Gating induces a conformational change in the outer vestibule of ENaC

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 li ttle is known about the mechanisms that control the gating of ENaC. We inve stigated the gating mechanism by introducing bulky side chains at a positio n adjacent to the extracellular end of the second membrane spanning segment (549, 520, and 529 in alpha, beta, and gamma ENaC, respectively). Equivale nt "DEG" mutations in related DEG/ENaC channels in Caenorhabditis elegans c ause swelling neurodegeneration, presumably by increasing channel activity. We found that the Na+ current was increased by mutagenesis or chemical mod ification of this residue and adjacent residues in alpha, beta, and gamma E NaC. This resulted from a change in the gating of ENaC; modification of a c ysteine at position 520 in beta ENaC increased the open state probability f rom 0.12 to 0.96. Accessibility to this side chain from the extracellular s ide 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 consisten t with a location for the DEG residue in the outer vestibule. The results s uggest that channel gating involves a conformational change in the outer ve stibule 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.