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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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 (h
ENaC)
from the human lung cell line H441, using RT-PCR. These splice variants
contain various structures in the extracellular domain, resulting
in premature truncation (h
ENaCx), 19-amino acid deletion
(h
ENaC
19), and 22-amino acid insertion (h
ENaC+22).
Wild-type h
ENaC and splice variants were functionally characterized
in Xenopus oocytes by coexpression with hENaC
- and
-subunits. Unlike wild-type h
ENaC,
undetectable or substantially reduced amiloride-sensitive currents were
observed in oocytes expressing these splice variants. Wild-type
h
ENaC was the most abundantly expressed h
ENaC 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 h
ENaC 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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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 h
ENaC with
altered function and suggest that alternate splicing of the h
ENaC
may be involved in the functional regulation of ENaC activity.
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MATERIALS AND METHODS |
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Isolation of full-length hENaC subunit.
The full-length coding region of h
ENaC 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 h
ENaC
(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).
Tissue distribution of hENaC.
To determine tissue distribution of h
ENaC 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 h
ENaC (h
ENaCx), 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
h
ENaC (h
ENaC
19) 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
h
ENaC (h
ENaC+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
h
ENaCs were separated on 10% SDS-polyacrylamide gel and transferred
to a polyvinylidene difluoride membrane. The transferred h
ENaCs 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 h
ENaC. 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 h
ENaC
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 h
ENaC 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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Cloning of alternative splice variants of hENaC.
To determine whether h
ENaC has alternatively spliced isoforms and,
if so, whether the splice variants display functional differences, we
examined the coding sequences of h
ENaC mRNAs. Using PCR primers that
were designed to amplify full-length coding sequences, we obtained and
characterized three novel splice variants of h
ENaC from the human
lung epithelial cell line, H441 (Fig. 1). Wild-type h
ENaC was
readily identified. The first splice variant, referred to as h
ENaCx,
contained a premature stop codon in the extracellular domain, whereas
the second (h
ENaC
19) and the third (h
ENaC+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 h
ENaCs were confirmed by restriction mapping
(Fig. 1B) and complete nucleotide sequencing analyses.
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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 h
ENaC cDNAs. Three different PCR primer
sets were used for coamplification of
1) h
ENaCx and h
ENaC,
2) h
ENaC
19 and h
ENaC,
and 3) h
ENaC+22 and h
ENaC from
H441 cells, respectively. The results show that the mRNA levels for
h
ENaCx, h
ENaC
19, and h
ENaC+22 were very low in
abundance compared with the wild-type h
ENaC in H441 cells (Fig.
2).
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hENaC
19 expression as translated proteins and
in Xenopus oocytes.
The h
ENaC
19 and wild-type h
ENaC 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 h
ENaC
19 and wild-type h
ENaC (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 h
ENaCs (19).
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hENaCx and
h
ENaC+22 expression in
Xenopus oocytes.
The cRNAs of h
ENaCx and h
ENaC+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
h
ENaCx (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 h
ENaC+22
(n = 17 oocytes), which was another
unexpected finding. Therefore, similar to what was done with the
h
ENaC
19 splice variant, we exchanged the extracellular region
of the wild-type h
ENaC with the homologous h
ENaC+22 region to
eliminate any undefined mutations and/or nucleotide sequencing errors in h
ENaC+22. In oocytes expressing this newly constructed h
ENaC+22 together with wild-type human
- and
-subunits,
amiloride-sensitive currents were negligible
(n = 16 oocytes), suggesting that the cysteine-rich domain of h
ENaC 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
h
ENaC 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 h
ENaC could
be derived from alternative RNA splicing mechanisms. To test this
hypothesis, exon-intron boundaries of the extracellular domain of
h
ENaC (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
h
ENaC
19 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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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 h
ENaC+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 h
ENaC+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 h
ENaCx splice variant
identified in this study represents another isoform of prematurely
truncated h
ENaC, and the lack of generation of amiloride-sensitive
currents in h
ENaCx-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
hENaC
19 or h
ENaC+22 could be explained by any of the
following mechanisms. First, the h
ENaC 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 h
ENaC+22. Second, the assembly of h
ENaC
with
- and
-subunits may not be proper, so that the final
assembled h
ENaC 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 h
ENaC splice variant-containing ENaC complexes is supported by the observations that there was no
increased ENaC activity even after injection of 25 ng of
h
ENaC
19 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 h
ENaC 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
h
ENaCs 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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