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

Abstract

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.Epithelial Na ϩ channels (ENaCs) 1 are expressed in a variety of tissues, including the distal nephron of the kidney, airway and alveolar epithelia in the lung, surface cells in the distal colon, urinary bladder epithelia, skin, and ducts within salivary and sweat glands (1). These transporters facilitate the movement of Na ϩ across the apical (or luminal) plasma membrane and have a critical role in extracellular fluid volume homeostasis, control of blood pressure, fetal lung maturation, and maintenance of airway fluids (1). ENaCs consist of at least three homologous subunits, termed ␣-, ␤-, and ␥ENaC, and are thought to form a tetrameric complex consisting of 2␣-, 1␤-, and 1␥-subunits (2-5), although one group has suggested a subunit stoichiometry of 3␣-, 3␤-, and 3␥-subunits (6). cDNAs encoding these Na ϩ channel subunits have been isolated and characterized from a variety of species. Each ENaC subunit has two predicted membrane-spanning domains. The amino-and carboxyl-terminal regions of the ENaCs are cytoplasmic, and each subunit has a large ectodomain (7-9). Domains of largely hydrophobic residues are located immediately following the putative first membrane-spanning domains and immediately preceding the second membrane-spanning domains of ␣-, ␤-and ␥rENaC (8, 10, 11). The hydrophobic domain immediately preceding the second membrane-spanning region of ENaC (referred to as the H2 domain) may insert in the membrane and form part of the channel pore (3,7,12).The diuretic drug amiloride is a prototypic inhibitor of epithelial Na ϩ channels. Several sites have been identified within the epithelial Na ϩ channel which participate in amiloride binding. Mutagenesis studies suggest that residues preceding the second ...

A Molecular Basis for Functional Peptide Mimicry of a Carbohydrate Antigen

“…A structure-based computer-screening approach was used to assemble an epitope map of the potential amino acids that could interact with the BR55-2 combining site (9,27). This mapping confirmed the participation of previously identified YRY and WRY motifs reacting with BR55-2 (28) and suggested a new putative sequence, FSLLW, that adopted an extended turn-like structure within the BR55-2 combining site.…”

“…Epitope Mapping of BR55-2 Combining Site-Using the positioned LeY structure within the BR55-2 combining site, we implemented the program Ligand-Design (LUDI (31) MSI/Biosym Technologies), as described previously (9,27,32), to search a fragment library and identify amino acid residue types able to interact with BR55-2, in a manner similar to that of LeY. The search was performed using standard default values and a fragment library supplied with the program.…”

Regulation of Epithelial Na+ Channels by Actin in Planar Lipid Bilayers and in the Xenopus Oocyte Expression System

“…Moreover, Rokaw et al (25) have recently presented evidence demonstrating that ␣-, ␤-, and ␥-ENaC are components of the epithelial Na ϩ channel biochemically isolated from A6 cells. Kieber-Emmons et al (34) have shown that the monoclonal antibody RA6.3, which was used to isolate biochemically a Na ϩ channel complex from A6 cells, recognizes an amiloridebinding site on ␣-ENaC. When these biochemically purified Na ϩ channels were incorporated into planar lipid bilayers, channel behavior (selectivity and single channel conductance) comparable to ␣␤␥-ENaC in bilayers was observed.…”

External Nickel Inhibits Epithelial Sodium Channel by Binding to Histidine Residues within the Extracellular Domains of α and γ Subunits and Reducing Channel Open Probability

“…Significant changes in Ni 2ϩ sensitivity were not observed with channels containing a mutation of these conserved histidine residues, suggesting that these conserved histidine residues are not essential for Ni 2ϩ inhibition of ENaC (Table I). We previously identified an amiloride-binding domain within the ECL of ␣ rat ENaC based on homology with an amiloride-binding site within an anti-amiloride antibody (14,40). Selected mutations within a six-residue tract (residues 278 -283, WYRFHY), including ␣His 282 , altered the amiloride sensitivity of channels composed solely of ␣-subunits reconstituted in planar lipid bilayers, although the amiloride sensitivity of ␣-␤-␥ ENaC containing an ␣His 282 mutation was unchanged (11,17).…”