A divergent CFTR homologue: highly regulated salt transport in
the euryhaline teleost F. heteroclitus
Thomas D.
Singer1,
Stephen J.
Tucker2,
William S.
Marshall3, and
Christopher F.
Higgins1
1 Nuffield Department of
Clinical Biochemistry and Imperial Cancer Research Fund Laboratories,
Institute of Molecular Medicine, John Radcliffe Hospital, University of
Oxford, Oxford OX3 9DS;
2 University Laboratory of
Physiology, Oxford OX1 3PT, United Kingdom; and
3 Department of Biology, Saint
Francis Xavier University, Antigonish, Nova Scotia, Canada B2G 2W5
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ABSTRACT |
The killifish,
Fundulus heteroclitus, is a euryhaline
teleost fish capable of adapting rapidly to transfer from freshwater (FW) to four times seawater (SW). To investigate osmoregulation at a
molecular level, a 5.7-kilobase cDNA homologous to human cystic
fibrosis transmembrane conductance regulator (hCFTR) was isolated from
a gill cDNA library from SW-adapted killifish. This cDNA encodes a
protein product (kfCFTR) that is 59% identical to hCFTR,
the most divergent form of CFTR characterized to date. Expression of
kfCFTR in Xenopus oocytes generated
adenosine 3',5'-cyclic monophosphate-activated,
Cl
-selective currents
similar to those generated by hCFTR. In SW-adapted killifish,
kfCFTR was expressed at high levels in the gill, opercular epithelium, and intestine. After abrupt exposure of FW-adapted killifish to SW, kfCFTR expression in the gill increased
severalfold, suggesting a role for kfCFTR in salinity adaptation. Under
similar conditions, plasma Na+
levels rose significantly after 8 h and then fell, although it is not
known whether these changes are directly responsible for the changes in
kfCFTR expression. The killifish provides a unique opportunity to understand teleost osmoregulation and the role of CFTR.
cystic fibrosis transmembrane conductance regulator; chloride
channel; killifish; gill; Xenopus
expression; osmoregulation; cystic fibrosis; Fundulus
heteroclitus
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INTRODUCTION |
THE EURYHALINE KILLIFISH, Fundulus
heteroclitus, lives in tidal marshes and estuaries
along the eastern coast of North America and has a remarkable capacity
to adapt rapidly to changes in salinity ranging from extremely dilute
[freshwater (FW); 0.1 mM NaCl] to extremely saline
[4× seawater (SW); ~2.0 M NaCl] (15). This ionic
adaptability makes the killifish a key model in understanding the
physiology of ionoregulation (reviewed in Refs. 17, 23, 38). SW-adapted
killifish, like marine teleosts, drink SW, absorb ions and water
through their intestine, and secrete the excess salt through
specialized mitochondria-rich "chloride" cells
present in their skin and gill epithelia. However, after transfer
to FW the killifish osmoregulates as a FW teleost, maintaining the same plasma NaCl levels as in SW.
The movement of Cl
across
the killifish skin and gill epithelium is believed to occur via
pathways similar to those of the mammalian airway epithelium (24).
Cl
secretion across the
opercular epithelium of teleosts is stimulated by adenosine
3',5'-cyclic monophosphate (cAMP) in response to hormones
such as
-adrenergic agonists, vasoactive intestinal polypeptide,
glucagon, and urotensin I and is blocked by diphenylamine-2-carboxylic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (11, 26). A low-conductance Cl
channel (~8.0 pS) is present in the apical membrane of primary cultures of opercular epithelium cells taken from SW-adapted killifish (26). These characteristics are similar to those of the human cystic
fibrosis transmembrane conductance regulator (hCFTR), a known
Cl
channel (1, 4, 13, 30).
To investigate osmoregulation in teleosts at a molecular level, a
CFTR homologue (designated kfCFTR) was cloned
from the gills of SW-adapted killifish. kfCFTR encodes a
protein that is the most divergent form of CFTR identified to date.
Heterologous expression of kfCFTR in
Xenopus oocytes demonstrated that this
homologue encodes a cAMP-activated
Cl
channel. In SW-adapted
killifish, kfCFTR was found to be most highly expressed in
the gill, opercular epithelium, and intestine. Abrupt exposure of
FW-adapted killifish to SW revealed increases in kfCFTR mRNA expression
in the gill and increased plasma
Na+ levels, implying a role for
kfCFTR in salinity adaptation. The killifish is an extremely adaptable
euryhaline teleost that provides a unique opportunity to investigate
the role of CFTR in an organism capable of highly regulated salt
secretion.
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MATERIALS AND METHODS |
Tissue samples. Killifish
(F. heteroclitus) gill tissues used
to make RNA for constructing a cDNA library were collected from fish
captured in esturine ponds near St. Andrews, New Brunswick, Canada, and
were maintained for 1-2 wk in 100% SW (~1,000 mosM). Killifish
tissue samples for Northern blot analysis were from fish captured in an
estuary near Antigonish, Nova Scotia, Canada. The killifish collected
from both locations were of the same genus and species and were both
collected from brackish water during summer months. Fish collected for
tissue distribution study were transferred directly to 100% SW and
held for at least 30 days. For SW transfer experiments, fish were first
transferred to 10% SW for 3 days and then transferred to FW and held
for at least 30 days. In both situations water temperature was between
20 and 24°C, with a constant light-to-dark photoperiod of 15:9 h.
Fish were fed twice daily a ration of tetra minimum flake
food and supplemented with frozen tubifex worms.
Extraction of RNA. Total RNA used for
cDNA library construction was isolated from St. Andrew's killifish
gill tissue by lysis in guanidinium isothyocyanate followed by CsCl
centrifugation (6). Poly(A)+ RNA
was isolated from total RNA using oligo(dT) cellulose chromatography (2). Total RNA used in Northern blot analysis was isolated separately
from different tissues from Antigonish killifish using a Qiagen RNeasy
kit, following the manufacturer's instructions.
Genomic library screening. A killifish
genomic library, constructed by Stratagene in the vector lambda Fix II,
was kindly supplied by Dr. D. Powers, Hopkins Marine Station, Stanford
University, CA. Plaque lifts of this library using ICN nylon membranes
were hybridized to a full-length dogfish shark CFTR (sCFTR) cDNA probe labeled with [32P]dCTP
by random priming (Promega Prime-a-gene) for 18 h at 42°C under low
stringency in 5× SSPE [1× SSPE = (in mM) 180 NaCl, 10 sodium phosphate, and 1 EDTA, pH 7.7] containing 5×
Denhardt's reagent (1× Denhardt's = 0.1% Ficoll, 0.1%
polyvinylpyrrolidone, and 0.1% bovine serum albumin), 0.5% sodium
dodecyl sulfate (SDS), 30% formamide, and 20 µg/ml denatured,
fragmented salmon sperm DNA. The membranes were washed at a final
stringency of 57°C in 2× SSC (1× SSC = 150 mM
NaCl and 15 mM sodium citrate) and 0.1% SDS and were exposed to either
phosphorimaging plates or autoradiographs. After screening 3 × 105 recombinants from an amplified
version of this library, a positive clone was recovered. A 1,636-base
pair (bp) genomic DNA fragment from this positive was cloned into a
pBluescript (Stratagene) plasmid, was fully sequenced on both strands,
and was found to contain a 173-bp region 91% identical to exon 22 of
hCFTR. This 173-bp fragment was used as a probe
to isolate a full-length cDNA (see below).
cDNA library construction/screening.
To clone a full-length kfCFTR cDNA, an
oligo(dT)-primed cDNA library was prepared using 2 µg of
poly(A)+ mRNA from the gill of St.
Andrew's SW-adapted killifish and was cloned into the Stratagene
vector lambda Zap II following the manufacturer's instructions. Plaque
lifts were hybridized for 18 h at 60°C under moderate stringency in
5× SSC, 10× Denhardt's reagent, 0.5% SDS, 100 µg/ml
tRNA, and 10% Dextran SO4, using as a probe the 173-bp kfCFTR genomic fragment (see above) labeled with
[32P]dCTP by random
priming (Promega Prime-a-gene). This probe, designated kfCFTR22, was
amplified from the genomic plasmid (see above) using the primer pair
The
membranes were washed at a final stringency of 60°C in 2×
SSC, 0.1% SDS and were autoradiographed. After screening 480,000 recombinants from the unamplified library, four positive clones were
isolated. The cDNA inserts from these lambda clones were rescued by in
vivo excision as pBluescript (Stratagene) plasmid recombinants. One of
the plasmids contained a 7,250-bp insert that was fully sequenced on
both strands.
Sequence analysis revealed that the insert contained the full-length
kfCFTR cDNA sequence. However, the sequence was interrupted by two intronlike regions of 1,274 and 393 bp corresponding precisely in location to introns 18 and 20 of hCFTR (42). Stop signals within these putative introns would disrupt the kfCFTR coding sequence.
In addition, this cDNA lacked the 126-bp region corresponding to
hCFTR exon 6b. Investigation by reverse
transcriptase-polymerase chain reaction of the same gill total RNA
sample used to construct the cDNA library revealed that these introns
are absent from bulk cellular kfCFTR RNA and that we had cloned a very
rare variant. In addition, the region homologous to hCFTR
exon 6b was shown to be part of the bulk of cellular RNA (data not
shown). On the basis of these results, the original 7,250-bp clone was
modified to "correct" these three differences: the two introns
were removed and the kfCFTR exon 6b was replaced through a
series of polymerase chain reaction amplifications, restriction
digests, and ligations. After these manipulations, the final cDNA clone
was fully sequenced on both strands to ensure that no mutations had
been introduced during the repair process. This final cDNA (5,709 bp)
contained 173 bp of the 5'-untranslated region (UTR), a 4,509-bp
open reading frame encoding kfCFTR, and a 1,027-bp 3'-UTR.
DNA sequencing. Sequencing was
performed using double-stranded templates with a combination of
specific oligonucleotides and the T3/T7 universal primers using the
Sequenase system (US Biochemical) and an ABI prism 377 auto sequencer.
DNA sequences were assembled and analyzed using Wisconsin Package
Genetics Computer Group software. The nucleotide and amino acid
sequence data have been deposited in the GenBank/EMBL Data Bank with
accession number AF000271.
Northern blot hybridization. Total RNA
(10 µg), isolated from the tissues of SW-adapted killifish collected
from Antigonish, was heated to 60°C for 5 min in a solution of 30%
deionized formamide, 2% formaldehyde, and 20 mM
3-(N-morpholino)propanesulfonic acid (MOPS). Glycerol-dye
buffer was added, and the RNA was fractionated at 60 V for 5 h on a 1%
agarose gel containing 20 mM MOPS and 6% formaldehyde. RNA size
markers (GIBCO BRL) were treated in an identical manner. The RNA was
transferred onto Hybond-N membrane (Amersham) by standard methods and
fixed by ultraviolet cross-linking (Stratagene). The membrane was
hybridized for 2 h at 65°C under high stringency in rapid-hyb
buffer (Amersham; following the manufacturer's instructions) with a
randomly primed (Promega Prime-a-gene),
[32P]dCTP-labeled
929-bp kfCFTR cDNA fragment amplified using the primer pair
This
929-bp fragment was used as a probe since it corresponded to the R
domain that is unique to CFTR and is unlikely to cross-hybridize with
related ATP-binding cassette (ABC) transporters (16). The membrane was
washed at a final stringency of 70°C in 0.1× SSC and 0.1%
SDS and was autoradiographed.
Transfer of killifish from FW to SW.
Killifish collected from Antigonish were held for at least 30 days in
FW, and then a group of eight fish were randomly netted from a large
holding tank and transferred in pairs to four 4.5-liter buckets also
containing FW. Air stones maintained oxygen levels. After 24 h, the
water in these buckets was replaced with 100% SW from a header tank over a period of 7 min. The transfer of fish was repeated for seven
groups of eight fish, with each group exposed to SW for a set period of
time (1, 3, 8, and 24 h and 2, 7, and 28 days). In addition, a group of
eight fish were transferred from the FW holding tank to the 4.5-liter
buckets also containing FW and sampled after 24 h. This FW control
group was intended to control for possible effects of the transfer
procedure. A final group of eight fish were sampled directly from the
FW holding tank. After the fish had been collected, they were double
pithed, and the blood was collected in capillary tubes after removal of
the caudal peduncle. Blood plasma was separated by centrifugation and
frozen at
20°C for later analysis of
Na+ levels by atomic absorption
(Varian AA-375). Na+ levels were
compared between groups by one-way analysis of variance followed by the
a posteriori sum-of-squares simultaneous test procedure. For each
treatment group, between four and eight fish were sampled for plasma
Na+.
Immediately after the fish blood was collected, gill arches from each
of the eight fish were removed, placed into separate cryovials, and
plunged into liquid nitrogen for subsequent RNA extraction. Total RNA
from the gills of between one and three individuals from each treatment
group was extracted and pooled. Northern blot hybridization was
conducted as above, using a single 10-µg sample from each group and a
randomly primed (Promega Prime-a-gene), [32P]dCTP-labeled
929-bp kfCFTR cDNA fragment as a probe. The blot was then
stripped with boiling 0.01% SDS and reprobed for loading control with
a randomly primed (Promega Prime-a-gene),
[32P]dCTP-labeled
700-bp killifish
-actin cDNA fragment amplified using the mouse
actin primer pair
The
hybridization signals for both kfCFTR and killifish
-actin [lower 1.5-kilobase (kb) band]
were quantified by densitometric scanning of autoradiographs (BioImage
densitometer, Millipore) using whole band analyzer software. Densities
collected as integrated intensities for kfCFTR were
normalized to the corresponding
-actin integrated
intensity and were expressed relative to values from fish transferred
from FW to FW.
Electrophysiology. For oocyte
expression, the kfCFTR and hCFTR open reading
frames were subcloned into the oocyte expression vector (pBF) (Dr.
Bernd Fakler, University of Tubingen, Germany; unpublished
observations), which provides the 5'- and
3'-UTRs of the Xenopus
-globin genes. Capped mRNA was synthesized by in vitro transcription
from the linearized cDNA. Oocyte preparation and maintenance was as
previously described (14). Stage V or VI oocytes were injected with
~1 ng of kfCFTR mRNA, in a final injection volume of 50 nl. Control
oocytes were injected with water. Whole cell currents were measured
24-48 h after injection using a two-electrode voltage clamp
essentially as described previously (14), except that the recording
solution was ND96 [in mM: 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid pH 7.5]. Stimulation of currents by cAMP was achieved by perfusion with recording solution containing 10 µM forskolin, 200 µM dibutyryl cAMP, and 100 µM 3-isobutyl-1-methylxanthine. This was
originally prepared as a 1,000× stock dissolved in dimethyl sulfoxide and was diluted as required. Experiments were performed at
room temperature (18-24°C).
 |
RESULTS |
The kfCFTR gene. A 5.7-kb cDNA
encoding the killifish CFTR homologue was cloned and
sequenced (see MATERIALS AND
METHODS). The cDNA has a single long open reading
frame encoding a protein of 1,503 amino acids. At the nucleotide level
the kfCFTR coding sequence is 62.2% identical to
hCFTR and 63.7% identical to dogfish shark CFTR
(sCFTR). The ATG triplet assigned as the translational initiation codon was based on homology with other forms of CFTR and the
similarity of the sequence surrounding this site to the consensus
sequence for translation initiation in higher eukaryotes (20). This
cDNA contains a 173-bp 5'-UTR, whereas the 1,027-bp 3'-UTR
contains a poly(A)+ addition
signal sequence, although no
poly(A)+ tail was found.
The kfCFTR protein displays less sequence identity to hCFTR than any
other CFTR homologue identified to date (Fig.
1). At the amino acid level the two
proteins show 59.0% identity and 77.3% similarity. The kfCFTR
homologue is similarly divergent from sCFTR, with 60.2% identity and
78.0% similarity. Figure 1 summarizes the sequence identity between
seven known CFTR protein homologues. Alignment of the kfCFTR amino acid
sequence with other published CFTR homologues (Fig.
2) reveals differences in the degree of
conservation of different domains of the protein. Table 1 shows sequence identity between hCFTR and
kfCFTR on a domain basis.

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Fig. 1.
Percentage amino acid sequence identity/similarity between cystic
fibrosis transmembrane conductance regulator (CFTR) homologues. These
sequences were taken from Refs. 7, 22, 30, 34, 35, and 37.
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Fig. 2.
Alignment of the deduced amino acid sequences of CFTR from 6 species.
Amino acids invariant between all species are highlighted in bold.
Residues identical in adjacent sequences are in capital letters, and
others are in lower case. The 12 membrane-spanning regions (TM1-TM12)
are boxed, the 2 nucleotide-binding domains are double underlined, and
the R domain is highlighted with a dashed line. Putative glycosylation
sites between TM7 and TM8 are underlined in each of the 6 sequences.
These sequences were taken from Refs. 7, 22, 30, 34, and 37.
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Table 1.
Percentage identity and similarity of individual domains of
killifish CFTR compared with equivalent domains of human CFTR
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Expression of the kfCFTR gene. To
study the pattern of kfCFTR expression, Northern analysis of
total RNA isolated from different SW-adapted killifish tissues was
performed using a 929-bp kfCFTR cDNA probe. The panel
directly below the autoradiograph shows controls for RNA loading of
each sample (Fig. 3). The predominant mRNA
species was ~7.5 kb in size, larger than the 6.5-kb mRNA of
hCFTR (30). A second smaller mRNA species of 5.5 kb present in gill, opercular epithelium, and posterior intestine may represent an
additional cross-reactive homologue or an alternatively spliced form.

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Fig. 3.
Tissue-specific expression of kfCFTR mRNA.
A: Northern blot of total RNA isolated
from different seawater (SW)-adapted killifish tissues probed with a
929-base pair (bp) kfCFTR cDNA fragment. Lanes were loaded
with 10 µg of total RNA from each tissue.
B: controls for RNA loading of each
sample. Positions of 28S and 18S ribosomal RNA molecules are indicated.
Post int, posterior intestine; O epith, opercular epithelium.
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kfCFTR is expressed at high levels in the gill, opercular
epithelium, and intestine of SW-adapted killifish, with moderate expression in the brain (Fig. 3). Low levels of expression, at least
20-fold less than the gill, were detected in the kidney, liver, spleen,
ovary, and testes on slightly overexposed autoradiographs (data not
shown). No expression was detected in either the eye or heart tissue.
kfCFTR expression and plasma
Na+ levels after
abrupt transfer from FW to SW.
To examine the effect of rapid transfer from FW to SW on kfCFTR
expression, FW-adapted killifish were exposed to SW and sampled after 1, 3, 8, and 24 h and 2, 7, and 28 days. Expression of
kfCFTR in the gills increased 8 h after transfer to SW (Fig.
4). Control fish transferred from FW to FW
showed levels of kfCFTR expression similar to the levels
from fish sampled directly from the FW holding tank, demonstrating that
expression changes were not due to stress of handling. Levels of
kfCFTR expression were quantitated from the Northern blot as
the ratio of kfCFTR to
-actin mRNA. A ninefold increase in
kfCFTR expression was seen 24 h after transfer to SW, which
decreased to a threefold higher level by 28 days compared with fish
transferred from FW to FW.

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Fig. 4.
Effect of rapid transfer from freshwater (FW) to SW on kfCFTR
expression. A: Northern blot of
10 µg of total RNA isolated from gill tissue of FW-adapted killifish
exposed to SW for indicated period of time, probed with a 929-bp
kfCFTR cDNA fragment. B:
same blot reprobed with a 700-bp killifish -actin cDNA fragment as a
control for RNA loading of each sample. FW/FW, FW to FW control fish;
d, days.
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Plasma Na+ levels were also
measured in these fish to verify that the response of the killifish
used to study kfCFTR expression was as expected. Plasma
Na+ levels reached a peak after 8 h of exposure to SW (226.3 ± 4.9 mM) and were significantly higher
than any other group (P < 0.05; Fig.
5). Fish transferred from FW to FW also
showed a significant increase in plasma
Na+ levels (188.7 ± 5.0 mM)
compared with fish sampled directly from the FW holding tank (164.5 ± 2.8 mM; P < 0.05).

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Fig. 5.
Effect of rapid transfer from FW to SW on plasma
Na+ levels. Plasma
Na+ concentration of FW-adapted
killifish exposed to SW for indicated period of time. Data points
without a common letter are significantly different at
P < 0.05. Each point represents mean ± SE of data obtained from 4-8 fish. Note: 28-day error bar is
within symbol.
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kfCFTR functions as a cAMP-activated
Cl
channel.
To assess whether the kfCFTR cDNA encodes a cAMP-activated
Cl
channel, it was
expressed in Xenopus oocytes. Using
the two-electrode voltage-clamp configuration, currents were measured
at +30 mV, since at these potentials no background currents were seen
in mock/uninjected oocytes. Basal currents were observed in oocytes injected with kfCFTR mRNA (3.88 ± 0.15 µA;
n = 8) compared with water-injected
oocytes (0.55 ± 0.15 µA;
n = 8; Fig.
6). These basal currents were of a
magnitude similar to those observed in oocytes injected with an
equivalent amount of hCFTR mRNA (4.16 ± 0.65 µA;
n = 6). In kfCFTR-expressing oocytes
these basal currents were increased significantly by cAMP stimulation
(16.55 ± 3.95 µA; n = 8; Fig.
6), equivalent to currents generated by expression of hCFTR on cAMP
stimulation (18.73 ± 3.15 mA; n = 6).

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Fig. 6.
Generation of cAMP-activated currents by kfCFTR. Histogram showing
currents recorded from control (water-injected) or kfCFTR cRNA-injected
oocytes, both before and 10 min after exposure to a cAMP agonist
cocktail. Currents were measured at +30 mV (holding potential 30
mV) and expressed as means ± SE (n = 8).
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Figure 7A
shows a representative current trace from an oocyte expressing kfCFTR
before stimulation, 10 min after addition of cAMP agonist, and 15 min
after removal of agonist. The stimulated kfCFTR currents exhibited a
linear current-voltage (I-V)
relationship with time- and voltage-independent characteristics (Fig.
7B). The effects of cAMP-dependent
activation could be reversed by removal of the agonist from the bath.

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Fig. 7.
Current-voltage relationship of kfCFTR channel activity.
A: typical current trace recorded from
an oocyte expressing kfCFTR before (basal) and 10 min after addition of
a cAMP agonist cocktail. Currents recorded 10 min after removal of
agonist are also shown (Wash). Currents were elicited by a series of
voltage steps from 110 to +30 mV in 10-mV steps from a holding
potential of 30 mV. B:
corresponding current-voltage relationship before, during, and after
cAMP stimulation.
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To assess the ion selectivity of kfCFTR, the bath
Cl
was replaced by the
impermeant anion gluconate (96 mM NaCl was substituted with 96 mM
sodium gluconate) after activation. This caused a 37.2 ± 2.8 mV
(n = 4) shift in the
reversal potential, from
33.8 ± 1.8 mV (NaCl) to 4.6 ± 1.9 mV (sodium gluconate), indicating a permeability
(P) ratio of
PCl/Pgluconate > 5.8. Figure 8 shows a
representative I-V relationship from
an agonist-stimulated oocyte before and after gluconate substitution.

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Fig. 8.
Anion selectivity of kfCFTR-generated currents. Representative
current-voltage relationship from an oocyte expressing kfCFTR recorded
in Cl -containing or
gluconate-containing extracellular medium. In both cases currents were
recorded in the presence of the cAMP agonist.
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DISCUSSION |
To study osmoregulation in the killifish, a homologue of the
hCFTR gene was characterized. The deduced amino acid
sequence of kfCFTR is the most divergent form of CFTR identified to
date, only 59.0% identical to hCFTR and considerably more divergent than the dogfish shark sequence that is 72.4% identical to hCFTR (22).
Despite this relatively low level of sequence conservation, function
has been conserved. Heterologous expression of kfCFTR in
Xenopus oocytes generated a
cAMP-regulated Cl
-selective
conductance with properties similar to those of hCFTR (3): a linear
I-V relationship that is time and
voltage independent and stimulated by cAMP.
This highly divergent kfCFTR homologue highlights those amino acids
that are conserved in all forms of CFTR. The first and second
nucleotide-binding domains (NBDs) are the most highly conserved domains, and the site of the most frequent mutation in humans,
F508,
is retained. The Walker A and B motifs located in each NBD of kfCFTR,
hallmarks of ATP binding proteins, are identical to the same motifs in
hCFTR except for four conservative substitutions: two located in Walker
A from NBD1 and two located in Walker B in NBD2. The presence of an R
domain demonstrates that kfCFTR is a true CFTR homologue, distinct from
other members of the ABC superfamily of transporters (16). The R domain
is the least highly conserved domain, although it retains eight
consensus protein kinase A (PKA) phosphorylation sites, similar to nine
for the R domain from both hCFTR (30) and sCFTR (22). This is
consistent with the model in which the R domain regulates channel
activation through phosphorylation by cAMP-dependent protein kinases.
In the putative transmembrane domains (TMDs), predicted
membrane-spanning segments TM6 and TM12, which are believed to line the
channel pore (5, 27), show the highest identity with those of hCFTR. The four cytoplasmic loops (CL) linking the TMs on the cytoplasmic side
of the membrane also show high levels of conservation. CL1 and CL2 are
thought to be involved in determining the open probability and
conductive states of the channel (39, 40), whereas CL3 and CL4 may
regulate channel opening and closing (31, 32). The extracellular loop
between TM7 and TM8 contains a single
N-linked glycosylation site (N-X-S/T)
in kfCFTR, compared with two sites for all other known CFTR homologues.
CFTR-like anion conductances have previously been measured in primary
cultures of opercular epithelium from SW-adapted killifish using
patch-clamp techniques (26). The functional and expression data
obtained here strongly suggest that kfCFTR is responsible for these
currents. Interestingly, the
Cl
conductance in the
opercular epithelium has been suggested to be regulated differently
from CFTR in mammalian airway epithelium (25, 26). First, killifish
opercular epithelium shows an
2-adrenergic-mediated downregulation of Cl
transport via intracellular Ca2+
(25). Second, the opercular epithelium transports
Cl
at a very high rate even
when unstimulated (26). These novel features of regulation were not
observed from the kfCFTR expressed in
Xenopus oocytes and may be due to
interactions of kfCFTR with endogenous regulatory elements present in
killifish opercular epithelium that are likely absent from
Xenopus oocytes. The kfCFTR homologue
characterized here will allow for closer examination of such functional
differences in relation to structure, with the use of the appropriate
expression systems.
The high expression of kfCFTR in the gill and opercular epithelium of
SW-adapted killifish is consistent with the finding that these tissues
contain an abundance of specialized
Cl
-secreting cells (18).
Using the vibrating probe technique, Foskett and Scheffy (12)
demonstrated that, in SW-adapted teleosts, Cl
movement was localized
to these cells. The gills and opercular epithelium that contain these
specialized cells are the tissues responsible for maintaining blood
NaCl levels significantly lower than those of the surrounding
environment in SW-adapted killifish. The current model for ion
transport by chloride cells in SW-adapted teleosts is similar to that
for mammalian airway epithelium (24). The driving force for
Cl
secretion is the
Na+ gradient established by the
Na+-K+-ATPase
located in the basolateral membrane (19), and a basolateral Na+-K+-2Cl
cotransporter generates increased intracellular
Cl
concentrations (9). The
gills and opercular epithelium act much like specialized salt-secreting
tissues such as the shark rectal gland (29) and the duck salt gland
(10), where CFTR is expressed at high levels and is thought to be the
primary Cl
channel.
Significantly, similar chloride cells in amphibian skin express an
anion conductance similar to CFTR (33), and these cells have been
compared with a subpopulation of cells in the human submucosal glands
where CFTR mRNA and protein expression is localized (8). The abundance
of these cells in the killifish gill and opercular epithelium make it
an ideal model for studying the role of CFTR and the chloride cell.
In addition to the expected high level of kfCFTR expression
in the gill and opercular epithelium, kfCFTR was also
expressed at high levels in the posterior intestine. Unlike the gill
and the opercular epithelium, the intestine of marine teleosts is mainly NaCl absorptive (21). This contrasts with the human intestine where CFTR is expressed at high levels in the secretory
crypts of the small intestine and distal colon (36). It will be
interesting to determine whether the intestinal form of kfCFTR
plays an absorptive rather than secretory role. Interestingly, in
the human sweat duct CFTR serves an absorptive role (28).
The killifish provides a unique opportunity to examine the role of CFTR
in an organism capable of highly regulated salt transport. kfCFTR
showed a rapid, ninefold increase in expression 24 h after FW-adapted killifish were exposed to SW followed by a drop in expression such that, after 28 days of SW exposure, they were only
threefold higher than fish transferred from FW to FW. Also associated
with the abrupt exposure to SW, plasma
Na+ levels increased significantly
from 188 to 226 mM in just 8 h and subsequently fell. A similar
transient 65 mosM increase in plasma osmolarity was observed after
10-15 h in killifish transferred from FW to SW (41). It is not yet
known whether plasma salt levels provide a direct signal for increased
kfCFTR gene expression.
The increases in kfCFTR expression strongly suggest a role
in SW adaptation and osmoregulation. The mechanism by which
kfCFTR gene expression increases during rapid SW adaptation
is not known and could be a result of transcriptional activation or a
reduction in mRNA turnover. Further investigation of the role of kfCFTR and its regulation at both the gene and protein level during rapid adaptation to changes in salinity will not only lead to an
understanding of the molecular mechanisms of teleost osmoregulation but
may also increase our understanding of hCFTR function and dysfunction.
 |
ACKNOWLEDGEMENTS |
We thank Drs. S. Grinstein, M. M. Manolson, and J. R. Riordan for
initial guidance; Soad Fahim, Noa Alon, and Zbyszko Grzelczak for
technical assistance; Drs. A. Harris and T. D. Bond for discussions and
critical reading of the manuscript; Dr. A. E. O. Trezise for advice on
cDNA library construction; the staff of the Huntsman Marine Science
Centre for assistance with specimen collection; F. Hamilton and Dr.
Ballantyne's 92-430 field course class for help in dissections of
tissues; Dr. D. Powers for supplying a sample of the
Fundulus heteroclitus genomic library; and Drs. J. R. Riordan and J. Marshall for providing dogfish shark CFTR probes.
 |
FOOTNOTES |
This work was supported by the Imperial Cancer Research Fund, Cystic
Fibrosis Research Trust, the Wellcome Trust, the Canadian Cystic
Fibrosis Foundation, and the Natural Sciences and Engineering Research
Council of Canada.
C. F. Higgins is a Howard Hughes International Research Scholar.
Present address of C. F. Higgins: Medical Research Council Clinical
Sciences Centre, Imperial College School of Medicine, Hammersmith
Hospital, Du Cane Rd., London W12 0NN, UK.
Present address of T. D. Singer and address for reprint requests: Dept.
of Anesthesiology, Vanderbilt University, 504 Oxford House, 1313 21st
Ave. South, Nashville, TN 37232-4125.
Received 4 August 1997; accepted in final form 3 November 1997.
 |
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