title: Gating induces a conformational change in the outer vestibule of ENaC.
abstract: The epithelial Na(+) channel (ENaC) is comprised of three homologous subunits (alpha, beta, and gamma). The channel forms the pathway for Na(+) absorption in the kidney, and mutations cause disorders of Na(+) homeostasis. However, little is known about the mechanisms that control the gating of ENaC. We investigated the gating mechanism by introducing bulky side chains at a position adjacent to the extracellular end of the second membrane spanning segment (549, 520, and 529 in alpha, beta, and gammaENaC, respectively). Equivalent "DEG" mutations in related DEG/ENaC channels in Caenorhabditis elegans cause swelling neurodegeneration, presumably by increasing channel activity. We found that the Na(+) current was increased by mutagenesis or chemical modification of this residue and adjacent residues in alpha, beta, and gammaENaC. This resulted from a change in the gating of ENaC; modification of a cysteine at position 520 in betaENaC increased the open state probability from 0. 12 to 0.96. Accessibility to this side chain from the extracellular side was state-dependent; modification occurred only when the channel was in the open conformation. Single-channel conductance decreased when the side chain contained a positive, but not a negative charge. However, alterations in the side chain did not alter the selectivity of ENaC. This is consistent with a location for the DEG residue in the outer vestibule. The results suggest that channel gating involves a conformational change in the outer vestibule of ENaC. Disruption of this mechanism could be important clinically since one of the mutations that increased Na(+) current (gamma(N530K)) was identified in a patient with renal disease.
authors: Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA. psnyder@blue.weeg.uiowa.edu
authors: P M
authors: PM
authors: Snyder
authors: D B
authors: DB
authors: Bucher
authors: D R
authors: DR
authors: Olson
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D004553:Electric Conductivity; D053503:Epithelial Sodium Channels; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology; D009865:Oocytes; D015222:Sodium Channels / Q000235:genetics* / Q000502:physiology*; D014982:Xenopus laevis
chemical_list: D053503:Epithelial Sodium Channels; D015222:Sodium Channels