Interaction of substrates with the intestinal brush border membrane Na/phosphate cotransporter

The interaction of Na+ and phosphate with the intestinal brush border membrane Na+/phosphate cotransporter was examined using stopped-flow tryptophan fluorescence and ion-exchange Dowex columns coupled to a light-activated microsecond timer (LAM timer) which measures exchange kinetics between protein-bound ions and the external medium Na+ or Na+ + H2PO4- induced tryptophan fluorescence quenching with apparent rate constants of 35 s-1 and 13 s-1, respectively. Dilution of substrate-bound cotransporter resulted in tryptophan fluorescence recovery consistent with cotransporter return to the substrate-free conformation. Recovery of the substrate-free conformation was slow (1.6 s-1) in the absence of phosphate, was accelerated by H2PO4 (7 s-1) and was inhibited by HPO4(2) (1.1 s-1). The effects of substrates on tryptophan fluorescence were sensitive to substrate site blockers consistent with tryptophan fluorescence monitoring cotransporter conformations and substrate-induced changes in conformation. Equivalent experiments using the LAM timer and either (22Na+) or Na+ + (32P) phosphate verified the rate constants for the substrate-induced quenching of tryptophan fluorescence, suggested that 2 Na+ 's were occluded by the cotransporter as part of the Na(+)-induced conformational change and that H2PO4 accelerated deocclusion of Na+. The association of phosphate with the cotransporter was also examined. Although cotransporter-bound phosphate was medium anion-insensitive, a cotransporter conformational change preceding the release of phosphate from the cotransporter was not observed. However, three lines of evidence suggest that release of phosphate from the cotransporter involved a unique cotransporter conformation which may suggest that phosphate was also occluded by the intestinal brush border Na+/phosphate cotransporter.