Evidence for the Existence of a Distinct SO–4-OH– Exchange Mechanism in the Human Proximal Colonic Apical Membrane Vesicles and Its Possible Role in Chloride Transport

Abstract

Recent studies have demonstrated that mutations in human downregulated in adenoma gene (DRA) result in congenital chloride diarrhea (CLD), and that DRA may be involved in chloride transport across the intestinal apical domains. DRA is highly homologous to sulfate transporters, but not to any member of the anion exchanger gene family (AEs). Our previous studies have characterized the existence of a distinct CI–-OH– (HCO3–) exchanger, with minimal affinity for sulfate in the human colonic apical membrane vesicles (AMV). However, the mechanism(s) of sulfate movement across the colonocyte plasma membranes in the human colon is not well understood. Current studies were undertaken to elucidate sulfate transport pathways in AMVs of human proximal colon. Purified AMV and rapid filtration 35SO–4 uptake techniques were used. Our results demonstrate the presence of a pH gradient-driven carrier-mediated SO–4-OH– exchange process in the human proximal colonic luminal membranes based on the following: a marked increase in the SO–4 uptake in the presence of an outwardly directed OH- gradient; a significant inhibition of SO–4 uptake by the membrane anion transport inhibitor, DIDS; demonstration of saturation kinetics (Km for SO–4 0.80 ± 0.17 mM and Vmax 649 ± 74 pmol/mg protein/10 sec); competitive inhibition of SO–4-OH– exchange by oxalate; SO–4 uptake was insensitive to alterations in the membrane potential; and inwardly directed Na+ gradient under non-pH gradient conditions did not stimulate SO–4 uptake. SO–4 uptake was significantly inhibited by increasing concentrations of chloride (1–10 mM) in the incubation media with a Ki, for CI– of 9.3 ± 1.4 mM. In contrast, OH–/HCO3– gradient-driven 36CI– uptake into these vesicles was unaffected by increasing concentrations of sulfate (10–50 mM). The above data indicate that two distinct transporters may be involved in SO–4 and CI– transport In the human intestinal apical membranes: an anion exchanger with high affinity for SO–4 and oxalate but low affinity for CI–, and a distinct CI–-OH– (HCO3–) exchanger with low affinity for SO–4.