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-subunit of the amiloride-sensitive
Na+ channel
Departments of 1 Medicine and of 2 Physiology and Biophysics, 3 Nephrology Research and Training Center, and 4 Neurobiology Research Center, University of Alabama, Birmingham, Alabama 35294
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The 
-subunit of the amiloride-sensitive epithelial
Na+ channel (ENaC) is critical
in forming an ion conductive pore in the membrane. We have identified
the wild-type and three splice variants of the human ENaC (hENaC)
from the human lung cell line H441, using RT-PCR. These splice variants
contain various structures in the extracellular domain, resulting
in premature truncation (hENaCx), 19-amino acid deletion
(hENaC
19), and 22-amino acid insertion (hENaC+22).
Wild-type hENaC and splice variants were functionally characterized
in Xenopus oocytes by coexpression with hENaC 
- and 
-subunits. Unlike wild-type hENaC,
undetectable or substantially reduced amiloride-sensitive currents were
observed in oocytes expressing these splice variants. Wild-type
hENaC was the most abundantly expressed hENaC mRNA species in all
tissues in which its expression was detected. These findings indicate that the extracellular domain is important to generate structural and
functional diversity of hENaC and that alternative splicing may play
a role in regulating hENaC activity.
human epithelial sodium channel -subunit; alternative splicing; lung cell line
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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THE SUBUNITS OF THE human epithelial
Na+ channel (hENaC) have been
recently cloned (17-19, 38), and it is known that mutations of
hENaC subunits are associated with Liddle's syndrome (11, 12, 28, 33)
and pseudohypoaldosteronism type 1 (5, 31). ENaC is a key component in
regulating the rate of transepithelial Na+ transport across epithelia
(21, 24, 25). ENaC is composed of at least three homologous subunits,
, , and , and the -subunit (ENaC) is critical to the
formation of an ion conductive membrane pore, whereas the -
and -subunits can greatly potentiate the level of expressed
Na+ currents in heterologous
expression systems (4, 18). In recent experiments in mice, gene
knockout of ENaC was lethal within 40 h of birth due to failure of
pulmonary fluid clearance, clearly demonstrating the critical role of
ENaC in forming a functional
Na+ channel complex in vivo
(13).
Electrophysiological studies of different epithelial systems have shown
a wide variability of ion selectivity, inhibitory profiles by amiloride
analogs, and/or single-channel conductances (1, 21, 24). The
reasons for this functional variability are poorly understood at
present. In the present study, we have characterized hENaC by
molecular cloning and functional expression studies in
Xenopus oocytes. The results
demonstrate three alternatively spliced variants of hENaC with
altered function and suggest that alternate splicing of the hENaC
may be involved in the functional regulation of ENaC activity.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Isolation of full-length hENaC subunit.
The full-length coding region of hENaC was obtained from the human
lung cell line H441, using RT-PCR. The H441 cells were obtained from
the American Type Culture Collection (NCI-H441; ATCC
HTB-174) and were previously shown to contain hENaC
(19). RT-PCR was performed as previously reported (22, 23); mRNAs were
extracted from cultured H441 cells using the Micro mRNA purification kit (Pharmacia Biotech). cDNA was synthesized by oligo(dT)-priming methods using avian myeloblastosis virus RT, and DNA was
amplified for 35-40 cycles in a programmable thermal controller
(PTC-100, MJ Research) with Taq DNA
polymerase (Boehringer Mannheim).
ENaC. The forward primer corresponded to nucleotide positions 82-97 (bp; numbered according to GenBank no. L29007),
modified at the 5' end to create a
BamH I restriction site, and the
reverse primer corresponded to bp 2096-2110 modified at the
5' end to create an Xho I
restriction site. PCR products were separated by electrophoresis on 1%
agarose gel, purified using GeneClean II (Bio 101), and cloned into the
pCR II vector (Invitrogen). The plasmid inserts were analyzed by
restriction enzyme analysis and/or nucleotide sequence
determination. Nucleotide sequences were determined by using an ABI 373 automated DNA sequencer at the University of Alabama at Birmingham
Center for AIDS Research DNA Sequencing Core. Both strands of the
inserts (prepared via Qiagen column) were sequenced. For in vitro
transcription and oocyte expression studies, the hENaC inserts were
subcloned into pcDNA3 after digestion with
BamH I and
Xho I.
Tissue distribution of hENaC.
To determine tissue distribution of hENaC splice variants, PCR was
performed on human cDNA libraries constructed from eight different
human tissues (QUICK-Screen human cDNA library panel; Clontech), which
were cloned using two different vectors, 
gt10 and TriplEx, with
the exception of brain tissue (which was cloned only in TriplEx
vector). PCR primers were designed so that the coamplified PCR products
derived from either control or splice variants could be readily
distinguishable on 2% agarose gel. To examine tissue distribution of
prematurely truncated hENaC (hENaCx), the forward primer
corresponded to bp 473-491 and the reverse primer corresponded to
bp 1010-1033. Tissue distribution of the 19-amino acid-deleted
hENaC (hENaC19) was examined using the forward primer
corresponding to bp 968-989 and the reverse primer corresponding
to bp 1171-1192. Tissue distribution of the 22-amino acid-inserted
hENaC (hENaC+22) was examined using the forward primer
corresponding to bp 1302-1325 and the reverse primer corresponding to bp 1459-1483.
In vitro transcription and translation.
The hENaCs cloned into pcDNA3 were linearized with
Xho I for the preparation of cRNA. The
full-length coding cDNAs of hENaC - and -subunits, kindly
provided by Dr. M. J. Welsh (University of Iowa, Iowa City, IA), were
subcloned into pcDNA3 and linearized with
Xho I. Linearized plasmid DNA was
purified by phenol-chloroform extraction. In vitro transcription was
carried out using the mMESSAGE mMACHINE T7 kit (Ambion) according to
the manufacturer's instructions. The integrity of in vitro transcribed
cRNA was analyzed by electrophoresing through 1.2% agarose-2.2 M
formaldehyde denaturing gel. In addition, the cRNA was translated in
vitro into biotin-labeled proteins in the presence of reticulocyte
lysates and biotin-lysine-tRNA (Boehringer Mannheim). Biotin-labeled
hENaCs were separated on 10% SDS-polyacrylamide gel and transferred
to a polyvinylidene difluoride membrane. The transferred hENaCs were
detected with a streptavidin-alkaline phosphatase conjugate. The color
reaction was initiated by adding substrate solution containing 420 µg/ml nitro blue tetrazolium and 188 µg/ml
5-bromo-4-chloro-3-indolyl phosphate in 100 mM Tris-150 mM NaCl-50 mM
MgCl2, and stopped by several
changes of distilled water.
Expression of hENaC in Xenopus oocytes.
Adult female Xenopus laevis were obtained from
Xenopus I (Ann Arbor, MI). Oocytes were isolated and defolliculated in
the presence of 2 mg/ml collagenase type A (Boehringer Mannheim). Stage
V-VI oocytes were selected by visual inspection and maintained at
18°C in ND-96 Ringer solution (in mM: 96 NaCl, 2.4 KCl, 2 CaCl2, 1.8 MgCl2, and 5 HEPES, pH 7.4)
supplemented with 5% horse serum (Life Technologies). Oocytes were
injected with 50 nl of the transcribed cRNAs (5 ng of cRNA for each
-, -, and -subunit) using a microinjector (Drummond Nanoject,
Drummond Scientific). Electrophysiological recordings were performed
2-3 days postinjection in ND-96 Ringer solution using a
two-electrode voltage-clamp system. The current-voltage relationship
was obtained by stepping for 500 ms from a holding potential of
40 mV to 120 to +20 mV, in 20-mV increments. To determine
Na+ selectivity of the expressed
ENaC activity, the external Na+
was replaced with either Li+ or
K+.
Exon-intron boundary of hENaC.
Genomic DNA was sequenced to determine exon-intron boundaries of the
extracellular domain of hENaC. Human genomic DNAs were obtained from
human B lymphoblast cell lines, using a QIAamp tissue kit (Qiagen). PCR
primers corresponding to either coding or intron regions of hENaC
were synthesized and used to amplify relevant genomic DNAs. Sequence
information regarding intron PCR primers was obtained from the report
by Chang et al. (5), who previously showed that hENaC was encoded by
at least 13 separate exons. Amplified genomic DNA was cloned into pCR
II and analyzed as described above.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Cloning of alternative splice variants of hENaC.
To determine whether hENaC has alternatively spliced isoforms and,
if so, whether the splice variants display functional differences, we
examined the coding sequences of hENaC mRNAs. Using PCR primers that
were designed to amplify full-length coding sequences, we obtained and
characterized three novel splice variants of hENaC from the human
lung epithelial cell line, H441 (Fig. 1). Wild-type hENaC was
readily identified. The first splice variant, referred to as hENaCx,
contained a premature stop codon in the extracellular domain, whereas
the second (hENaC19) and the third (hENaC+22) splice
variants contained a deletion of 19 amino acids and an addition of 22 amino acids in the extracellular domain, respectively. Nucleotide
sequences of these hENaCs were confirmed by restriction mapping
(Fig. 1B) and complete nucleotide sequencing analyses.
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ENaCx was the shortest isoform
(1793-bp PCR product) due to deletions at two extracellular regions: bp
767-957 (191-bp deletion) and 1061-1117 (57-bp deletion). The
hENaC19 (1984 bp) had a 57-bp deletion at nucleotides
1061-1117; hENaC+22 was the longest splice isoform (2107 bp),
with a 66-bp insertion at nucleotide position 1441. The wild-type
hENaC, 2041 bp, was nearly identical to the previously published
hENaC sequence (19, 38).
Minor nucleotide changes were detected in the cloned hENaCs compared
with the previously published hENaC sequence (19). There was one
nucleotide change from A to G at bp 587 in hENaCx, which would
change the corresponding amino acid from threonine to alanine. There
were two nucleotide changes in hENaC19:
1) C to A at bp 1309 without an
amino acid change and 2) A to G at bp 2069 with an amino acid change from threonine to alanine. The wild-type hENaC had six nucleotide changes:
1) G to A at bp 164 with an amino
acid change from glutamic acid to lysine,
2) T to C at bp 375 with an amino
acid change from methionine to threonine, 3) T to G at bp 393 with an amino
acid change from leucine to arginine,
4) G to A at bp 1222 without an
amino acid change, 5) C to T at bp
1575 with an amino acid change from serine to phenylalanine, and
6) A to G at bp 2069 with an amino
acid change from threonine to alanine. There were two nucleotide
changes in hENaC+22: 1) A to G at
bp 1897 without an amino acid change and
2) A to G at bp 2069 with an amino
acid change from threonine to alanine. These nucleotide changes could
represent a cloning artifact that occurred during sequencing or DNA
amplification or accurate nucleotide changes occurring in cells during
alternative RNA splicing or RNA editing.
Tissue expression of hENaC.
To confirm that the nucleotide deletion and insertion observed in the
splice variants were not due to a cloning artifact, we designed PCR
primers that would amplify the area containing either the deletion or
insertion sites from the hENaC cDNAs. Three different PCR primer
sets were used for coamplification of
1) hENaCx and hENaC,
2) hENaC19 and hENaC,
and 3) hENaC+22 and hENaC from
H441 cells, respectively. The results show that the mRNA levels for
hENaCx, hENaC19, and hENaC+22 were very low in
abundance compared with the wild-type hENaC in H441 cells (Fig.
2).
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ENaC splice variants with wild-type hENaC
from 15 different human cDNA libraries showed that the wild-type hENaC was the most abundantly expressed hENaC mRNA species in all
tissues in which its expression was detected. The highest level of
hENaC expression was observed in kidney and lung. Moderate levels of
hENaC expression were detected in liver and pancreas, and weak
expression was detected in heart and placenta. Negligible levels of
expression were detected in brain and skeletal muscle. This pattern of
hENaC expression is similar to the previous reports based on
Northern blot analysis (19, 38). The general expression level of
hENaCx, hENaC19, and hENaC+22 was negligible in most cDNA libraries except in the heart and lung cDNA libraries (Fig. 2B).
hENaC19 expression as translated proteins and
in Xenopus oocytes.
The hENaC19 and wild-type hENaC were transcribed in vitro
with T7 RNA polymerase to generate cRNAs. These cRNAs were either injected into Xenopus oocytes for
functional expression or translated with the reticulocyte lysate system
to confirm their ability to make a protein. For example, in vitro
translation of hENaC19 and wild-type hENaC (as shown in
Fig. 3) produced proteins of 74 ± 2 and
77 ± 2 kDa, respectively, consistent with the expected size of 19-amino acid-deleted or wild-type hENaCs (19).
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ENaC cRNAs were expressed in
Xenopus oocytes in combination with
human - and -subunits of ENaC, which are known to potentiate the
level of expressed Na+ currents in
oocytes (4, 18). After injection of these cRNAs into oocytes,
amiloride-sensitive currents were observed in oocytes expressing
wild-type hENaC and ENaC - and -subunit cRNAs (Fig. 4). Amiloride-sensitive currents at
100 mV holding potential averaged 1,479 ± 193 nA with
96 mM NaCl and 2,240 ± 245 nA with 96 mM LiCl in the bath,
respectively (n = 11 oocytes in each
series). Replacement of the bath solution with 96 mM KCl did not
generate any amiloride-sensitive currents, consistent with previous
reports by other laboratories (4, 18). It is worth noting that the two
amino acid changes (methionine to threonine and leucine to arginine)
observed in the first transmembrane domain of the wild-type hENaC
did not affect the ion selectivity of
Li+ > Na+ >>
K+, suggesting that these two
amino acids in the first membrane-spanning domain are not
critically involved in determining the ion selectivity of the hENaC
complex. It has previously been shown that the short segment before the
second transmembrane domain and the second transmembrane domain of the
ENaC are critical regions for ion conduction and selectivity (27,
39).
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ENaC, substantially reduced
levels of amiloride-sensitive currents were observed in oocytes with hENaC19 and equal amounts of ENaC - and -subunit cRNAs
(n = 18 oocytes); amiloride-sensitive
currents at 100 mV averaged 119 ± 30 nA with 96 mM
NaCl in the bath. We also tried to express hENaC19 cRNAs with
25 ng cRNA, an amount that was fivefold greater than the normal amount
of -subunit (5 ng cRNA) that was injected into each oocyte. Despite
the increased amount of injected cRNA, there was not any increase in
the amiloride-sensitive currents (n = 4 oocytes). Furthermore, we coexpressed hENaC19 cRNA with carboxy terminus-truncated -subunits (K100 construct, Oh and Warnock, unpublished data) together with wild-type hENaC
-subunit, but still substantially reduced levels of
amiloride-sensitive currents (179 ± 38 nA;
n = 11 oocytes) were
observed. The carboxy terminus-truncated ENaC -subunit is a
gain-of-function mutation that has been shown to increase expressed
amiloride-sensitive ENaC activity in oocytes by at least threefold
(26). The possible dominant negative effect of the hENaC19 on
wild-type ENaC expression was examined by coexpressing a 1:1 mixture of
wild-type hENaC and hENaC19 together with - and
-subunits. The results showed that there was no dominant negative
effect of the hENaC19 on wild-type ENaC expression;
amiloride-sensitive currents at 100 mV averaged 972 ± 206 nA with 96 mM NaCl in the bath (n = 20 oocytes).
We were surprised to find very little amiloride-sensitive current in
oocytes expressing the hENaC19 splice variant. Therefore, to
eliminate any undefined mutations and/or nucleotide sequencing errors, we exchanged a 501-bp fragment of the extracellular region of
the wild-type hENaC with the homologous hENaC19 region
(444 bp; encompassing the 57-bp deletion site) after digestion of both plasmids with EcoN I and
Sac II. After confirmation of the
successful exchange of sequences between the wild-type hENaC and
hENaC19 by restriction mapping and sequencing analyses, cRNA
was synthesized in vitro from the linearized new hENaC19
construct (19-amino acid-deleted construct based on the wild-type
hENaC sequences) and the new wild-type hENaC (control hENaC
construct based on the hENaC19 sequences). The cRNAs were
then expressed in oocytes together with hENaC - and -subunit
cRNAs. Large amiloride-sensitive currents were observed in oocytes
expressing the new wild-type hENaC; amiloride-sensitive currents at
100 mV holding potential averaged 900 ± 77 nA with 96 mM NaCl in the bath (n = 8 oocytes). In contrast, an extremely low level of amiloride-sensitive currents was
observed in oocytes expressing the new hENaC19
(n = 26 oocytes). Therefore, it is
likely that the loss of function in the hENaC19 is due to the
specific deletion of 19 amino acids from the extracellular domain of
hENaC and not from undetected nucleotide errors and/or minor
nucleotide differences between the wild-type hENaC and hENaC19. In this respect, it is interesting to note that
artificial modification of the extracellular domain involving different
regions from the deleted 19 amino acids in hENaC, such as deletion
of a putative amiloride-binding site (3, 14) or insertion of a FLAG
epitope (6), can produce Na+ currents in oocytes, further
strengthening the hypothesis that the 19 amino acids deleted in the
hENaC19 splice variant have a critical role in the assembly
and/or functional expression of the ENaC complex.
To confirm further the importance of the 19 amino acids deleted in the
hENaC19, we have constructed a rat homologue
(rENaC19) to hENaC19 by substituting the
corresponding 19 amino acids with 2 novel amino acids using
PCR-directed in vitro mutagenesis. Coexpression of wild-type rat -,
-, and -subunits produced large
amiloride-sensitive currents at 100 mV holding potential, averaging 3,063 ± 726 nA with 96 mM NaCl in the bath
(n = 5 oocytes). In contrast, greatly
reduced amiloride-sensitive currents were observed in oocytes
expressing rENaC19 in combination with wild-type rat - and -subunits; amiloride-sensitive currents at 100
mV averaged 403 ± 144 nA with 96 mM NaCl in the bath
(n = 8 oocytes; Fig.
5).
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hENaCx and
hENaC+22 expression in
Xenopus oocytes.
The cRNAs of hENaCx and hENaC+22 were synthesized in vitro, and
their functional competence was tested in the
Xenopus oocyte expression system.
Amiloride-sensitive currents were undetectable in oocytes expressing
hENaCx (n = 12 oocytes), which is
consistent with previous observations (16). Interestingly, often
undetectable or very small (~20 nA) and unstable amiloride-sensitive
currents were observed in oocytes expressing hENaC+22
(n = 17 oocytes), which was another
unexpected finding. Therefore, similar to what was done with the
hENaC19 splice variant, we exchanged the extracellular region
of the wild-type hENaC with the homologous hENaC+22 region to
eliminate any undefined mutations and/or nucleotide sequencing errors in hENaC+22. In oocytes expressing this newly constructed hENaC+22 together with wild-type human - and -subunits,
amiloride-sensitive currents were negligible
(n = 16 oocytes), suggesting that the cysteine-rich domain of hENaC where 22 amino acids are inserted is
also a critical region for normal function of the ENaC complex.
Determination of exon-intron splicing junctions in
hENaC.
Alternative splicing of the primary RNA transcript is one of the key
mechanisms for generating structural and functional diversity of many
membrane proteins (29). At present, the genomic structure of the
hENaC has not been published, although a single gene has been
localized to chromosome 12 by several laboratories (19, 20, 37).
Functional isoforms corresponding to splice variants of hENaC could
be derived from alternative RNA splicing mechanisms. To test this
hypothesis, exon-intron boundaries of the extracellular domain of
hENaC (emphasizing the area encompassing the 57-bp deletion site)
were determined using genomic DNA-PCR and subsequent nucleotide
sequence analysis. The results demonstrated an exon-intron splice
junction at the 5' end of the 57-bp deletion site (Fig. 6A). Two
more exon-intron splice junctions were identified downstream from the
57-bp deletion site. Detailed examination of the 57-bp deletion region
demonstrated conserved nucleotide sequences for the 5' and
3' splice sites and a pyrimidine-rich tract near the 3'
splice site, which are the consensus sequences recognized by spliceosomes (29). Therefore, as a mechanism for generating the
hENaC19 splice variant, it can be speculated that these 57 bp
can be spliced out under certain circumstances using an internal
splicing site of an exon, which is one well-known RNA splicing pattern
(29).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The present study demonstrates the presence of three novel splice
variants of the extracellular domain of hENaC subunits, which appear
to be derived from alternative RNA splicing mechanism. Furthermore,
functional expression studies of these splice variants demonstrate that
they generate highly reduced or often undetectable amiloride-sensitive
Na+ currents in
Xenopus oocytes, indicating the
importance of the extracellular domain in the expression of functional
ENaC complexes in the membrane. Similar observations have been made in
an ENaC-related invertebrate gene, degenerin, which has been found to
contain an extracellular regulatory domain; this regulatory domain is not found in any of the mammalian ENaC proteins (7).
Caenorhabditis elegans, which express a mutant
degenerin, MEC-4, with a nine-amino acid deletion at this regulatory
domain, undergo neurodegeneration, suggesting that this region
negatively regulates degenerin function (8). Therefore, it seems that
the ENaC gene superfamily contains an important regulatory domain in
the extracellular region, which can cause either gain of function or
loss of function. The present study also emphasizes the importance of
carefully examining the full-length coding regions of ENaC mRNAs,
because rather subtle changes that may not be detected by commonly used
mRNA detection methods (i.e., Northern blot and in situ hybridization
analyses) may produce dramatically altered function in a cell. In this
respect, there have been several reports demonstrating the presence of mRNAs in certain cells or tissues without any corresponding proteins and/or functional activity of the mRNAs (9, 10, 32). It is
possible that relatively minor changes, such as the alternatively spliced 57-bp deletion or 66-bp insertion described herein, could account for the loss of functional activity in these other examples.
The presence of splice variants of the ENaC subunit has recently
been described in rat taste tissues (16) and in a chick cochlear cDNA
library (15). In rat taste tissue, two -subunit splice variants were
found with nucleotide deletions that introduced a premature stop codon
at the extracellular domain and resulted in prematurely truncated
proteins. Interestingly, these two splice variants share the same
splicing site with the hENaC+22 variant (Fig.
6B). One of these splice variants
from rat taste tissue was functionally characterized to show that the
truncated protein that lacks the second transmembrane domain failed to
generate amiloride-sensitive Na+
currents when expressed in Xenopus
oocytes. Killick and Richardson (15) have found a splice variant of the
-subunit of chick ENaC with the addition of two exons of 163 and 276 bp into the extracellular domain. The second exon of this splice
variant is introduced at the same splicing site as the hENaC+22
variant (Fig. 6B), suggesting that
this splicing site is commonly used in various species to produce
splice variants of the ENaC subunit. Taken together, these findings
indicate that in addition to transcriptional regulation of ENaC subunit
genes, alternative RNA splicing might represent an additional mechanism
for the regulation of ENaC activity.
Linkage analysis has demonstrated an association of ENaC gene mutations
with pseudohypoaldosteronism type 1, an inherited human disorder
characterized by salt wasting, hyperkalemic acidosis, and
unresponsiveness to mineralocorticoids, consistent with
loss-of-function mutations of the ENaC complex (5, 31). Chang et al.
(5) found two different premature truncations of the hENaC occurring either before the first transmembrane domain or before the second transmembrane domain. Expression of these mutations in
Xenopus oocytes generated no
amiloride-sensitive currents (5), consistent with the pathophysiology
of pseudohypoaldosteronism type 1. The hENaCx splice variant
identified in this study represents another isoform of prematurely
truncated hENaC, and the lack of generation of amiloride-sensitive
currents in hENaCx-expressing oocytes is consistent with the
observation by Chang et al. (5).
Although the mechanisms for physical assembly of ENaC complexes and
targeting and maintenance of ENaCs in the plasma membrane have not yet
been defined, the loss of ENaC activity in oocytes expressing
hENaC19 or hENaC+22 could be explained by any of the
following mechanisms. First, the hENaC splice variant-containing ENaCs may not function properly in the plasma membrane due to the
critical involvement of the missing 19 amino acids or the disrupted
cysteine-rich domain in hENaC+22. Second, the assembly of hENaC
with - and -subunits may not be proper, so that the final
assembled hENaC splice variant-containing ENaC complexes cannot be
inserted into the membrane and/or are unstable in the cytoplasm. The possibility of improper assembly of ENaC complexes and/or abnormal insertion of hENaC splice variant-containing ENaC complexes is supported by the observations that there was no
increased ENaC activity even after injection of 25 ng of
hENaC19 cRNA into the oocytes (a fivefold increase compared
with the usual amount) and that coexpression with carboxy
terminus-truncated -subunit and wild-type -subunit did not result
in any increased ENaC activity. It has been shown that this truncation
mutation of the -subunit described in the original pedigree with
Liddle's syndrome can cause abnormally regulated, highly activated
ENaC complexes when expressed in oocytes (26, 30, 40). A recent approach described by Firsov et al. (6), who expressed FLAG epitope
tagged ENaC in oocytes and quantitated the expression level of ENaC at
the cell surface using
125I-labeled anti-FLAG monoclonal
antibody, may be useful in the future to distinguish between assembly
and impaired function of ENaC expressed at the cell surface.
ENaC expression is not restricted to
Na+-absorptive epithelial cells.
In this study we have detected weak-to-moderate levels of hENaC mRNA
expression in liver, pancreas, heart, and placenta, where ENaC activity
has not been reported (Fig. 2). Interestingly, the expression of ENaC
mRNAs in nonepithelial tissues has been consistently reported by
several laboratories (15, 18, 19, 22, 38). The patch-clamp technique
has also been used to demonstrate the presence of ENaC-like channel
activities in vascular smooth muscle cells (34), thyroid cells (35),
human B lymphocytes (2), and brain endothelial cells (36). If ENaC
mRNAs are processed to make functional ENaC proteins in these cells,
then their physiological roles may be very different from the classical role of ENaCs in absorptive epithelia.
Identification of alternatively spliced variants of the ENaC subunit
in human (this study) and in other species (15, 16) indicates a
possible heterogeneity of multimeric ENaC structure and, possibly,
varied functional roles of ENaCs in different tissues. Under certain
circumstances, it is possible that certain splice variants of the
ENaC subunit can be preferentially produced via alternative RNA
splicing to serve as a regulatory component for ENaC activity. In this
respect, the three nonfunctional hENaC splice variants described
herein, which show a loss of channel function in oocytes, may play a
role as a negatively acting component for ENaC activity, for example,
in a salt excess environment. In the future, it will be important to
determine the physiological significance of splice variants of
hENaCs and understand the factors that regulate the expression of
these three splice variants.
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ACKNOWLEDGEMENTS |
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We thank Dr. M. J. Welsh for providing the hENaC clones and Dr. M. W. Quick for helping us to set up the oocyte expression system. We also thank Martha Yeager for secretarial support.
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FOOTNOTES |
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This work was supported in part by National Institutes of Health Grants NS-34877 (to Y. Oh) and DK-19407 and DK-53161 (to D. G. Warnock) and by grants from the National Kidney Foundation (to J. K. Tucker).
Address for reprint requests: Y. Oh, Dept. of Medicine, Division of Nephrology, Sparks Center 865, University of Alabama at Birmingham, Birmingham, AL 35294 (FAX: 205-934-1147; E-mail: yoh{at}nrtc.dom.uab.edu).
Received 29 August 1997; accepted in final form 8 January 1998.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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1.
Benos, D. J.,
S. Cunnigham,
R. R. Baker,
K. B. Beason,
Y. Oh,
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120:
31-113,
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