We have generated two site-directed mutants, K504E and K515E, in the α subunit of an amiloride-sensitive bovine epithelial Na+ channel, αbENaC. The region in which these mutations lie is in the large extracellular loop immediately before the second membrane-spanning domain (M2) of the protein. We have found that when membrane vesicles prepared from Xenopus oocytes expressing either K504E or K515E αbENaC are incorporated into planar lipid bilayers, the gating pattern, cation selectivity, and amiloride sensitivity of the resultant channel are all altered as compared to the wild-type protein. The mutated channels exhibit either a reduction or a complete lack of its characteristic burst-type behavior, significantly reduced Na+:K+ selectivity, and an approximately 10-fold decrease in the apparent inhibitory equilibrium dissociation constant (K(i)) for amiloride. Single-channel conductance for Na+ was not affected by either mutation. On the other hand, both K504E and K515E αbENaC mutants were significantly more permeable to K+, as compared to wild type. These observations identify a lysine-rich region between amino acid residues 495 and 516 of αbENaC as being important to the regulation of fundamental channel properties.