EFFECTS OF INHIBITORS ON ANION-EXCHANGERS IN RABBIT RENAL BRUSH-BORDER MEMBRANE-VESICLES

Cl-formate, Cl-oxalate, and SO4-CO3 exchange participate in Cl and org anic anion transport across the brush border membrane of the rabbit pr oximal tubule. To determine the functional similarity of these transpo rters to each other and to band 3, we characterized, in isolated membr ane vesicles, the inhibition of these transporters by compounds known to inhibit erythrocyte band 3. 4,4'-Dinitro-2,2'-disulfonic stilbene ( DNDS), diphenylamine-2-carboxylate (DPC), flufenamate, and 4-aceto-4'- isothiocyano-2,2'-disulfonic stilbene (SITS) were effective inhibitors of Cl-oxalate and SO4-CO3 exchange, suggesting at least some common s tructural motifs between these exchangers and band 3. Cl-formate excha nge was relatively insensitive to DNDS and DPC but sensitive to flufen amate (IC50 = 43 mu M). Sensitivity to DNDS but not 4-amino-4'-amino-2 ,2'-disulfonic stilbene, a feature of band 3, was seen only for the SO 4-CO3 exchanger. None of the exchangers had significant affinity for d ipyridamole, furosemide, or probenecid. Finally, the presence of DPC o r flufenamate increased the IC50 for reversible inhibition by DNDS, co nsistent with at least a partial overlap between the disulfonic stilbe ne and diphenylamine carboxylate binding sites of the Cl-oxalate excha nger. We next examined the effect of irreversible SITS binding, The Cl -oxalate exchanger was inhibited 90%, consistent with its high affinit y for reversible inhibition by stilbenes. SITS pretreatment caused 50% inhibition of the Cl-formate exchanger, consistent with the reduced a ffinity of this exchanger for reversible binding of stilbenes. Despite the high sensitivity of the SO4-CO3 exchanger to reversible inhibitio n by stilbenes, SITS pretreatment caused <20% irreversible inhibition of this exchanger. Finally, we characterized the stilbene inhibition o f the Cl-oxalate exchanger in more detail. The presence of oxalate inc reased the IC50 for reversible inhibition by DNDS or SITS, implying th at oxalate can directly compete at the reversible stilbene binding sit e of the Cl-oxalate exchanger. However, oxalate could not protect agai nst covalent inactivation of the Cl-oxalate exchanger by SITS, indicat ing the presence of a separate site for irreversible binding of disulf onic stilbenes. These results suggest a dissociation between the sensi tivities of proximal tubule anion exchangers to reversible and irrever sible inhibition by disulfonic stilbenes. In contrast to band 3, the C l-oxalate exchanger must possess separate sites for reversible and irr eversible interaction with stilbenes, with only the former overlapping the substrate binding site.