Sodium absorption in epithelial cells is rate-limited by the epithelial sodium channel (ENaC) activity in lung, kidney, and the distal colon. Pathophysiological conditions, such as cystic fibrosis and Liddle syndrome, result from water-electrolyte imbalance partly due to malfunction of ENaC regulation. Because the quaternary structure of ENaC is yet undetermined, the bases of pathologically linked mutations in ENaC subunits α, β, and γ are largely unknown. Here, we present a structural model of heterotetrameric ENaC α1βα2γ that is consistent with previous cross-linking results and site-directed mutagenesis experiments. By using this model, we show that the disease-causing mutation αW493R rewires structural dynamics of the intersubunit interfaces α1β and α2γ. Changes in dynamics can allosterically propagate to the channel gate. We demonstrate that cleavage of the γ-subunit, which is critical for full channel activation, does not mediate activation of ENaC by αW493R. Our molecular dynamics simulations led us to identify a channel-activating electrostatic interaction between α2Arg-493 and γGlu-348 at the α2γ interface. By neutralizing a sodium-binding acidic patch at the α1β interface, we reduced ENaC activation of αW493R by more than 2-fold. By combining homology modeling, molecular dynamics, cysteine cross-linking, and voltage clamp experiments, we propose a dynamics-driven model for the gain-of-function in ENaC by αW493R. Our integrated computational and experimental approach advances our understanding of structure, dynamics, and function of ENaC in its disease-causing state.