The Na1 Binding Site In The Human Sodium-Phosphate Cotransporter Napi-Iia

The levels of inorganic phosphate (Pi) in humans are tightly regulated to prevent pathologies such as bone loss and vascular calcification. Sodium-coupled Pi transporters of the SLC34 solute carrier family (NaPi-II) are responsible for intestinal Pi absorption and renal Pi reabsorption. These transporters move divalent Pi into the cell using the transmembrane sodium electrochemical gradient. All three isoforms, NaPi-IIa, IIb, and IIc, are thought to bind three sodium ions during the transport cycle, although NaPi-IIc co-transports only two of those ions, whereas the other isoforms (IIa, IIb) co-transport three sodium ions and are consequently electrogenic. Although extensive functional studies have provided valuable insights into this family of transporters, unfortunately no three-dimensional structural data is available. Recently, we proposed a structural model of NaPi-IIa based on a crystal structure of a sodium-coupled dicarboxylate transporter VcINDY. With this model, we successfully predicted the regions of the protein that mediate binding of the phosphate anion and two of the sodium ions (Na2 and Na3), but it was not possible to identify the position of the sodium ion that binds first to the empty transporter (Na1). Here, we used molecular modeling techniques to refine the earlier model by adjusting the target-template sequence alignment for transmembrane helices 2, 5 and 6. We obtained two alternative structural models with different implications for a putative Na1 binding site, and with possible improvements to the coordination in the predicted Na3 binding site. These alternatives were assessed by biochemical and electrophysiological analysis of site-specific mutants of human NaPi-IIa. The experimental results strongly favor one of the two models, and identify residues with a key functional role in the transport process. The improved NaPi-IIa model provides a firmer foundation for a molecular-level understanding of sodium-coupled Pi uptake by SLC34 transporters.