Brian E. Peerce

Examination of the substrate stoichiometry of the intestinal Na+/phosphate cotransporter

SummaryThe substrate stoichiometry of the intestinal Na+/phosphate cotransporter was examined using two measures of Na+-dependent phosphate uptake: initial rates of uptake with [32P] phosphate and phosphate-induced membrane depolarization using the potential-sensitive dye diSC3(5). Isotopic phosphate measures electrogenic and electroneutral Na+-dependent phosphate uptake, while phosphate-induced membrane depolarization measures electrogenic phosphate uptake. Using these measures of Na-dependent phosphate uptake, three parameters were compared: substrate affinity; phenylglyoxal sensitivity and labeling; and inhibiton by mono- and di-fluorophosphates. Na+/phosphate cotransport was found to have similar Na+ activations (apparentK0.5s of 28 and 25mm), apparentKms for phosphate (100 and 410 μm), andK0.5s for inhibition by phenylglyoxal (70 and 90 μm) using isotopic phosphate, uptake and membrane depolarization, respectively. Only difluorophosphate inhibited Na+-dependent phosphate uptake below 1mm at pH 7.4.Difluorophosphate also protected a 130-kDa polypeptide from FITC-PG labeling in the presence of Na+ with apparentK0.5 for phosphate of 200 μm; similar to the apparentKm for phosphate uptake, andK0.5 for phosphate protection against FITC-PG inhibition of Na+-dependent phosphate uptake and FITC-PG labeling of the 130-kDa polypeptide. These results indicate that the intestinal Na+/phosphate cotransporter is electrogenic at pH 7.4, that H2PO4− is the transport-competent species, and that the 130-kDa polypeptide is an excellent candidate for the intestinal Na+/phosphate cotransporter.

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.

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

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.

Examination of the molecular mechanism of SH reagent-induced inhibition of the intestinal brush-border membrane Na+/phosphate cotransporter

SH residues on the rabbit intestinal brush-border membrane Na+/phosphate cotransporter were examined using a variety of SH specific reagents, proteolytic digestion and HPLC separation of SH-labeled cotransporter, and partial reaction assays. Of the seven SH-containing peptide fragments on the non-denatured non-reduced cotransporter six peptides were labeled: five SH-containing peptides were labeled with acrylodan or IAF (iodoacetamidofluorescein) and three peptides were labeled with IAEDANS. One SH-containing peptide was labeled with IAEDANS or fluorescein maleimide only. Selective SH labeling conditions employing acrylodan and IAEDANS were used to identify the environments of these SH-containing peptides in the native cotransporter. The nature of SH reagent-induced inhibition of Na(+)-dependent phosphate uptake was examined using substrate-induced conformational changes, and substrate-induced changes in IAEDANS and acrylodan fluorescence of the SH-labeled Na+/phosphate cotransporter. The results indicate that five of the SH-labeled peptides sense the Na(+)-induced conformational change, three peptides sense the Na++ difluorophosphate-induced conformational change, and one peptide senses only the Na++ monofluorophosphate-induced conformational change. Five of the SH-labeled peptides are passive participants in the substrate-induced conformational changes with only SH 51 involved in cotransporter function. Alkylation of SH 51 resulted in a cotransporter conformation which differed from the substrate-mediated conformations and was characterized by increased monofluorophosphate sensitivity.

A 40-kDa polypeptide from papain digestion of the rabbit intestinal Na+/phosphate cotransporter retains Na+ and phosphate cotransport

The rabbit intestinal brush border membrane Na+/phosphate cotransporter was digested with a variety of proteolytic enzymes. Limited papain digestion generated a 40-kDa polypeptide (P40) which retained putative substrate site markers, fluorescein isothiocyanatophenyl glyoxal and eosin n-acetyl imidazole. P40 retained Na+- and phosphate-selective tryptophan fluorescence quenching, pH sensitivity of ion-induced conformational changes, and tight Na+ and H(2)PO(4)(-) binding. Reconstituted into proteoliposomes, P40 catalyzed Na+-dependent phosphate uptake. The N-terminus of P40 was blocked. An internal sequence of P40 demonstrated that it was derived from NaPi II b. These results suggest that P40 may be a useful model system for studies of the molecular mechanism of Na+-dependent phosphate cotransport and a starting point for structural studies.