EDITORIAL FOCUS
Molecular cloning and transmembrane structure of hCLCA2 from
human lung, trachea, and mammary gland
Achim D.
Gruber1,
Kevin D.
Schreur2,
Hong-Long
Ji2,
Catherine M.
Fuller2, and
Bendicht U.
Pauli1
1 Cancer Biology Laboratories,
Department of Molecular Medicine, Cornell University College of
Veterinary Medicine, Ithaca, New York 14853;
and 2 Department of Physiology
and Biophysics, University of Alabama, Birmingham, Alabama 35294
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ABSTRACT |
The CLCA family of
Ca2+-activated
Cl
channels has recently
been discovered, with an increasing number of closely related members isolated from different species. Here we report the cloning of the
second human homolog, hCLCA2, from a human lung cDNA library. Northern
blot and RT-PCR analyses revealed additional expression in trachea and
mammary gland. A primary translation product of 120 kDa was cleaved
into two cell surface-associated glycoproteins of 86 and 34 kDa in
transfected HEK-293 cells. hCLCA2 is the first CLCA homolog for which
the transmembrane structure has been systematically studied.
Glycosylation site scanning and protease protection assays revealed
five transmembrane domains with a large, cysteine-rich, amino-terminal
extracellular domain. Whole cell patch-clamp recordings of
hCLCA2-transfected HEK-293 cells detected a slightly outwardly rectifying anion conductance that was increased in the presence of the
Ca2+ ionophore ionomycin and
inhibited by DIDS, dithiothreitol, niflumic acid, and tamoxifen.
Expression in human trachea and lung suggests that hCLCA2 may play a
role in the complex pathogenesis of cystic fibrosis.
calcium-activated chloride channel; cystic fibrosis
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INTRODUCTION |
ION CHANNELS PLAY a crucial role in many diseases, most
notably in cystic fibrosis, where a genetic defect of the cystic
fibrosis transmembrane conductance regulator (CFTR) is responsible for the disturbed ion transport (3, 19, 23, 25, 26, 30). CFTR is a
multifunctional transport protein that functions not only as an
epithelial Cl
channel but
also as a regulator of other ion channels and cellular pathways (10,
29, 31). Several studies have indicated that, in addition to CFTR, a
Ca2+-activated secretory pathway
for Cl
may play an
important role in modulating the disease severity in various tissues of
cystic fibrosis patients and CFTR knockout mice (1, 4, 18, 20, 27, 33,
35). However, little is known about the molecular basis of the channels involved.
A new family of proteins has recently been discovered that mediate a
Ca2+-activated
Cl
conductance in a variety
of tissues. Four members of this family have been identified, including
bovine lung endothelial cell adhesion molecule-1 (Lu-ECAM-1) (8, 40),
bovine Ca2+-activated
Cl
channel (CaCC or bCLCA1)
(6), murine CLCA1 (mCLCA1) (9), and human CLCA1 (hCLCA1) (11).
Patch-clamp studies with transfected human embryonic kidney (HEK-293)
cells have shown that bCLCA1, mCLCA1, and hCLCA1 mediate a
Ca2+-activated
Cl
conductance that can be
inhibited by the anion channel blocker DIDS and the reducing agent
dithiothreitol (DTT). The protein size, structure, and processing seem
to be similar among different CLCA family members and have been studied
in most detail for Lu-ECAM-1 (8). The Lu-ECAM-1 open reading frame
(ORF) encodes a precursor glycoprotein of 130 kDa that is processed to
a 90-kDa amino-terminal cleavage product and a group of 30- to 40-kDa
glycoproteins that are glycosylation variants of a single polypeptide
derived from its carboxy terminus. Both subunits are associated with
the outer cell surface, but only the 90-kDa subunit is thought to be
anchored to the cell membrane via four transmembrane domains (8). Based on hydrophobicity analyses, analogous structural models have been suggested for mCLCA1 and hCLCA1 (9, 11). Although the protein processing and function appear to be conserved among CLCA homologs, significant differences exist in their tissue expression patterns. For
example, bovine Lu-ECAM-1 is expressed primarily in vascular endothelia
(40), bCLCA1 is exclusively detected in the trachea (6), and hCLCA1 is
selectively expressed in a subset of human intestinal epithelial cells
(11). Thus the emerging picture is that of a multigene family with
members that are highly tissue specific, similar to the ClC family of
voltage-gated Cl
channels
(14). A role for CLCA homologs in the complex ion-trafficking disorder
of cystic fibrosis has been speculated, based on observations that the
cellular expression patterns of bCLCA1, mCLCA1, and hCLCA1 overlap with
that of CFTR in the respective tissues (6, 9, 11, 12). Before now no
Ca2+-activated
Cl
channel had been cloned
from human lung, the most severely affected organ in cystic fibrosis.
Here we describe the cloning of hCLCA2, the second hCLCA family member,
and provide a detailed account of the membrane topology of this new
Cl
channel, using
glycosylation site scanning and protease protection assays (28, 38).
hCLCA2 is selectively expressed in lung, trachea, and mammary gland.
Its transient expression in HEK-293 cells reveals a
Ca2+-activated
Cl
conductance, which is
similar to that of previously cloned CLCA family members.
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MATERIALS AND METHODS |
Isolation and cloning of the hCLCA2
cDNA. A human lung cDNA library (Clontech) was screened
as described (11) using
[
-32P]dCTP
nick-labeled Lu-ECAM-1 cDNA as probe. For hybridization and washing,
low-stringency conditions were applied [2 washes with 2×
standard sodium citrate (SSC), 0.1% SDS at 55°C for 20 min,
followed by 2 washes with 0.4× SSC, 0.1% SDS at 40°C for 10 min]. Positive phage clones were amplified, cloned into
pBluescript (Stratagene), and sequenced. Automated sequencing with
initial plasmid-derived primers followed by internal gene-specific
primers was performed by the Cornell University DNA Sequencing Facility using dRhodamine terminator cycle sequencing on an ABI prism 377 DNA
sequencer (PE Applied Biosystems). Missing 5' and 3' ends of the isolated cDNA species were completed using the rapid
amplification of cDNA ends technique (RACE; Life Technologies). For
expression studies, the 2,832-bp hCLCA2 ORF was PCR amplified from
human trachea poly(A)+ RNA
(Clontech) following reverse transcription with Superscript RNase
H+ RT (Life Technologies) and
random hexamer priming. PCR was performed with Pwo DNA polymerase
[Boehringer; initial denaturation at 94°C for 3 min, 35 cycles at 94°C for 50 s, 58°C for 30 s, and 72°C for
2 min, with a time increment of 3 s/cycle for each extension step (72°C), followed by a final extension step of 72°C
for 8 min]. Primer sequences were
5'-
TACAACATGACCCAAAGGAGC-3' (upstream) and
5'-
GACACTTTGGATATTTATTTATAATAATTTTGTTC-3 (downstream), with Not I linkers
underlined. PCR products were gel purified, incubated with
Not I, and cloned into the expression vector pcDNA3.1 (Invitrogen). Four different full-length PCR products were sequenced to control for potential PCR-induced sequence errors.
Northern blot and RT-PCR analyses.
Human multiple tissue Northern blots (Clontech) contained 2 µg
poly(A)+ RNA per lane of heart,
brain, placenta, lung, liver, skeletal muscle, kidney, pancreas,
spleen, thymus, prostate, testis, ovary, small intestine, colon mucosa,
peripheral blood leukocytes, stomach, thyroid, spinal cord, lymph node,
trachea, adrenal gland, or bone marrow. In addition, total RNA was
isolated (Trizol method, Life Technologies) from MCF-10A cells at 80%
confluency (American Type Culture Collection). MCF-10A total RNA (20 µg/lane) and human mammary gland
poly(A)+ RNA (2 µg/lane,
Clontech) were resolved on a formaldehyde gel, blotted onto
nitrocellulose, and hybridized with the
[
-32P]dCTP
nick-labeled (RTS RadPrime, Life Technologies) hCLCA2 cDNA as described
(11). For the study on the hCLCA2 expression pattern, highly stringent
washing conditions were employed (2 washes with 2× SSC, 0.1% SDS
at 55°C for 20 min followed by 2 washes with 0.1× SSC, 0.1%
SDS at 65°C for 20 min). Autoradiographs were exposed using an
intensifying screen at
70°C for up to 8 days. Stripping of
the blots and rehybridization with a probe for the housekeeping gene
elongation factor-1
(EF-1
) were performed to control
for RNA quality and loading amounts as described previously (13). RT-PCR was performed using the above-mentioned conditions and primers
to detect hCLCA2 expression in
poly(A)+ RNA samples from human
lung, trachea, mammary gland, intestine, and spleen (Clontech) and in
total RNA isolated from MCF-10A cells. PCR products were gel purified
(QIAquick gel extraction kit; Qiagen), cloned into the pGem-T vector
(Promega), and sequenced. In all RT-PCR assays, water, substituting for
RNA in the reverse transcription, served as a negative control. A cDNA
fragment of EF-1
was amplified to control for conditions of reverse
transcription and PCR (13).
In vitro translation, construction of glycosylation
mutants, and protease protection assay. The hCLCA2 ORF
cloned into the expression vector pcDNA3.1 was transcribed and
translated with the TNT T7 coupled reticulocyte lysate system (Promega)
in the presence of
L-[35S]methionine
(Amersham). Reactions were carried out at 30°C for 90 min with and
without canine pancreatic microsomal membranes (Promega). Samples were
analyzed by 8% SDS-PAGE, followed by drying of the gel and exposure to
film for 8 h.
Six cDNA constructs were generated with an asparagine (AAT or AAC) to
glutamine (CAA) mutation that abolishes a consensus glycosylation site
(NxS/T
QxS/T). These mutations (N150Q, N292Q, N522Q, N637Q,
N822Q, and N938Q) were positioned between adjacent potential
transmembrane domains as determined by hydrophobicity analysis
(Kyte-Doolittle algorithm, 19 amino acid residues per window). The
constructs were generated by overlap extension PCR as described (11)
and cloned into the Not I site of
pcDNA3.1, using Not I
site-incorporated PCR primers. Correct sequences of the products were
verified by sequencing. The six constructs were in vitro translated in
the absence or presence of microsomal membranes and resolved on an 8%
ProSieve 50 Tris/glycine SDS-polyacrylamide gel (FMC Bioproducts) that
enabled optimal resolution in the high-molecular-mass range. Molecular masses were estimated using molecular mass standards and a digital image analysis system (AlphaImager; AlphaInnotech). In
addition, protease protection assays were performed as described (26).
Briefly, in the presence of microsomal membranes in vitro translated
and 35S-labeled wild-type hCLCA2
was incubated with proteinase K (Sigma; 100 µg/ml) for 60 min on ice
with or without detergent present (0.5% Nonidet P-40). The reaction
was stopped by adding 5 mM phenylmethylsulfonyl fluoride, and the
products were analyzed by 12% SDS-PAGE, drying of the gel, and
exposure to film.
Expression of Myc-tagged protein constructs in HEK-293
cells. Two Myc-tagged hCLCA2 constructs
were generated by inserting a partial sequence of the human c-Myc
protein (EQKLISEEDL) (5) near the amino or carboxy terminus of hCLCA2
(between amino acids 33 and 34 or 725 and 726), using
overlap extension PCR as described (11), and cloned into pcDNA3.1.
Correct sequences were verified by sequencing. DNA constructs were
transfected into 70% confluent HEK-293 cells via the Lipofectamine
Plus method (Life Technologies) using 20 µl Plus reagent, 30 µl
lipid, and 4 µg DNA/100-mm dish in a 3-h incubation. Cells were lysed
48 h later in the presence of protease inhibitors (1% aprotinin, 1 µM leupeptin, 2 mM phenylmethylsulfonyl fluoride). Lysates were
resolved via 10% SDS-PAGE, blotted, and probed with mouse anti-human
c-Myc antibody 9E10 (Calbiochem), followed by enhanced
chemiluminescence detection (ECL; Amersham). Surface expression of the
proteins was shown by surface biotinylation (Biotin NHS,
Vector; 100 µg/ml) of transfected nonpermeabilized HEK-293 cells 48 h
after transfection (20 min at 37°C, followed by extensive washing
with PBS). Immunoprecipitation with antibody 9E10, incubation with
protein G beads, boiling of the beads in SDS loading buffer, and
SDS-PAGE were followed by blotting and detection with
peroxidase-conjugated streptavidin and ECL. To estimate the extent of
hCLCA2 glycosylation, 9E10 immunoprecipitates were incubated with
N-glycanase (0.3 U/40 µl sample;
Genzyme) at 37°C for 18 h before sample denaturation and loading of
the gel.
Electrophysiology. The hCLCA2 cDNA was
transiently transfected into HEK-293 cells, which lack an endogenous
Ca2+-activated
Cl
conductance (9, 11, 41).
To control for transfection efficiency and to identify transfected
cells, the same cells were cotransfected with a green fluorescent
protein reporter vector (EGFP, Clontech). Parental HEK-293 cells were
cultured in DMEM with 10% fetal bovine serum in the
absence of antibiotics. Cells grown on collagen-coated glass coverslips
placed in the bottom of 35-mm dishes were transfected with 5 µl
Lipofectamine, 0.5 µg of hCLCA2 cloned into pcDNA3.1, and 0.5 µg of
EGFP during a 2- to 3-h incubation period (Life Technologies). After
transfection, cells were allowed to recover for 24 h before patch-clamp
recording. In all cases, the cells that were cotransfected with hCLCA2
and EGFP and that fluoresced green were also expressing a
Ca2+-sensitive
Cl
conductance, whereas no
currents were observed in mock-transfected (EGFP alone) or
untransfected cells (see also Refs. 9 and 11). To record channel
activities under whole cell conditions, cells were superfused at
1-2 ml/min with bath solution (in mM: 112 N-methyl-D-glucamine chloride, 30 sucrose, 2 CaCl2, 2 MgCl2, and 5 HEPES, pH 7.4). Borosilicate glass electrodes (tip resistance 6-9 M
) were
filled with an identical solution plus 5 mM ATP. In some experiments designed to examine the effect of 4 µM ionomycin, the pipette solution also contained 1 mM EGTA and 0.366 mM
CaCl2. The free Ca2+ concentration under these
conditions was calculated to be ~25 nM. After seal formation (>1
G
) and establishment of the whole cell recording configuration,
cells were clamped at +20 mV and currents recorded at room temperature
using an Axopatch 200A (Axon Instruments, Foster City, CA) connected to
a personal computer through a TL1 interface (Axon) with 12-bit
resolution. The records were sampled at 5-10 kHz and filtered at
1-2 kHz with a four-pole Bessel filter. The current-voltage
relationship of hCLCA2 was determined using 300-ms voltage steps from a
holding potential of +20 mV to potentials from
100 to +100 mV at
10-mV intervals. To normalize membrane currents for differences in cell
size, the capacitative current transiently recorded in response to a
10-mV hyperpolarizing pulse was integrated and divided by the given voltage to give total membrane capacitance for each cell.
Nucleotide sequence accession number.
The GenBank accession number for the hCLCA2 sequence is AF043977.
 |
RESULTS |
Identification and cloning of hCLCA2.
A human lung cDNA library was screened with Lu-ECAM-1 cDNA as probe in
an attempt to isolate a CLCA homolog from human lung. After the
sequencing of the positive clones and completion of the 5' and
3' cDNA ends by the RACE technique, a single 3.6-kb cDNA species
was identified and named hCLCA2. Sequence accuracy was verified by
sequencing of four different full-length RT-PCR products from human
trachea mRNA generated by the highly accurate Pwo DNA polymerase. The nucleotide sequence shared high degrees of identity with those of
Lu-ECAM-1 (86%), bCLCA1 (85%), mCLCA1 (76%), and hCLCA1 (63%). Northern blot analyses under highly stringent conditions yielded bands
of the expected size of 3.6 kb in trachea and mammary gland, whereas
all other tissues tested were negative (Fig.
1). Although isolated from a
lung cDNA library, hCLCA2 was not detected in the lung by Northern blot
hybridization. However, the more sensitive RT-PCR revealed its
expression in lung in addition to trachea and mammary gland, suggesting
a significantly lower expression level in the lung. Because of RNA
analyses from whole tissue extracts, the cell types expressing hCLCA2
could not be identified. However, hCLCA2 was also detected in the
nonmalignant human mammary epithelial cell line MCF-10A using both
Northern blot and RT-PCR analyses (Fig. 1), suggesting epithelial
expression at least in the mammary gland. All PCR products were
sequenced, and sequence identities with the cDNA isolated from the lung
library together with the observed signal size of 3.6 kb on the RNA
blots indicated that both the RNA blot and RT-PCR signals in fact
represented hCLCA2.

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Fig. 1.
Northern blot hybridization and RT-PCR analyses of hCLCA2 tissue
expression pattern. Poly(A)+ RNA
(2 µg/lane) from human tissues and mammary epithelial cell
line MCF-10A was hybridized with a
32P-labeled probe representing the
hCLCA2 open reading frame (ORF; top
left). Absence of a signal for intestinal hCLCA1
indicates specificity for hCLCA2. Blots were stripped and rehybridized
with a probe for elongation factor-1 (EF-1 ) as an internal
control (bottom left). Exposure
times were 48 h (hCLCA2) and 24 h (EF-1 ). Top
right: RT-PCR analysis of hCLCA2 expression in human
tissues and cell line MCF-10A with primers flanking entire ORF (2,838 bp). A 219-bp fragment of EF-1 was amplified to control for RNA
quality and RT-PCR conditions (bottom
right). Negative controls starting from reverse
transcription included sterile water instead of RNA.
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Characterization of the hCLCA2
protein. The ORF of the hCLCA2 cDNA encodes a 943-amino
acid polypeptide with high levels of amino acid sequence identity with
Lu-ECAM-1 (76%), bCLCA1 (76%), mCLCA1 (69%), and hCLCA1 (51%; Fig.
2). The polypeptide is preceded by a
canonical signal sequence with a predicted signal peptidase cleavage
site between amino acids 31 and 32 (32). The predicted size of the
full-length protein (104 kDa) is consistent with the results of an in
vitro translation assay yielding a primary translation product of
~105 kDa (Fig.
3A). In
the presence of microsomal membranes, the protein was glycosylated in
vitro to a 120-kDa glycoprotein. To ascertain whether the hCLCA2
protein is cleaved into two subunits in mammalian cells as reported for
other CLCA homologs (8, 9, 11), two cDNA constructs were generated with
a c-Myc tag within the amino or carboxy terminus, respectively
(constructs "m1" and "m2") and transfected into HEK-293
cells. In fact, immunoblots of cell lysates probed with an anti-Myc
antibody identified an 86-kDa protein when the tag was inserted near
the amino terminus (m1) and a 34-kDa protein when the tag was situated
near its carboxy terminus (m2; Fig.
3B), confirming a similar cleavage
in hCLCA2. The presence of protease inhibitors in the lysis buffer
suggests that the observed cleavage did not occur after lysis of the
cells. To study the extent of glycosylation of each subunit,
immunoprecipitates of both Myc-tagged constructs from transfected
HEK-293 cells were deglycosylated by
N-glycanase treatment. The 86- and
34-kDa glycoproteins were reduced in size by 11 and 2.5 kDa, proposing
approximately four and one glycosylation sites, respectively (Fig.
3B). Detection of the two Myc-tagged
constructs in anti-Myc antibody immunoprecipitates from
surface-biotinylated, nonpermeabilized HEK-293 cells suggests that both
the 86- and 34-kDa proteins are expressed on the surface of the
transfected cells (Fig. 3C).

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Fig. 2.
Predicted hCLCA2 amino acid sequence aligned (Clustal method) with all
known CLCA homologs. Identical amino acids are indicated by dots;
dashes represent gaps. Major hydrophobic regions spanning 19 or more
amino acid residues are overlined (S, signal sequence;
1-5, transmembrane domains).
Cysteine residues conserved in amino-terminal extracellular domain are
bold and enlarged. Consensus sites are marked for
N-linked glycosylation (*),
phosphorylation by protein kinase C (PKC) (:), and phosphorylation by
cAMP-dependent protein kinase ( ). Arrow, conserved consensus site
for monobasic proteolytic cleavage; ::, 2 adjacent PKC sites. GenBank
accession numbers are AF039400 (hCLCA1), U36455 (bCLCA1 or CaCC),
AF001261 (Lu-ECAM-1), and AF047838 (mCLCA1). Lu-ECAM-1, lung
endothelial cell adhesion molecule-1; h, human; b, bovine; m,
murine.
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Fig. 3.
Biochemical analysis of the hCLCA2 protein.
A: in vitro translation without
( M) and with (+M) processing and glycosylation by microsomal
membranes. Glycosylation site scanning was performed by in vitro
translation of 6 glycosylation knockouts [N150Q to N938Q; wild
type (wt)]. Unglycosylated constructs uniformly ran at 105 kDa
( M), whereas glycosylated proteins were 120 kDa (wt, N292Q,
N637Q, N938Q) or 118 kDa (N150Q, N522Q, N822Q) in size (+M; 8% gel).
In a protease protection assay, three fragments of 18, 21, and 30 kDa
were protected from degradation by proteinase K in absence of detergent
( D), and the polypeptide was fully degraded in
presence of detergent (+D; 12% gel). In all experiments,
L-[35S]methionine-labeled
proteins were detected by SDS-PAGE, drying of the gel, and
exposure to film for 8 h. B:
immunoblot detection of Myc-tagged hCLCA2 constructs overexpressed
in HEK-293 cells. Tags were placed near the amino terminus (m1) or
within the carboxy-terminal cleavage product (m2; see Fig. 5). The
120-kDa precursor protein was processed into 2 proteins of 86 kDa (m1)
and 34 kDa (m2). The somewhat weaker band below the 34-kDa band (2nd
lane) probably represents an incomplete glycosylation variant of this
protein, which may not be visible in the less abundant 120-kDa
precursor due to lower resolution in the high-molecular-mass range of
this blot. Bands at 65 and 67 kDa represent endogenous c-Myc. Cell
lysates including protease inhibitors were resolved by 10% SDS-PAGE 48 h after transfection, transferred to a membrane, and probed with
anti-Myc antibody 9E10. Deglycosylation with
N-glycanase (+G) reduced the molecular
masses from 86 to 75 kDa (m1) and from 33.8 to 31.4 kDa (m2).
C: analysis of surface expression of
Myc-tagged hCLCA2 constructs m1 and m2. Transfected HEK-293 cells were
surface biotinylated, washed extensively with PBS, and lysed in the
presence of protease inhibitors, followed by immunoprecipitation with
anti-Myc antibody 9E10, SDS-PAGE, and probing with horseradish
peroxidase-conjugated streptavidin. Both the 86- and 34-kDa proteins
were biotinylated and therefore associated with the apical cell
membrane.
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To elucidate the transmembrane topology of hCLCA2, glycosylation site
scanning and protease protection assays were performed as described for
other channel proteins (28, 38). Potential transmembrane regions were
determined using a hydrophobicity analysis (Fig.
4). Based on this prediction, six
glycosylation knockout cDNA constructs were generated, each having a
single asparagine-to-glutamine (N
Q) mutation that abolishes a
consensus glycosylation site between two adjacent potential
transmembrane domains (N150Q, N292Q, N522Q, N637Q, N822Q, and N938Q).
Following in vitro translation and glycosylation, the products were
analyzed using a high-resolution PAGE. Mutation of three of the six
sites (N150Q, N522Q, and N822Q) led to a reduction in size of the
respective glycoproteins by ~2 kDa, indicating usage of these sites
and therefore extracellular location (Fig. 3A). However, lack of size reduction
of the remaining constructs did not necessarily prove intracellular
location of their mutated sites, because an extracellular consensus
glycosylation site may not have been used due to sterical hindrance.
Therefore, a protease protection assay was performed to determine the
sizes of the extracellular domains, complementing the information
derived from the glycosylation site scanning. In principle,
extracellular loops are protected from proteolysis due to their
translocation into the lumen of lipid microsomes, functionally
representing the endoplasmic reticulum. The calculated sizes of each
possible extracellular domain of hCLCA2 are given in Fig. 4 with ~2
kDa to be added per adjacent transmembrane domain. Wild-type hCLCA2 was
in vitro translated in the presence of microsomal membranes and
digested with proteinase K in the absence or presence of detergent.
Three degradation products of 18, 21, and 30 kDa were protected from
proteolysis in the absence of detergent (Fig.
3A), indicating their extension into
the microsomes. In the presence of detergent, the protein was fully
degraded. Both the glycosylation data and the sizes of protected
extracellular domains are consistent with a five-transmembrane topology
(Fig. 5). In the proposed model, the
fragments protected from proteolysis correspond in size to the first
extracellular domain (30 kDa, resulting from 27.7 kDa plus one
transmembrane segment, tm1), the second extracellular domain (18 kDa,
resulting from 13.7 kDa plus two transmembrane segments, tm2 and tm3),
and the third extracellular domain (21 kDa, resulting from 16.8 kDa
plus two transmembrane segments, tm4 and tm5). The size of the 21-kDa
fragment also indicates degradation and therefore intracellular
location of the carboxy-terminal tail of ~2 kDa. Given the number and
locations of consensus glycosylation sites of the primary hCLCA2
polypeptide (Fig. 2), this transmembrane model is also consistent with
the extent of glycosylation of the two 86- and 34-kDa subunits as
detected by N-glycanase treatment (Fig. 3B). Accordingly, the 86-kDa
subunit contains three glycosylation sites within the first (N74, N97,
and N150) and one within the second extracellular loop (N522), whereas
only one site (N822) is present within the extracellular loop of the
34-kDa cleavage product (Fig. 5).

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Fig. 4.
Hydrophobicity plot of hCLCA2 amino acid sequence (Kyte-Doolittle
algorithm, 19 amino acid residues/window). Hydrophobic domains are
given as positive values (SS, signal sequence; tm1 to tm5,
transmembrane regions). Numbers and bars above curve indicate
calculated molecular masses between transmembrane domains, including 2 kDa per potential glycosylation site for interpretation of results of
protease protection assay. Names of glycosylation knockout mutants
(NxQ) indicate locations of their abolished glycosylation sites. Sites
where a Myc tag was placed in constructs m1 and m2 are indicated with
arrows under curve. Arrowhead, consensus site for monobasic proteolytic
cleavage. Units are in kcal/mol (vertical axis) and first amino acid
residue/window (horizontal axis).
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Fig. 5.
Proposed transmembrane topology of hCLCA2 with transmembrane domains
numbered 1-5. Dashed lines, locations of c-Myc tag in constructs
m1 and m2. Sites of asparagine-linked glycosylation (Nx) are indicated
by treelike drawing. Cx, cysteine residues conserved among all known
CLCA homologs; Px, consensus sites for phosphorylation by protein
kinase C, with sites conserved among all known homologs underlined;
arrow, conserved consensus site for monobasic proteolytic cleavage.
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When compared with the amino acid sequences of previously cloned
homologs, hCLCA2 shares a number of interesting sequence motifs. For
example, the pattern of cysteine residues present within the large
amino-terminal, extracellular domain of all previously cloned homologs
is conserved in hCLCA2 (Fig. 2). Also conserved is the consensus site
for monobasic proteolytic cleavage following arginine residue 675 (7),
the location of which is consistent with the sizes of the cleavage
products of 86 and 34 kDa. Analyses of the predicted intracellular
domains of hCLCA2 revealed seven consensus phosphorylation sites for
protein kinase C (PKC; Fig. 5) but none for
Ca2+/calmodulin protein kinase II
or cAMP-dependent protein kinase.
Electrophysiological characterization of
hCLCA2. Electrophysiological studies for hCLCA2 were
conducted in a manner analogous to those reported for bCLCA1, mCLCA1,
and hCLCA1, which have all been shown to be associated with activation
of a Ca2+-sensitive
Cl
conductance. When
transiently expressed in HEK-293 cells, hCLCA2 was associated with an
outwardly rectified current that was activated by ionomycin. In
contrast, nontransfected cells, cells transfected with the EGFP vector
alone (mock-transfected), or transfected cells in the absence of
ionomycin were not associated with any significant current (Fig.
6). The outwardly rectifying
current-voltage relationship exhibited by hCLCA2-transfected HEK-293
cells exposed to 2 mM Ca2+ in the
pipette was absent from vector alone transfected cells (Fig.
7) (see also Refs. 9, 11, 39). This current
was sensitive to DIDS (300 µM), DTT (2 mM), niflumic acid (NFA; 100 µM), and tamoxifen (10 µM) (Fig. 8).
The average current recorded at +100 mV was 9.60 ± 2.87 pA/pF and
was reduced to 0.15 ± 1.60 (SE) pA/pF (n = 5) in the presence of DIDS.
Exposure to DTT reduced the mean current from 9.70 ± 6.42 to 1.96 ± 2.09 pA/pF (n = 6). Similarly, both NFA and tamoxifen reduced the current from a mean of 6.24 ± 4.75 to 0.64 ± 0.96 pA/pF
(n = 6) and from 12.05 ± 3.85 to
1.02 ± 1.68 pA/pF (n = 5), respectively (Fig. 9). In contrast, no
significant current was recorded from cells that were either
untransfected or transfected with the EGFP vector alone. In the case of
untransfected cells, the average current recorded in the presence of 2 mM Ca2+ in the pipette was 1.57 ± 0.72 pA/pF (n = 8), whereas, in
mock-transfected cells, the current in the presence of
Ca2+ was 0.97 ± 0.39 pA/pF
(n = 10). When the pipette solution
contained low Ca2+ (~25 nM) with
2 mM Ca2+ in the bath, perfusion
of the Ca2+ ionophore ionomycin
through the bath also activated the current (Figs. 6 and 9). Under
these conditions, average currents in vector alone transfected and
untransfected cells in the presence of ionomycin were 1.52 ± 1.83 (n = 5) and 0.22 ± 1.02 pA/pF
(n = 8), respectively. In
hCLCA2-transfected cells, addition of ionomycin increased the current
from 1.7 ± 1.04 to 10.77 ± 3.8 pA/pF
(n = 7, P < 0.001). These results suggest
that expression of hCLCA2 in HEK-293 cells is associated with the
appearance of a Ca2+-sensitive
Cl
conductance.

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Fig. 6.
Representative data collected from individual cells illustrating
Cl current
(ICl)
expression in hCLCA2-transfected HEK-293 cells. In presence of low (25 nM) internal Ca2+ and high (2 mM)
external Ca2+, neither
untransfected nor mock-transfected [green fluorescent protein
reporter vector (EGFP) alone] HEK-293 cells showed significant
current expression following exposure to ionomycin (IONO). In contrast,
in hCLCA2-transfected cells, ionomycin exposure resulted in appearance
of an outwardly rectified
ICl.
|
|

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Fig. 7.
Attenuation of hCLCA2 current expression by
ICl inhibitors.
In presence of 2 mM intracellular (pipette) and extracellular
Ca2+ (bath), hCLCA2-transfected
HEK-293 cells consistently expressed
Cl currents with a maximum
amplitude between 300 and 500 pA. Subsequent exposure to 300 mM DIDS, 2 mM dithiothreitol (DTT), 100 mM niflumic acid (NFA), or 10 mM tamoxifen
resulted in near-complete inhibition of expressed current. Data are
taken from individual cells.
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|

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Fig. 8.
Current-voltage relationship of hCLCA2 expressed in HEK-293 cells.
Representative data collected from individual cells showing effect of
inhibitors on whole cell currents in presence of 2 mM intracellular
Ca2+. Currents recorded from
untransfected or EGFP alone transfected cells were negligible (see
text). A: current expressed in
hCLCA2-transfected cells displayed slight outward rectification with
maximal activation of 12.06 pA/pF measured at +100 mV ( ). Subsequent
perfusion with 300 µM DIDS resulted in inhibition of the current to
0.66 pA/pF ( ).
B-D:
as above, using inhibitors DTT (2 mM,
B) yielding 10.12 pA/pF ( ) and
2.41 pA/pF ( ), NFA (100 µM, C)
yielding 14.98 pA/pF ( ) and 1.78 pA/pF ( ), and tamoxifen (10 µM, D) yielding 14.95 pA/pF ( )
and 1.96 pA/pF ( ).
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Fig. 9.
Summary of effects of ionomycin and inhibitors on hCLCA2 current
expression recorded at +100 mV. In presence of low cytosolic free
Ca2+ (~25 nM), exposure of EGFP
alone transfected and untransfected HEK-293 cells in presence of
ionomycin yielded currents of 1.52 ± 1.83 (n = 5) and 0.22 ± 1.02 (SE) pA/pF
(n = 8), respectively. Exposure of
hCLCA2-transfected cells to ionomycin resulted in an increase in
current from 1.69 ± 1.04 to 10.77 ± 3.8 pA/pF
(n = 7;
* P < 0.001). When internally
perfused with solutions containing 2 mM
Ca2+, transfected cells exhibited
a maximal current of 9.27 ± 4.91 pA/pF
(n = 22; * P < 0.001).
Exposure to DIDS (300 µM), DTT (2 mM), NFA (100 µM), or tamoxifen
(10 µM) inhibited the maximally activated currents to 0.152 ± 1.6, 1.95 ± 2.09, 0.64 ± 0.96, or 1.02 ± 1.68 pA/pF,
respectively.
|
|
 |
DISCUSSION |
A novel family of Ca2+-activated
Cl
channels has recently
been introduced by our laboratories (6, 8, 9, 11, 12). The family
members cloned thus far are the bovine Lu-ECAM-1 (8, 40), the bovine
Ca2+-sensitive
Cl
channel (CaCC or bCLCA1)
(6), the murine mCLCA1 (9), and a first human homolog, hCLCA1, which is
exclusively expressed in the intestine (11). Here we have described a
second human family member that is expressed in human lung, trachea,
and mammary gland. hCLCA2 shares many of the structural and functional
peculiarities of its homologs. For example, the sizes and extent of
glycosylation of the primary in vitro translation products of CLCA
homologs are conserved within the family. In mammalian cells, the
hCLCA2 primary translation product was cleaved into 86-kDa
amino-terminal and 34-kDa carboxy-terminal polypeptides that are both
associated with the outer cell surface. The corresponding cleavage
products are 90 and 38 kDa for Lu-ECAM-1 (8), 90 and 37-41 kDa for
hCLCA1 (11), and 90 and 32-38 kDa for mCLCA1 (9). It is unclear whether there are any structural or functional relationships between the larger amino-terminal and the smaller carboxy-terminal polypeptides of the CLCA homologs, although studies with amino- and
carboxy-terminal-truncated constructs of bCLCA1 have suggested that the
carboxy-terminal cleavage product is dispensable for channel function
(15). Also conserved among all homologs are a pattern of
amino-terminal, extracellular cysteine residues, a consensus
recognition site for monobasic proteolytic cleavage that is consistent
with the sizes of the two cleavage products, and consensus sites for
phosphorylation by PKC, although their respective functional
significance remains to be established.
Detailed biochemical analyses on the structure of hCLCA2 revealed a
five-transmembrane topology with three transmembrane domains within the
86-kDa amino-terminal and two within the 34-kDa carboxy-terminal cleavage products. This result is at variance with the models proposed
for earlier cloned CLCA homologs, where, based on the much less
reliable hydrophobicity analyses alone, four transmembrane regions have
been suggested, all located within the larger amino-terminal cleavage
product (6, 8, 9, 11). A consequence of this difference is the
intracellular location of the predicted cleavage site between the two
hCLCA2 subunits and extracellular locations for the proposed models for
Lu-ECAM-1, mCLCA1, and hCLCA1. Therefore, the data on the CLCA homologs
other than hCLCA2 need to be reevaluated, and analogous biochemical
studies will have to be performed for each family member. Given similar
functional characteristics among CLCA homologs, it is likely that the
established hCLCA2 transmembrane topology will serve as the prototype
for all members of this channel family.
Measurements on the Ca2+-activated
Cl
conductance of hCLCA2
were performed in transfected HEK-293 cells. This cell line was chosen
because it is devoid of any intrinsic
Ca2+-activated
Cl
conductance (41).
Consistent with previous findings on CLCA homologs, the
Ca2+ ionophore ionomycin elicited
an increase in whole cell current in hCLCA2-expressing HEK-293 cells.
This current was sensitive to standard inhibitors of
Cl
channels, such as DIDS,
NFA, and tamoxifen. Although Ca2+
was present in the bath at unphysiological concentrations (2 mM),
recent evidence suggests that ionomycin may release
Ca2+ from the endoplasmic
reticulum store exclusively, in which case its concentration only
increases in the range of 200-500 nM (34). The data presented here
do not unequivocally prove that hCLCA2 forms an anion channel itself
but would also be consistent with a role of hCLCA2 as a regulator of an
as-yet-unidentified, endogenous channel that by itself is not sensitive
to Ca2+. However, it has been
shown that the closely related bovine tracheal bCLCA1 forms a genuine
channel protein when reconstituted into planar lipid bilayers (16, 24).
Under the conditions used in the present study, we did not observe any
time dependence of activation of the
Ca2+-sensitive current. Although
time-dependent activation of
Ca2+-sensitive
Cl
currents has been
previously reported in epithelial cells (2, 37), the lack of such a
characteristic may reflect the use of a heterologous expression system
and the loss of associated proteins that confer this property (17).
Alternatively, hCLCA2 may not underlie the time-dependent
Ca2+-sensitive current recorded
from native airway cells, even though it is expressed in that tissue.
The observation that the hCLCA2-associated current is also sensitive to
the anti-estrogen tamoxifen is also consistent with a role for the
expressed protein as an anion channel. Several other anion channels,
notably a volume-regulated channel described in Ehrlich ascites tumor
cells (22), a Ca2+-activated
Cl
current identified in
arterial endothelial cells (21), and a member of the ClC family of
Cl
channels (39), were also
shown to be sensitive to this compound. Considering the expression of
hCLCA2 in human breast epithelium, it remains to be established whether
its sensitivity to tamoxifen plays any role in the effectiveness of
this drug against breast cancer.
The function of hCLCA2 as a mediator of a
Ca2+-activated
Cl
current and its
expression in human lung and trachea warrant future investigations
aimed at its potential involvement in the complex ion-secretory
disorder of cystic fibrosis. Especially intriguing is the question
whether it may form a viable alternate
Cl
channel that could be
exploited for pharmacological targeting to circumvent the defect of the
CFTR Cl
channel. Studies in
other systems, including animal models of cystic fibrosis, have shown
that a Ca2+-sensitive
Cl
conductance is present
in cystic fibrosis cells and may even be upregulated. This observation
is particularly relevant in the cystic fibrosis knockout mouse model,
where expression of an as-yet-unidentified Ca2+- and DIDS-sensitive
Cl
conductance is thought
to rescue the cystic fibrosis mouse from significant airway disease
(10, 27, 36). In the same CFTR (
/
) mouse, lethal intestinal pathology is associated with
absence of a Ca2+-activated
pathway for Cl
secretion,
whereas expression of a
Ca2+-sensitive
Cl
conductance in the
murine intestine is thought to compensate for the lack of CFTR function
and rescue the intestinal phenotype (4, 36). However, to what extent
the Ca2+-sensitive
Cl
conductance may
substitute for the defective CFTR in human cystic fibrosis is unclear,
especially since data obtained in CFTR
(
/
) mice may not be readily extrapolated to human cystic
fibrosis due to their significantly different cystic fibrosis
phenotypes (3, 4, 27, 30). A future challenge will be to establish whether differences in tissue-specific channels between species contribute to the differences observed between the phenotypes of cystic
fibrosis patients and murine CFTR knockouts.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Dale Benos for his assistance in the
Cl
conductance studies of
hCLCA2 and Dr. Benos and Dr. Randy Elble for their constructive
discussions. Heather Archibald is commended for excellent technical assistance.
 |
FOOTNOTES |
This work was supported by National Institutes of Health Grants
CA-47668 and CA-71626 (B. U. Pauli), DK-53090 (C. M. Fuller and D. Benos), and 5T32HL-07703-08 (K. D. Schreur), funds from the Cystic
Fibrosis Foundation (C. M. Fuller), and a fellowship from the German
Research Council (A. D. Gruber).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: B. U. Pauli,
Cancer Biology Laboratories, Dept. of Molecular Medicine, Cornell Univ.
College of Veterinary Medicine, Ithaca, NY 14853-6401 (E-mail:
bup1{at}cornell.edu).
Received 10 December 1998; accepted in final form 4 March 1999.
 |
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