Mechanism of coupling between Cl- and OH- transport in renal brush-border membranes.
The coupling mechanism for Cl- and H+/OH- transport in renal brush-border vesicles was examined from intravesicular pH changes following imposed H+ and Cl- gradients. Vesicles were loaded with 6-carboxyfluorescein and exposed to H+ gradients and Cl-, gluconate, or sulfate gradients, each with and without a K+/valinomycin voltage clamp. Parallel experiments were performed with vesicles equilibrated with 10 mM HCO3- or 5 mM formate. Rate of H+/OH- transport was determined from the initial rate of change in 6-carboxyfluorescein fluorescence, vesicle buffer capacity and the relationship between fluorescence and vesicle pH. In contrast to gluconate or sulfate, Cl- caused enhanced H+/OH- transport under all conditions. This difference was eliminated with voltage clamping in the presence of gluconate, SO4(2-), or HCO3-, but not in the presence of formate. These findings were not affected by the method of preparation of the vesicles. Electrically coupled Cl-/OH- transport was not inhibited by 100 microM DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonate) or 100 microM DBDS (4,4'-dibenzamidostilbene-2,2'-disulfonate). SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate) was found to be a protonophore at concentrations greater than 500 microM. As a control for the method, we demonstrated amiloride inhibitable, electroneutral Na+-H+ exchange (H+ flux = 107 +/- 9 nmol/s per mg, 100 mM Na+) and electroneutral, DBDS inhibitable Cl(-)-HCO3- exchange in sealed human red blood cell ghosts. Therefore, electroneutral Cl(-)-OH- or HCO3- exchange does not measurably contribute to Cl- transport in the proximal tubule brush border. Cl(-)-formate exchange with formic acid recycling appears to be the only electroneutral coupling mechanism between Cl- and OH- transport demonstrable in renal brush-border membrane vesicles.
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D005260:Female; D005561:Formates / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006878:Hydroxides / Q000378:metabolism*; D007474:Ion Exchange; D007668:Kidney / Q000648:ultrastructure*; D008871:Microvilli / Q000378:metabolism*; D011817:Rabbits; D013267:Stilbenes / Q000494:pharmacology
D002712:Chlorides; D005561:Formates; D006878:Hydroxides; D013267:Stilbenes; C030544:formic acid; C031356:hydroxide ion