Peptide Inhibition of Constitutively Activated Epithelial Na+ Channels Expressed in Xenopus Oocytes

Ji, Hong; Fuller, Catherine M.; Benos, Dale J.

doi:10.1074/jbc.274.53.37693

Cited by 16 publications

(21 citation statements)

References 53 publications

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“…We proposed the hypothesis that the COOH-terminal chains of ␤ and ␥ could act as intrinsic channel blockers by serving as an inactivation moiety. Our evidence, obtained in both bilayers (7,8) and heterologous expression systems (9), supports this type of mechanism. We tested the hypothesis that the functional gating particle comprised the COOH-terminal tails of both the ␤-and ␥-ENaC subunits associated as a two-strand, antiparallel ␤-sheet.…”

supporting

confidence: 65%

Intrinsic gating mechanisms of epithelial sodium channels

Fuller

Benos

2002

American Journal of Physiology-Cell Physiology

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The hypothesis that there is a highly conserved, positively charged region distal to the second transmembrane domain in ␣-ENaC (epithelial sodium channel) that acts as a putative receptor site for the negatively charged COOH-terminal ␤-and ␥-ENaC tails was tested in mutagenesis experiments. After expression in Xenopus oocytes, ␣-ENaC constructs in which positively charged arginine residues were converted into negatively charged glutamic acids could not be inhibited by blocking peptides. These observations provide insight into the gating machinery of ENaC.Liddle mutant; amiloride; inside-out patch; voltage clamp; post-M2 region LIDDLE'S SYNDROME is a form of hereditary hypertension produced by mutations within the epithelial sodium channel (ENaC) (1, 17). These mutations result in constitutive channel activation. Both an increase in functional channel number and an increase in singlechannel open probability (P o ) have been reported (5-9, 14, 15). The initial description of Liddle's syndrome identified truncation mutations in the COOH-terminal polypeptide chain of the ␤-ENaC (and subsequently the ␥-ENaC) subunit as being causative for constitutive channel activation (3, 5-9, 14, 15). We proposed the hypothesis that the COOH-terminal chains of ␤ and ␥ could act as intrinsic channel blockers by serving as an inactivation moiety. Our evidence, obtained in both bilayers (7,8) and heterologous expression systems (9), supports this type of mechanism. We tested the hypothesis that the functional gating particle comprised the COOH-terminal tails of both the ␤-and ␥-ENaC subunits associated as a two-strand, antiparallel ␤-sheet. Support for this idea was threefold: 1) the inhibitory effects of adding COOH-terminal ␤-and ␥-ENaC 30-amino acid residue peptides together with ENaC comprising wild-type ␣-and COOH-terminally truncated ␤-and ␥-subunits produced a greater than additive inhibition of the channel; 2) circular dichroism studies showed that the 30-mer ␤ and ␥ peptides formed a ␤-sheet; and 3) when the isoleucines and valines within the 30-mer peptides were replaced by the ␤-sheet, breaking amino acids proline or aspartic acid, the resulting peptides were unable to affect basalactivated ENaC (8). The paradigm that we have developed for ENaC gating is as follows. Because ␣-ENaC itself forms a functional sodium channel (4), there must be an intrinsic gating mechanism in ␣-ENaC alone. We hypothesized that calcium is intimately involved in this process and have presented evidence to this effect (2). The overall gating properties of ␣-ENaC vs. ␣␤␥-ENaC in bilayers do not differ (7). Because the elimination of the cytoplasmic COOH-terminal tails of either or both of the ␤-and ␥-subunits substantially increases single-channel P o (5-9), there must be at least two separate gating processes, one inherent to ␣-ENaC alone and one conferred onto the complex by the ␤-and ␥-subunits.To further elucidate the mechanism underlying the COOH-terminal ␤-and ␥-ENaC tail block of ENaC, we tested the hypothesis that a highly conserved region...

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supporting

confidence: 65%

Intrinsic gating mechanisms of epithelial sodium channels

Fuller

Benos

2002

American Journal of Physiology-Cell Physiology

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“…The vitelline membrane was then manually removed from the shrunken oocytes using fine forceps (9). The devitellinized oocyte was immediately transferred to a recording chamber (Warner Instruments, Hamden, CT) and perfused with a Li ϩ -rich intracellular solution before patch clamping the cell.…”

Section: Methodsmentioning

confidence: 99%

“…Patch Clamp Studies-Inside-out configuration of the patch clamp recording was used as described previously (9). The reference electrode was a Ag/AgCl pellet bathed in the bath solution (in mM, 140 lithium glutamate, 0.5 EGTA, 10 Hepes, pH 7.4).…”

Section: Methodsmentioning

confidence: 99%

“…Many of the described mutations result in truncation of the C termini of either the ␤-or ␥-subunit of ENaC. It is likely that the increased channel activity seen in patients with Liddle's disease is due to both increased ENaC cell surface expression and increased channel open probability (9). Ismailov et al (8) demonstrated that the addition of trypsin (0.5 mg/ml) to the putative intracellular side of lymphocyte Na ϩ channels incorporated into planar lipid bilayers increased the single channel open probability (P o ) in normal individuals as compared with that seen in lymphocyte Na ϩ channels obtained from patients with Liddle's disease.…”

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confidence: 99%

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The Serine Protease Trypsin Cleaves C Termini of β- and γ-Subunits of Epithelial Na+ Channels

Jovov

Berdiev

Fuller

et al. 2002

Journal of Biological Chemistry

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Both extracellular and intracellular proteases can activate epithelial Na؉ channels (ENaC). The mechanism by which serine proteases activate ENaC is unknown. We investigated the effect of the serine protease trypsin on in vitro translated and immunopurified ␣-, ␤-, and ␥-rENaC subunits. Immunopurified subunit proteins were exposed to increasing concentrations of trypsin ranging from 0.002 to 2 g/ml in Tris-buffered saline buffer for 2 h. The proteolytic mixture was subjected to SDS-PAGE and analyzed by autoradiography. Our results demonstrate that the ␤-and ␥-subunits of ENaC were most susceptible to trypsin proteolysis, and exposure to as little as 0.002 g/ml trypsin resulted in a reduction in the size of the ␤-and ␥-transcripts by 7-8 kDa. By using N-and C-terminally truncated ␤-and ␥-subunits, we determined that trypsin cleaved the C termini of both subunits, resulting in a channel structure resembling that seen in Liddle's disease. Exposure to 2 g/ml trypsin completely digested all three subunits. Our results suggest different susceptibilities of proteolytic sites of ENaC subunits to trypsin. Thus, we propose that limited intracellular proteolysis may be one of the potential physiological mechanisms of sodium channel regulation.Epithelial sodium channels (ENaC) 1 play a key role in the regulation of sodium balance, extracellular fluid volume, blood pressure, and fluid reabsorption. The activity of ENaC is tightly regulated in order to ensure ion and volume homeostasis of the extra-and intracellular milieu. This regulation is under the control of several hormones and intracellular factors by mechanisms that are not yet completely understood (1). The idea that proteases can modulate the activity of epithelial amiloride-sensitive Na ϩ channels is not new. Both extracellular and intracellular effects of proteases on epithelial amiloride-sensitive Na ϩ channels have been reported. Garty and Edelman (2) observed that extracellular trypsin, at a concentration of 1 mg/ml, induced an irreversible inhibition of the sodium transport in toad urinary bladder and that this effect could be prevented by amiloride. Lewis and Alles (3) studied the effects of proteases such as kallikrein, urokinase, and plasmin and observed that these proteases converted a normally highly selective amiloride-sensitive Na ϩ channel into a nonselective cation channel. Chraibi et al. (4) found that trypsin, at a concentration of 2 g/ml, increased the amiloride-sensitive current up to 20-fold when added to the bathing solution of ENaCexpressing Xenopus oocytes. Jovov et al. (5) demonstrated that A6 cells (derived from Xenopus laevis kidney) express and secrete a kallikrein-like serine protease from their apical side. This serine protease has been cloned and named channel-activating protease (CAP1) (6). Masilamani et al. (7) suggested that activation of Na ϩ currents by aldosterone is mediated by CAP1 that in turn cleaves ␥-ENaC in the early portion of extracellular loop (7). Until now, this hypothesis has not been tested. An intracellular effe...

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“…Such a domain in ␣ENaC binds the SH3 domain within ␣-spectrin and has been implicated in localizing the channel to the luminal membrane in epithelia. In addition, the COOH termini of ␤-and ␥ENaC may impact ENaC open probability by promoting channel closing (24,25). All of these COOH-terminal domains, described previously (15), are more distal than the membrane reactive domain we recently identified in the COOH tail of ␥ENaC.…”

mentioning

confidence: 99%