Abstract
The extracellular regions of epithelial Na(+) channel subunits are highly ordered structures composed 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 epithelial Na(+) channel 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 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 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.
Highlights
There are regulatory interactions between Epithelial Naϩ channels (ENaCs) and extracellular factors
We found that mature forms of the  and ␥ subunit were seen with Wild type (WT) ENaC
The lack of change in ENaC surface expression when the mouse ␣ subunit knuckle domain is deleted, in association with the markedly impaired Naϩ self-inhibition response, suggests that channel activation reflects an increase in its open probability
Summary
Results: Mutations of multiple ␣ subunit knuckle residues activate ENaC by suppressing the inhibitory effect of Naϩ. Conclusion: Interactions between the ␣ subunit knuckle and palm/finger domains regulate ENaC. Deletion of the peripheral knuckle domain of the ␣ subunit in the ␣␥ trimer results in channel activation, reflecting an increase in channel open probability due to a loss of the inhibitory effect of external Na؉ (Na؉ selfinhibition). The introduction of Cys residues at homologous sites within either the  or ␥ subunit knuckle domain resulted in little or no change in Na؉ self-inhibition. Our results suggest that multiple residues in the ␣ 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
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