Epithelial sodium channels (ENaC) play an essential role in maintaining total body fluid and electrolyte homeostasis. As such, abnormal expression of ENaC at the cell surface is linked to several important human diseases. Although the stability of ENaC subunits has been extensively studied by protein biochemical analysis, the half-life of the functional channel in the apical membrane remains controversial. Because the functional stability of the multisubunit channel may be more physiologically relevant than the stability of individual subunit proteins, we performed studies of functional ENaC channels using A6 epithelial cells, a Xenopus laevis distal nephron cell line. We recorded single-channel activity in over 400 cells with the translation blockers cycloheximide (CHX) or puromycin, as well as the intracellular protein trafficking inhibitors brefeldin A (BFA) or nocodazole. Our cell-attached, single-channel recordings allow us to quantify the channel density in the apical membrane, as well as to determine channel open probability (Po) from control (untreated) cells and from cells at different times of drug treatment. The data suggest that the half-life of ENaC channels is approximately 3.5 h following puromycin, BFA, and nocodazole treatment. Furthermore, these three drugs had no significant effect on the Po of ENaC for at least 6 h after exposure. A decrease in apical channel number and Po was observed following 2 h of CHX inhibition of protein synthesis, and the apparent channel half-life was closer to 1.5 h following CHX treatment. Treatment of cells with the translation inhibitors does not alter the expression of the protease furin, and therefore changes in protease activity cannot explain changes in ENaC Po. Confocal images show that BFA and nocodazole both disrupt most of the Golgi apparatus after 1-h exposure. In cells with the Golgi totally disrupted by overnight exposure to BFA, 20% of apical ENaC channels remained functional. This result suggests that ENaC is delivered to the apical membrane via a pathway that might bypass the Golgi vesicular trafficking pathway, or that there might be two pools of channels with markedly different half-lives in the apical membrane.
" PM as high - mannose - bearing variants but acquire with time both detergent insolubility and more complex glycans ( Ghosh et al . 1999 ) . In addition , other ion channels have been sug - gested to bypass the Golgi apparatus . The epithelial sodium channel ( ENaC ) is at least partly delivered to the apical membrane via an unconventional route ( Yu et al . 2008 ) . Likewise , subunits of the Kv4 potassium channel co - localize with the IC protein ERGIC - 53 but do not seem to follow the same route through the Golgi cisternae as the vesicular stomatitis G protein but are instead transported via a Golgi - independent pathway requiring the channel - interacting protein KChlP1 ( Flowerdew and Burg"
[Show abstract][Hide abstract] ABSTRACT: Studies carried out during the last 2 decades have dramatically increased our knowledge of the pathways and mechanisms of intracellular membrane traffic, most recently due to the developments in light microscopy and in vivo imaging of fluorescent fusion proteins. These studies have also revealed that certain molecules do not behave according to the classical transportation rules first documented in cell biology textbooks in the 1980s and 1990s. Initially, unconventional mechanisms of secretion that do not involve passage of cargo through the stacked Golgi cisternae were thought to confer on cells the ability to discard excess amounts of protein products. With time, however, more physiological mechanisms and roles have been proposed for an increasing number of secretory processes that bypass the Golgi apparatus.
Cell and Tissue Research 04/2012; 352(1). DOI:10.1007/s00441-012-1409-5 · 3.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The epithelial Na(+) channel (ENaC) is a major regulator of salt and water reabsorption in a number of epithelial tissues. Abnormalities in ENaC function have been directly linked to several human disease states including Liddle's syndrome, psuedohypoaldosteronism, and cystic fibrosis and may be implicated in states as diverse as salt-sensitive hypertension, nephrosis, and pulmonary edema. ENaC activity in epithelial cells is highly regulated both by open probability and number of channels. Open probability is regulated by a number of factors, including proteolytic processing, while ENaC number is regulated by cellular trafficking. This review discusses current understanding of apical membrane delivery, cell surface stability, endocytosis, retrieval, and recycling of ENaC and the molecular partners that have so far been shown to participate in these processes. We review known sites and mechanisms of hormonal regulation of trafficking by aldosterone, vasopressin, and insulin. While many details of the regulation of ENaC trafficking remain to be elucidated, knowledge of these mechanisms may provide further insights into ENaC activity in normal and disease states.
American journal of physiology. Renal physiology 06/2008; 296(1):F10-24. DOI:10.1152/ajprenal.90248.2008 · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Proteins leave the endoplasmic reticulum (ER) for the plasma membrane via the classical secretory pathway, but routes bypassing the Golgi apparatus have also been observed. Apical and basolateral protein secretion in epithelial Madin-Darby canine kidney (MDCK) cells display differential sensitivity to Brefeldin A (BFA), where low concentrations retard apical transport, while basolateral transport still proceeds through intact Golgi cisternae. We now describe that BFA-mediated retardation of glycoprotein and proteoglycan transport through the Golgi apparatus induces surface transport of molecules lacking Golgi modifications, possessing those acquired in the ER. Low concentrations of BFA induces apical Golgi bypass, while higher concentrations were required to induce basolateral Golgi bypass. Addition of the KDEL ER-retrieval sequence to model protein cores allowed observation of apical Golgi bypass in untreated MDCK cells. Basolateral Golgi bypass was only observed after the addition of BFA or upon cholesterol depletion. Thus, in MDCK cells, an apical Golgi bypass route can transport cargo from pre-Golgi organelles in untreated cells, while the basolateral bypass route is inducible.
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