Potassium is an important macronutrient required for plant growth, whereas sodium (Na+) can be toxic at high concentrations. The wheat K+ uptake transporter HKT1 has been shown to function in yeast and oocytes as a high affinity K+-Na+ cotransporter, and as a low affinity Na+ transporter at high external Na+. A previous study showed that point mutations in HKT1, which confer enhancement of Na+ tolerance to yeast, can be isolated by genetic selection. Here we report on the isolation of mutations in new domains of HKT1 showing further large increases in Na+ tolerance. By selection in a Na+ ATPase deletion mutant of yeast that shows a high Na+ sensitivity, new HKT1 mutants at positions Gln-270 and Asn-365 were isolated. Several independent mutations were isolated at the Asn-365 site. N365S dramatically increased Na+ tolerance in yeast compared with all other HKT1 mutants. Cation uptake experiments in yeast and biophysical characterization in Xenopus oocytes showed that the mechanisms underlying the Na+ tolerance conferred by the N365S mutant were: reduced inhibition of high affinity Rb+ (K+) uptake at high Na+ concentrations, reduced low affinity Na+ uptake, and reduced Na+ to K+ content ratios in yeast. In addition, the N365S mutant could be clearly distinguished from less Na+-tolerant HKT1 mutants by a markedly decreased relative permeability for Na+ at high Na+ concentrations. The new mutations contribute to the identification of new functional domains and an amino acid in a loop domain that is involved in cation specificity of a plant high affinity K+ transporter and will be valuable for molecular analyses of Na+ transport mechanisms and stress in plants.
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