Structure-Guided Identification of Critical Residues in the Vacuolar Na⁺,K⁺/H⁺ Antiporter NHX1 from Arabidopsis thaliana

Cation/Proton Antiporters (CPA) acting in all biological membranes help regulate the volume and pH of cells and of intracellular organelles. A key issue with these proteins is their structure-function relationships since they present intrinsic regulatory features that rely on structural determinants, including pH-sensitivity and the stoichiometry of ion exchange. Crystal structures are only available for prokaryotic CPA, whereas the eukaryotic ones have been modeled using the former as templates. Here we show an updated and improved structural model of the tonoplast-localized K+,Na+/H+ antiporter NHX1 of Arabidopsis as a representative of the vacuolar NHX family that is key to the accumulation of K+ into plant vacuoles. Conserved residues judged as functionally important were mutated and the resulting protein variants were tested for activity in the yeast Saccharomyces cerevisiae. Results indicate that residue N184 in the ND-motif characteristic of CPA1 could be replaced by the DD-motif of CPA2 family members with minimal consequences on activity, yet this residue may help to regulate the optimal pH range of the exchanger. Attempts to alter the electroneutrality of AtNHX1 by different combinations of amino acid replacements at N184, R353 and R390 residues resulted in inactive or partly active proteins with differential ability to control the vacuolar pH of the yeast.