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Identification of the renal Na+/H+ exchanger with N,N′-dicyclohexylcarbodiimide (DCCD) and amiloride analogues

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Summary

Dicyclohexylcarbodiimide (DCCD) and the 5-ethylisopropyl-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 α-subunit of the Na+, K+-ATPase. [14C]-Br-EIPA was mainly incorporated into a brushborder 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.

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  1. Max-Planck-Institut für Biophysik, D-6000, Frankfurt/Main 70, Federal Republic of Germany

    Thomas Friedrich, Jutta Sablotni & Gerhard Burckhardt

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  1. Thomas Friedrich
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Friedrich, T., Sablotni, J. & Burckhardt, G. Identification of the renal Na+/H+ exchanger with N,N′-dicyclohexylcarbodiimide (DCCD) and amiloride analogues. J. Membrain Biol. 94, 253–266 (1986). https://doi.org/10.1007/BF01869721

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