The hypothesis that there is a highly conserved, positively charged region distal to the second transmembrane domain in α-ENaC (epithelial sodium channel) that acts as a putative receptor site for the negatively charged COOH-terminal β- and γ-ENaC tails was tested in mutagenesis experiments. After expression in Xenopus oocytes, α-ENaC constructs in which positively charged arginine residues were converted into negatively charged glutamic acids could not be inhibited by blocking peptides. These observations provide insight into the gating machinery of ENaC.