SITE-DIRECTED MUTAGENESIS REDUCES THE NA-AFFINITY K+ TRANSPORTER( AFFINITY OF HKT1, AN NA+ ENERGIZED HIGH)

HKT1 encodes a high affinity Na+ coupled K+ transporter expressed in t he cortical cells of Triticum aestivum roots. To identify regions of t he protein involved in the binding and transport of Na+ and K+, mutati ons were introduced into a domain of HKT1 containing 16 amino acids th at are highly conserved across a range of putative K+ transport protei ns from different phyla. Two mutations had a significant effect on the functional characteristics of the transporter. A yeast growth assay s howed that concentrations of NaCl between 2.5 to 50 mM stimulated the growth of yeast expressing HKT1 containing the E464Q substitution, but not the growth of yeast expressing HKT1, Kinetic analysis confirmed t hat the E464Q mutation lowered the affinity of HKT1 for Na+ but did no t affect its affinity for K+. A second mutation in the same region F46 3L aas created that also lowered the affinity of the transporter for N a+ The importance of these highly conserved amino acid residues is hig hlighted by the fact that they have remained conserved through evoluti on, The results of this mutational analysis suggest that this domain i n HKT1 plays a role in the binding and transport of Na+, (C) 1998 Fede ration of European Biochemical Societies.