Examination of the substrate stoichiometry of the intestinal Na+/phosphate cotransporter.
The 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 inhibition by mono- and di-fluorophosphates. Na+/phosphate cotransport was found to have similar Na+ activations (apparent K0.5's of 28 and 25 mM), apparent Km's for phosphate (100 and 410 microM), and K0.5's for inhibition by phenylglyoxal (70 and 90 microM) using isotopic phosphate uptake and membrane depolarization, respectively. Only difluorophosphate inhibited Na+-dependent phosphate uptake below 1 mM at pH 7.4. Difluorophosphate also protected a 130-kDa polypeptide from FITC-PG labeling in the presence of Na+ with apparent K0.5 for phosphate of 200 microM; similar to the apparent Km for phosphate uptake, and K0.5 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.
Peerce
B E
BE
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77550.
D000818:Animals; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000378:metabolism*; D005459:Fluorides / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000378:metabolism*; D007700:Kinetics; D008564:Membrane Potentials; D008871:Microvilli / Q000378:metabolism; D010658:Phenylglyoxal / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism* / Q000494:pharmacology; D011817:Rabbits; D012964:Sodium / Q000378:metabolism*; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters
D002352:Carrier Proteins; D010710:Phosphates; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters; C012980:fluorophosphate; D012964:Sodium; D010658:Phenylglyoxal; D005459:Fluorides