Functional Roles of Clusters of Hydrophobic and Polar Residues in the Epithelial Na+ Channel Knuckle Domain

The extracellular regions of epithelial Na+ channel subunits are highly ordered structures composed of domains formed by alpha helices and beta strands. Deletion of the peripheral knuckle domain of the alpha subunit in the alpha beta gamma 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 beta or gamma subunit knuckle domain within the alpha beta gamma trimer dramatically reduces epithelial Na+ channel function and surface expression, and impairs subunit maturation. We systematically mutated individual alpha 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 toward the palm domain, whereas a group of polar residues are predicted to orient their side chains toward the space between the knuckle and finger domains. Among the mutants causing the greatest suppression of Na+ self-inhibition were alpha P521C, alpha I529C, and alpha S534C. The introduction of Cys residues at homologous sites within either the beta or gamma subunit knuckle domain resulted in little or no change in Na+ self-inhibition. Our results suggest that multiple residues in the gamma 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.