title: Gain-of-Function Mutation W493R in the Epithelial Sodium Channel Allosterically Reconfigures Intersubunit Coupling.
abstract: 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.
authors.affiliation: From the Program in Molecular and Cellular Biophysics, Curriculum in Bioinformatics and Computational Biology, Department of Biochemistry and Biophysics, and.
authors.forename: Mahmoud
authors.initials: M
authors.lastname: Shobair
authors.affiliation: From the Program in Molecular and Cellular Biophysics, Department of Biochemistry and Biophysics, and.
authors.forename: Onur
authors.initials: O
authors.lastname: Dagliyan
authors.affiliation: From the Program in Molecular and Cellular Biophysics, Department of Biochemistry and Biophysics, and.
authors.forename: Pradeep
authors.initials: P
authors.lastname: Kota
authors.affiliation: Cystic Fibrosis and Pulmonary Diseases Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599.
authors.forename: Yan L
authors.initials: YL
authors.lastname: Dang
authors.affiliation: Cystic Fibrosis and Pulmonary Diseases Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599.
authors.forename: Hong
authors.initials: H
authors.lastname: He
authors.affiliation: Cystic Fibrosis and Pulmonary Diseases Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599.
authors.forename: M Jackson
authors.initials: MJ
authors.lastname: Stutts
authors.affiliation: From the Program in Molecular and Cellular Biophysics, Curriculum in Bioinformatics and Computational Biology, Department of Biochemistry and Biophysics, and Cystic Fibrosis and Pulmonary Diseases Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 dokh@unc.edu.
authors.forename: Nikolay V
authors.identifier: http://orcid.org/0000-0002-8225-4025
authors.initials: NV
authors.lastname: Dokholyan
mesh_terms: D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D053503:Epithelial Sodium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D020836:Protein Structure, Quaternary; D051381:Rats; D012964:Sodium / Q000737:chemistry* / Q000378:metabolism; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship
chemical_list: D053503:Epithelial Sodium Channels; D012964:Sodium
keywords: allosteric activation; channelopathies; electrophysiology; ion channel; molecular docking; molecular dynamics; protein-protein interaction; tetramer model