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Original Article

^a From the Institut de Pharmacologie et de Toxicologie, Université de Lausanne, CH-1005 Lausanne, Switzerland
Institut de Pharmacologie et de Toxicologie, Bugnon 27, CH-1005 Lausanne, Switzerland.Fax: 4121 692 5355;

laurent.schild{at}ipharm.unil.ch

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 , one β, and one 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 subunits, which precedes the second putative transmembrane helix (M2). A residue in the subunit (S589) 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 , β, and subunits (G587, βG529, S541) 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 S589 mutations. We conclude that the residues G587 to S589 and homologous residues in the β and subunits form the selectivity filter, which tightly accommodates Na⁺ and Li⁺ ions and excludes larger ions like K⁺.

Key Words: epithelial Na⁺ channel • Xenopus oocyte • pore • selectivity • ion channel

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