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*
Institut de Pharmacologie et de Toxicologie,
Université de Lausanne, Switzerland
Institut Nationale de la Santé et de la
Recherche Médicale U478, Institut
Fédératif de
Recherche, Paris Cedex, France.
Correspondence to Dr. Bernard C. Rossier, Institut de Pharmacologie et de Toxicologie, Rue du Bugnon 27, CH-1005 Lausanne, Switzerland. Phone: +41 21 6925351; Fax: +41 21 6925355; E-mail: Bernard.Rossier{at}ipharm.unil.ch or Dr. Edith Hummler, Institut de Pharmacologie et de Toxicologie, Rue du Bugnon 27, CH-1005 Lausanne, Switzerland. Phone: +41 21 6925357; Fax: +41 21 6925355; E-mail: Edith.HummlerBeermann{at}ipharm.unil.ch
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-, ß-, and 
-ENaC
subunits in Xenopus oocytes also showed that mCAP1 induces a
significant increase in ENaC-mediated current accompanied by a decrease of
channel molecules at the cell surface. It is proposed that this novel mouse
channel activating protease may act as a regulator of ENaC within the
kidney. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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spectrin (3), Nedd-4
(4), and proteins such as
K-Ras2 and sgk kinase (reviewed in reference
(5). We have previously
identified an epithelial serine protease (xCAP1) that activates the
amiloride-sensitive sodium channel ENaC in a Xenopus kidney
epithelial A6 cell line
(6,7).
xCAP1 is a 329-residue protein belonging to the serine protease family. The
coexpression of xCAP1 with ENaC in the Xenopus oocyte increases the
activity of the sodium channel by two- to three- fold. This effect of xCAP1 is
inhibited by aprotinin, a serine protease inhibitor
(6). According to its sequence
analysis, xCAP1 is thought to be a secreted and/or a
glycosyl-phosphatidyl-inositol (GPI)-anchored protein. We proposed that ENaC
activity could be regulated by the activity of an extracellular protease
expressed at the apical surface of the kidney cell. This defined a novel
extracellular signaling pathway for ion channels that has not yet been studied
in the mammalian kidney. The most closely related mammalian protein to xCAP1
is a human serine protease, prostasin, which has been identified in semen
(8) and which shares 53%
homology with the amphibian gene product xCAP1. Tissue-specific expression of
prostasin mRNA revealed the presence of transcripts in prostate, liver,
testes, salivary glands, kidney, lung, pancreas, and colon and in a proximal
tubule kidney cell line (8).
The specific distribution of prostasin mRNA along the human nephron was not
reported and, specifically, it was not shown whether prostasin is expressed in
the distal part of the nephron. This is an important issue, since sodium
balance is achieved by the regulation of ENaC in the distal nephron, namely,
in the cells of distal convoluted tubules and collecting ducts, which is the
main target for aldosterone response
(1). The amphibian A6 cells,
derived from the principal cells of the collecting duct of the
Xenopus kidney, express ENaC, which is controlled by aldosterone
(9) and activated by xCAP1
(6). This raises the question
of whether the mammalian prostasin is the gene product corresponding to the
Xenopus CAP1. The aim of this study was to identify a mammalian CAP1 homologue in cells of the cortical collecting duct. To achieve this goal, we selected a novel established clonal mouse collecting duct cell line, mpkCCDc14, which faithfully reproduces the expected properties of collecting duct cells (10). As in A6 cells, this cell line has a high transepithelial electrical resistance and expresses an amiloride-sensitive transepithelial sodium transport that is responsive to physiologic concentrations of aldosterone. In the present report, we describe the identification of a mouse serine protease homologous to xCAP1, termed mCAP1, which has properties similar to the Xenopus counterpart. We propose that this homologue is responsible for regulating ENaC activity in the mammalian kidney.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Identification and Isolation of a Novel Full-Length Murine Serine
Protease cDNA
Reverse transcription (RT)-PCR was performed on total RNA from
mpkCCDc14 cells. Briefly, 3 µg of total RNA was treated with
DNase I (Boehringer Mannheim, Germany) followed by reverse transcription using
Superscript II (Life Technologies) and random primers (Pharmacia). PCR was
performed using 1/10 of the cDNA digested with RNase H (Life Technologies) at
55°C for 15 min. PCR was done in 50-µl reactions containing 1.5 mM
MgCl2, 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 150 µM of each
deoxynucleoside 5'-triphosphate, 5% DMSO, 0.5 µM of each primer, and
2.5 U of Taq DNA polymerase (Boehringer Mannheim). Forty cycles were
run using primers D1 and D2 each consisting of 30 s at 94°C, 30 s at
48°C, and 1 min at 72°C. After the last cycle, elongation was allowed
to proceed for 7 min at 72°C. A second PCR was performed using 1/20 of the
first PCR reaction with nested primers D1 and D3 under the same conditions.
The 512-bp amplified PCR product was separated on a 1.2% agarose gel,
extracted, and purified (Qiagen, Chatsworth, CA), subcloned into pT7Blue
Vector (Blunt Cloning Kit, Novagen, Madison, WI), and sequenced. The primers
used were as follows: D1 (sense) 5'-AA(AG)TT(CT)CCITGGCA(AG)GT-3',
nt +118 to +134; D2 (antisense) 5'-CC(AG)CA(CT)TC(AG)TCICCCCA-3',
nt +743 to +727; D3 (antisense) 5'-CCIGC(AG)CA(AGT)ATCAT(AG)TC-3',
nt +629 to +613; according to xCAP1 (5' and 3').
Rapid Amplification of cDNA Ends
For 5' rapid amplification of cDNA ends (5'-RACE), after
reverse transcription of mpkCCDc14 total mRNA (as described above),
mCAP1-specific reverse primers RC1 (5'-CTTCACCTCATACGCTTCC-3', nt
position +297 to +279) and RC2 (5'-GTTGCCATCGTAGGTGATG-3', nt
position +201 to +183) were used sequentially with adapter-specific primer
dC-5R (5'-GCATGCTCGAGCGGCCGCAACCCCCCCCCCCCCCCCCC-3') and 5R
(5'-GCATGCTCGAGCGGCCGCAAC-3'), according to the manufacturer's
instructions (Life Technologies). For 3'-RACE, reverse transcription was
performed using an oligo(dT) adapter primer
(5'-CGAGATCTATGCGGCCGCTTTTTTTTTTTTTTTTTTTT-3'). mCAP1-specific
primers RC3 (5'-TCAGTGAGCCTCCAGACC-3', nt position +535 to +552)
and RC4 (5'-CTGTAGCTGCCTGTACAA-3', nt position +600 to +617) were
used. To amplify the full-length cDNA from mpkCCDc14 cells, primer
1 (sense 5'-TGCCTTCAAAACCAGCCTTC-3', nt position -73 to -54) and
primer 2 (antisense 5'-TGTCCTTGGGTGTGCTGTG-3', nt position +1083
to +1065; mCAP1; accession no. AF 188613) covering the open reading frame of
mCAP1 were used according to the manufacturer's instructions (High Fidelity
Expand PCR System, Boehringer, Germany). The 1156-bp PCR product was purified
by agarose gel electrophoresis, cloned into pT7Blue Vector (Blunt Cloning Kit,
Novagen), and confirmed by sequencing.
Northern Blot Analysis
Total RNA (20 µg) extracted from various mouse tissues were run on a
0.8% denaturing glyoxal agarose gel and blotted onto nylon membranes
(Hybond-N, Amersham). Membranes were hybridized with random-primed
32P-labeled probes for mCAP1 (512 bp: nt +163 to +674),
rENaC, and rat GAPDH.
RT-PCR Analysis
Total RNA was prepared from whole mouse kidney, confluent
mpkCCDc14 cells, and microdissected tubules. Thin kidney slices
from 1-mo-old mice (n = 3) fed with a standard diet were incubated in
Dulbecco's modified Eagle's medium:Ham's F12 (1:1) supplemented with 0.1%
collagenase (Boehringer Mannheim) for 1 h at 37°C. S2 (proximal convoluted
tubule [PCT]) and S3 (pars recta [PR]) segments from proximal convoluted
tubules, medullary and cortical ascending limbs of Henle's loop, distal
convoluted tubules, and cortical (CCD) and inner medullary collecting ducts
were microdissected as described
(11). Single microdissected
tubules (0.3 to 0.5 mm long) from each segment were pooled (five to eight
single tubules) and stored at -80°C before use. Total RNA was extracted
from whole kidney, cultured cells, and microdissected tubules using the
RNA-PLUS extraction kit (Bioprobe Systems, Montreuil-sous-bois, France). The
RNA were reverse-transcribed with Moloney murine leukemia virus reverse
transcriptase (Life Technologies, Eragny, France) at 42°C for 45 min. Two
hundred fifty nanograms of whole kidney and mpkCCDc14 cell cDNA and
non-reverse-transcribed RNA were amplified for 32 cycles in 100 µl of total
volume containing 1.5 mM MgCl2, 50 mM KCl, 20 mM Tris-HCl, pH 8.4,
40 µM dNTP, 1 U Taq polymerase, 30 pmol of mCAP1 primer 1 and RC1.
Microdissected tubule cDNA were amplified for 38 cycles using the same buffer
containing 4 pmol of mCAP1 primers and 0.45 pmol of ß-actin primers
(5'-CGTGGGCCGCCCTAGGCACCA-3' and
5'-TTGGCCTTAGGGTTCAGGGGGG3' as described previously)
(10). The thermal cycling
program was as follows: 94°C for 30 s, 57°C for 30 s, and 72°C for
1 min. Amplification products were run on 3.5% agarose gel, stained with
ethidium bromide, and photographed, or were run on 4% polyacrylamide gel and
autoradiographed. The identity of the amplified products was controlled by
digestion with NciI (Life Technologies).
Electrophysiologic Measurements in Xenopus Oocytes
For functional expression studies, mCAP1 cDNA was subcloned into pSD5
expression vector and in vitro-transcribed
(7). Expression studies were
performed in stage V/VI occytes isolated from Xenopus laevis
(Noerdhoek, South Africa). Routinely, oocytes were injected with 0.33 ng of
each cRNA coding for the rat -, ß-, and -ENaC subunits in
the presence or absence of 2 ng of cRNA coding for mCAP1 in a total volume of
100 nl. Oocytes were incubated overnight in modified Barth saline solution in
the presence or absence of 100 µg/ml aprotinin. Electrophysiologic
measurements were performed 24 to 48 h after cRNA injection using the
two-electrode voltage-clamp technique, and the amiloride-sensitive current
(INa) was measured in the presence of 120 mM of
Na+ in frog Ringer's solution with 5 µM amiloride at a holding
potential of -100 mV. The oocytes were perfused with 2 µg/ml trypsin during
2 to 3 min and INa was remeasured. Amiloride sensitivity
of injected sodium channel subunits has been measured with 0.001, 0.01, 0.1,
1, and 10 µM amiloride in the presence of 120 mM Na+. Cell
surface expression has been performed as described
(12). Briefly, 1 ng of tagged
rat -, ß-, and -ENaC cRNA were expressed in oocytes either
coinjected with 2 ng of mCAP1 cRNA or water-injected. The density of the
channel was then estimated by binding of iodinated anti-FLAG (M2) monoclonal
antibody (Sigma, Buchs, Switzerland).
Statistical Analyses
All results are reported as means ± SEM. Comparing independent sets
of data, unpaired t tests were used to determine significance. In
experiments in which oocytes were perfused with trypsin, paired t
tests were used. P < 0.01 was considered significant. n
represents the number of experiments performed.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Identification of the Murine Homologue for xCAP1
RT-PCR analysis of mpkCCDc14 cells using degenerated
oligonucleotides to xCAP1 revealed a 512-bp PCR product. A 1768-bp cDNA
(without polyA+) was obtained by 5'- and 3'-RACE
(Figure 2). This clone
contained an open reading frame of 339 amino acids. Alignment of the predicted
amino acid sequence (Genetic Computer Group, Inc., Madison, WI) revealed that
the deduced protein belongs to the serine protease family. It shows high
homology with the trypsin-like protease family. It shared 50% homology with
the channel activating protease xCAP1, 46% with the mouse trypsinogen
precursor, and 80% with the human prostasin
(Figure 2). The N terminus
exhibits a putative cleavable signal peptide and the C terminus a hydrophobic
domain with a presumed GPI membrane-anchoring site known to act as a dominant
apical target signal in polarized epithelial cells
(6). Therefore, this new mouse
serine protease was named mCAP1 for mouse channel activating protease 1.
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Tissue and Intrarenal mCAP1 mRNA Expression
Northern blot analysis revealed strong hybridization signals (about 1.8 kb)
in kidney, lung, and salivary glands and a weak signal in distal colon, as
well as in mpkCCDc14 cells
(Figure 3), and in skin,
stomach, duodenum, small intestine, colon, bladder, and prostate (data not
shown). There was no detectable hybridization signal in liver, heart, and
brain (Figure 3), or in spleen,
testis, ovary, pancreas, and muscle (data not shown). Hybridization with mouse
-ENaC subunit (lower band) revealed coexpression with mCAP1 in the
relevant tissues (kidney, mpkCCDc14, colon, lung, salivary gland).
The upper band corresponds to a cross-hybridization to a 28S RNA
(Figure 3).
|
To map precisely the distribution of mCAP1 along the nephron, we used RT-PCR on microdissected nephron segments. The sets of mCAP1 primers used permitted detection of amplified products in both whole mouse kidney and cultured mpkCCDc14 cells. The specificity of the amplified products was confirmed by their digestion with the NciI restriction enzyme (Figure 4A). Because the intrarenal distribution of proteases is not known, we have analyzed the expression of mCAP1 along the mouse nephron (Figure 4B). The convoluted (PCT) and straight terminal portion (PR) of the proximal tubule exhibited the highest mCAP1 transcript abundance when compared with the levels of ß-actin transcripts. Substantial amounts of mCAP1 transcripts were also detected in cortical ascending limbs of Henle's loop and CCD (Figure 4B). In contrast, very low levels of mCAP1 expression were detected in medullary ascending limbs of Henle's loop and inner medullary collecting duct as well as in distal convoluted tubules.
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Functional Analysis of mCAP1 in Xenopus Oocytes
Coexpression of mCAP1 with each of the ENaC subunits from Xenopus
(xENaC), rat (rENaC), and human (hENaC) led to an approximately sixfold
significant (P < 0.01, unpaired t test) increase in the
macroscopic amiloride-sensitive current (xENaC INa: 0.2
± 0.02 µA [n = 21] versus 1.01 ± 0.04 µA
[n = 20]; rENaC INa: 0.75 ± 0.04 µA
[n = 19] versus 5.03 ± 0.16 µA [n = 21];
hENaC INa: 2.41 ± 0.07 µA [n = 21]
versus 12.12 ± 0.26 µA [n = 21])
(Figure 5A). In the absence of
-, ß-, and -ENaC subunits, mCAP1 injected into oocytes did
not generate measurable amiloride-sensitive current (data not shown).
|
We then compared the amiloride sensitivity of ENaC channel activity
consisting of rat -, ß-, and -ENaC subunits coinjected with
mCAP1 cRNA into Xenopus oocytes. The Kiamil of
ENaC activity alone (Kiamil: 0.106 ± 0.008 µM;
n = 18) did not significantly differ (P > 0.1, unpaired
t test) from the Kiamil in oocytes coexpressing all ENaC
subunits and mCAP1 (Kiamil: 0.91 ± 0.005 µM;
n = 17). We conclude that the concentration of amiloride used in our
assay is sufficient to block effectively ENaC-dependent sodium transport (over
95%) and that the effect of mCAP1 is therefore not underestimated.
Next, we tested the effects of trypsin and aprotinin on amiloride-sensitive
ENaC-mediated sodium current in Xenopus oocytes injected with all
three subunits of the rat (-, ß-, and -ENaC) in the
presence (Figure 5B, lanes 5 to
8) or absence of mCAP1 cRNA (Figure
5B, lanes 1 to 4). In the absence of aprotinin, mCAP1 induced a
five- to sixfold increase of the amiloride-sensitive sodium current
(INa) (Figure
5B, lane 1 versus lane 5). Trypsin (2 µg/ml) was able
to activate ENaC in the absence of mCAP1
(Figure 5B, lane 1
versus lane 2) but did not further increase the
INa stimulated by mCAP1
(Figure 5B, lane 5
versus lane 6). When oocytes were incubated overnight with 100
µg/ml aprotinin, the increase of INa by mCAP1 was
inhibited (Figure 5B, lane 5
versus lane 7). The inhibitory effect of aprotinin was only effective
in the presence of mCAP1, as it was not observed in its absence
(Figure 5B, lane 1
versus lane 3). This finding suggests that the oocyte has very little
endogenous proteolytic activity able to activate ENaC. The effect of aprotinin
on INa could be reversed by trypsin in noninjected or
injected mCAP1 oocytes (Figure
5B, lane 3 versus lane 4 and lane 7 versus lane
8). The lack of additive effect of trypsin and mCAP1 and the inhibition of
mCAP1 activation by aprotinin and its reversal by trypsin strongly suggested
that mCAP1 and trypsin acted through a common proteolytic pathway. mCAP1 could
increase the amiloride-sensitive sodium current mediated by ENaC by two
mechanisms: either by changing the electrophysiologic properties of the
channel (conductance, ion selectivity, gating kinetics) or by increasing its
cell-surface expression.
Coexpression of FLAG-tagged -, ß-, and -ENaC subunits
and mCAP1 cRNA in oocytes produced a fourfold increase (4.4 ± 0.7;
n = 25) in INa
(Figure 5C, left panel). This
increase of INa using the tagged ENaC subunits was lower
than the increase using nontagged subunits. Higher basal currents measured
under those conditions might be responsible for this difference in decreasing
the effect of the protease
(13). We also observed a
twofold decrease (1.8 ± 0.1; n = 25) in expression of the
channel molecules at the cell surface
(Figure 5C, right panel). These
results suggested that the intrinsic channel activity was increased in the
presence of mCAP1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In a previous study (6), we showed that xCAP1 increased the amiloride-sensitive sodium current but induced a small but nonsignificant decrease (-20%) in the binding of anti-FLAG antibodies. Because the increase in INa could not be explained by a change in the single channel conductance or in the cell surface expression of ENaC, we suggest that the increase in INa was due to an increase in the overall open probability of the channel molecules present at the oocyte surface (12,13,15).
Like xCAP1, mCAP1 appears to activate preexisting channels located in the plasma membrane, because the number of molecules expressed at the surface is not increased by mCAP1 but is significantly decreased (by 50%). According to this assay, we propose that mCAP1 has a major effect on the overall open probability of the channel if one assumes that mCAP1 has no effect on ion selectivity and/or single channel conductance.
mCAP1 Is Probably the Mouse Counterpart of Human Prostasin
According to the data presented in this study, mCAP1 mRNA transcripts are
expressed at high levels in epithelial tissues that are known to be the site
of an amiloride-sensitive transepithelial sodium transport (CCD, colon, lung,
salivary gland, skin, bladder). mCAP1 is also expressed at low levels in other
epithelial tissues, such as the stomach, duodenum, and small intestine, where
ENaC might be expressed under some special pathophysiologic conditions. It is
not yet known whether human prostasin can activate ENaC in the kidney, the
colon, or the lung. However, preliminary experiments have shown that it is the
case in the Xenopus oocyte system (unpublished observation). On the
basis of tissue-specific expression and functional studies, we would therefore
like to propose that mCAP1, xCAP1, and prostasin are the corresponding genes
for the three species studied so far.
Regulation of ENaC Activity and Sodium Transport
Using isolated microdissected tubules, the results from RT-PCR experiments
clearly indicate that mCAP1 is expressed at high levels in the proximal tubule
and to a lesser extent in the distal part of the nephron and the CCD
(Figure 4). Although we have
not analyzed the expression of mCAP1 at the protein level, the high expression
of mCAP1 in the proximal tubule could indicate a local mode of action. In the
rat, ENaC transcripts and ENaC activity are specifically found in the S3
segment of the proximal tubule, and the presence of an amiloride-sensitive
sodium transport supports this finding
(16). In the present study,
mCAP1 appears to be expressed all along the proximal tubule (from S1 to S3).
Another interesting and more intriguing possibility is that mCAP1 is secreted
into the tubular fluid of the proximal tubule to activate ENaC expression in
cells of the distal part of the nephron. Such a distant mode of action has
also been proposed for other secreted proteases such as kallikrein
(17). The second possibility
predicts that mCAP1 is synthesized in cells of the CCD, then targeted to the
apical membrane to control in situ the activity of ENaC. This is
supported by: (1) the detection of mCAP1 mRNA transcripts in the
collecting duct and in a highly differentiated cell line deriving from mouse
CCD; (2) the presence of serine protease activity in the apical
membrane of these cells, which can be inhibited by aprotinin; and (3)
the regulation of ENaC activity in oocytes. Both modes of action are not
mutually exclusive. The regulation of each of these two pathways could be
different in function of various physiologic or pathophysiologic stimuli.
Although it is not excluded that other serine protease may activate ENaC
in vivo, the cloning of mCAP1 will allow us to study the physiologic
role of this serine protease in vivo by gene targeting and to assess
its physiologic relevance in the control of sodium balance, blood volume, and
BP.
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Acknowledgments |
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Footnotes |
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American Society of Nephrology
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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rENaC) mediates its localization at the apical membrane.
EMBO J 13:4440
-4450, 1994[Medline]
subunit in A6 renal cells. J Am Soc
Nephrol 8:1813
-1822, 1997[Abstract]
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M. N. Helms, L. Liu, Y.-Y. Liang, O. Al-Khalili, A. Vandewalle, S. Saxena, D. C. Eaton, and H.-P. Ma Phosphatidylinositol 3,4,5-Trisphosphate Mediates Aldosterone Stimulation of Epithelial Sodium Channel (ENaC) and Interacts with {gamma}-ENaC J. Biol. Chem., December 9, 2005; 280(49): 40885 - 40891. [Abstract] [Full Text] [PDF]
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E. Hummler and V. Vallon Lessons from Mouse Mutants of Epithelial Sodium Channel and Its Regulatory Proteins J. Am. Soc. Nephrol., November 1, 2005; 16(11): 3160 - 3166. [Abstract] [Full Text] [PDF]
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J. Loffing and L. Schild Functional Domains of the Epithelial Sodium Channel J. Am. Soc. Nephrol., November 1, 2005; 16(11): 3175 - 3181. [Full Text] [PDF]
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 O. Olivieri, A. Castagna, P. Guarini, L. Chiecchi, G. Sabaini, F. Pizzolo, R. Corrocher, and P. G. Righetti Urinary Prostasin: A Candidate Marker of Epithelial Sodium Channel Activation in Humans Hypertension, October 1, 2005; 46(4): 683 - 688. [Abstract] [Full Text] [PDF]
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A. Adebamiro, Y. Cheng, J. P. Johnson, and R. J. Bridges Endogenous Protease Activation of ENaC: Effect of Serine Protease Inhibition on ENaC Single Channel Properties J. Gen. Physiol., September 26, 2005; 126(4): 339 - 352. [Abstract] [Full Text] [PDF]
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 C. Leyvraz, R.-P. Charles, I. Rubera, M. Guitard, S. Rotman, B. Breiden, K. Sandhoff, and E. Hummler The epidermal barrier function is dependent on the serine protease CAP1/Prss8 J. Cell Biol., August 1, 2005; 170(3): 487 - 496. [Abstract] [Full Text] [PDF]
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U. Hasler, M. Vinciguerra, A. Vandewalle, P.-Y. Martin, and E. Feraille Dual Effects of Hypertonicity on Aquaporin-2 Expression in Cultured Renal Collecting Duct Principal Cells J. Am. Soc. Nephrol., June 1, 2005; 16(6): 1571 - 1582. [Abstract] [Full Text] [PDF]
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 C. Planes, C. Leyvraz, T. Uchida, M. A. Angelova, G. Vuagniaux, E. Hummler, M. Matthay, C. Clerici, and B. Rossier In vitro and in vivo regulation of transepithelial lung alveolar sodium transport by serine proteases Am J Physiol Lung Cell Mol Physiol, June 1, 2005; 288(6): L1099 - L1109. [Abstract] [Full Text] [PDF]
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R. P. Hughey, J. B. Bruns, C. L. Kinlough, and T. R. Kleyman Distinct Pools of Epithelial Sodium Channels Are Expressed at the Plasma Membrane J. Biol. Chem., November 19, 2004; 279(47): 48491 - 48494. [Abstract] [Full Text] [PDF]
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Z. Tong, B. Illek, V. J. Bhagwandin, G. M. Verghese, and G. H. Caughey Prostasin, a membrane-anchored serine peptidase, regulates sodium currents in JME/CF15 cells, a cystic fibrosis airway epithelial cell line Am J Physiol Lung Cell Mol Physiol, November 1, 2004; 287(5): L928 - L935. [Abstract] [Full Text] [PDF]
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R. P. Hughey, J. B. Bruns, C. L. Kinlough, K. L. Harkleroad, Q. Tong, M. D. Carattino, J. P. Johnson, J. D. Stockand, and T. R. Kleyman Epithelial Sodium Channels Are Activated by Furin-dependent Proteolysis J. Biol. Chem., April 30, 2004; 279(18): 18111 - 18114. [Abstract] [Full Text] [PDF]
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G. M. Verghese, Z. Y. Tong, V. Bhagwandin, and G. H. Caughey Mouse Prostasin Gene Structure, Promoter Analysis, and Restricted Expression in Lung and Kidney Am. J. Respir. Cell Mol. Biol., April 1, 2004; 30(4): 519 - 529. [Abstract] [Full Text] [PDF]
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 B. C. Rossier The Epithelial Sodium Channel: Activation by Membrane-Bound Serine Proteases Proceedings of the ATS, January 1, 2004; 1(1): 4 - 9. [Abstract] [Full Text] [PDF]
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R. A. Caldwell, R. C. Boucher, and M. J. Stutts Serine protease activation of near-silent epithelial Na+ channels Am J Physiol Cell Physiol, January 1, 2004; 286(1): C190 - C194. [Abstract] [Full Text] [PDF]
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