Identification of an amiloride binding domain within the alpha-subunit of the epithelial Na+ channel

Limited information is available regarding domains within the epithelial Na+ channel (ENaC) which participate in amiloride binding, We previously utilized the anti-amiloride antibody (BA7.1) as a surrogate amiloride receptor to delineate amino acid residues that con??? tact amiloride, and identified a putative amiloride binding domain WYRFHY (residues 278-283) within the extracellular domain of alpha rENaC. Mutations were generated to examine the role of this sequence in amiloride binding, Functional analyses of wild type (wt) and mutant alpha rENaCs were performed by cRNA expression in Xenopus oocytes and by reconstitution into planar lipid bilayers, Wild type alpha rENaC was inhibited by amiloride with a K-i of 169 nM. Deletion of the entire WYRFHY tract (alpha rENaC Delta 278-283) resulted in a loss of sensitivity of the channel to submicromolar concentrations off amiloride (K-i = 26.5 mu M). Similar results were obtained when either alpha rENaC or alpha rENaC Delta 278-283 were co-expressed with wt beta- and alpha rENaC (K-i values of 155 nM and 22.8 mu m, respectively), Moreover, alpha rENaC H282D was insensitive to submicromolar concentrations of amiloride (K-i = 6.52 mu M), whereas alpha rENaC H282R was inhibited by amiloride with a K-i of 29 nM. These mutations do not alter ENaC Na+:K+ selectivity nor single-channel conductance,These data suggest that residues within the tract WYRFHY participate in amiloride binding, Our results, in conjunction with recent studies demonstrating that mutations within the membrane-spanning domains of alpha rENaC and mutations preceding the second membrane-spanning domains of alpha-, beta-, and gamma rENaC alters amiloride's K-i, suggest that selected regions of the extracellular loop of alpha rENaC may be in close proximity to residues within the channel pore.