The N-terminal domain allosterically regulates cleavage and activation of the epthelial sodium channel

Pradeep Kota, Ginka Buchner, Hirak Chakraborty, Yan L. Dang, Hong He, Guilherme J.M. Garcia, Jan Kubelka, Martina Gentzsch, M. Jackson Stutts, Nikolay V. Dokholyan

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

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 Tyr370 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.

Original languageEnglish (US)
Pages (from-to)23029-23042
Number of pages14
JournalJournal of Biological Chemistry
Volume289
Issue number33
DOIs
StatePublished - Aug 15 2014

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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