The epithelial sodium channel (ENaC) is comprised of three homologous subunits. Channels composed solely of α and β subunits (αβ channels) exhibit a very high open probability (Po) and reduced sensitivity to amiloride, in contrast to channels composed of α and subunits or of all three subunits (i.e., α and αβ channels). A mutant channel comprised of α and β subunits, and a chimeric subunit where the region immediately preceding (β12 and wrist) and encompassing the second transmembrane domain (TM2) was replaced with the corresponding region of the β subunit (-βTM2) displayed characteristics reminiscent of αβ channels, including a reduced potency of amiloride block and a loss of Na⁺ self-inhibition (reflecting an increased Po). Substitutions at key pore-lining residues of the -βTM2 chimera enhanced the Na⁺ self-inhibition response, whereas key subunit substitutions reduced the response. Furthermore, multiple sites within the TM2 domain of the subunit were required to confer high amiloride potency. In summary, we have identified novel pore-lining residues of the subunit of ENaC that are important for proper channel gating and its interaction with amiloride.