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Plasma membrane insertion of epithelial sodium channels occurs with dual kinetics

  • Ion channels, receptors and transporters
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Abstract

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.

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Acknowledgments

We are grateful to Dr. Manuel Criado (Instituto de Neurociencias de Alicante) for his suggestion of using the bungarotoxin system to follow ENaC trafficking and for sharing the V201 construct and to Dr. Diana Wesch for performing preliminary FRAP experiments and transferrin stainings  in Xenopus oocytes. This work was funded by grants from Instituto de Salud Carlos III–Subdirectorate General for Evaluation and Promotion of Research Grants (project PI12/ 00428) and Spanish Ministry of Economy Grants Consolider-Ingenio CSD 2008–0005 and BFU2010-16265 and co-financed by the European Regional Development Funds, “A Way of Making Europe,” from the European Union. T.G. is funded by the Ramon y Cajal Program (Spanish Ministry of Economy, Spain). The confocal microscopy facility was supported partially by the Insular Council of Tenerife and grants MCT-FEDER 2003/2004 and IMBRAIN-FP7-REGPOT-2012-31637, awarded to the Institute of Biomedical Technologies and the Center of Biomedical Research of the Canary Islands at ULL.

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  1. Department of Physiology, Institute of Biomedical Technologies and Centre for Biomedical Research of the Canary Islands (CIBICAN), University of La Laguna, 38071, La Laguna, Spain

    Rafaela González-Montelongo, Diego Alvarez de la Rosa & Teresa Giraldez

  2. Department of Biochemistry and Molecular Biology, University of Oviedo, 33006, Oviedo, Spain

    Francisco Barros

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  1. Rafaela González-Montelongo
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  3. Diego Alvarez de la Rosa
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Correspondence to Diego Alvarez de la Rosa or Teresa Giraldez.

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González-Montelongo, R., Barros, F., Alvarez de la Rosa, D. et al. Plasma membrane insertion of epithelial sodium channels occurs with dual kinetics. Pflugers Arch - Eur J Physiol 468, 859–870 (2016). https://doi.org/10.1007/s00424-016-1799-4

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