Gain-of-Function Mutation W493R in the Epithelial Sodium Channel Allosterically Reconfigures Intersubunit Coupling

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 , , and are largely unknown. Here, we present a structural model of heterotetrameric ENaC (12) that is consistent with previous cross-linking results and site-directed mutagenesis experiments. By using this model, we show that the disease-causing mutation W493R rewires structural dynamics of the intersubunit interfaces (1) and (2). Changes in dynamics can allosterically propagate to the channel gate. We demonstrate that cleavage of the -subunit, which is critical for full channel activation, does not mediate activation of ENaC by W493R. Our molecular dynamics simulations led us to identify a channel-activating electrostatic interaction between (2)Arg-493 and Glu-348 at the (2) interface. By neutralizing a sodium-binding acidic patch at the (1) interface, we reduced ENaC activation of W493R 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 W493R. Our integrated computational and experimental approach advances our understanding of structure, dynamics, and function of ENaC in its disease-causing state.