Non-coordinate Regulation of Endogenous Epithelial Sodium Channel (ENaC) Subunit Expression at the Apical Membrane of A6 Cells in Response to Various Transporting Conditions

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145

Alveolar hypoxia may impair sodium-dependent alveolar fluid transport and induce pulmonary edema in rat and human lung, an effect that can be prevented by the inhalation of ␤ 2 -agonists. To investigate the mechanism of ␤ 2 -agonist-mediated stimulation of sodium transport under conditions of moderate hypoxia, we examined the effect of terbutaline on epithelial sodium channel (ENaC) expression and activity in cultured rat alveolar epithelial type II cells exposed to 3% O 2 for 24 h. Hypoxia reduced transepithelial sodium current and amiloridesensitive sodium channel activity without decreasing ENaC subunit mRNA or protein levels. The functional decrease was associated with reduced abundance of ENaC subunits (especially ␤ and ␥) in the apical membrane of hypoxic cells, as quantified by biotinylation. cAMP stimulation with terbutaline reversed the hypoxia-induced decrease in transepithelial sodium transport by stimulating sodium channel activity and markedly increased the abundance of ␤-and ␥-ENaC in the plasma membrane of hypoxic cells. The effect of terbutaline was prevented by brefeldin A, a blocker of anterograde transport. These novel results establish that hypoxiainduced inhibition of amiloride-sensitive sodium channel activity is mediated by decreased apical expression of ENaC subunits and that ␤ 2 -agonists reverse this effect by enhancing the insertion of ENaC subunits into the membrane of hypoxic alveolar epithelial cells.

Section: Discussionmentioning

confidence: 99%

Hypoxia and β2-Agonists Regulate Cell Surface Expression of the Epithelial Sodium Channel in Native Alveolar Epithelial Cells

Planès

Blot‐Chabaud

Matthay

et al. 2002

151

145

“…Thus, our results were not influenced by possible dynamic rearrangement of channel subunits during the experiment. This becomes particularly important considering the recent findings of others suggesting that in the plasma membrane, ENaC stoichiometry and possibly oligomerization is not fixed (26).…”

Section: Discussionmentioning

confidence: 99%

Fluorescence Resonance Energy Transfer Analysis of Subunit Stoichiometry of the Epithelial Na+ Channel

Staruschenko

Medina

Patel

et al. 2004

Activity of the epithelial Na ؉ channel (ENaC) is ratelimiting for Na ؉ (re)absorption across electrically tight epithelia. ENaC is a heteromeric channel comprised of three subunits, ␣, ␤, and ␥, with each subunit contributing to the functional channel pore. The subunit stoichiometry of ENaC remains uncertain with electrophysiology and biochemical experiments supporting both a tetramer with a 2␣:1␤:1␥ stoichiometry and a higher ordered channel with a 3␣:3␤:3␥ stoichiometry. Here we used an independent biophysical approach based upon fluorescence resonance energy transfer (FRET) between differentially fluorophore-tagged ENaC subunits to determine the subunit composition of mouse ENaC functionally reconstituted in Chinese hamster ovary and COS-7 cells. We found that when all three subunits were co-expressed, ENaC contained at least two of each type of subunit. Findings showing that ENaC subunits interact with similar subunits in immunoprecipitation studies are consistent with these FRET results. Upon native polyacrylamide gel electrophoresis, moreover, oligomerized ENaC runs predominantly as a single species with a molecular mass of >600 kDa. Because single ENaC subunits have a molecular mass of ϳ90 kDa, these results also agree with the FRET results. The current results as a whole, thus, are most consistent with a higher ordered channel possibly with a 3␣:3␤:3␥ stoichiometry.Ion channels are integral membrane proteins that form selective pores in the plasma membrane through which ions move down their electrochemical gradients. The epithelial Na ϩ channel (ENaC) 1 is a member of the ENaC/Deg gene superfamily (1-4). This family contains several ion channels (e.g. ASIC, BNaC, DEG, DRASIC, ENaC, and FaNaCH) of similar structure that serve diverse physiological functions from modulating mechanosensory transduction and neuronal signaling to control of electrolyte movement across epithelial barriers.Functional ENaC, similar to most other ion channels, is a heteromeric protein complex containing several distinct channel subunits. Four ENaC subunits have been identified, ␣, ␤, ␥, and ␦ (1, 5, 6). The first three subunits are widely expressed and together form the channel resident to the luminal plasma membrane of epithelial cells capable of electrogenic Na ϩ (re-)absorption. Consistent with this idea are findings showing that co-expression of only two of these three subunits produces little to no current in heterologous expression systems (1,5,7,8). The ␦ENaC subunit localizes to the brain and reproductive tissues where it is believed to substitute for ␣ENaC in the functional channel (6). Importantly, the stoichiometric relationship between ENaC subunits is uncertain.Each ENaC subunit has a cytosolic NH 2 and COOH terminus with two transmembrane domains separated by a single large extracellular loop with all three ENaC subunits, ␣, ␤, and ␥, contributing the functional pore (1, 5, 9, 10). Several laboratories have tested ENaC subunit stoichiometry with a biophysical approach ultimately using MacKinnon's formulation to est...

“…At the appropriate chase time, cells were rinsed with PBS and lysed in detergent solution (6). After a brief centrifugation to remove nuclei, lysates were immunoprecipitated with anti-␤-ENaC or -V5 antibodies, and antibody-antigen complexes were collected using fixed Staphylococcus aureus (Pansorbin; Calbiochem) or protein G-coupled Sepharose (Sigma), respectively.…”

Section: Cell Lines and Adenoviruses-mdck Type I Cells Were Maintainementioning

confidence: 99%

“…While biochemical studies from several laboratories universally report a short half-life for the total pool of newly synthesized ENaC subunits (typically 1-2 h) (2-7), there is considerable dispute regarding the stability of ENaC subunits that have reached the plasma membrane. Some studies report rapid decay of all three cell surface subunits in polarized MDCK and A6 cells (4, 5), whereas we and others have observed long half-lives of ␣-and ␥-ENaC that were biotinylated at the apical plasma membrane of A6 cells (6,8). We use the term noncoordinate regulation to describe this differential turnover of ENaC subunits and have proposed possible molecular models for this observation based on experimental results from many laboratories (6, 9).…”

mentioning

confidence: 99%

Differential Current Decay Profiles of Epithelial Sodium Channel Subunit Combinations in Polarized Renal Epithelial Cells

Mohan

Bruns

Weixel³

et al. 2004

Self Cite

In many epithelial tissues in the body, the rate of Na ؉ reabsorption is governed by the activity of the epithelial sodium channel (ENaC). The assembly, trafficking, and turnover of the three ENaC subunits (␣, ␤, and ␥) is complex and not well understood. Recent experiments suggest that ENaC must be proteolytically cleaved for maximal activity and may explain the discrepancies reported in prior biochemical approaches focused on quantitating the trafficking and half-life of full-length subunits. As an alternative approach to examining the dynamics of ENaC subunits, we have generated doxycycline-repressible replication-defective recombinant adenoviruses encoding individual epitope-tagged mouse ENaC subunits and expressed these in polarized MDCK I cells. Co-infection with these viruses encoding all three subunits generates robust amiloride-sensitive currents in polarized MDCK cells. Significant current was also observed in cells expressing ␣-and ␥-mENaC in the absence of ␤-mENaC. These currents did not appear to result from association with endogenous canine ␤-ENaC. Treatment of ␣␤␥-expressing cells with cycloheximide (CHX) resulted in the rapid inhibition (within 3 h) of ϳ50 -80% of the initial current; however, a sizable fraction of the initial current remained even after 6 h of CHX. By contrast, CHX addition to cells expressing only ␣-and ␥-mENaC resulted in rapid decay in current with no residual fraction. Our data suggest that ENaC channels of differing stoichiometries are differentially trafficked and degraded and provide support for the possibility that noncoordinate trafficking of ENaC subunits may function in vivo as a mechanism to modulate ENaC activity.The epithelial sodium channel (ENaC) 1 is an apical heterotetramer containing ␣, ␤, and ␥ subunits that serves as the rate-limiting step in sodium reabsorption in a number of tissues (reviewed in Ref. 1). Given the clinical importance of ENaC activity, the assembly, trafficking, and response to hormonal stimulation of ENaC subunits have been subjects of considerable study and debate. While biochemical studies from several laboratories universally report a short half-life for the total pool of newly synthesized ENaC subunits (typically 1-2 h) (2-7), there is considerable dispute regarding the stability of ENaC subunits that have reached the plasma membrane. Some studies report rapid decay of all three cell surface subunits in polarized MDCK and A6 cells (4, 5), whereas we and others have observed long half-lives of ␣-and ␥-ENaC that were biotinylated at the apical plasma membrane of A6 cells (6,8). We use the term noncoordinate regulation to describe this differential turnover of ENaC subunits and have proposed possible molecular models for this observation based on experimental results from many laboratories (6, 9).Considerable data suggest that activation of ENaC channels is regulated by proteolytic enzymes (10 -12). Recently, new evidence has emerged that suggests that activation of ENaC involves direct furin-mediated cleavage of ␣-and ␥-ENaC subunits wi...