Permeability properties of ENaC selectivity filter mutants

The epithelial Na+ channel (ENaC), located in the apical membrane of tight epithelia, allows vectorial Na+ absorption. The amiloride-sensitive ENaC is highly selective for Na+ and Li+ ions. There is growing evidence that the short stretch of amino acid residues (preM2) preceding the putative second transmembrane domain M2 forms the outer channel pore with the amiloride binding site and the narrow ion-selective region of the pore. We have shown previously that mutations of the alpha S589 residue in the preM2 segment change the ion selective making the channel permeant to K+ ions. To understand the molecular basis of this important change in ionic selectivity, we have substituted alpha S589 with amino acids of different sizes and physicochemical properties. Here, we show that the molecular cutoff of the channel pore for inorganic and organic cations increases with the size of the amino acid residue at position alpha 589, indicating that alpha S589 mutations enlarge the pore at the selectivity, filter. Mutants with an increased permeability to large cations show a decrease in the ENaC unitary conductance of small cations such as Na+ and Li+. These findings demonstrate the critical role of the pore size at the alpha S589 residue for the selectivity properties of ENaC. Out, data are consistent with the main chain carbonyl oxygens of the alpha S589 residues lining the channel pore at the selectivity filter with their side chain pointing away from the pore lumen. We propose that the alpha S589 side chain is oriented toward the subunit-subunit interface and that substitution of alpha S589 by larger residues increases the pore diameter by adding extravolume at the subunit-subunit interface.