Side chain orientation of residues lining the selectivity filter of epithelial Na+ channels.

Abstract Epithelial Na+ channels (ENaCs) selectively conduct Na+ and Li+ but exclude K+. A three-residue tract ((G/S)XS) present within all three subunits has been identified as a key structure forming a putative selectivity filter. We investigated the side chain orientation of residues within this tract by analyzing accessibility of the introduced sulfhydryl groups to thiophilic Cd2+. Xenopus oocytes were used to express wild-type or mutant mouse αβγENaCs. The blocking effect of external Cd2+ was examined by comparing amiloride-sensitive Na+ currents measured by two-electrode voltage clamp in the absence and presence of Cd2+ in the bath solution. The currents in mutant channels containing a single Cys substitution at the first or third position within the (G/S)XS tract (αG587C, αS589C, βG529C, βS531C, γS546C, and γS548C) were blocked by Cd2+ with varying inhibitory constants (0.06-13 mm), whereas the currents in control channels were largely insensitive to Cd2+ at concentrations up to 10 mm. The Cd2+ blocking effects were fast, with time constants in the range of seconds, and were only partially reversible. The blocked currents were restored by 10 mm dithiothreitol. Mutant channels containing alanine or serine substitutions at these sites within the α subunit were only poorly and reversibly blocked by 10 mm Cd2+. These results indicate that the introduced sulfhydryl groups face the conduction pore and suggest that serine hydroxyl groups within the selectivity filter in wild-type ENaCs face the conduction pore and may contribute to cation selectivity by participating in coordination of permeating cations.