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Plasmin and chymotrypsin have distinct preferences for channel activating cleavage sites in the ? subunit of the human epithelial sodium channel.

ABSTRACT: Proteolytic activation of the epithelial sodium channel (ENaC) involves cleavage of its ? 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 ?ENaC (K189) that corresponds to a plasmin cleavage site (K194) in mouse ?ENaC. We mutated this site to alanine (K189A) and expressed human wild-type (wt) ???ENaC and ???(K189A)ENaC in Xenopus laevis oocytes. The ?(K189A) mutation reduced but did not abolish activation of ENaC whole cell currents by plasmin. Mutating a putative prostasin site (?(RKRK178AAAA)) had no effect on the stimulatory response to plasmin. In contrast, a double mutation (?(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 ?ENaC, which may be relevant for tissue-specific proteolytic ENaC activation.

SUBMITTER: Haerteis S

PROVIDER: S-EPMC3457690 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Plasmin and chymotrypsin have distinct preferences for channel activating cleavage sites in the γ subunit of the human epithelial sodium channel.

Haerteis Silke S Krappitz Matteus M Diakov Alexei A Krappitz Annabel A Rauh Robert R Korbmacher Christoph C

The Journal of general physiology 20120910 4

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 hu ...[more]

PMID: 22966015

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Project description:BackgroundWater and solute transport across epithelia can occur via the transcellular or paracellular pathways. Tight junctions play a key role in mediating paracellular ion reabsorption in the kidney. In the renal collecting duct, which is a typical absorptive tight epithelium, coordination between transcellular sodium reabsorption and paracellular permeability may prevent the backflow of reabsorbed sodium to the tubular lumen along a steep electrochemical gradient.MethodsTo investigate whether transcellular sodium transport controls tight-junction composition and paracellular permeability via modulating expression of the transmembrane protein claudin-8, we used cultured mouse cortical collecting duct cells to see how overexpression or silencing of epithelial sodium channel (ENaC) subunits and claudin-8 affect paracellular permeability. We also used conditional kidney tubule-specific knockout mice lacking ENaC subunits to assess the ENaC's effect on claudin-8 expression.ResultsOverexpression or silencing of the ENaC γ-subunit was associated with parallel and specific changes in claudin-8 abundance. Increased claudin-8 abundance was associated with a reduction in paracellular permeability to sodium, whereas decreased claudin-8 abundance was associated with the opposite effect. Claudin-8 overexpression and silencing reproduced these functional effects on paracellular ion permeability. Conditional kidney tubule-specific ENaC γ-subunit knockout mice displayed decreased claudin-8 expression, confirming the cell culture experiments' findings. Importantly, ENaC β-subunit or α-subunit silencing or kidney tubule-specific β-ENaC or α-ENaC knockout mice did not alter claudin-8 abundance.ConclusionsOur data reveal the specific coupling between ENaC γ-subunit and claudin-8 expression. This coupling may play an important role in preventing the backflow of reabsorbed solutes and water to the tubular lumen, as well as in coupling paracellular and transcellular sodium permeability.

Dataset Information

Plasmin and chymotrypsin have distinct preferences for channel activating cleavage sites in the ? subunit of the human epithelial sodium channel.

Publications

Plasmin and chymotrypsin have distinct preferences for channel activating cleavage sites in the γ subunit of the human epithelial sodium channel.

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