Expression of the Human Colonic Na+ Channel α-Subunit in Xenopus Oocytes

The α subunit of voltage-gated Na+ channels of brain, skeletal muscle, and cardiomyocytes is functionally modulated by the accessory β1, but not the β2 subunit. In the present study, we used β1/β2 chimeras to identify molecular regions within the β1 subunit that are responsible for both the increase of the current density and the acceleration of recovery from inactivation of the human heart Na+ channel (hH1). The channels were expressed in Xenopus oocytes. As a control, we coexpressed the β1/β2 chimeras with rat brain IIA channels. In agreement with previous studies, the β1 extracellular domain sufficed to modulate IIA channel function. In contrast to this, the extracellular domain of the β1 subunit alone was ineffective to modulate hH1. Instead, the putative membrane anchor plus either the intracellular or the extracellular domain of the β1 subunit was required. An exchange of the β1 membrane anchor by the corresponding β2 subunit region almost completely abolished the effects of the β1 subunit on hH1, suggesting that the β1 membrane anchor plays a crucial role for the modulation of the cardiac Na+ channel isoform. It is concluded that the β1 subunit modulates the cardiac and the neuronal channel isoforms by different molecular interactions: hH1 channels via the membrane anchor plus additional intracellular or extracellular regions, and IIA channels via the extracellular region only.

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Human Nedd4 interacts with the human epithelial Na+ channel: WW3 but not WW1 binds to Na+-channel subunits

Biochemical Journal ◽

10.1042/bj3450503 ◽

2000 ◽

Vol 345 (3) ◽

pp. 503-509 ◽

Cited By ~ 26

Author(s):

Tracy J. FARR ◽

Sarah J. CODDINGTON-LAWSON ◽

Peter M. SNYDER ◽

Fiona J. MCDONALD

Keyword(s):

Binding Sites ◽

Xenopus Oocytes ◽

Na Channel ◽

Channel Activity ◽

Α Subunit ◽

Ww Domains ◽

Ubiquitin Protein Ligase ◽

Human Lung Cells ◽

Epithelial Na Channel ◽

Proline Rich Region

The epithelial Na+ channel (ENaC) regulates Na+ absorption in epithelial tissues including the lung, colon and sweat gland, and in the distal nephrons of the kidney. When Na+-channel function is disrupted, salt and water homoeostasis is affected. The cytoplasmic regions of the Na+-channel subunits provide binding sites for other proteins to interact with and potentially regulate Na+-channel activity. Previously we showed that a proline-rich region of the α subunit of the Na+ channel bound to a protein of 116 kDa from human lung cells. Here we report the identification of this protein as human Nedd4, a ubiquitin-protein ligase that binds to the Na+-channel subunits via its WW domains. Further, we show that WW domains 2, 3 and 4 of human Nedd4 bind to the α, β and γ Na+-channel subunits but not to a mutated β subunit. In addition, when co-expressed in Xenopus oocytes, human Nedd4 down-regulates Na+-channel activity.

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Protease Modulation of the Activity of the Epithelial Sodium Channel Expressed in Xenopus Oocytes

The Journal of General Physiology ◽

10.1085/jgp.111.1.127 ◽

1998 ◽

Vol 111 (1) ◽

pp. 127-138 ◽

Cited By ~ 141

Author(s):

Ahmed Chraïbi ◽

Véronique Vallet ◽

Dmitri Firsov ◽

Solange Kharoubi Hess ◽

Jean-Daniel Horisberger

Keyword(s):

Sodium Channel ◽

Xenopus Oocytes ◽

Epithelial Sodium Channel ◽

Cell Surface Expression ◽

K Channel ◽

Na Channel ◽

Extracellular Proteases ◽

Open Probability ◽

Α Subunit ◽

Channel Protein

We have investigated the effect of extracellular proteases on the amiloride-sensitive Na+ current (INa) in Xenopus oocytes expressing the three subunits α, β, and γ of the rat or Xenopus epithelial Na+ channel (ENaC). Low concentrations of trypsin (2 μg/ml) induced a large increase of INa within a few minutes, an effect that was fully prevented by soybean trypsin inhibitor, but not by amiloride. A similar effect was observed with chymotrypsin, but not with kallikrein. The trypsin-induced increase of INa was observed with Xenopus and rat ENaC, and was very large (∼20-fold) with the channel obtained by coexpression of the α subunit of Xenopus ENaC with the β and γ subunits of rat ENaC. The effect of trypsin was selective for ENaC, as shown by the absence of effect on the current due to expression of the K+ channel ROMK2. The effect of trypsin was not prevented by intracellular injection of EGTA nor by pretreatment with GTP-γS, suggesting that this effect was not mediated by G proteins. Measurement of the channel protein expression at the oocyte surface by antibody binding to a FLAG epitope showed that the effect of trypsin was not accompanied by an increase in the channel protein density, indicating that proteolysis modified the activity of the channel present at the oocyte surface rather than the cell surface expression. At the single channel level, in the cell-attached mode, more active channels were observed in the patch when trypsin was present in the pipette, while no change in channel activity could be detected when trypsin was added to the bath solution around the patch pipette. We conclude that extracellular proteases are able to increase the open probability of the epithelial sodium channel by an effect that does not occur through activation of a G protein-coupled receptor, but rather through proteolysis of a protein that is either a constitutive part of the channel itself or closely associated with it.

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NaCl-dependent expression of amiloride-blockable Na+ channel in Xenopus oocytes

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1992.262.2.g244 ◽

1992 ◽

Vol 262 (2) ◽

pp. G244-G248 ◽

Cited By ~ 2

Author(s):

C. Asher ◽

D. Singer ◽

R. Eren ◽

O. Yeger ◽

N. Dascal ◽

...

Keyword(s):

Xenopus Oocytes ◽

The Other ◽

Na Channel ◽

Channel Activity ◽

High Affinity ◽

Other Hand ◽

Exogenous Rna ◽

Lower Intestine ◽

Regulation Of Transport

RNA was isolated from chicken lower intestine (both colon and coprodeum) and injected into Xenopus oocytes. 22Na+ fluxes measured after 1-4 days demonstrated the induction of an amiloride-blockable pathway. The Na+ transporter expressed by the exogenous RNA had a high affinity to amiloride (inhibitory constant less than 0.1 microM), but was insensitive to ethylisopropyl amiloride, i.e., it is likely to be the apical Na+ channel. Functional channels were readily expressed in oocytes injected with RNA derived from chickens fed a low-NaCl diet. On the other hand, no channel activity was detected in oocytes injected with RNA isolated from chickens fed a high-NaCl diet. Thus the previously reported regulation of transport by the dietary NaCl intake involves modulations in the level of mRNA that codes either for the Na+ channel or a posttranscriptional regulator of the channel.

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Long-term regulation of renal Na-dependent cotransporters and ENaC: response to altered acid-base intake

AJP Renal Physiology ◽

10.1152/ajprenal.2000.279.3.f459 ◽

2000 ◽

Vol 279 (3) ◽

pp. F459-F467 ◽

Cited By ~ 36

Author(s):

Gheun-Ho Kim ◽

Stephen W. Martin ◽

Patricia Fernández-Llama ◽

Shyama Masilamani ◽

Randall K. Packer ◽

...

Keyword(s):

Collecting Duct ◽

Na Channel ◽

Thick Ascending Limb ◽

Acid Base Balance ◽

Acid Base ◽

Opposite Pattern ◽

Α Subunit ◽

Base Balance ◽

Acid Loading

Increased systemic acid intake is associated with an increase in apical Na/H exchange in the renal proximal tubule mediated by the type 3 Na/H exchanger (NHE3). Because NHE3 mediates both proton secretion and Na absorption, increased NHE3 activity could inappropriately perturb Na balance unless there are compensatory changes in Na handling. In this study, we use semiquantitative immunoblotting of rat kidneys to investigate whether acid loading is associated with compensatory decreases in the abundance of renal tubule Na transporters other than NHE3. Long-term (i.e., 7-day) acid loading with NH4Cl produced large decreases in the abundances of the thiazide-sensitive Na-Cl cotransporter (TSC/NCC) of the distal convoluted tubule and both the β- and γ-subunits of the amiloride-sensitive epithelial Na channel (ENaC) of the collecting duct. In addition, the renal cortical abundance of the proximal type 2 Na-dependent phosphate transporter (NaPi-2) was markedly decreased. In contrast, abundances of the bumetanide-sensitive Na-K-2Cl cotransporter of the thick ascending limb and the α-subunit of ENaC were unchanged. A similar profile of changes was seen with short-term (16-h) acid loading. Long-term (7-day) base loading with NaHCO3resulted in the opposite pattern of response with marked increases in the abundances of the β- and γ-subunits of ENaC and NaPi-2. These adaptations may play critical roles in the maintenance in Na balance when changes in acid-base balance occur.

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On the Interaction between Amiloride and Its Putative α-Subunit Epithelial Na+Channel Binding Site

Journal of Biological Chemistry ◽

10.1074/jbc.m503500200 ◽

2005 ◽

Vol 280 (28) ◽

pp. 26206-26215 ◽

Cited By ~ 26

Author(s):

Ossama B. Kashlan ◽

Shaohu Sheng ◽

Thomas R. Kleyman

Keyword(s):

Binding Site ◽

Na Channel ◽

Α Subunit ◽

Epithelial Na Channel

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Regulation of a cloned epithelial Na+ channel by its β- and γ-subunits

AJP Cell Physiology ◽

10.1152/ajpcell.1997.273.6.c1889 ◽

1997 ◽

Vol 273 (6) ◽

pp. C1889-C1899 ◽

Cited By ~ 37

Author(s):

Mouhamed S. Awayda ◽

Albert Tousson ◽

Dale J. Benos

Keyword(s):

Fluorescence Intensity ◽

Expression System ◽

Fold Increase ◽

Na Channel ◽

Β Subunit ◽

Open Probability ◽

Animal Pole ◽

Α Subunit ◽

Amino Acid Peptide ◽

Xenopus Oocyte Expression

Using the Xenopus oocyte expression system, we examined the mechanisms by which the β- and γ-subunits of an epithelial Na+channel (ENaC) regulate α-subunit channel activity and the mechanisms by which β-subunit truncations cause ENaC activation. Expression of α-ENaC alone produced small amiloride-sensitive currents (−43 ± 10 nA, n = 7). These currents increased >30-fold with the coexpression of β- and γ-ENaC to −1,476 ± 254 nA ( n = 20). This increase was accompanied by a 3.1- and 2.7-fold increase of membrane fluorescence intensity in the animal and vegetal poles of the oocyte, respectively, with use of an antibody directed against the α-subunit of ENaC. Truncation of the last 75 amino acids of the β-subunit COOH terminus, as found in the original pedigree of individuals with Liddle’s syndrome, caused a 4.4-fold ( n = 17) increase of the amiloride-sensitive currents compared with wild-type αβγ-ENaC. This was accompanied by a 35% increase of animal pole membrane fluorescence intensity. Injection of a 30-amino acid peptide with sequence identity to the COOH terminus of the human β-ENaC significantly reduced the amiloride-sensitive currents by 40–50%. These observations suggest a tonic inhibitory role on the channel’s open probability ( P o) by the COOH terminus of β-ENaC. We conclude that the changes of current observed with coexpression of the β- and γ-subunits or those observed with β-subunit truncation are likely the result of changes of channel density in combination with large changes of P o.

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