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J Gen Physiol
2002 Aug 01;1202:191-201. doi: 10.1085/jgp.20028598.
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Synergistic activation of ENaC by three membrane-bound channel-activating serine proteases (mCAP1, mCAP2, and mCAP3) and serum- and glucocorticoid-regulated kinase (Sgk1) in Xenopus Oocytes.
Vuagniaux G
,
Vallet V
,
Jaeger NF
,
Hummler E
,
Rossier BC
.
Abstract
Sodium balance is maintained by the precise regulation of the activity of the epithelial sodium channel (ENaC) in the kidney. We have recently reported an extracellular activation of ENaC-mediated sodium transport (I(Na)) by a GPI-anchored serine protease (mouse channel-activating protein, mCAP1) that was isolated from a cortical collecting duct cell line derived from mouse kidney. In the present study, we have identified two additional membrane-bound serine proteases (mCAP2 and mCAP3) that are expressed in the same cell line. We show that each of these proteases is able to increase I(Na) 6-10-fold in the Xenopus oocyte expression system. I(Na) and the number (N) of channels expressed at the cell surface (measured by binding of a FLAG monoclonal I(125)-radioiodinated antibody) were measured in the same oocyte. Using this assay, we show that mCAP1 increases I(Na) 10-fold (P < 0.001) but N remained unchanged (P = 0.9), indicating that mCAP1 regulates ENaC activity by increasing its average open probability of the whole cell (wcP(o)). The serum- and glucocorticoid-regulated kinase (Sgk1) involved in the aldosterone-dependent signaling cascade enhances I(Na) by 2.5-fold (P < 0.001) and N by 1.6-fold (P < 0.001), indicating a dual effect on N and wcP(o). Compared with Sgk1 alone, coexpression of Sgk1 with mCAP1 leads to a ninefold increase in I(Na) (P < 0.001) and 1.3-fold in N (P < 0.02). Similar results were observed for mCAP2 and mCAP3. The synergism between CAPs and Sgk1 on I(Na) was always more than additive, indicating a true potentiation. The synergistic effect of the two activation pathways allows a large dynamic range for ENaC-mediated sodium regulation crucial for a tight control of sodium homeostasis.
Figure 1. . mCAP1, 2, and 3 are membrane-bound serine proteases. (A) Structural properties of the three mCAPs (mCAP1, mCAP2, and mCAP3). The amino acid sequence of each protein was scanned using ProfileScan algorithm to confirm the presence of domains indicated. Numbers delineate the location of each domain. (B) A representative phylogenetic tree of the GPI-anchored mCAP1 and type II transmembrane mCAP2 and mCAP3 serine protease family proteins. Multiple alignment was generated by the clustal W program (mCAP1, AAG1705; hProstasin, Q16651; xCAP1, AAB969054; mCAP2, AY043240; hTMPRSS4-1, CAC60389; hTMPRSS4–2; MT-SP2, Q9NRS4; mTMPRSS2; epitheliasin, AAF97867; hTMPRSS2, AAC51784; mTMPRSS3, CAC83350; hTMPRSS3, P57727; mCAP3; epithin, AAD02230; hMT-SP1, AAF00109; xMT-SP1, BAB08218). (C) Amino acid alignment of the serine protease catalytic domain of mCAP1, mCAP2, and mCAP3 with mouse trypsinogen precursor (1–246), was performed using Pileup program (Genetic Computer Group). Gray and black boxes indicate similar and identical residues, respectively. The catalytic triad (H, D, and S, asterisks), the amino acids implicated in the P1 substrate specificity (closed circle), and the position for Na+ sensibility (open circle) are indicated.
Figure 2. . Coexpression of all three serine proteases with ENaC. Multiple tissue Northern blot containing 20 μg of RNA/lane were hybridized with cDNA fragments of the rat αENaC, mouse CAP1, CAP2, CAP3, and GAPDH genes, as described in materials and methods. mRNA transcript lengths are indicated.
Figure 3. . Functional analysis of mCAP1–3 in Xenopus oocytes. (A) Comparison of the effect of mCAP1, mCAP2, and mCAP3 on INa in Xenopus oocytes. Oocytes were injected with rat ENaC subunits in the presence of either water (open bar, lane 1) or increasing amounts (2, 4, and 8 or 12 ng) of mCAP1 (closed bar, lanes 2–4), mCAP2 (light gray bar, lanes 5–8), or mCAP3 (dark gray bars, lanes 9–12). §§, P < 0.01 versus conditions with other amounts of the same cRNA protease. n ≥ 12 measured oocytes. (B) Effect of preincubation with aprotinin (lanes 3, 4, 7, 8, 11, 12, 15, 16) or perfusion of trypsin (gray bars, lanes 2, 4, 6, 8, 10, 12, 14, 16) on oocytes injected with either rENaC and water (lane 1–4) or rENaC together with 4 ng mCAP1 (lane 5–8), 8 ng mCAP2 (lane 9–12), and 2 ng mCAP3 (lane 13–16). n ≥ 12 measured oocytes.
Figure 4. . Synergistic activation of mCAP1–3 and Sgk1 on ENaC activity. (A) Oocytes were injected with cRNA coding either for α, β, and γFLAG-tagged rENaC subunits (rENaCf) and water (lane 1) or rENaCf together with mCAP1 (lane 2), mCAP2 (lane 3), mCAP3 (lane 4), Sgk1 (lanes 5–8), or both Sgk1 and mCAP1 (lane 6), Sgk1 and mCAP2 (lane 7), and Sgk1 and mCAP3 (lane 8). INa was measured in absence (open bar) and after perfusion of trypsin (closed bar) and normalized to INa in oocytes injected with rENaC alone. n ≥ 15 measured oocytes per experimental condition taken from Tables I–III. (B) Normalized oocyte cell surface expression of rENaCf in oocytes experiments described in A. Water, lane 1; mCAP1, lane 2; mCAP2, lane 3; mCAP3, lane 4; Sgk1, lane 5; mCAP1 + Sgk1, lane 6; mCAP2 + Sgk1, lane 7; mCAP3 + Sgk1, lane 8. ***, P < 0.001 versus rENaCf + water.
Figure 5. . Model of CAPs and Sgk1 action on ENaC. (A) CAPs increase the open probability of ENaC channels. The substrate of CAPs may be ENaC or protein(s) associated with ENaC. (B) Model of Sgk1 action on ENaC. Sgk1 principally increases the number of active channels at the cell surface by diminishing the removal of the channels from the cell surface and by increasing its Po. (C) CAPs proteases and Sgk1 act synergically on the channel open probability and cell surface expression.
Adachi,
Activation of epithelial sodium channels by prostasin in Xenopus oocytes.
2001, Pubmed,
Xenbase
Adachi,
Activation of epithelial sodium channels by prostasin in Xenopus oocytes.
2001,
Pubmed
,
Xenbase
Alvarez de la Rosa,
The serum and glucocorticoid kinase sgk increases the abundance of epithelial sodium channels in the plasma membrane of Xenopus oocytes.
1999,
Pubmed
,
Xenbase
Bens,
Corticosteroid-dependent sodium transport in a novel immortalized mouse collecting duct principal cell line.
1999,
Pubmed
Bridges,
Na+ transport in normal and CF human bronchial epithelial cells is inhibited by BAY 39-9437.
2001,
Pubmed
Canessa,
Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits.
1994,
Pubmed
,
Xenbase
Chen,
Epithelial sodium channel regulated by aldosterone-induced protein sgk.
1999,
Pubmed
,
Xenbase
Chraïbi,
Protease modulation of the activity of the epithelial sodium channel expressed in Xenopus oocytes.
1998,
Pubmed
,
Xenbase
Dang,
Residue 225 determines the Na(+)-induced allosteric regulation of catalytic activity in serine proteases.
1996,
Pubmed
Debonneville,
Phosphorylation of Nedd4-2 by Sgk1 regulates epithelial Na(+) channel cell surface expression.
2001,
Pubmed
,
Xenbase
Donaldson,
Regulation of the epithelial sodium channel by serine proteases in human airways.
2002,
Pubmed
,
Xenbase
Firsov,
Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: a quantitative approach.
1996,
Pubmed
,
Xenbase
Firsov,
The heterotetrameric architecture of the epithelial sodium channel (ENaC).
1998,
Pubmed
,
Xenbase
Hooper,
Type II transmembrane serine proteases. Insights into an emerging class of cell surface proteolytic enzymes.
2001,
Pubmed
Kim,
Cloning and chromosomal mapping of a gene isolated from thymic stromal cells encoding a new mouse type II membrane serine protease, epithin, containing four LDL receptor modules and two CUB domains.
1999,
Pubmed
Lin,
Molecular cloning of cDNA for matriptase, a matrix-degrading serine protease with trypsin-like activity.
1999,
Pubmed
Loffing,
Aldosterone induces rapid apical translocation of ENaC in early portion of renal collecting system: possible role of SGK.
2001,
Pubmed
,
Xenbase
Masilamani,
Aldosterone-mediated regulation of ENaC alpha, beta, and gamma subunit proteins in rat kidney.
1999,
Pubmed
Narikiyo,
Regulation of prostasin by aldosterone in the kidney.
2002,
Pubmed
,
Xenbase
Náray-Fejes-Tóth,
sgk is an aldosterone-induced kinase in the renal collecting duct. Effects on epithelial na+ channels.
1999,
Pubmed
,
Xenbase
Palmer,
Conductance and gating of epithelial Na channels from rat cortical collecting tubule. Effects of luminal Na and Li.
1988,
Pubmed
Palmer,
Gating of Na channels in the rat cortical collecting tubule: effects of voltage and membrane stretch.
1996,
Pubmed
Perona,
Structural basis of substrate specificity in the serine proteases.
1995,
Pubmed
Perona,
Evolutionary divergence of substrate specificity within the chymotrypsin-like serine protease fold.
1997,
Pubmed
Pácha,
Regulation of Na channels of the rat cortical collecting tubule by aldosterone.
1993,
Pubmed
Robert-Nicoud,
Transcriptome of a mouse kidney cortical collecting duct cell line: effects of aldosterone and vasopressin.
2001,
Pubmed
Rubera,
A conditional allele at the mouse channel activating protease 1 (Prss8) gene locus.
2002,
Pubmed
Schechter,
On the size of the active site in proteases. I. Papain.
1967,
Pubmed
Snyder,
Serum and glucocorticoid-regulated kinase modulates Nedd4-2-mediated inhibition of the epithelial Na+ channel.
2002,
Pubmed
Takeuchi,
Reverse biochemistry: use of macromolecular protease inhibitors to dissect complex biological processes and identify a membrane-type serine protease in epithelial cancer and normal tissue.
1999,
Pubmed
Vallet,
Cell-surface expression of the channel activating protease xCAP-1 is required for activation of ENaC in the Xenopus oocyte.
2002,
Pubmed
,
Xenbase
Vallet,
An epithelial serine protease activates the amiloride-sensitive sodium channel.
1997,
Pubmed
,
Xenbase
Vallet,
Epithelial sodium channel regulatory proteins identified by functional expression cloning.
1998,
Pubmed
,
Xenbase
Vuagniaux,
Activation of the amiloride-sensitive epithelial sodium channel by the serine protease mCAP1 expressed in a mouse cortical collecting duct cell line.
2000,
Pubmed
,
Xenbase
Wallrapp,
A novel transmembrane serine protease (TMPRSS3) overexpressed in pancreatic cancer.
2000,
Pubmed
Webster,
Characterization of sgk, a novel member of the serine/threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum.
1993,
Pubmed
Yu,
Molecular cloning, tissue-specific expression, and cellular localization of human prostasin mRNA.
1995,
Pubmed
Zheng,
Apical sorting of bovine enteropeptidase does not involve detergent-resistant association with sphingolipid-cholesterol rafts.
1999,
Pubmed
