Abstract
Liddle’s mutation increases ENaC surface expression by decreasing endocytosis of active channels
The FASEB journal, Vol.20(4), pp.A795-A795
03/2006
DOI: 10.1096/fasebj.20.4.A795
Abstract
In Liddle’s syndrome, mutations in a PPPxYxxL motif increase Na+ transport by increasing ENaC surface expression. This sequence fits the consensus of both an endocytosis motif and a Nedd4‐2 binding motif. We hypothesized that Liddle’s syndrome mutations might increase surface expression by disrupting endocytosis of active channels. We used a novel assay based on ENaC proteolytic cleavage; cleavage in the extracellular domains of α and γ ENaC results in channel activation. We mutated the α and γ cleavage sites to prevent channel activation by endogenous proteases, and coexpressed them with either wild type or Liddle’s (R566X) β ENaC in Fischer rat thyroid epithelial cells. The resulting channels did not produce sodium current under basal conditions. However, addition of trypsin to the apical membrane activated the channels. Following removal of trypsin, the amiloride sensitive sodium current was monitored as an assay of removal of activated channels from the cell surface. We found that the rate of endocytosis was rapid, reaching a plateau by 30 min. In contrast, the endocytosis rate was significantly reduced when the channel contained a Liddle’s syndrome mutation. To confirm these findings biochemically, we expressed the mutant channel in HEK‐293 cells. Following trypsin cleavage, we quantitated the rate of disappearance of cleaved channels from the cell surface using a biotinylation assay. Consistent with our functional studies, a Liddle’s mutation in β ENaC reduced endocytosis. We conclude that Liddle’s syndrome mutations increase ENaC surface expression in part by reducing endocytosis of active channels.
Supported by American Heart Association Heartland affiliate postdoctoral fellowship 0425623Z
Details
- Title: Subtitle
- Liddle’s mutation increases ENaC surface expression by decreasing endocytosis of active channels
- Creators
- Rajesh Kabra - University of IowaKristin K. Knight - University of IowaPeter M. Snyder - University of Iowa
- Resource Type
- Abstract
- Publication Details
- The FASEB journal, Vol.20(4), pp.A795-A795
- Publisher
- Federation of American Societies for Experimental Biology
- DOI
- 10.1096/fasebj.20.4.A795
- ISSN
- 0892-6638
- eISSN
- 1530-6860
- Number of pages
- 1
- Grant note
- American Heart Association Heartland (0425623Z)
- Language
- English
- Date published
- 03/2006
- Academic Unit
- Molecular Physiology and Biophysics; Cardiovascular Medicine; Fraternal Order of Eagles Diabetes Research Center; Medicine Administration; Internal Medicine
- Record Identifier
- 9984303003002771
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