Functional roles of clusters of hydrophobic and polar residues in the epithelial Na+ channel knuckle domain

Abstract The extracellular regions of epithelial Na+ channel (ENaC) subunits are highly ordered structures comprised of domains formed by α helices and β strands. Deletion of the peripheral knuckle domain of the α subunit in the αβγ trimer results in channel activation, reflecting an increase in channel open probability due to a loss of the inhibitory effect of external Na+ (Na+ self-inhibition). In contrast, deletion of either the β or γ subunit knuckle domain within the αβγ trimer dramatically reduces ENaC function and surface expression, and impairs subunit maturation. We systematically mutated individual α subunit knuckle domain residues, and assessed functional properties of these mutants. Cysteine substitutions at 14 of 28 residues significantly suppressed Na+ self-inhibition. The side chains of a cluster of these residues are non-polar and are predicted to be directed towards the palm domain, whereas a group of polar residues are predicted to orient their side chains towards the space between knuckle and finger domains. Among the mutants causing the greatest suppression of Na+ self-inhibition are αP521C, αI529C and αS534C. The introduction of Cys residues at homologous sites within either the β or γ subunit knuckle domain resulted in little or no change in Na+ self-inhibition. Our results suggest that multiple residues in the α subunit knuckle domain contribute to the mechanism of Na+ self-inhibition by interacting with palm and finger domain residues via two separate and chemically distinct motifs.