Renal basolateral membrane anion transporter characterized by a fluorescent disulfonic stilbene

Abstract

The fluorescence enhancement of 4,4'-dibenzamido-2,2'-disulfonic stilbene (DBDS) upon binding to membranes was used to examine proximal tubule stilbene binding sites. Equilibrium binding studies of DBDS to renal brush border (BBMV) and basolateral membrane vesicles (BLMV) were performed using a fluorescence enhancement technique developed for red blood cells (A.S. Verkman, J.A. Dix and A.K. Solomon, J. Gen. Physiol. 81:421-449, 1983). In the absence of transportable anions, DBDS bound reversibly to a single class of sites on BLMV isolated from rabbit (Kd = 3.8 microM) and rat (3.2 microM); 100 microM dihydro-4,4'-diisothiocyano-2,2'-disulfonic stilbene (H2DIDS) blocked greater than 95% of binding. H2DIDS inhibitable DBDS binding was not detected using rat or rabbit BBMV. In rabbit BLMV, DBDS Kd doubled with 10 mM SO4, 50 mM HCO3 and 100 mM Cl, but was not altered by Na or pH (6-8). In stopped-flow experiments the exponential time constant for DBDS binding slowed with SO4, HCO3 and Cl, but was unaffected by Na. These results are consistent with competitive binding of DBDS and anions at an anion transport site. To relate DBDS binding data to anion transport inhibition we used 35SO4 uptake to characterize several modes of rabbit BLM anion transport: H/SO4 and Na/SO4 cotransport, and Cl/SO4 countertransport. Each transport process was electroneutral and was inhibited by H2DIDS, furosemide, probenecid, chlorothiazide and DBDS. The apparent KI's for DBDS (3-20 microM) were similar to Kd for DBDS binding. These studies define a class of anion transport sites on the proximal tubule basolateral membrane measurable optically by a fluorescent stilbene.