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J Biol Chem, Vol. 274, Issue 14, 9648-9655, April 2, 1999

Antiidiotypic Antibody Recognizes an Amiloride Binding Domain within the  Subunit of the Epithelial Na⁺ Channel

Thomas Kieber-Emmons, Chaomei Lin^§¶, Mary H. Foster^§, and Thomas R. Kleyman^§¶**

From the Departments of ^§ Medicine,  Pathology, and ^** Physiology, University of Pennsylvania, and the ^¶ Veterans Affairs Medical Center, Philadelphia, Pennsylvania 19104

We previously raised an antibody (RA6.3) by an antiidiotypic approach which was designed to be directed against an amiloride binding domain on the epithelial Na⁺ channel (ENaC). This antibody mimicked amiloride in that it inhibited transepithelial Na⁺ transport across A6 cell monolayers. RA6.3 recognized a 72-kDa polypeptide in A6 epithelia treated with tunicamycin, consistent with the size of nonglycosylated Xenopus laevis ENaC. RA6.3 specifically recognized an amiloride binding domain within the -subunit of mouse and bovine ENaC. The deduced amino acid sequence of RA6.3 was used to generate a three-dimensional model structure of the antibody. The combining site of RA6.3 was epitope mapped using a novel computer-based strategy. Organic residues that potentially interact with the RA6.3 combining site were identified by data base screening using the program LUDI. Selected residues docked to the antibody in a manner corresponding to the ordered linear array of amino acid residues within an amiloride binding domain on the -subunit of ENaC. A synthetic peptide spanning this domain inhibited the binding of RA6.3 to ENaC. This analysis provided a novel approach to develop models of antibody-antigen interaction as well as a molecular perspective of RA6.3 binding to an amiloride binding domain within ENaC.

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