Plasma membrane insertion of epithelial sodium channels occurs with dual kinetics
The epithelial sodium channel (ENaC) constitutes the rate-limiting step for Na(+) transport across electrically tight epithelia. Regulation of ENaC activity is critical for electrolyte and extracellular volume homeostasis, as well as for lung liquid clearance and colon Na(+) handling. ENaC activity is tightly controlled by a combination of mechanisms involving changes in open probability and plasma membrane abundance. The latter reflects a combination in channel biosynthesis and trafficking to and from the membrane. Studying ENaC trafficking with different techniques in a variety of expression systems has yielded inconsistent results, indicating either fast or slow rates of insertion and retrieval, which range from the order of minutes to several hours. Here, we use Xenopus oocytes as ENaC expression system to study channel insertion rate in the membrane using two different techniques under comparable conditions: (1) confocal microscopy coupled to fluorescence recovery after photobleaching (FRAP) measurements; and (2) fluorescent bungarotoxin (BTX) binding to ENaC subunits modified to include BTX binding sites (BBSs) in their extracellular domain, a technique that has not been previously used to study ENaC trafficking. Our confocal-FRAP data indicate a fast rate of ENaC incorporation to the membrane in a process conditioned by channel subunit composition. On the other hand, BTX binding experiments indicate much slower channel insertion rates, with matching slow ENaC retrieval rates. The data support a model that includes fast recycling of endocytosed ENaC with parallel incorporation of newly synthesized channels at a slower rate.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2016 |
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| Erschienen: |
2016 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:468 |
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| Enthalten in: |
Pflugers Archiv : European journal of physiology - 468(2016), 5 vom: 15. Mai, Seite 859-70 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
González-Montelongo, Rafaela [VerfasserIn] |
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| Links: |
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| Themen: |
Bungarotoxins |
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| Anmerkungen: |
Date Completed 10.04.2017 Date Revised 13.11.2018 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1007/s00424-016-1799-4 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM257471294 |
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| 100 | 1 | |a González-Montelongo, Rafaela |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Plasma membrane insertion of epithelial sodium channels occurs with dual kinetics |
| 264 | 1 | |c 2016 | |
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| 500 | |a Date Revised 13.11.2018 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a The epithelial sodium channel (ENaC) constitutes the rate-limiting step for Na(+) transport across electrically tight epithelia. Regulation of ENaC activity is critical for electrolyte and extracellular volume homeostasis, as well as for lung liquid clearance and colon Na(+) handling. ENaC activity is tightly controlled by a combination of mechanisms involving changes in open probability and plasma membrane abundance. The latter reflects a combination in channel biosynthesis and trafficking to and from the membrane. Studying ENaC trafficking with different techniques in a variety of expression systems has yielded inconsistent results, indicating either fast or slow rates of insertion and retrieval, which range from the order of minutes to several hours. Here, we use Xenopus oocytes as ENaC expression system to study channel insertion rate in the membrane using two different techniques under comparable conditions: (1) confocal microscopy coupled to fluorescence recovery after photobleaching (FRAP) measurements; and (2) fluorescent bungarotoxin (BTX) binding to ENaC subunits modified to include BTX binding sites (BBSs) in their extracellular domain, a technique that has not been previously used to study ENaC trafficking. Our confocal-FRAP data indicate a fast rate of ENaC incorporation to the membrane in a process conditioned by channel subunit composition. On the other hand, BTX binding experiments indicate much slower channel insertion rates, with matching slow ENaC retrieval rates. The data support a model that includes fast recycling of endocytosed ENaC with parallel incorporation of newly synthesized channels at a slower rate | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 4 | |a Dynamin | |
| 650 | 4 | |a ENaC | |
| 650 | 4 | |a Endocytosis | |
| 650 | 4 | |a Exocytosis | |
| 650 | 4 | |a Fluorescence | |
| 650 | 7 | |a Bungarotoxins |2 NLM | |
| 650 | 7 | |a Epithelial Sodium Channels |2 NLM | |
| 650 | 7 | |a Sodium Channel Blockers |2 NLM | |
| 700 | 1 | |a Barros, Francisco |e verfasserin |4 aut | |
| 700 | 1 | |a Alvarez de la Rosa, Diego |e verfasserin |4 aut | |
| 700 | 1 | |a Giraldez, Teresa |e verfasserin |4 aut | |
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