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Dietary Na + restriction promotes release of an inhibitory tract from the γ ENaC subunit
Author(s) -
Carattino Marcelo D,
Mueller Gunhild M,
Palmer Lawrence G,
Frindt Gustavo,
Hughey Rebecca P,
Kleyman Thomas R
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.1068.1
Subject(s) - furin , epithelial sodium channel , cleavage (geology) , protein subunit , xenopus , chemistry , microbiology and biotechnology , biochemistry , enzyme , medicine , biology , sodium , gene , paleontology , organic chemistry , fracture (geology)
Maturation of ENaC involves furin‐dependent cleavage of the α and γ subunits. Cleavage of the γ subunit by furin at a single site (γRKRK143) and subsequent cleavage by a second protease at a distal site increases ENaC activity. We previously demonstrated that prostasin cleaves the γ subunit at a polybasic tract (γRKRK186). Deletion of the 43 residue tract between the furin and prostasin sites functionally mimics the effect of double cleavage. To determine whether this tract dissociates from processed γ subunits, we generated a polyclonal antibody (Ab) against this region (anti‐γ43). This Ab precipitates both full‐length (93 kDa) and furin‐processed (75 kDa) γ subunits with a C‐terminal V5 tag expressed in Xenopus oocytes. Co‐expression of prostasin resulted in a 70 kDa form detected with an anti‐V5 Ab. The 70 kDa band was not precipitated with anti‐γ43, suggesting that the intervening tract between the furin and prostasin sites had been released. ENaCs in kidney homogenates from rats fed a normal or low Na + diet were precipitated with either anti‐γ43 or an anti‐γ C‐terminal (anti‐γ) Ab, and then immunoblotted with the anti‐γ Ab. Low Na + diet increased γ subunit cleavage detected with the anti‐γ Ab. Anti‐γ43 Ab precipitated the full‐length but not the cleaved form of the γ subunit in both groups of rats. Our results suggest that the inhibitory tract dissociates from the γ subunit in kidneys from rats on a low Na + diet.