IDENTIFICATION OF AN AMILORIDE BINDING DOMAIN WITHIN THE ALPHA-SUBUNIT OF THE EPITHELIAL NA+ CHANNEL

Limited information is available regarding domains within the epitheli al Na+ channel (ENaC) which participate in amiloride binding, We previ ously utilized the anti-amiloride antibody (BA7.1) as a surrogate amil oride receptor to delineate amino acid residues that con??? tact amilo ride, and identified a putative amiloride binding domain WYRFHY (resid ues 278-283) within the extracellular domain of alpha rENaC. Mutations were generated to examine the role of this sequence in amiloride bind ing, Functional analyses of wild type (wt) and mutant alpha rENaCs wer e performed by cRNA expression in Xenopus oocytes and by reconstitutio n into planar lipid bilayers, Wild type alpha rENaC was inhibited by a miloride with a K-i of 169 nM. Deletion of the entire WYRFHY tract (al pha rENaC Delta 278-283) resulted in a loss of sensitivity of the chan nel to submicromolar concentrations off amiloride (K-i = 26.5 mu M). S imilar results were obtained when either alpha rENaC or alpha rENaC De lta 278-283 were co-expressed with wt beta- and alpha rENaC (K-i value s 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 th e tract WYRFHY participate in amiloride binding, Our results, in conju nction with recent studies demonstrating that mutations within the mem brane-spanning domains of alpha rENaC and mutations preceding the seco nd membrane-spanning domains of alpha-, beta-, and gamma rENaC alters amiloride's K-i, suggest that selected regions of the extracellular lo op of alpha rENaC may be in close proximity to residues within the cha nnel pore.