Cathepsin B is secreted apically from Xenopus 2F3 cells and cleaves the epithelial sodium channel (ENaC) to increase its activity.
The epithelial sodium channel (ENaC) plays an important role in regulating sodium balance, extracellular volume, and blood pressure. Evidence suggests the α and γ subunits of ENaC are cleaved during assembly before they are inserted into the apical membranes of epithelial cells, and maximal activity of ENaC depends on cleavage of the extracellular loops of α and γ subunits. Here, we report that Xenopus 2F3 cells apically express the cysteine protease cathepsin B, as indicated by two-dimensional gel electrophoresis and mass spectrometry analysis. Recombinant GST ENaC α, β, and γ subunit fusion proteins were expressed in Escherichia coli and then purified and recovered from bacterial inclusion bodies. In vitro cleavage studies revealed the full-length ENaC α subunit fusion protein was cleaved by active cathepsin B but not the full-length β or γ subunit fusion proteins. Both single channel patch clamp studies and short circuit current experiments show ENaC activity decreases with the application of a cathepsin B inhibitor directly onto the apical side of 2F3 cells. We suggest a role for the proteolytic cleavage of ENaC by cathepsin B, and we suggest two possible mechanisms by which cathepsin B could regulate ENaC. Cathepsin B may cleave ENaC extracellularly after being secreted or intracellularly, while ENaC is present in the Golgi or in recycling endosomes.
Department of Physiology, Emory University School of Medicine and the Center for Cell and Molecular Signaling, Atlanta, GA 30345, USA. aalli@emory.edu
Abdel A
AA
Alli
John Z
JZ
Song
Otor
O
Al-Khalili
Hui-Fang
HF
Bao
He-Ping
HP
Ma
Alia A
AA
Alli
Douglas C
DC
Eaton
D000818:Animals; D002401:Cathepsin B / Q000235:genetics / Q000378:metabolism*; D011992:Endosomes / Q000235:genetics / Q000378:metabolism*; D053503:Epithelial Sodium Channels / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D006056:Golgi Apparatus / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D017433:Protein Structure, Secondary; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
D053503:Epithelial Sodium Channels; D011993:Recombinant Fusion Proteins; C533912:CTSB protein, human; D002401:Cathepsin B