W493R Gain of Function Mutation in Atypical Cystic Fibrosis Rewires the Epithelial Sodium Channel Dynamics

Abstract

Sodium absorption in epithelial cells is rate-limited by the epithelial sodium channel (ENaC) activity in lungs, 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's molecular regulation. The molecular mechanism(s) of pathologically implicated mutations in ENaC subunits are largely unknown due to absence of structural models for ENaC's oligomers. Here, we propose a dynamics-driven mechanism of the gain of function mutation αW493R implicated in atypical cystic fibrosis. We utilize a combination of discrete molecular dynamics simulations (DMD) of the extracellular region of ENaC's heterotrimer αβγ and functional data from whole-cell electrophysiology experiments. Structure-function studies suggest that tetramers and trimers are the major characterized functional oligomeric states of ENaC. Using the crystal structure of the acid sensing ion channel, ENaC's structural homolog in the ENaC/degenerin mechanosensitive ion channel family, we have constructed homology models of ENaC subunits alpha, beta and gamma, in addition to the heterotrimers αβγ, αγβ and the heterotetramer αβαγ. Electrophysiology data show constitutive activity of αW493Rβγ in oocytes indicated by higher open probability and elevated basal activity compared to WT. Our DMD simulation provides an allosteric framework, which agrees with experimental data. W493R rewires the electrostatically mediated inter-residue interaction network in close proximity to W493, resulting in widening of the pore geometry in the outer mouth of the pore in the extracellular region. Rewiring effect of inter-residue interactions in 493R mutant pocket allosterically propagates across the channel resulting in a more stabilized global conformational ensemble of the channel. These findings predict a novel mechanism of ENaC's constitutive activity, in which changes in local dynamics can affect the relative population of the channel's active states and its open probability.