IMMUNOCHEMICAL CHARACTERIZATION OF NA+ H+ EXCHANGER ISOFORM NHE4/

Mammalian Na+/H+ exchangers (NHEs) are a family of transport proteins (NHE1-NHE5). To date, the cellular and subcellular localization of NHE 4 has not been characterized using immunochemical techniques. We purif ied 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. Ii; reacted specifically with both the fusion protein and to a 60- to 65-kDa polypeptide expressed in NHE4-transfected LAP1 cells. By Western blot analysis, NHE4 was identified as a 65- to 70-kDa prot ein that was expressed most abundantly in stomach and in multiple addi tional epithelial and nonepithelial rat tissues including skeletal mus cle, heart, kidney, uterus, and liver Subcellular localization of NHE4 in the kidney was evaluated by Western blot analysis of membrane frac tions isolated by Percoll gradient centrifugation. NHE4 was found to c ofractionate with the basolateral markers NHE1 and Na+-K+-ATPase rathe r than the luminal marker gamma-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- to 70-kDa protein with a broad tissue distribution. In two types of epithelial cells, kidney and stomach, NHE4 is localized to the baso lateral membrane.