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Acute inhibition of the epithelial Na+ channel (ENaC) by epidermal growth factor (EGF) involves the PY motif and putative ERK phosphorylation site
Author(s) -
Falin Rebecca Anne,
Cotton Calvin U
Publication year - 2006
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.20.4.a346-b
Subject(s) - phosphorylation , epithelial sodium channel , epidermal growth factor , mapk/erk pathway , mutant , protein subunit , microbiology and biotechnology , chemistry , biology , receptor , biochemistry , gene , sodium , organic chemistry
Liddle’s syndrome (LS) is caused by a number of mutations in the C‐terminus of the β or γ subunit of ENaC resulting in hyperactive Na + absorption in the renal collecting duct (CD). A mouse model for LS that expresses the truncated β subunit was crossed with a mouse that expresses GFP specifically in the CD, allowing us to isolate primary CD cells by FACS. It was found that EGF inhibited short‐circuit current (I SC ) by 29 ± 5 % in wild‐type primary cells but only by 6 ± 3 % in Liddle’s cells. EGF elicits acute inhibition of Na + absorption in an ERK‐dependent manner. In order to elucidate the role of specific regions on the β subunit C‐terminus, MDCK cell lines expressing ENaC with mutations in the PY motif (P616L) and ERK phosphorylation site (T613A), as well as the truncation mutant (R564stop), were created using the Phoenix retroviral system. To differentiate between channels containing endogenous and mutant subunits, the β G525C mutation was employed. Channels containing endogenous β subunit were blocked with 1 μM BNZ and the acute inhibition of mutant channels by EGF subsequently determined. Mutant channels were blocked by 10 mM AML. EGF inhibited AML‐sensitive I SC in control cells 45 ± 3%, while the R564stop, P616L and T613A mutants were inhibited 12 ± 4, 12 ± 2, and 19 ± 5 %, respectively. We conclude that the PY motif and ERK phosphorylation site have a key role in the acute inhibition of Na + transport by EGF. (Supported by the AHA & NIH)