Immunochemical characterization of na+/h+ exchanger isoform nhe4. Pizzonia, John H., Daniel Biemesderfer, Ali K. Abu-Alfa, Ming-Shiou Wu, Markus Exner, Paul Isenring, Peter Igarashi, and Peter S. Aronson. Section of Nephrology, Department of Internal Medicine, Yale University School of Medicine, P.O. Box 208029, New Haven, CT 06520 -8029.
APStracts 5:0121F, 1998.
Mammalian Na+/H+ exchangers (NHEs) are a family of transport proteins (NHE1-NHE5). To date, the cellular and subcellular localization of NHE4 has not been characterized using immunochemical techniques. We purified a fusion protein containing a portion of rat NHE4 (amino acids 565-675) to use as immunogen. A monoclonal antibody (11H11) was selected by ELISA. It reacted specifically with both the fusion protein and to a 60-65 kDa polypeptide expressed in NHE4-transfected LAP1 cells. By Western blot analysis, NHE4 was identified as a 65-70 kDa protein that was expressed most abundantly in stomach and in multiple additional epithelial and non-epithelial rat tissues including skeletal muscle, heart, kidney, uterus and liver. Subcellular localization of NHE4 in the kidney was evaluated by Western blot analysis of membrane fractions isolated by Percoll gradient centrifugation. NHE4 was found to cofractionate with the basolateral markers NHE1 and Na+,K+-ATPase rather than the luminal marker g-glutamyl transferase. In stomach, NHE4 was detected by immunoperoxidase labeling on the basolateral membrane of cells at the base of the gastric gland. We conclude that NHE4 is a 65-70 kDa protein with a broad tissue distribution. In two types of epithelial cells, kidney and stomach, NHE4 is localized to the basolateral membrane. 

Received 7 February 1997; accepted in final form 2 July 1998.
APS Manuscript Number F44-7.
Article publication pending Am. J. Physiol. (Renal Physiology).
ISSN 1080-4757 Copyright 1998 The American Physiological Society.
Published in APStracts on 30 July 1998