title: The N-terminal domain allosterically regulates cleavage and activation of the epithelial sodium channel.
abstract: The epithelial sodium channel (ENaC) is activated upon endoproteolytic cleavage of specific segments in the extracellular domains of the α- and γ-subunits. Cleavage is accomplished by intracellular proteases prior to membrane insertion and by surface-expressed or extracellular soluble proteases once ENaC resides at the cell surface. These cleavage events are partially regulated by intracellular signaling through an unknown allosteric mechanism. Here, using a combination of computational and experimental techniques, we show that the intracellular N terminus of γ-ENaC undergoes secondary structural transitions upon interaction with phosphoinositides. From ab initio folding simulations of the N termini in the presence and absence of phosphatidylinositol 4,5-bisphosphate (PIP2), we found that PIP2 increases α-helical propensity in the N terminus of γ-ENaC. Electrophysiology and mutation experiments revealed that a highly conserved cluster of lysines in the γ-ENaC N terminus regulates accessibility of extracellular cleavage sites in γ-ENaC. We also show that conditions that decrease PIP2 or enhance ubiquitination sharply limit access of the γ-ENaC extracellular domain to proteases. Further, the efficiency of allosteric control of ENaC proteolysis is dependent on Tyr(370) in γ-ENaC. Our findings provide an allosteric mechanism for ENaC activation regulated by the N termini and sheds light on a potential general mechanism of channel and receptor activation.
authors: Departments of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599; Departments of Molecular and Cellular Biophysics, and University of North Carolina, Chapel Hill, North Carolina 27599.
authors: Pradeep
authors: P
authors: Kota
authors: Department of Chemistry, University of Wyoming, Laramie, Wyoming 82071, and.
authors: Ginka
authors: G
authors: Buchner
authors: Departments of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599.
authors: Hirak
authors: H
authors: Chakraborty
authors: Departments of Cystic Fibrosis and Pulmonary Diseases Research and Treatment Center, University of North Carolina, Chapel Hill, North Carolina 27599.
authors: Yan L
authors: YL
authors: Dang
authors: Departments of Cystic Fibrosis and Pulmonary Diseases Research and Treatment Center, University of North Carolina, Chapel Hill, North Carolina 27599.
authors: Hong
authors: H
authors: He
authors: Biotechnology & Bioengineering Center, Department of Otolaryngology and Communication Sciences, Medical College of Wisconsin, Milwaukee, Wisconsin 53226.
authors: Guilherme J M
authors: GJM
authors: Garcia
authors: Department of Chemistry, University of Wyoming, Laramie, Wyoming 82071, and.
authors: Jan
authors: J
authors: Kubelka
authors: Departments of Cystic Fibrosis and Pulmonary Diseases Research and Treatment Center, University of North Carolina, Chapel Hill, North Carolina 27599,; Departments of Cell Biology and Physiology and University of North Carolina, Chapel Hill, North Carolina 27599.
authors: Martina
authors: M
authors: Gentzsch
authors: Departments of Cystic Fibrosis and Pulmonary Diseases Research and Treatment Center, University of North Carolina, Chapel Hill, North Carolina 27599.
authors: M Jackson
authors: MJ
authors: Stutts
authors: Departments of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599; Departments of Molecular and Cellular Biophysics, and University of North Carolina, Chapel Hill, North Carolina 27599; Departments of Cystic Fibrosis and Pulmonary Diseases Research and Treatment Center, University of North Carolina, Chapel Hill, North Carolina 27599,. Electronic address: dokh@unc.edu.
authors: Nikolay V
authors: NV
authors: Dokholyan
mesh_terms: D000494:Allosteric Regulation / Q000502:physiology; D000818:Animals; D053503:Epithelial Sodium Channels / Q000737:chemistry* / Q000235:genetics; D056004:Molecular Dynamics Simulation; D009154:Mutation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D051381:Rats
chemical_list: D053503:Epithelial Sodium Channels
keywords: Allosteric Regulation; Electrophysiology; Gamma ENaC; Ion Channel; Molecular Dynamics; Protein Conformation; Structural Model