Epithelial sodium channel pore region - Structure and role in gating

Epithelial sodium channels (ENaC) have a crucial role in the regulation of extracellular fluid volume and blood pressure. To study the structure of th e pore region of ENaC, the susceptibility of introduced cysteine residues t o sulfhydryl-reactive methanethiosulfonate derivatives ((a-aminoethyl)metha nethiosulfonate hydrobromide (MTSEA) and [(2-(trimethylammonium)ethyl]metha nethiosulfonate bromide (MTSET)) and to Cd2+ was determined. Selected mutan ts within the amino-terminal portion (alpha Val(569)-alpha Trp(582)) of the pore region responded to MTSEA, MTSET, or Cd2+ with stimulation or inhibit ion of whole cell Na+ current. The reactive residues were not contiguous bu t were separated by 23 residues where substituted cysteine residues did not respond to the reagents and line one face of an cu-helix. The activation o f (alpha S580C beta gamma mENaC by MTSET was associated with a large increa se in channel open probability. Within the carboxyl-terminal portion (alpha Ser(583)-alpha Ser(592)) of the pore region, only one mutation (alpha S583 C) conferred a rapid, nearly complete block by MTSEA, MTSET, and Cd2+, wher eas several other mutant channels were partially blocked by MTSEA or Cd2+ b ut not by MTSET. Our data suggest that the outer pore of ENaC is formed by an cr-helix, followed by an extended region that forms a selectivity filter . Furthermore, our data suggest that the pore region participates in ENaC g ating.