On the Molecular Basis of Ion Permeation in the Epithelial Na⁺ Channel

The epithelial Na⁺ channel (ENaC) is highly selective for Na⁺ and Li⁺ over K⁺ and is blocked by the diuretic amiloride. ENaC is a heterotetramer made of two α, one β, and one γ homologous subunits, each subunit comprising two transmembrane segments. Amino acid residues involved in binding of the pore blocker amiloride are located in the pre-M2 segment of β and γ subunits, which precedes the second putative transmembrane α helix (M2). A residue in the α subunit (αS589) at the NH₂ terminus of M2 is critical for the molecular sieving properties of ENaC. ENaC is more permeable to Li⁺ than Na⁺ ions. The concentration of half-maximal unitary conductance is 38 mM for Na⁺ and 118 mM for Li⁺, a kinetic property that can account for the differences in Li⁺ and Na⁺ permeability. We show here that mutation of amino acid residues at homologous positions in the pre-M2 segment of α, β, and γ subunits (αG587, βG529, γS541) decreases the Li⁺/Na⁺ selectivity by changing the apparent channel affinity for Li⁺ and Na⁺. Fitting single-channel data of the Li⁺ permeation to a discrete-state model including three barriers and two binding sites revealed that these mutations increased the energy needed for the translocation of Li⁺ from an outer ion binding site through the selectivity filter. Mutation of βG529 to Ser, Cys, or Asp made ENaC partially permeable to K⁺ and larger ions, similar to the previously reported αS589 mutations. We conclude that the residues αG587 to αS589 and homologous residues in the β and γ subunits form the selectivity filter, which tightly accommodates Na⁺ and Li⁺ ions and excludes larger ions like K⁺.