Selectivity Filter of Epithelial Na+ channels Is Linked to the Mechanism of Na+ Self‐inhibition

Extracellular Na + regulates epithelial Na + channels (ENaCs) by a process referred to as Na + self‐inhibition, which reflects an allosteric reduction of ENaC open probability. The mechanism by which external Na + inhibits ENaC has not been elucidated. We investigated a potential involvement of ENaC selectivity filter in this process. The Na + self‐inhibition responses of mouse αβγ ENaCs with a single mutation of the filter‐forming residues (G/S‐X‐S), as well as wild type channels were examined by analyzing the current decay following a rapid increase in the extracellular Na + concentration by two‐electrode voltage clamp in Xenopus oocytes. We found that cysteine substitution of any residue within the α GSS sequence greatly accelerated the current decay compared to that of wild type, with estimated time constants of 3.3 ± 0.2 sec (n=7) for αG587C; 2.4 + 0.1 sec (n=12) for αS588C; 3.4 ± 0.4 sec (n=7) for αS589C and 6.5 ± 0.2 sec (n=7) for wild type. The magnitude of the self‐inhibition response was increased by all mutations with an averaged inhibition of 83% for αG587C; 67% for αS588C; 59% for αS589C and 43% for wild type. In contrast, a more conservative substitution (αG587S or αS589T) that does not alter Li + /Na + and K + /Na + selectivity resulted in no enhancement of Na + self‐inhibition. These results suggest that a normal Na + self‐inhibition response depends on the functional integrity of the selectivity filter of ENaC, and that the filter is linked in some manner to the mechanism of Na + self‐inhibition. (Supported by NIH DK54354)