Structural and Functional Characterization of the Vacuolar-Type Na+, K+/H+ Antiporter NHX1 from Rice (Oryza sativa L.)

Plant vacuolar-type Na⁺, K⁺/H⁺ antiporters (NHXs) play important roles in pH and K⁺ homeostasis and osmotic balance under normal physiological conditions. Under salt stress, vacuolar-type NHX enhances salt tolerance by compartmentalizing Na⁺ into vacuoles. However, the ion transport mechanism of vacuolar-type NHX remains poorly understood due to the absence of resolved protein crystal structures. To investigate the ion transport mechanism for vacuolar-type NHX, the three-dimensional structure of rice vacuolar-type NHX1 (OsNHX1) was established through homology modeling and AlphaFold3.0. The OsNHX1 model contains thirteen transmembrane segments according to hydrophobic characteristics and empirical and phylogenetic data. Furthermore, this study validated the OsNHX1 model via functional experiments, revealing a set of key charged amino acids essential for its activity. Mapping these amino acids onto the OsNHX1 model revealed that its pore domain exhibits a transmembrane charge-compensated pattern similar to that of NHE1 while also displaying a distinct charge distribution on either side of the pore domain. Comparative analysis of the key amino acid sites responsible for ion transport in the crystal structure of OsSOS1 and NHE1 revealed that OsNHX1 employs a unique ion transport mechanism. This study will enhance our understanding of the function and catalytic mechanism of OsNHX1 and other plant vacuolar-type NHXs.