Identification of the human NHE-1 form of Na+-H+ exchanger in rabbit renal brush border membranes

• Published in: The Journal of clinical investigation 1993-05, Vol.91 (5), p.2097-2102
• Main Authors: WEINMAN, E. J, STEPLOCK, D, CORRY, D, SHENOLIKAR, S
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Ammonium Chloride - pharmacology; Animals; Biological and medical sciences; Blotting, Western; Brain - metabolism; Carrier Proteins - isolation & purification; Carrier Proteins - metabolism; Cell Fractionation; Cell Line; Cell Membrane - metabolism; Cricetinae; Cricetulus; Electrophoresis, Polyacrylamide Gel; Humans; Investigative techniques, diagnostic techniques (general aspects); Kidney - drug effects; Kidney - metabolism; Kinetics; Medical sciences; Microvilli - drug effects; Microvilli - metabolism; Molecular Sequence Data; Molecular Weight; Muscles - metabolism; Organ Specificity; Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques; Rabbits; Sodium-Hydrogen Exchangers; Urinary system
• ISSN: 0021-9738; 1558-8238
• DOI: 10.1172/JCI116433

To study the relation between the human Na(+)-H+ exchanger (NHE-1) and the renal brush border membrane (BBM) Na(+)-H+ exchanger, polyclonal antibodies to synthetic peptides representing a putative external (Ab-E) and an internal cytosolic domain (Ab-I) of human NHE-1 were generated in rabbits. Western immunoblot analyses indicated that both antibodies recognized a 97-kD protein in rabbit renal BBM but not basolateral membranes (BLM). Octyl glucoside-extracted rabbit renal BBM proteins also contained the 97-kD polypeptide as did a fraction eluted from an anion-exchange column with 0.2 M NaCl (fraction A). A fraction eluting between 0.2 and 0.4 M NaCl (fraction B) did not contain this protein. Prior reconstitution studies have indicated that Na(+)-H+ exchange activity is higher significantly in fraction B than fraction A. Administration of NH4Cl for 3-7 d to rabbits, a stimulus known to increase renal BBM Na(+)-H+ exchange activity, did not result in a change in expression of the 97-kD protein in either renal BBM or BLM. The results indicate that affinity-purified polyclonal antibodies to two separate domains of the human Na(+)-H+ exchanger recognize a 97-kD protein in rabbit renal BBM but not BLM. The dissociation between recognition of the 97-kD protein using antibodies and the majority of functional Na(+)-H+ exchange activity after chromatographic fractionation of solubilized BBM proteins and in native BBM after administration of NH4Cl suggest that rabbit renal BBM contains more than one form of Na(+)-H+ exchanger.