Prostasin interacts with the epithelial Na+ channel and facilitates cleavage of the γ-subunit by a second protease
| Autor: | Marcelo D. Carattino, Lawrence G. Palmer, Gunhild M. Mueller, Rebecca P. Hughey, Thomas R. Kleyman, Gustavo Frindt, Anna C. Rued |
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| Rok vydání: | 2014 |
| Předmět: |
Epithelial sodium channel
Male Proteases Consensus site Physiology medicine.medical_treatment Protein subunit Cleavage (embryo) Rats Sprague-Dawley Xenopus laevis medicine Animals Humans Sodium Chloride Dietary Epithelial Sodium Channels Furin Protease biology Serine Endopeptidases Molecular biology Amiloride Protein Subunits HEK293 Cells biology.protein Oocytes Female medicine.drug |
| Zdroj: | American journal of physiology. Renal physiology. 307(9) |
| ISSN: | 1522-1466 |
| Popis: | During maturation, the α- and γ-subunits of the epithelial Na+channel (ENaC) undergo proteolytic processing by furin. Cleavage of the γ-subunit by furin at the consensus site γRKRR143and subsequent cleavage by a second protease at a distal site strongly activate the channel. For example, coexpression of prostasin with ENaC increases both channel function and cleavage at the γRKRK186site. We generated a polyclonal antibody that recognizes the region 144–186 in the γ-subunit (anti-γ43) to determine whether prostasin promotes the release of the intervening tract between the putative furin and γRKRK186cleavage sites. Anti-γ43 precipitated both full-length (93 kDa) and furin-processed (83 kDa) γ-subunits from extracts obtained from oocytes expressing αβHA-γ-V5 channels, but only the full-length (93 kDa) γ-subunit from oocytes expressing αβHA-γ-V5 channels and either wild-type or a catalytically inactive prostasin. Although both wild-type and catalytically inactive prostasin activated ENaCs in an aprotinin-sensitive manner, only wild-type prostasin bound to aprotinin beads, suggesting that catalytically inactive prostasin facilitates the cleavage of the γ-subunit by an endogenous protease in Xenopus oocytes. As dietary salt restriction increases cleavage of the renal γ-subunit, we assessed release of the 43-mer inhibitory tract on rats fed a low-Na+diet. We found that a low-Na+diet increased γ-subunit cleavage detected with the anti-γ antibody and dramatically reduced the fraction precipitated with the anti-γ43 antibody. Our results suggest that the inhibitory tract dissociates from the γ-subunit in kidneys from rats on a low-Na+diet. |
| Databáze: | OpenAIRE |
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