Characterization of the selectivity filter of the epithelial sodium channel

The epithelial sodium channel (ENaC) is composed of three homologous subuni ts termed alpha, beta, and gamma. Previous studies suggest that selected re sidues within a hydrophobic region immediately preceding the second membran e-spanning domain of each subunit contribute to the conducting pore of ENaC . We probed the pore of mouse ENaC by systematically mutating all 24 amino acids within this putative pore region of the alpha-subunit to cysteine and co-expressing these mutants with wild type beta- and gamma-subunits of mou se ENaC in Xenopus laevis oocytes, Functional characteristics of these muta nts were examined by two-electrode voltage clamp and single channel recordi ng techniques. Two distinct domains were identified based on the functional changes associated with point mutations. An amino-terminal domain (alpha-V al(569)-alpha-Gly(579)) showed minimal changes in cation selectivity or ami loride sensitivity following cysteine substitution. In contrast, cysteine s ubstitutions within the carboxyl-terminal domain (alpha-Ser(580)-alpha-Ser( 592)) resulted in significant changes in cation selectivity and moderately altered amiloride sensitivity. The mutant channels containing alpha G587C o r alpha S589C were permeable to K+, and mutation of a GSS tract (positions alpha 587-alpha 589) to GYG resulted in a moderately K+-selective channel. Our results suggest that the C-terminal portion of the pore region within t he alpha-subunit contributes to the selectivity filter of ENaC.