Inhibition of Na+-H+ exchange by N,N'-dicyclohexylcarbodiimide in isolated rat renal brush border membrane vesicles.
The inactivation of rat renal brush border membrane Na+-H+ exchange by the covalent carboxylate reagent N,N'-dicyclohexylcarbodiimide (DCCD) was studied by measuring 1 mM Na+ influx in the presence of a pH gradient (pHi = 5.5; pHo = 7.5) and H+ influx in the presence of a Na+ or Li+ gradient ([Na+]i = 150 mM; [Na+]o = 1.5 mM). In the presence of DCCD, the rate of Na+ uptake decreased exponentially with time and transport inhibition was irreversible. At all DCCD concentrations the loss of activity was described by a single exponential, consistent with one critical DCCD-reactive residue within the Na+-H+ exchanger. Among several carbodiimides the most hydrophobic carbodiimide, DCCD, was also the most effective inhibitor of Na+-H+ exchange. With 40 nmol of DCCD/mg of protein, at 20 degrees C for 30 min, 75% of the amiloride-sensitive 1 mM Na+ uptake was inhibited. Neither the equilibrium Na+ content nor the amiloride-insensitive Na+ uptake was significantly altered by the treatment. The Na+-dependent H+ flux, measured by the change in acridine orange absorbance, was also decreased 80% by the same DCCD treatment. If 150 mM NaCl, 150 mM LiCl, or 1 mM amiloride was present during incubation of the brush border membranes with 40 nmol of DCCD/mg of protein, then Li+-dependent H+ flux was protected 50, 100, or 100%, respectively, compared to membranes treated with DCCD in the absence of Na+-H+ exchanger substrates. The combination of DCCD and an exogenous nucleophile, e.g. ethylenediamine and glycine methyl ester, increased Na+-dependent H+ flux in the presence of 80 nmol of DCCD/mg of protein, compared to the transport after DCCD treatment alone. These findings suggest that the Na+-H+ exchanger contains a single carboxylate residue in a hydrophobic region of the protein, and the carboxylate and/or a nearby endogenous nucleophilic group is critical for exchange activity.
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002234:Carbodiimides / Q000494:pharmacology*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008094:Lithium / Q000494:pharmacology; D008871:Microvilli / Q000187:drug effects / Q000378:metabolism; D011522:Protons; D051381:Rats; D012964:Sodium / Q000378:metabolism* / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers
D002234:Carbodiimides; D002352:Carrier Proteins; D011522:Protons; D017923:Sodium-Hydrogen Exchangers; D004024:Dicyclohexylcarbodiimide; D000584:Amiloride; D008094:Lithium; D012964:Sodium