Gamma subunit second transmembrane domain contributes to epithelial sodium channel gating and amiloride block

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 γ-subunits or of all three subunits (i.e., αγ- and αβγ-channels). A mutant channel comprised of α- and β-subunits, and a chimeric γ-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 (γ-βTM2), displayed characteristics reminiscent of αβ-channels, including a reduced amiloride potency of block and a loss of Na(+) self-inhibition (reflecting an increased Po). Substitutions at key pore-lining residues of the γ-βTM2 chimera enhanced the Na(+) self-inhibition response, whereas key γ-subunit substitutions reduced the response. Furthermore, multiple sites within the TM2 domain of the γ-subunit were required to confer high amiloride potency. In summary, we have identified novel pore-lining residues of the γ-subunit of ENaC that are important for proper channel gating and its interaction with amiloride.