Effect of substrates and pH on the intestinal Na+/phosphate cotransporter: evidence for an intervesicular divalent...

Intervesicular divalent phosphate-induced inhibition of the intestinal brush-border membrane Na+/phosphate cotransporter was examined using Na(+)-dependent phosphate uptake, substrate-induced tryptophan fluorescence quenching, and the apparent pKa values for substrate-induced conformational changes. In right-side-out (RSO) reconstituted proteoliposomes, only monovalent phosphate inhibited Na(+)-dependent phosphate uptake in the absence of pre-equilibration. Addition of divalent phosphate to inside-out (ISO) proteoliposomes resulted in 80 +/- 5% inhibition of Na(+)-dependent phosphate uptake in the absence of pre-equilibration. The nature of divalent phosphate-induced inhibition of cotransporter function was examined using cotransporter partial reaction assays based on substrate-induced conformational changes reported as changes in tryptophan fluorescence. Na+ but not K+ induced a quenching of tryptophan fluorescence with a K0.5 of 25 mM and an apparent Hill coefficient of 1.8. Monovalent phosphate (difluorophosphate) induced a further quenching of tryptophan fluorescence with a K0.5 of 53 microM. Divalent phosphate (monofluorophosphate) had no effect on tryptophan fluorescence, but inhibited the difluorophosphate-induced quenching of tryptophan fluorescence. The Na+ to Na++ divalent phosphate (monofluorophosphate) conformation and the Na+ to Na++ monovalent phosphate (difluorophosphate) conformations were compared using tryptophan quench reagents. These transitions had different apparent pKa values and different phenylglyoxal sensitivities consistent with monovalent phosphate and divalent phosphate interacting with the cotransporter at separate sites.