Second Transmembrane Domains of ENaC Subunits Contribute to Ion Permeation and Selectivity

Abstract

Epithelial sodium channels (ENaC) are composed of three structurally related subunits (α, β, and γ). Each subunit has two transmembrane domains termed M1 and M2, and residues conferring cation selectivity have been shown to reside in a pore region immediately preceding the M2 domains of the three subunits. Negatively charged residues are interspersed within the M2 domains, and substitution of individual acidic residues within human α-ENaC with arginine essentially eliminated channel activity in oocytes, suggesting that these residues have a role in ion permeation. We examined the roles of M2 residues in contributing to the permeation pore by individually mutating residues within the M2 domain of mouse αENaC to cysteine and systematically characterizing functional properties of mutant channels expressed in Xenopus oocytes by two-electrode voltage clamp. The introduction of cysteine residues at selected sites, including negatively charged residues (αGlu595, αGlu598, and α-Asp602) led to a significant reduction of expressed amiloride-sensitive Na+ currents. Two mutations (αE595C and αD602C) resulted in K+-permeable channels whereas multiple mutations altered Li+/Na+ current ratios. Channels containing αD602K or αD602A also conducted K+ whereas more conservative mutations (αD602E and αD602N) retained wild type selectivity. Cysteine substitution at the site equivalent to αAsp 602 within β mENaC (βD544C) did not alter either Li +/Na+ or K+/Na+ current ratios, although mutation of the equivalent site within γ mENaC (γD562C) significantly increased the Li+/Na+ current ratio. Mutants containing introduced cysteine residues at αGlu595, αGlu598, αAsp602, or αThr607 did not respond to externally applied sulfhydryl reagent with significant changes in macroscopic currents. Our results suggest that some residues within the M2 domain of αENaC contribute to the channel's conduction pore and that, in addition to the pore region, selected sites within M2 (αGlu 595 and αAsp602) may have a role in conferring ion selectivity.