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Correspondence to: Laurent Schild, Institut de Pharmacologie et de Toxicologie, Bugnon 27, CH-1005 Lausanne, Switzerland., laurent.schild{at}ipharm.unil.ch (E-mail), Fax: 4121 692 5355; (fax)
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 ß, 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 ß 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 , ß, and subunits (G587, ßG529, 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 ßG529 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 ß 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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