Identification of the renal Na+/H+ exchanger with N,N'-dicyclohexylcarbodiimide (DCCD) and amiloride analogues.
Dicyclohexylcarbodiimide (DCCD) and the 5-ethyl-isopropyl-6-bromo-derivative of amiloride (Br-EIPA) have been used as affinity and photoaffinity labels of the Na+/H+ exchanger in rat renal brush-border membranes. Intravesicular acidification by the Na+/H+ exchanger was irreversibly inhibited after incubation of vesicles for 30 min with DCCD. The substrate of the antiporter, Na+, and the competitive inhibitor, amiloride, protected from irreversible inhibition. The Na+-dependent transport systems for sulfate, dicarboxylates, and neutral, acidic, and basic amino acids were inhibited by DCCD, but not protected by amiloride. An irreversible inhibition of Na+/H+ exchange was also observed when brush-border membrane vesicles were irradiated in the presence of Br-EIPA. Na+ and Li+ protected. [14C]-DCCD was mostly incorporated into three brush-border membrane polypeptides with apparent molecular weights of 88,000, 65,000 and 51,000. Na+ did not protect but rather enhanced labeling. In contrast, amiloride effectively decreased the labeling of the 65,000 molecular weight polypeptide. In basolateral membrane vesicles one band was highly labeled by [14C]DCCD that was identified as the alpha-subunit of the Na+,K+-ATPase. [14C]-Br-EIPA was mainly incorporated into a brush-border membrane polypeptide with apparent molecular weight of 65,000. Na+ decreased the labeling of this protein. Similar to the Na+/H+ exchanger this Na+-protectable band was absent in basolateral membrane vesicles. We conclude that a membrane protein with an apparent molecular weight of 65,000 is involved in rat renal Na+/H+ exchange.
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology*; D000818:Animals; D002234:Carbodiimides / Q000494:pharmacology*; D002352:Carrier Proteins / Q000378:metabolism*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008871:Microvilli / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D017923:Sodium-Hydrogen Exchangers; D013329:Structure-Activity Relationship
D002234:Carbodiimides; D002352:Carrier Proteins; D017923:Sodium-Hydrogen Exchangers; D004024:Dicyclohexylcarbodiimide; D000584:Amiloride