Characterization of the Selectivity Filter of the Epithelial Sodium Channel*

  1. Thomas R. Kleyman§
  1. From the Departments of Medicine and Physiology, School of Medicine, University of Pennsylvania and the Veteran Affairs Medical Center, Philadelphia, Pennsylvania 19104

Abstract

The epithelial sodium channel (ENaC) is composed of three homologous subunits termed α, β, and γ. Previous studies suggest that selected residues within a hydrophobic region immediately preceding the second membrane-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 α-subunit to cysteine and co-expressing these mutants with wild type β- and γ-subunits of mouse ENaC inXenopus laevis oocytes. Functional characteristics of these mutants were examined by two-electrode voltage clamp and single channel recording techniques. Two distinct domains were identified based on the functional changes associated with point mutations. An amino-terminal domain (α-Val569–α-Gly579) showed minimal changes in cation selectivity or amiloride sensitivity following cysteine substitution. In contrast, cysteine substitutions within the carboxyl-terminal domain (α-Ser580–α-Ser592) resulted in significant changes in cation selectivity and moderately altered amiloride sensitivity. The mutant channels containing αG587C or αS589C were permeable to K+, and mutation of a GSS tract (positions α587–α589) to GYG resulted in a moderately K+-selective channel. Our results suggest that the C-terminal portion of the pore region within the α-subunit contributes to the selectivity filter of ENaC.

  • Abbreviations:
    ENaC
    epithelial sodium channel
    mENaC
    mouse ENaC
    • Received November 4, 1999.
    • Revision received December 30, 1999.
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