1 Department of Internal
Medicine, The amiloride-sensitive epithelial sodium channel (ENaC) is
composed of three subunits:
sodium transport; Xenopus oocyte; patch clamp; gene expression; alternate transcripts
THE MAJOR ROUTE FOR
Na+ transport in kidney collecting
duct cells is via aldosterone-responsive, amiloride-inhibitable
Na+ channels. These channels are
also expressed throughout the airway epithelia and distal colon
epithelia and in sweat ducts. Recently, several members of a family of
amiloride-sensitive epithelial Na+
channel (ENaC) proteins were cloned from rat and human
tissue (3, 5, 19, 20, 23, 24, 40). The channel, containing Recessive and dominant mutations of the ENaC complex cause human
disease. Type 1 pseudohypoaldosteronism, with salt wasting, hypotension, and hyperkalemia, can occur as a consequence of homozygous inactivating mutations in any of the three subunits (7, 34). Liddle's
syndrome, an autosomal dominant form of salt-sensitive hypertension, is
secondary to an activating mutation in 5' Rapid amplification of cDNA ends.
Two sets of modified human cDNAs were obtained for 5' rapid
amplification of cDNA ends (5'-RACE). The double-stranded human kidney Marathon-Ready cDNA (Clontech, Palo Alto, CA) has an adapter sequence 5'-CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT ligated to both ends. The single-stranded 5'-RACE-Ready human lung cDNA (Clontech) has an anchor sequence
5'-CACGAATTCACTATCGATTCTGGAACCTTCAGAGG-NH2 ligated to its 3' end. Gene-specific reverse primers Genomic PCR.
To amplify intervening sequences between exon 1A, exon 1B, and exon 2, primers DNA sequencing.
Amplified fragments from PCR reactions were cloned into pCR II 2.0 or
2.1 (Invitrogen, San Diego, CA), and individual clones were sequenced.
DNA sequencing was performed using dye terminator cycle sequencing
chemistry with Amplitaq DNA polymerase FS enzyme and was
analyzed on a 373A stretch fluorescent automated sequencer (Applied
Biosystems, Foster City, CA). For identification of transcription start
sites, cDNA templates were sequenced by the dideoxy chain termination
method using Sequenase 2.0 (Stratagene, La Jolla, CA) and
[ Cell line and RNA preparation.
A human lung tumor cell line H441 [American Type Culture
Collection (ATCC), Rockville, MD] was grown as a monolayer in
RPMI 1640 medium supplemented with 8.5% bovine calf serum, 8.5% fetal bovine serum (FBS), 20 mM
L-glutamine, 5 µg/ml insulin,
5 µg/ml transferrin, and 5 ng/ml selenium. A human colon carcinoma
line HT-29 (ATCC) was grown as a monolayer in McCoy's 5A medium
supplemented with 10% FBS and 20 mM glutamine. A human lung cystic
fibrosis cell line, IB3-1 (41), was grown in LHC-8 (Biofluids,
Rockville, MD) supplemented with 5% FBS. A human cortical collecting
duct (hCCD) cell line (26) was grown in 1:1 DMEM-Ham's F-12
supplemented with 2% FBS, 5 nM triiodothyronine
(T3), 100 nM dexamethasone, 5 µg/ml insulin, 5 µg/ml transferrin, and 5 ng/ml selenium. RNA prepared from H441, hCCD, HT-29, and IB3-1 cells and from human lung, colon, and kidney tissue were used for Northern analysis or
ribonuclease protection assays (RPA). Total RNA was prepared by
solubilization of monolayers or homogenized tissue in guanidinium thiocyanate buffer followed by extraction, first with
Tris-saturated and then with water-saturated phenol, and
precipitated with isopropanol (8).
Ribonuclease protection assay.
To assess the relative abundance of each transcript in various
tissues, each of the four cDNAs identified by 5'-RACE was used to
construct templates for RPA. 1) The first was an
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
,
, and
. The human
-ENaC
subunit is expressed as at least two transcripts (N. Voilley, E. Lingueglia, G. Champigny, M. G. Mattei, R. Waldmann, M. Lazdunski, and
P. Barbry. Proc. Natl. Acad. Sci. USA
91: 247-251, 1994). To determine the origin of these transcripts,
we characterized the 5' end of the
-ENaC gene. Four
transcripts that differ at their first exon were identified. Exon 1A
splices to exon 2 to form the 5' end of
-ENaC1, whereas exon
1B arises separately and continues into exon 2 to form
-ENaC2. Other
variant mRNAs,
-ENaC3 and
-ENaC4, are formed by activating
5' splice sites within exon 1B. Although
-ENaC3 and -4 did not
change the open reading frame for
-ENaC,
-ENaC2 contains upstream
ATGs that add 59 amino acids to the previous (
-ENaC1) protein. To
address the significance of these isoforms, both proteins were
expressed in Xenopus oocytes. The cRNA
for each
-ENaC transcript when combined with
- and
-ENaC cRNA
reconstituted a low-conductance ion channel with amiloride-sensitive currents of similar characteristics. We have thus identified variant
-ENaC mRNAs that lead to functional ENaC peptides.
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
-,
-,
and
-subunits, when reconstituted in
Xenopus oocytes, is amiloride
sensitive, highly selective for
Na+, and has a single-channel
conductance of 4-10 pS (3, 20). The mRNAs for these subunits are
regulated by glucocorticoids, mineralocorticoids, and dietary
Na+ intake and are expressed in
epithelia where amiloride-sensitive Na+ transport has been identified
(6, 10, 19, 29, 35, 39), providing strong evidence that these channels
represent the highly selective low-conductance epithelial
Na+ channel.
- or
-ENaC (13, 31). These
findings suggest that some patients with salt-sensitive hypertension
may have subtle defects in ENaC function or regulation (30). To begin
to evaluate this possibility in some detail, we have chosen to examine
-ENaC mRNA expression. In human kidney and lung, transcripts of at
least two different sizes have been previously described for
-ENaC
(24, 38), but their molecular structure, regulation, or significance
has not been established. We now identify four separate transcripts for
-ENaC that arise by separate initiation of transcription and
alternate splicing within the first exon. These 5' variant mRNAs lead
to distinct and functional
-ENaC peptides.
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
3
[5'-TCCTCCGCCGTGGGCTGCTG; +133 to +114 from the original
initiation codon; see Figs. 1 and 2 (24, 38)] and
4
[5'-CCCTGGAGTGGACTGTGGAGGGCTAG; +56 to +31 from the
original initiation codon; see Figs. 1 and 2 (24, 38)] were used
singly or sequentially with anchor- or adapter-specific primers in PCR
reactions using Taq polymerase.
Typically, the amplification occurred for 35 cycles each at 94°C
for 30 s, with annealing at 61-69°C for 30 s and extension at
72°C for 3 min.
7 (5'-AGGCCGCTGCACCTGTCAG),
8
(5'-CCTGGGGCAGAGACAGAATC), and
4 were used. To amplify genomic
DNA corresponding to the 5' portion of exon 1A, primers
18
(5'-GAGGGGGTGGCGAGGAATCA) and
19
(5'-CTGGGCAGGGGCTTTAGACG) were used; 100 ng of human genomic DNA
were used in each amplification reaction, with denaturing at 94°C
for 30 s, annealing at 63-67°C for 30 s, and extension at
72°C for 3-4 min for a total of 35-40 cycles.
-35S]dATP with
primers
21 (5'-CTCGAGCTGTGTCCTGATTC) and
23
(5'-TCAGGCCCTGCAGAGAAGAGAGAAGAGGTC). Ribonuclease
protected products were run alongside sequenced products on a
sequencing gel. Transcription factor binding motifs within the human
-ENaC (
-hENaC) gene were identified with MacDNASIS (Hitachi
software, San Bruno, CA).
-ENaC2
cDNA that included the 5' end of exon 2 and the 3' portion
of exon 1B (nt +446 to +806, see Fig. 2) ligated into pCR II antisense to the T7 polymerase promoter. This is predicted to protect a 361-nt
fragment corresponding to
-ENaC2 and a 188-nt fragment corresponding
to
-ENaC1, -3, and -4. 2) The
second was an
-ENaC1 cDNA that included the 5' end of exon 2 and the 3' portion of exon 1A (nt +614 to +860, see Fig. 2)
ligated into pBluescript II
SK
(Stratagene) antisense
to the T3 promoter. This is
predicted to protect a 247-nt fragment corresponding to
-ENaC1 and a
188-nt fragment corresponding to
-ENaC2, -3, and -4. 3) The third was an
-ENaC3 cDNA
fragment that included the 5' end of exon 2 and the contiguous
portion of exon 1B seen in this form (nt +277 to +536, see Fig. 2)
cloned into pCR II antisense to the T7 promoter. This is predicted to
protect a 260-nt fragment corresponding to
-ENaC3, a 150-nt fragment
corresponding to
-ENaC2, and a 110-nt fragment corresponding to
-ENaC1, -2, and -4. 4) The fourth
was an
-ENaC4 cDNA fragment that included the 5' end of exon 2 and the contiguous portion of exon 1B seen in this form (nt +18 to +253, see Fig. 2) cloned into pBluescript II
SK
antisense to the T7
promoter. This is predicted to protect a 236-nt fragment corresponding
to
-ENaC4 and a 153-nt fragment corresponding to
-ENaC1, -2, and
-3.
18
and
19 (see Figs. 2 and 6). A second fragment that extended to the
Bsu36 I site within exon 1A was
created by restriction digestion of the
18-
19 fragment (see Figs.
2 and 6). Both constructs were ligated into pCR II antisense to the T7
polymerase promoter.
Northern analysis.
H441 RNA samples were denatured, resolved on a 1.5% agarose
and 6% formaldehyde gel, and then transferred to nylon membranes (Zetaprobe-GT, Bio-Rad, Hercules, CA). A cDNA probe (-common) that
extends from exon 2 to a Sma I site
~1,100 bp downstream of the initiation codon was prepared from an
-ENaC cDNA clone (gift from M. J. Welsh, University of Iowa). An
exon 1A-specific probe was prepared from the 3' 350 nt of exon
1A. A cDNA fragment prepared by restriction digestion of a
5'-RACE
-ENaC2 clone that began at +128 and ended at a
Sac I site (+563) was used to identify transcripts that included the 3' portion of exon 1B. A
Pst
I-Asp718 I fragment derived from exon
2 was used as an exon 2-specific probe. Random primer-extended
[
-32P]dCTP-labeled
cDNA probes were hybridized to immobilized RNA in 1 mM EDTA, 0.25 M
NaH2PO4,
pH 7.2, and 7% SDS at 65°C for 12 h. The blots were then washed in
1 mM EDTA, 40 mM
NaH2PO4,
pH 7.2, and 5% SDS at 65°C for 30-90 min. To identify
transcripts that included the 5' portion of exon 1B, an
Hph
I-Xho I fragment was used as a
template for the preparation of a single-stranded antisense DNA probe
by extension from a specific primer,
21 (short double-stranded DNA
probes from this region did not identify either transcript). This
radiolabeled DNA probe was hybridized in 5× standard sodium
citrate (SSC), 7% SDS, 20 mM
NaH2PO4,
pH 7.2, and 1× Denhardt's solution at 60°C for 16 h and then
washed first with 3× SSC, 5% SDS, 25 mM
NaH2PO4,
pH 7.5, and 10× Denhardt's and then with 1× SSC and 1%
SDS at 60°C.
In vitro translation.
A full-length -ENaC1 in pcDNA3 (Invitrogen, Carlsbad, CA) with the
last 24 bp that encode the COOH-terminal octapeptide replaced with the
FLAG epitope (
-1-FLAG) was obtained from M. J. Welsh (University of Iowa). The cloned 5' variant
-ENaC (exon 1B)
was spliced to the 5' end of
-ENaC1 using overlapping primers
in a PCR reaction to generate
-1-FLAG. The 5'-untranslated
region (UTR) in both constructs was under 15 bp, and the translation stop followed immediately after the FLAG epitope. Sequential
transcription and translation of
-1-FLAG and
-2-FLAG were
performed alongside pcDNA3 and H2O
controls using the TnT reticulocyte lysate system (Promega, Madison, WI) following the manufacturer's instructions. [35S]methionine was
used to label the synthesized peptides, and the products were
visualized by SDS-PAGE and autoradiography.
Expression in Xenopus oocytes.
The coding regions for the four hENaC subunits [(-2),
(-1),
, and
] were subcloned into the plasmid pGEM-HE. This
vector contains the 5'- and 3'-UTRs of the
Xenopus
-globin gene flanking the
cloning site and was engineered specifically to enhance expression of
in vitro transcribed cRNAs in Xenopus
oocytes (21). Each cDNA was transcribed and capped in vitro using the
Message Machine kit (Ambion). The cRNAs were combined into two groups
[
(-2)
and
(-1)
] so that 50-nl injections
carried 2.5 ng of each hENaC subunit.
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RESULTS |
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Two groups have reported the primary structure of human -ENaC mRNA
and its predicted protein (24, 38). The cloned
-ENaC cDNA included
103 nt of 5'-UTR, the complete 3'-UTR, and an open reading
frame of 2010 nt that encodes a 669-amino acid protein. Both groups
reported that the cloned cDNA hybridized to two transcripts in several
human tissues, but the identify of these transcripts was not
established.
To identify the nature of the alternate transcripts previously
reported, we set out to map the organization of the -hENaC gene
beginning with the 5' end. We first determined the nucleotide sequence of the 5' end of
-ENaC mRNA in human kidney and lung by 5'-RACE using reverse primers (
3 and
4) designed to
anneal to
-ENaC RNA just downstream of the previously described
translation initiation codon. Several clones were sequenced from both
tissues, and remarkable heterogeneity was noted, with at least four
possible cDNA forms identified. One form with a 5'-UTR of 709 nt
(
-ENaC1, Fig. 1)
corresponded to that previously described in kidney and lung but
extended several hundred bases upstream (24, 38) and was identified in
13% of clones. The other three forms diverged from
-ENaC1 at
54 to the original translation start codon and were consistent
with alternately initiated transcripts. The most abundant form
(
-ENaC2, Fig. 1), representing ~74% of the clones, added an
additional 535 nt 5' to the point of divergence. Two other forms,
-ENaC3 and -4, appeared to be internally deleted versions of
-ENaC2 consistent with splice variants and were found infrequently.
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To understand the genomic organization at the 5' end of the
-ENaC gene, specifically to determine the exon-intron organization in this region, forward primers (
7 and
8) corresponding to the 5' portions of form
-ENaC1 and
-ENaC3 mRNA were used with
downstream primers
4 or
3 to amplify intervening sequences from
genomic DNA (Fig. 1). Sequence analysis confirmed that a 665-nt intron, starting and ending with consensus GT and AG,
respectively, was spliced out of
-ENaC1. This intron ends at
54 to the original translation start codon and corresponds to
the point of divergence between forms
-ENaC1 and
-ENaC2 (Fig. 1).
The intron sequence (intron 1) thus separates exon 1A (5' end of
-ENaC1) from exon 2. Sequence corresponding to
-ENaC2 arises
within this intron sequence and therefore represents an alternate first
exon (exon 1B). This alternately transcribed sequence continues
unspliced into exon 2. Forms
-ENaC3 and -4 were identified as
variants of exon 1B spliced to exon 2, created by activation of
5' splice sites within exon 1B (Figs. 1 and
2). Of the four transcripts,
-ENaC2
contained four in-frame translation initiation codons that would be
predicted to extend the length of the open reading frame by up to 59 amino acids, creating a variant protein
-ENaC2 (Figs. 1 and 2). All
other transcripts keep the original open reading frame intact
(
-ENaC1).
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To examine these alternate transcripts in greater detail, we performed RPA in selected tissues (Fig. 3). This was intended to confirm the existence of these variant mRNAs and to assess the relative proportion of each of these in kidney, colon, and lung, principal sites of amiloride-sensitive Na+ transport. Most studies used normal human kidney and lung tissue as well as IB3-1, HT-29, hCCD, and H441 cells. Given the complexity of these transcripts, it was not possible to design a single RPA template to distinguish all transcripts simultaneously. Therefore, separate RPA templates were constructed from each of the identified cDNA forms, designed to distinguish one of the transcripts from the other three. With the use of this method, each of the four transcripts was readily identified in all tissues examined except hCCD (Fig. 3, A-D).
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We next determined if any or all of these transcripts corresponded in
size to that previously identified by Northern analysis (24, 38). To
examine this issue, a series of cDNA probes were used for Northern
blotting of H441 RNA. An -common cDNA probe corresponding to exon 2 and downstream sequences identified two equally abundant transcripts of
3.9-4.0 and 3.5-3.6 kb in H441 RNA (Fig.
4E).
These are probably the same as the 3.8- and 3.4-kb transcripts reported
by Voilley et al. (38) and the 3.9- and 3.2-kb transcripts reported by
McDonald et al. (24). By using a variety of short exon-delimited
probes, we show that both transcripts contain exon 2 (Fig.
4D), whereas an exon 1A-specific
probe hybridized to the 3.9- to 4.0-kb transcript alone (Fig.
4A). A 100-nt 5'-terminal exon
1B probe that corresponds to
-ENaC2, -3, and -4 hybridized to both
transcripts (Fig. 4B), whereas a
3'-terminal exon 1B probe (Fig.
4C) that corresponds to
-ENaC2
and -3 hybridized to the 3.9- to 4.0-kb transcript alone. These results
clearly indicate that, although exon 2 is contained within both
transcripts,
-ENaC1, -2, and -3 mRNAs correspond to the larger
transcript alone. Taken together, the data in Figs. 2-4 also
suggest that
-ENaC4 mRNA corresponds to the 3.5- to 3.6-kb
transcript. Because the relative amount of the 3.5- to 3.6-kb
transcript identified by the exon 2 probe is much greater than that
identified by the 5'-terminal exon 1B probe, we cannot exclude
the presence of other transcripts that contain exon 2 but do not have
portions of exon 1A or 1B within them.
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To clarify whether exon 1A and 1B have distinct transcription start sites, we performed RPAs on total RNA from selected tissues using a genomic fragment that included the terminal 20 nt of exon 1A and 5' portions of exon 1B. Two protected fragments were seen, suggesting that exon 1B is initiated separately. The more abundant protected fragment, a 385-nt fragment, predicted a transcription initiation site for exon 1B 53 nt downstream of the 5' splice site of exon 1A (Fig. 5A). This transcription start site was seen in kidney tissue and lung tissue and H441 and IB3-1 cells. A longer, much weaker protected fragment was also seen, suggesting that some mRNA species extended the entire length of exon 1B and into exon 1A. To confirm these findings and to accurately localize the transcription start site, a second shorter riboprobe that extended from the same 5' end to the Xho I site (100 nt downstream of the putative transcription start site) was used. The more abundant protected fragment was resolved on a sequencing gel to two closely migrating bands and indicated transcription start sites 53 and 55 nt downstream of the 5' splice site of exon 1A (Fig. 5B). A protected fragment that was 71 nt longer indicated the existence of an mRNA species that included exon 1B and begins in exon 1A.
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Working separately and as part of the Human Genome Project, a group of
investigators had isolated and begun to characterize a PAC clone from
human chromosome 12 that contained the 5' of the -ENaC gene.
Using the nucleotide sequence information from this clone (Raju
Kucherlapati and Kate Montgomery, Albert Einstein College of Medicine,
Bronx, NY, personal communication), we amplified a genomic fragment
that included the 5' portion of exon 1A and the putative 5'
flanking region. When used as a riboprobe with RNA from kidney and
H441, a protected fragment ~470 nt long was seen,
suggesting that the transcription start site for exon 1A mapped within
this fragment (Fig.
6A). A
second, shorter riboprobe was constructed using sequence from the
5' end to accurately localize a single transcription start site
for exon 1A (Fig. 6B) 724 nt upstream of the principal transcription start site for exon 1B.
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The predicted open reading frame for -ENaC2 is 2187 bases long and
encodes an additional 59 amino acids 5' and in-frame to that
encoded by
-ENaC1 (Fig.
7A). The
predicted translation start codon in
-ENaC2 has a more favorable
consensus sequence for translation initiation compared with
-ENaC1
(Fig. 7B) and should yield a protein
with a molecular mass of 82 kDa. We tested whether an
-ENaC2
construct could make the corresponding protein in an in vitro
transcription-translation system using reticulocyte membranes. A single
band of the predicted size was seen with
-ENaC2, confirming that the
5'-most ATG is used as the translation initiation codon for
-ENaC2. In contrast, in this in vitro system, two translated products are seen with
-ENaC1, one corresponding to the longest open
reading frame prediction of 76 kDa and the other indicating a second
downstream translation start site.
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To establish that these -ENaC peptide isoforms are functional,
transcripts encoding these peptides were expressed in
Xenopus oocytes together with
- and
-ENaC subunits and Na+
transport was analyzed. Figure 8 shows
representative whole cell currents and corresponding current-voltage
(I-V)
relationships recorded from oocytes injected with water (Fig. 8,
A and
B) or ENaC (Fig. 8,
C-H).
The currents from water-injected oocytes are relatively small (<0.5
µA at
60 mV) and have a negative reversal potential
(Erev),
indicating predominant contributions from endogenous K+ and
Cl
channels. Bath
application of 10 µM amiloride had no effect on these currents (data
not shown). The contribution of hENaC to the total oocyte current was
determined by bath application of 10 µM amiloride as depicted in Fig.
8,
C-H.
In an oocyte expressing hENaC, the
Erev is generally
slightly positive, indicating contribution of a channel type with a
positive Erev.
Addition of amiloride to the bath reduces the current amplitude and
shifts the Erev to negative values, resulting in a current trace and
I-V
relationship that is similar to those from water-injected oocytes. The
amiloride-sensitive currents that are due to hENaC expression are
derived by subtracting the postamiloride values from the preamiloride
values (Fig. 8, G and
H). The
Erev of the
amiloride-sensitive currents is typically near +10 mV. Because ENaC is
known to be highly Na+ selective,
the calculated
Erev would be
substantially more positive than +10 mV, if one assumes typical
intracellular ionic concentrations. It has been suggested, however,
that the overexpression of ENaC in oocytes results in markedly elevated
intracellular Na+ concentrations
(5, 23), and this is the likely explanation for the observed
Erev. Expression
of either
-ENaC form along with the
- and
-subunits resulted
in time-independent, amiloride-sensitive currents with average current
magnitudes and
Erev values that were indistinguishable (Fig. 8H).
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A representative example of the single-channel currents recorded from
an oocyte expressing -2
-ENaC is shown in Fig.
9A. Single-channel currents from
-1
-ENaC expressing oocytes were similar. Both displayed low conductance, slow kinetics, and variable open probabilities with no obvious differences in any of these parameters. The
I-V
relationships for these single-channel recordings were linear between
80 and
20 mV, with a slope conductance of ~7 pS for both groups
(Fig. 9B). This value for slope
conductance is consistent with previous reports of ENaC single-channel
recordings at room temperature where
Li+ was used as the charge carrier
(12).
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DISCUSSION |
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We have characterized four -ENaC transcripts in various human
tissues that arise by utilization of alternate first exons and by
alternate splicing. Exon 1A and exon 2 are spliced together after
removal of a 665-nt intron 1 to form the 5' end of
-ENaC1 (Fig. 1). Interestingly, the exon 1B sequence is contained within intron 1 and is transcribed and processed unspliced into exon 2 to form
-ENaC2 (Fig. 1). Other variant mRNAs are formed by activation of
5' splice sites within exon 1B, adding to the heterogeneity of
RNA transcripts.
The analysis of the open reading frame of -ENaC2 transcript reveals
several in-frame ATGs upstream of the previously reported translation
start codon. The most proximal start site adds a further 59 amino acids
at the NH2 terminus, creating a
variant protein (
-ENaC2) with a predicted molecular mass of 81.8 kDa
compared with
-ENaC1 of 75.7 kDa. To assess the potential
significance of this transcript, its expression was examined in several
tissues (Fig. 3,
A-D).
Although a cell line established from hCCD (26) failed to express
-ENaC, every other tissue examined showed evidence for mRNAs that
encode
-ENaC1 protein (transcripts
-ENaC1, -3, -4) and
-ENaC2
protein (transcript
-ENaC2). Both forms were equally expressed in
lung and colon, whereas
-ENaC2 was greater than the sum of the other
transcripts in kidney. The relative proportion of the transcripts also
varied within the examined cell lines; for example,
-ENaC1 was the
most abundant transcript in IB3-1 and H441 cell lines (Fig. 3,
A and
B).
The topology for rat -ENaC has been determined by several
laboratories, and the model indicates that there are two
membrane-spanning domains (M1 and M2), intracellular
NH2 and COOH terminals, and a
single extracellular loop (4, 28, 32). Hydropathy analyses (16) of
-ENaC1 and
-ENaC2 predict a similar topology (data not shown),
suggesting that the two peptides differ in the length of the
cytoplasmic NH2-terminal segment.
Specifically, we can detect no additional hydrophobic segment that
would suggest a third transmembrane domain nor can we detect a short
hydrophobic core of apolar residues characteristic of a signal sequence
(2) at the NH2 terminus of
-ENaC2.
Sequence analysis of the
NH2-terminal cytoplasmic domain
reveals two potential sites for myristoylation but no sites for
phosphorylation or other posttranslational modifications. A motif
PGLM[K/E]GNK[L/R]EEQ that is absent in
bovine -ENaC (Fig. 9 and Ref. 11) is represented once in
-ENaC1
and rat
-ENaC and repeated twice in human
-ENaC2 (Fig.
10A).
The role of the NH2-terminal
domain in the function of the Na+
channel is not known. However, some possibilities are suggested by
analogy with other integral membrane proteins. For example, heteromeric
membrane proteins like the Shaker
K+ channels and acetylcholine
receptor complex contain structural elements in the
NH2-terminal domain that regulate
subunit assembly (17, 37). The
NH2-terminal region of
-ENaC
may also contain sites interacting with cytoskeletal elements or
regulated by intracellular second messengers, and these interactions
may be different for each of the
NH2-terminal variants we have
identified. The mechanisms by which molecules such as G proteins, actin
filaments, and the protein kinases C and A interact with ENaC remain to
be elucidated (1). Such interactions that regulate channel assembly or
function are likely to be epithelial cell specific and may not be
demonstrable in the Xenopus expression
system.
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Although they may exist, 5' variant transcripts have not been
reported in other mammalian species. Alternate transcripts that have
been described in mouse and rat appear to be splice variants that
differ in their origin and in the nature of their translated products
from what we have identified in humans. The rat splice variant,
-rENaCa, initially identified by RT-PCR in taste tissues, is also
expressed as a minor transcript in kidney and lung. This splice variant
introduces a premature stop codon before the second transmembrane
domain of the translated protein and when expressed in
Xenopus oocytes does not generate an
amiloride-sensitive Na+ current
(18). The mouse
-ENaC gene is expressed as a 3.5-kb transcript in
kidney, colon, and lung. In addition, a 1.2-kb transcript was
identified only in the prenatal and early postnatal mouse colon and to
the exclusion of the normal transcript (9). This transcript hybridizes
to cDNA fragments from the 3' portion of
-ENaC but not to
5' fragments that encode the
NH2 terminus, the first
transmembrane domain, and the proximal portion of the extracellular domain. Although not directly tested, the translated product of this
transcript is thus unlikely to reconstitute an
Na+ channel. These data clearly
show that there are species-specific variations in the nature of the
-ENaC transcripts expressed with likely widely different
consequences.
The fact that human -ENaC mRNA heterogeneity is determined in part
by distinct transcription start sites raises questions regarding the
nature of the promoter(s) for these two transcripts. Generally,
promoters characterized by a TATA box are found within the first
25-50 nt of the transcription start site. TATA-less promoters are
typically GC-rich in the 5' flanking region and may have GC boxes
with which TFIID and other TATA-binding proteins interact to initiate
transcription (27). The
-hENaC gene is TATA-less and contains two GC
boxes immediately upstream of the transcription start site (36). In the
-ENaC gene, we have amplified and sequenced ~500 nt of genomic
sequence directly upstream of exon 1B (within exon 1A) and have not
identified a TATA box or a GC box. Sequences directly upstream of exon
1A are GC rich and contain one motif with homology to a GC box (Fig.
10B). It is possible that sequences
within exon 1A function as promoter elements for exon 1B, with
sequences further upstream functioning as promoter elements for exon
1A, thus providing separate regulatory control. In this regard, several
transcription factor binding motifs are identified within these
regions, including AP2, Sp1, PEA3, and nuclear
factor-
B motifs (Fig. 10B).
Alternatively, both transcripts may be directed by a common promoter
upstream of exon 1A and transcriptional regulation provided by distinct
trans-acting factors. Separately, translational control of each
-ENaC peptide may be provided by the
length, composition, and secondary structure of its 5'-UTR (15).
Whether the variant 5'-UTRs of
-ENaC impose constraints on
translational efficiency is yet to be determined.
The biophysical characteristics of -ENaC1 and
-ENaC2 when
coexpressed with
- and
-subunits in oocytes are very similar (Figs. 8 and 9). Both isoforms produce small-conductance channels with
slow kinetics, typical of ENaC expressed in oocytes (5, 33, 38) and of
naturally occurring ENaC channels in the mammalian collecting duct (25,
39). Although the functional difference between these isoforms is not
evident in the Xenopus expression system, we speculate that in its natural state in human epithelial cells these variants may confer distinct properties to the channel that
could have an impact on tightly regulated
Na+ transport.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Raju Kucherlapati and Kate Montgomery for sharing
prepublication nucleotide sequence information from a genomic clone that contained portions of the human -ENaC gene. We also thank Michael Welsh for
-ENaC1 and
- and
-ENaC cDNA, E. R. Liman for
pGem-HE, Pam Zeitlin for IB3-8 cells, Prof. Pierre Ronco for hCCD
cells, and Andrew Russo and Curt Sigmund for critical reading of the
manuscript. We acknowledge the DNA synthesis and sequencing services
provided by the University of Iowa DNA core facility.
![]() |
FOOTNOTES |
---|
This work was supported in part by grants from the Cystic Fibrosis Foundation Gene Therapy Center at the University of Iowa, by the March of Dimes Foundation, by an American Heart Association grant-in-aid, and by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-52617.
The nucleotide sequences reported in this paper have been submitted to GenBank with accession number U81961.
Portions of this work have been presented at the American Society of Nephrology annual meeting in 1996 and 1997.
Address for reprint requests: C. P. Thomas, Division of Nephrology, Dept. of Internal Medicine, Univ. of Iowa Hospitals and Clinics, 200 Hawkins Drive, Iowa City, IA 52242-1081.
Received 14 October 1997; accepted in final form 2 February 1998.
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