Inhibition of H+/organic cation antiport by carboxyl reagents in rabbit renal brush-border membrane vesicles.

The inactivation of the renal H+/organic cation antiporter by the covalent carboxyl reagent N,N'-dicyclohexylcarbodiimide (DCCD) was studied by measuring tetraethylammonium (TEA) uptake in brush border membrane vesicles isolated from rabbit renal cortex. Pretreatment of membrane vesicles with DCCD resulted in an irreversible inhibition of H+ gradient-dependent TEA uptake in a dose-dependent manner with IC50 of 10 microM. The inhibition was not due to the disruption of the vesicles or a faster collapse of an imposed H+ gradient. The transport system was significantly protected from DCCD inhibition by substrates, TEA, choline and amiloride, but not by N1-methylnicotinamide. Kinetic analysis indicated that the inhibition by DCCD was due to an increase in Km and a decrease in Vmax. The hydrophilic carbodiimides, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide and another carboxyl reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline also inhibited the H(+)-dependent TEA uptake with a lower potency than did DCCD. These results suggest that carboxyl groups are essential for the H+/organic cation antiport and that they may be located at or near the substrate (organic cation) binding sites of the transporter in rabbit brush border membranes.