Simultaneous occlusion of Na+ and phosphate by the intestinal brush border membrane Na+/phosphate cotransporter

The molecular mechanism of Na+ and phosphate transport by the intestinal brush border membrane Na+/phosphate cotransporter was examined using anion and cation exchange columns interfaced to a light-activated microsecond timer. The nature of ion:cotransporter interaction was examined for sensitivity to itself versus non-substrates, and sensitivity to amino acid specific reagents. Two possible ion:cotransporter interactions were examined. (a) ion bound, where ion exchanges rapidly with medium ion but not non-substrates, and (b) ion occluded, where ion exchanges slowly with medium ion. The Na+/phosphate cotransporter was found to occlude Na+ or Na+ and H2PO4, but not H2PO4 alone or HPO4. Phosphate occlusion had an absolute requirement for Na+ with K+ or Cs+ unable to substitute. Phosphate occlusion distinguished between phosphate and sulfate. Deocclusion from the fully loaded cotransporter was consistent with the release of 2 Na+'s prior to phosphate. These results predict two ternary cotransporter conformations differing in the Na+ sensitivity of their phosphate affinity.