Epithelial Sodium Channel (ENaC) Tagged with Bungarotoxin Binding Site to Study Channel Plasma Membrane Expression and Trafficking
Transepithelial sodium reabsorption through ENaC plays a key role in electrolyte and extracellular volume homeostasis and regulation of blood pressure. ENaC is a heterotrimeric ion channel formed by α, β and γ subunits. Channel activity is tightly regulated by many local and circulating factors, which in turn control channel cell surface expression (N) and open probability (Po). ENaC upregulation resulting from decreased endocytosis or enhanced delivery to the membrane have been described. However, there are several technical challenges in the study of ENaC trafficking, including low levels of plasma membrane expression. The aim of this study was to develop a new tool to directly study the dynamics of ENaC trafficking to and from the plasma membrane. To that end we inserted an α‐bungarotoxin (BTX) binding site (BBS) in the extracellular domain of the a subunit and used fluorescently labeled BTX to study ENaC plasma membrane expression and trafficking in Xenopus laevis oocytes. Mutant α BBSβγ channels do not shown any functional abnormality in the absence or presence of the toxin. Confocal imaging of Alexa Fluor 488 (AF488)‐labeled BTX showed specific labeling in plasma membrane of oocytes expressing α BBSβγ channels. ENaC retrieval was detected by imaging AF488‐BTX labeled oocytes at different time points whereas ENaC insertion rate was detected by blocking α BBSβγ‐expressing oocytes with unlabeled BTX and then staining with AF488‐BTX at different times. Our results suggest that tagged α BBS subunits provide a useful tool to study plasma membrane ENaC abundance and trafficking to and from the membrane..
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2015 |
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| Erschienen: |
2015 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:29 |
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| Enthalten in: |
The FASEB Journal - 29(2015) |
| Beteiligte Personen: |
Gonzalez‐Montelongo, Rafaela [VerfasserIn] |
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| BKL: |
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| Anmerkungen: |
© Federation of American Societies for Experimental Biology |
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| Umfang: |
1 |
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| doi: |
10.1096/fasebj.29.1_supplement.969.18 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| 520 | |a Transepithelial sodium reabsorption through ENaC plays a key role in electrolyte and extracellular volume homeostasis and regulation of blood pressure. ENaC is a heterotrimeric ion channel formed by α, β and γ subunits. Channel activity is tightly regulated by many local and circulating factors, which in turn control channel cell surface expression (N) and open probability (Po). ENaC upregulation resulting from decreased endocytosis or enhanced delivery to the membrane have been described. However, there are several technical challenges in the study of ENaC trafficking, including low levels of plasma membrane expression. The aim of this study was to develop a new tool to directly study the dynamics of ENaC trafficking to and from the plasma membrane. To that end we inserted an α‐bungarotoxin (BTX) binding site (BBS) in the extracellular domain of the a subunit and used fluorescently labeled BTX to study ENaC plasma membrane expression and trafficking in Xenopus laevis oocytes. Mutant α BBSβγ channels do not shown any functional abnormality in the absence or presence of the toxin. Confocal imaging of Alexa Fluor 488 (AF488)‐labeled BTX showed specific labeling in plasma membrane of oocytes expressing α BBSβγ channels. ENaC retrieval was detected by imaging AF488‐BTX labeled oocytes at different time points whereas ENaC insertion rate was detected by blocking α BBSβγ‐expressing oocytes with unlabeled BTX and then staining with AF488‐BTX at different times. Our results suggest that tagged α BBS subunits provide a useful tool to study plasma membrane ENaC abundance and trafficking to and from the membrane. | ||
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