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

Abstract

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+.