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

Abstract

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.

Mammalian ion‐coupled solute transporters.

“…Semenza and colleagues (Schmidt et al 1983) isolated the rabbit small intestinal Na+/glucose co-transporter as a 72 kDa polypeptide (by SDS-PAGE) to near homogeneity. Peerce & Clarke (1990) also purified the transporter. These efforts, however, did not result in the isolation of the transporter in a form suitable for amino acid sequencing.…”

Phosphophloretin sensitivity of rabbit renal NaPi-IIa and NaPi-Ia

“…NaPi-Ia and NaPi-IIb have increased Na ϩ affinity with decreasing pH and appear to preferentially or exclusively transport H 2 PO 4 (10,25,28,32,41,44). Comparisons of the structures of the two proteins have not been reported.…”

“…NaPi-IIb is thought to transport only H 2 PO 4 , may be inhibited by HPO 4 at an internal regulatory site (10,32), has a substrate stoichiometry of 2, and increases its Na ϩ affinity with decreasing pH (10,32,33,41). NaPi-IIa is more sensitive to phosphites (foscarnet) and less sensitive to arsenate (43) than NaPi-IIb (3,24).…”

Inhibition of renal Na+‐Pi cotransporter by mercuric chloride: Role of sulfhydryl groups

“…Additionally, if an essential group is at or near the substrate binding site(s) on the cotransporter, the transport may be protected from inhibition by the substrate or its analogues. Studies of the intestinal Na+ /D-glucose cotransporter have identified several essential groups, including: disulfide bonds [8,9], sulfhydryl [10,11], lysine [12], tyrosine [13,14], and amino groups [15,161. Studies of L-proline transport have also demonstrated the presence of lysine and tyrosine groups [17] at the transporter (imino carrier) site. Modification of arginine [18,19] and tyrosine [20] residues as well as sulfhydryl groups [201 has resulted in the inhibition of Pi transport in renal BBMV.…”