On the interaction between amiloride and its putative α-subunit epithelial Na+ channel binding site

The epithelial Na+ channel ( ENaC) belongs to the structurally conserved ENaC/Degenerin superfamily. These channels are blocked by amiloride and its analogues. Several amino acid residues have been implicated in amiloride binding. Primary among these are alpha Ser-583, beta Gly-525, and gamma Gly-542, which are present at a homologous site within the three subunits of ENaC. Mutations of the beta and gamma glycines greatly weakened amiloride block, but, surprisingly, mutation of the serine of the alpha subunit resulted in moderate (< 5-fold) weakening of amiloride K-i. We investigated the role of alpha Ser-583 in amiloride binding by systematically mutating alpha Ser-583 and analyzing the mutant channels with two-electrode voltage clamp. We observed that most mutations had moderate effects on amiloride block, whereas those introducing rings showed dramatic effects on amiloride block. In addition, mutations introducing a beta-methyl group at this site altered the electric field of ENaC, affecting both amiloride binding and the voltage dependence of channel gating. We also found that the His mutation, in addition to greatly weakening amiloride binding, appends a voltage-sensitive gate within the pore of ENaC at low pH. Because diverse residues at alpha 583, such as Asn, Gln, Ser, Gly, Thr, and Ala, have similar amiloride binding affinities, our results suggest that the wild type Ser side chain is not important for amiloride binding. However, given that some alpha Ser-583 mutations affect the electrical properties of the channel whereas those introducing rings greatly weaken amiloride block, we conclude that amiloride binds at or near this site and that alpha Ser-583 may have a role in ion permeation through ENaC.