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The N-terminal domain allosterically regulates cleavage and activation of the epithelial sodium channel. J Biol Chem 2014 Aug 15;289(33):23029-42

Date

06/30/2014

Pubmed ID

24973914

Pubmed Central ID

PMC4132802

DOI

10.1074/jbc.M114.570952

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.

Author List

Kota P, Buchner G, Chakraborty H, Dang YL, He H, Garcia GJ, Kubelka J, Gentzsch M, Stutts MJ, Dokholyan NV

Author

Guilherme Garcia PhD Assistant Professor in the Biomedical Engineering department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Allosteric Regulation
Animals
Epithelial Sodium Channels
Molecular Dynamics Simulation
Mutation
Protein Structure, Secondary
Protein Structure, Tertiary
Proteolysis
Rats
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