Molecular Cloning and Functional Expression of a Novel Amiloride-sensitive Na+ Channel

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...

supporting

confidence: 64%

“…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.…”

mentioning

confidence: 99%

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

Staruschenko

Medina

Patel

et al. 2004

“…A fourth ENaC subunit, ␦-ENaC, has been cloned from a human kidney library (5). In heterologous expression systems, ␦-ENaC has functional similarities with ␣-ENaC, but its physiological role is still unclear (6).…”

mentioning

confidence: 99%

Atomic Force Microscopy Reveals the Architecture of the Epithelial Sodium Channel (ENaC)

Stewart

Haerteis

Diakov

et al. 2011

The epithelial sodium channel (ENaC) is a member of the ENaC/degenerin superfamily. ENaC is a heteromultimer containing three homologous subunits (␣, ␤, and ␥); however, the subunit stoichiometry is still controversial. Here, we addressed this issue using atomic force microscopy imaging of complexes between isolated ENaC and antibodies/Fab fragments directed against specific epitope tags on the ␣-, ␤-and ␥-subunits. We show that for ␣-, ␤-and ␥-ENaC alone, pairs of antibodies decorate the channel at an angle of 120°, indicating that the individual subunits assemble as homotrimers. A similar approach demonstrates that ␣␤␥-ENaC assembles as a heterotrimer containing one copy of each subunit. Intriguingly, all four subunit combinations also produce higher-order structures containing two or three individual trimers. The trimer-of-trimers organization would account for earlier reports that ENaC contains eight to nine subunits.The epithelial sodium channel (ENaC) 5 is a member of the ENaC/degenerin superfamily, which also includes acid-sensing ion channels (ASICs; reviewed in Ref. 1). ENaC mediates Na ϩ entry across the apical membranes of many absorptive epithelia, including the alveolar epithelium, salivary duct, distal colon, and sweat glands. ENaC plays a particularly important role in Na ϩ transport across the aldosterone-sensitive distal nephron (reviewed in Ref. 2), which is responsible for the regulation of Na ϩ and K ϩ excretion, and thus plays an important role in the control of blood pressure. ENaC is the target of potassiumsparing diuretics such as amiloride and spironolactone used in the treatment of cardiovascular disease. Whereas amiloride is a direct blocker of the channel, the mineralocorticoid receptor antagonist spironolactone inhibits ENaC activity by preventing its stimulation by aldosterone.ENaC is a heteromultimer containing three homologous subunits (␣, ␤, and ␥), which have 30 -40% amino acid identity (3, 4). A fourth ENaC subunit, ␦-ENaC, has been cloned from a human kidney library (5). In heterologous expression systems, ␦-ENaC has functional similarities with ␣-ENaC, but its physiological role is still unclear (6). Each ENaC subunit contains two transmembrane domains, a large extracellular loop, and short intracellular N and C termini. Full ENaC activity requires coexpression of all three subunits (4). In the past, it has been proposed that ENaC is assembled from either four (7-9) or eight to nine subunits (10, 11). However, the recently published crystal structure of ASIC 1a (12) suggests that ENaC is likely to be a trimer (13), although this has not been demonstrated.We have developed a method, based on AFM imaging, for determining the stoichiometry and arrangement of subunits within multimeric proteins (reviewed in Ref. 14). The method involves engineering specific epitope tags onto each protein subunit and expressing the proteins exogenously in a suitable cell line (usually tsA 201). Membrane fractions from the transfected cells are solubilized in detergent, and the proteins are isola...

“…Another ENaC core unit, human ␣ subunit with 37% amino acid identity to ␦ subunit, failed to increase the current amplitude by the application of capsazepine, and unexpectedly, capsazepine caused a slight reduction of the hENaC␣␤␥ current. It has been described that there are differences in Na ϩ permeability (␣:␦ ϭ ϳ2:0.6 as I Li ϩ /I Na ϩ ) and the amiloride sensitivity (␣:␦ ϭ 0.1:2.6 M as IC 50 ) between ␣ and ␦ subunits (4,6,7,10). Because the chemical agents influencing strongly either the ␣ or ␦ subunit have been poorly investigated, capsazepine is a potentially powerful tool for the electrophysiological analysis of ENaC␦ and the elucidation of the clinical ENaC␦ function in humans.…”

Section: Discussionmentioning

confidence: 99%

“…There is an overall ϳ37% amino acid identity between the ␣, ␤, ␥, and ␦ subunits. ENaC␣ is expressed mainly in epithelia, such as the kidney, lung, and colon, and binds with these ␤ and ␥ subunits to play a pathophysiological role (2,4), whereas the ␦ subunit is widely distributed throughout the brain and is also expressed in the heart, kidney, and pancreas (10,11). More recently, we have demonstrated that protons activate ENaC␦, indicating that it may contribute to pH sensation and/or pH regulation in the human brain (11).…”

mentioning

confidence: 99%

Capsazepine Is a Novel Activator of the δ Subunit of the Human Epithelial Na+ Channel

Yamamura¹,

Ugawa²,

Ueda³

et al. 2004

The amiloride-sensitive epithelial Na ؉ channel (ENaC) regulates Na ؉ homeostasis into cells and across epithelia. So far, four homologous subunits of mammalian ENaC have been isolated and are denoted as ␣, ␤, ␥, and ␦. The chemical agents acting on ENaC are, however, largely unknown, except for amiloride and benzamil as ENaC inhibitors. In particular, there are no agonists currently known that are selective for ENaC␦, which is mainly expressed in the brain. Here we demonstrate that capsazepine, a competitive antagonist for transient receptor potential vanilloid subfamily 1, potentiates the activity of human ENaC␦␤␥ (hENaC␦␤␥) heteromultimer expressed in Xenopus oocytes. The inward currents at a holding potential of ؊60 mV in hENaC␦␤␥-expressing oocytes were markedly enhanced by the application of capsazepine (>1 M), and the capsazepine-induced current was mostly abolished by the addition of 100 M amiloride. The stimulatory effects of capsazepine on the inward current were concentrationdependent with an EC 50 value of 8 M. Neither the application of other vanilloid compounds (capsaicin, resiniferatoxin, and olvanil) nor a structurally related compound (dopamine) modulated the inward current. Although hENaC␦ homomer was also significantly activated by capsazepine, unexpectedly, capsazepine had no effect on hENaC␣ and caused a slight decrease on the hENaC␣␤␥ current. In conclusion, capsazepine acts on ENaC␦ and acts together with protons. Other vanilloids tested do not have any effect. These findings identify capsazepine as the first known chemical activator of ENaC␦.