Dataset Information

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 ?- and ?-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 ?-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 ?-helical propensity in the N terminus of ?-ENaC. Electrophysiology and mutation experiments revealed that a highly conserved cluster of lysines in the ?-ENaC N terminus regulates accessibility of extracellular cleavage sites in ?-ENaC. We also show that conditions that decrease PIP2 or enhance ubiquitination sharply limit access of the ?-ENaC extracellular domain to proteases. Further, the efficiency of allosteric control of ENaC proteolysis is dependent on Tyr(370) in ?-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.

SUBMITTER: Kota P

PROVIDER: S-EPMC4132802 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Kota Pradeep P Buchner Ginka G Chakraborty Hirak H Dang Yan L YL He Hong H Garcia Guilherme J M GJM Kubelka Jan J Gentzsch Martina M Stutts M Jackson MJ Dokholyan Nikolay V NV

The Journal of biological chemistry 20140628 33

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 expe ...[more]

PMID: 24973914

Similar Datasets

Project description:Proteolytic activation of the epithelial sodium channel (ENaC) involves cleavage of its γ subunit in a critical region targeted by several proteases. Our aim was to identify cleavage sites in this region that are functionally important for activation of human ENaC by plasmin and chymotrypsin. Sequence alignment revealed a putative plasmin cleavage site in human γENaC (K189) that corresponds to a plasmin cleavage site (K194) in mouse γENaC. We mutated this site to alanine (K189A) and expressed human wild-type (wt) αβγENaC and αβγ(K189A)ENaC in Xenopus laevis oocytes. The γ(K189A) mutation reduced but did not abolish activation of ENaC whole cell currents by plasmin. Mutating a putative prostasin site (γ(RKRK178AAAA)) had no effect on the stimulatory response to plasmin. In contrast, a double mutation (γ(RKRK178AAAA;K189A)) prevented the stimulatory effect of plasmin. We conclude that in addition to the preferential plasmin cleavage site K189, the putative prostasin cleavage site RKRK178 may serve as an alternative site for proteolytic channel activation by plasmin. Interestingly, the double mutation delayed but did not abolish ENaC activation by chymotrypsin. The time-dependent appearance of cleavage products at the cell surface nicely correlated with the stimulatory effect of chymotrypsin on ENaC currents in oocytes expressing wt or double mutant ENaC. Delayed proteolytic activation of the double mutant channel with a stepwise recruitment of so-called near-silent channels was confirmed in single-channel recordings from outside-out patches. Mutating two phenylalanines (FF174) in the vicinity of the prostasin cleavage site prevented proteolytic activation by chymotrypsin. This indicates that chymotrypsin preferentially cleaves at FF174. The close proximity of FF174 to the prostasin site may explain why mutating the prostasin site impedes channel activation by chymotrypsin. In conclusion, this study supports the concept that different proteases have distinct preferences for certain cleavage sites in γENaC, which may be relevant for tissue-specific proteolytic ENaC activation.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets