xylem, suggesting that K transport may be indirectly affected. The expression of AtHKT1 was modulated not only by the concentrations of Na and K but also by the osmolality of non-ionic compounds.3. The conserved Arg near the middle of the M2D segment was essential for the K^+ transport of KtrB. We propose the existence of the salt bridge(s) between the positive residues in the M2D and the conserved negative residues in the pore region, which reduces an electrostatic barrier against cation permeation caused by the positive residue(s) and that stabilizes the transporter configuration.4. Mutation of a conserved His-157 in the second pore loop of KtrB, drastically reduced the activity of the K^+ transporter KtrABE system from Synechocystis PCC 6803. The result suggests that His-157 plays an essential role in the K^+ transport activity of the transporter system.5. H transport activity of one of Synechocystis Na/H transporters was measured using the inverted membrane vesicle of E. coli expressing the protein. We have identified the charged residues essential for the K transport.