Physiol Rev 85: 1061–1092, 2005;
doi:10.1152/physrev.00016.2004.
Structure and Function of CLCA Proteins
MATTHEW E. LOEWEN AND GEORGE W. FORSYTH
Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
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I. Introduction to CLCA Proteins
II. Cloning the CLCA Genes
A. Bovine CLCAs
B. Human and mouse CLCAs
C. Porcine CLCAs
D. Miscellaneous
III. CLCA Protein Structure
A. Human CLCAs
B. Mouse CLCAs
C. Bovine CLCAs
D. Porcine pCLCA1
IV. Endogenous Tissue Expression Sites
A. Human CLCAs
B. Murine CLCAs
C. Bovine CLCAs
D. Porcine pCLCA1
E. Other CLCAs
F. Correlating tissue expression sites to CLCA groups
V. Functional Responses to Expression of CLCA Proteins
A. Human CLCAs
B. Mouse CLCAs
C. Bovine CLCAs
D. Porcine pCLCA1
E. Other CLCAs
VI. Pathophysiological Connections to CLCA Expression
A. CLCA in asthma
B. CLCA function in cystic fibrosis
C. CLCA in the normal epithelium
D. CLCA in CF epithelium
VII. CLCA Proteins in Oncology
A. Cell cycle control
B. Cell adhesion and tumor metastases
VIII. CLCA, Vascular Tone, and Hypertension
A. Hypertension
IX. CLCA, Bestrophins, and Retinopathy
X. Summary
Loewen, Matthew E., and George W. Forsyth. Structure and Function of CLCA Proteins. Physiol Rev 85:
1061–1092, 2005; doi:10.1152/physrev.00016.2004.—CLCA proteins were discovered in bovine trachea and named for
a calcium-dependent chloride conductance found in trachea and in other secretory epithelial tissues. At least four
closely located gene loci in the mouse and the human code for independent isoforms of CLCA proteins. Full-length
CLCA proteins have an unprocessed mass ratio of ⬃100 kDa. Three of the four human loci code for the synthesis
of membrane-associated proteins. CLCA proteins affect chloride conductance, epithelial secretion, cell-cell adhesion, apoptosis, cell cycle control, mucus production in asthma, and blood pressure. There is a structural and
probable functional divergence between CLCA isoforms containing or not containing ␤4-integrin binding domains.
Cell cycle control and tumor metastasis are affected by isoforms with the binding domains. These isoforms are
expressed prominently in smooth muscle, in some endothelial cells, in the central nervous system, and also in
secretory epithelial cells. The isoform with disrupted ␤4-integrin binding (hCLCA1, pCLCA1, mCLCA3) alters
epithelial mucus secretion and ion transport processes. It is preferentially expressed in secretory epithelial tissues
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0031-9333/05 $18.00 Copyright © 2005 the American Physiological Society
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including trachea and small intestine. Chloride conductance is affected by the expression of several CLCA proteins.
However, the dependence of the resulting electrical signature on the expression system rather than the CLCA protein
suggests that these proteins are not independent Ca2⫹-dependent chloride channels, but may contribute to the
activity of chloride channels formed by, or in conjunction with, other proteins.
I. INTRODUCTION TO CLCA PROTEINS
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II. CLONING THE CLCA GENES
The CLCA gene family members are named for the
species from which they were cloned, and numbered
within each species in chronological order of discovery.
The history of the cloning of the CLCA gene family will be
summarized, and cross-species homologs with probable
structural and functional similarities will be identified.
The phylogenetic relationship between some of the better
understood CLCA proteins is presented in Figure 1.
A. Bovine CLCAs
The first CLCA cDNA sequence (bCLCA1) was reported
in 1995 (39). The authors were pursuing the gene coding for
a 140-kDa bovine tracheal protein that had been identified
with by Ca2⫹/calmodulin-dependent protein kinase-regulated chloride conductance. The 140-kDa protein was
thought to be a homotetramer of identical 38-kDa subunits,
linked together by disulfide bonds. Reduction of the 140-kDa
FIG. 1. Phylogenetic tree of representative clones of the CLCA gene
family. The tree developed using Clustalw followed by Phylip software
identifies three major CLCA structural groups. The scale is substitution
per site expressed as a percent using nucleic acid sequence. Each arm is
labeled accordingly.
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Secretory epithelial cells possess ion channels that
are asymmetrically distributed between the basolateral
and apical membrane. Vectoral ion movement through a
subset of these channels produces osmotic gradients that
drive net fluid movement into the lumen of secretory
tissues. Conductive chloride channels located on the apical membrane of secretory epithelial cells are of special
interest due to their role as regulatory targets. Regulation
of chloride conductance channels by intracellular second
messengers has been considered to be the primary level
of control for epithelial fluid secretion.
The predominant chloride conductance of most secretory tissues occurs via the cAMP-dependent cystic
fibrosis transmembrane regulator protein (CFTR). However, many tissues also have an independent chloride
conductance that can be activated by elevating the concentration of intracellular Ca2⫹. Activity of such a protein
has been functionally linked to fluid secretion in the trachea and in the small intestine. Upon their discovery in
1995, the members of the CLCA protein family were
thought to be this elusive calcium-dependent chloride
conductance. Nearly a decade later, research into CLCA
protein biology is proceeding in interesting and unexpected directions.
Attempts to understand CLCA protein function have
been complicated by uncertainty about a unifying molecular basis for the actions of family members. Different
CLCA proteins have several apparently unrelated, and
sometimes conflicting, functional roles. The first discovered member of the family was implicated with chloride
conductance in secretory epithelial tissues (39). However,
another original CLCA protein, LuECAM-1, functions in
tumor cell adhesion and metastasis (1–3, 50). This role for
LuECAM-1 in tumor adhesion appears to conflict with
significant tumor suppressor activity reported for other
CLCA proteins (49, 79, 112). CLCA proteins have also
been connected recently to the regulation of mucus production in the asthmatic airway (88, 135), and to ion
transport in the airway of cystic fibrosis patients (83, 84).
Other roles include CLCA involvement in chloride transport affecting vascular smooth muscle tone and blood
pressure, and in retinal functions that are disrupted in
Best’s vitelliform macular dystrophy (9, 73, 94, 124).
The history of CLCA cloning will be surveyed, followed by physiological effects observed upon heterologous CLCA isoform expression. Predictions for protein
structure and function will connect some basic biophysi-
cal results of CLCA isoform expression with the pathophysiology of associated disease processes. The targeting
of CLCA proteins for pharmacological modulation of related disease conditions will also be discussed.
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bCLCA1 and bCLCA2, although the mouse and bovine
isoforms are full-length membrane-associated proteins.
hCLCA3 was cloned from human spleen cDNA and has
been found in several tissues.
Gob-5 or mCLCA3, the mouse ortholog to hCLCA1,
was cloned from mouse gut cDNA (103). This mCLCA3
ortholog, as well as mCLCA1/2, mCLCA4, and related
mouse ESTs 4732440A06, AI747448 and AI504701 (mCLCA5,
6, and ?) colocalize on mouse chromosome 3 H2-H3 (108,
158, 160).
The human hCLCA2, hCLCA1, hCLCA4, and hCLCA3
genes align consecutively in the same orientation, encompassing 232 kb of chromosome 1 with no other genes
interspersed (76). Gene clustering can be a mechanism
for controlling temporal gene expression by internal intron or bidirectional promoters (95, 208). An area 1,617 bp
upstream of the first intron codes for 24 different transcription factor binding sites.
B. Human and Mouse CLCAs
Clones similar to bCLCA1 and -2 were reported in
1998 from human genome and mouse lung library screening. The human CLCA1 gene and its putative promoter
region span 31,902 bp of chromosome 1 at p22-p31 (75). A
promoter region containing a TATA box precedes the
predicted site of transcriptional initiation by 22 nucleotides. Fifteen exons from 90 to 604 bp are interspersed
with 14 introns of 170 to 5,651 bp. The hCLCA1 cDNA was
found independently in 1999 by screening an EST database with bCLCA1 sequence. The EST clone, named
HCaCC1, is identical to hCLCA1 (4). The 2,745 bp open
reading frame (ORF) of hCLCA1 was amplified in polymerase chain reaction (PCR) to clone a full-length cDNA
for functional expression.
A bCLCA2 probe was used to identify mCLCA1 in a
mouse lung cDNA library (67). mCLCA1 was also identified in a EST database using a probe based on bovine
bCLCA1 sequence (160). This group localized mCLCA1 to
mouse chromosome 3 at the H2-H3 band. mCLCA1 is the
murine ortholog of truncated hCLCA3 (Fig. 1) (158).
Several CLCA isoforms were reported in 1999.
mCLCA2 was cloned from mouse mammary gland in a
suppression subtractive hybridization on the involuting
mammary gland (107). mCLCA2 maps closely to mCLCA1
in the mouse genome and may even be a splice variant of
mCLCA1 (158). The hCLCA2 cDNA was cloned from a
human lung library using a bCLCA1 cDNA probe (78). As
with mCLCA2, hCLCA2 was expressed at high levels in
the mammary gland, but the murine counterpart of
hCLCA2 is the hypothetical mouse protein 4732440A06
(158), now called mCLCA5 (54). hCaCC2 and hCaCC3
(now hCLCA4 and hCLCA3) were discovered independently at the same time (4).
hCLCA3 is a short CLCA protein with a premature
stop codon (78). It is most similar to mCLCA1/2 and to
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C. Porcine CLCAs
A monoclonal antibody selected to inhibit chloride
conductance in ileal brush-border vesicles was used to
identify pCLCA1 cDNA in a porcine ileal gene expression
library (61, 63). Cloning and expression of the full-length
cDNA was reported in 2000 (68). The screening strategy
used to identify this isoform connects pCLCA1 to chloride
conductance.
D. Miscellaneous
Reports of a calcium-activated chloride conductance
in smooth muscle (85, 137) led to the discovery of
mCLCA4 (48). mCLCA4 is most similar to the prematurely
truncated hCLCA3, but mCLCA4 is a full-length integral
membrane protein.
Incomplete segments of CLCA cDNA have been
cloned from other species. A portion of a rat ortholog
rCLCA1 has been cloned from rat pancreas cDNA (179). A
partial canine CLCA, cCLCA1, cloned from dog retinal
pigment epithelium with primers specific for the porcine
pCLCA1 has also been reported, and expression levels
have been investigated (117). An hCLCA5 cDNA has been
reported recently (1). However, little information on its
cloning or function is available.
III. CLCA PROTEIN STRUCTURE
A. Human CLCAs
1. hCLCA1
The cDNA for hCLCA1 encodes a 914-amino acid protein with a calculated molecular mass of 100.9 kDa (75). A
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protein produced a 36- to 38-kDa polypeptide lacking chloride channel activity in lipid bilayers (39, 155). A candidate
cDNA was identified by screening a bovine tracheal cDNA
library with polyclonal antibody to this polypeptide. The
identified cDNA was described as coding for a calciumactivated chloride channel (CaCC), but is now called
bCLCA1 (39, 59, 60, 146).
A second bovine CLCA sequence (Lu ECAM-1or
bCLCA2) was identified by screening a gene expression
library with monoclonal antibody that blocked an adhesion-receptor/ligand pair interaction between lung-metastatic melanoma cells and lung matrix-modulated bovine
aortic endothelial cells (50, 211–214). Expression of the
Lu ECAM-1 cDNA produced a protein with properties of a
lung endothelial cell adhesion molecule(50). Lu ECAM-1
is now termed bCLCA2 (59, 146).
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FIG. 2. Proposed membrane topology of human CLCA orthologs. A:
1) hCLCA1 membrane topology, with four transmembrane domains and
small COOH-terminal secreted cleavage product predicted from epitope
insertion (75). 2) Alternative single transmembrane topology to accommodate von Willebrand A domain folding, as proposed by Whitaker et al.
(197). B: five transmembrane domains of hCLCA2 as proposed by Gruber et
al. (80). Three of the five transmembrane domains locate to the NH2terminal fragment. C: prematurely truncated hCLCA3 is a secreted protein
with stop codon insertion before first transmembrane domain (78).
(73). CLCA expression also modulates chloride conductance, leading to predictions that some chloride conductance channels may be hetero-oligomeric structures.
A metal ion-dependent adhesion site (MIDAS) motif
is a noncontiguous amino acid sequence that organizes
into a divalent cation binding structure through normal
VWA domain folding. MIDAS motifs bind magnesium or
calcium and participate in stabilizing protein-protein interactions. CLCA folding, based on sequence homology to
a collagen receptor, could form a VWA domain with
MIDAS function (197). The MIDAS motif could be a site
for calcium binding in CLCA proteins to interact with and
alter the conductivity of chloride ion channels. However,
MIDAS motif precedents in other proteins involve extra-
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signal sequence and four putative transmembrane domains
are predicted from hydropathy data. There are 9 potential
sites for asparagine-linked glycosylation, 13 consensus sites
for protein kinase C (PKC) phosphorylation, and 3 consensus sites for phosphorylation by Ca2⫹/calmodulin-dependent
kinase II. There are no PKA or tyrosine phosphorylation
consensus sequences (75). In vitro translation of the human
CLCA1 resulted in a 100-kDa product that increased in size
to 125 kDa after glycosylation.
Membrane topology predictions have been based on
detection of the c-myc epitope (EQKLISEEDL) inserted at
different regions within the protein. However, epitope insertion may be affecting protein conformation, as CLCA proteins are cleaved by monobasic proteases with conformation-dependent cleavage specificity, and several of the
tagged constructs were not processed normally (44, 164).
Insertion of the epitope between amino acids 366/367 or
492/493 resulted in a 90-kDa polypeptide, apparently interfering with the ability of the smaller cleaved ⬃40-kDa product to interact with the larger 90-kDa subunit. Other epitope
placements blocked proteolytic cleavage (75). A model
based on the c-myc epitope insertion and cell surface biotinylation has an extracellular NH2 and COOH terminus and
four transmembrane domains (80, 146) (Fig. 2).
An alternative model for hCLCA1 structure and relationship to the membrane has also been predicted without
direct supporting data (Fig. 2A) (197). Using SMART predictions based on sequence homology to the ␣2-integrin
collagen receptor domain, the CLCA family members are
suggested to consist of a central (extracellular) von Willebrand factor domain A (VWA domain), leaving only a
single transmembrane domain near the COOH terminus
(197). The significant disparity between the alternate
models involves predictions about the folding and topography of the NH2-terminal half of the protein. This region
of the protein is predicted to contain either two helical
transmembrane domains (TM1 and TM2), or to be extracellular with secondary and tertiary structure forming a
VWA domain. The TM3 and TM4 domains and the cytoplasmic loop between these domains containing protein
kinase consensus sites are not excluded by either conformation of the NH2-terminal folding.
VWA domains are recognized for involvement in protein-protein interactions. The voltage-gated calcium channel is a precedent for VWA domain involvement in ion
channel subunit interactions. The properties of this channel are modulated by interaction of the pore-forming ␣1subunit with the ␣2 VWA domain-containing subunit complex (86, 197). There is parallel evidence for CLCA interaction with other proteins. Hetero-oligomeric CLCA
interactions affect cell binding and mitogenic signaling
via integrins (1–3), as well as ion channel activity of the
␣1-subunit of the large-conductance potassium channel
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cellular polypeptide structure, while effects of Ca2⫹ ionophores on chloride conductance associated with CLCA
expression invoke changes in intracellular Ca2⫹ concentration as the basis for CLCA regulation of chloride con-
ductance. Without structural data, there is no direct evidence for VWA domain formation and function in CLCA
proteins in spite of significant amino acid sequence identity and modeling compatibility (see Fig. 3).
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FIG. 3. Alignment of the closest mammalian orthologs mCLCA3 (gob-5) and hCLCA1 with pCLCA1. The amino acid sequence identity for
pCLCA1:hCLCA1 was 78%, pCLCA1:mCLCA3 was 73%, and hCLCA1:mCLCA3 was 75% as determined by Clustalw software. The von Willebrand
A domain, the predicted major transmembrane domains, and phosphorylation consensus sequences on the major cytoplasmic loop are
identified.
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2. hCLCA2
4. hCLCA4
The hCLCA2 ortholog is predicted to form a 943amino acid polypeptide (80). It contains a canonical signal
sequence with a peptidase cleavage site. A primary translation product of 105 kDa from an in vitro translation
assay increases to 120 kDa after glycosylation. Proteolytic
processing at the monobasic proteolytic 673/674 cleavage
site leaves an NH2-terminal c-myc epitope tag in an 86kDa protein and a 34-kDa COOH terminus. Deglycosylation reduced the size corresponding to four glycosylation
sites on the 86-kDa fragment and one on the smaller
34-kDa fragment. Loss of glycosylation in mutants N150Q
and N522Q indicated that these regions are extracellular
(80). However, these results are compatible with either
multiple transmembrane domains or an extracellular NH2terminal VWA domain. A N822Q mutation indicated an
extracellular loop in the smaller cleaved product between
transmembrane domains 4 and 5.
Findings from a microsomal protein protection assay
digesting all internal loops gave results in agreement with
the interpreted five transmembrane domain structure of
hCLCA2. It should also be noted that both cleavage products were detected in surface-biotinylated, nonpermeablized HEK293 cells, suggesting that both the larger and
the smaller products localize to the cell surface. In this
model the internal loops contained seven PKC phosphorylation sites, but there were no consensus sites for Ca2⫹/
calmodulin protein kinase II or cAMP-dependent protein
kinase (80). hCLCA2, as well as several other CLCA proteins, contain a ␤4-integrin binding domain (1) whose
significance is discussed below.
There are no current structural data for the hCLCA4
clone.
The hCLCA3 cDNA contains two internal stop
codons (78). Sequential ORFs code for the NH2-terminal
262 amino acids, and a second polypeptide from amino
acid 266 to 461. Together, the two ORFs code for approximately the first one-third of the full-length CLCA protein.
The NH2-terminal polypeptide terminates before potential
transmembrane domains, and the second fragment lacks
a signal sequence, although its amino acid sequence includes the first two predicted transmembrane domains of
other CLCA family members. In vitro synthesis produced
30- and 22-kDa products corresponding to calculated size
for ORF1 and ORF2, respectively, but only the NH2-terminal polypeptide was detected in transfected HEK293
cells. The secreted ORF1 product was also detected upon
immunoblotting the supernatant from transfected cells
(78). Mouse homologs mCLCA1/2 and mCLCA4 lack the
internal stop codons, implying that there may be no function for the truncated, secreted hCLCA3 product.
Physiol Rev • VOL
1. mCLCA1/2
The in vitro translated size of 100 kDa for this
902-amino acid protein increases to 125 or 130 kDa with
glycosylation. Posttranslational processing gives NH2terminal 90-kDa and COOH-terminal 38/32-kDa components. A general four transmembrane domain structure
is proposed for this protein, leaving the COOH terminus
without a transmembrane domain (67). There is little
structural data available for mCLCA2. The mCLCA2
protein may be a splice variant of mCLCA1 (158), sharing significant cDNA sequence identity with mCLCA1
and hCLCA3. It is reported to produce similar processed products as found for mCLCA1 when expressed
in HEK293 cells (49).
2. mCLCA3
This mouse CLCA is the murine counterpart of
hCLCA1. The mCLCA3 cDNA codes for a 100-kDa protein in an in vitro translation assay, and increases to 110
kDa after glycosylation with microsomal membranes
(109). Microsomal protease treatment produced a 35kDa product corresponding to an extracellular NH2
terminus. An NH2-terminal 90-kDa product was identified in transiently transfected HEK293 and COS-7 cells
and in the mouse large and small intestine, with the
expression being more intense in the small intestine.
An unexplained 45-kDa product which may be a fragment of a truncated portion of the amino terminus of
the protein with similarities to the secreted 37 kDa
amino terminus of hCLCA3 was also detected in the
large, but not small intestine (109).
3. mCLCA4
The primary structure of mCLCA4 is similar to
mCLCA1 and 2. It is 909 amino acids in length, with a
conserved cleaved NH2-terminal signal and a second
monobasic cleavage site which results in a 90- and 30- to
40-kDa product (48). The primary structure also contains
most of the calcium/calmodulin kinase II (CaMKII) sites
found in mCLCA1 and mCLCA2.
4. mCLCA5 and mCLCA6
The two proteins have predicted mass ratios of 103.6
and 101.9 kDa, respectively (54). Uncleaved glycosylated
proteins expressed in tsA201 cells were 125 kDa. Process-
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3. hCLCA3
B. Mouse CLCAs
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ing by monobasic cleavage produced COOH-terminal glycosylated fragments of 35 kDa after subtraction of the
EGFP tag (54). Several consensus acceptor sites for protein kinase A (PKA), PKC and CaMKII occur in the amino
acid sequence, but the significance of these sites is uncertain without more information on the transmembrane
orientation of the expressed proteins. Conserved ␤4-integrin binding motifs on NH2- and COOH-fragments of
mCLCA5 are altered so as to be nonfunctional in
mCLCA6.
C. Bovine CLCAs
Cloned bovine isoforms are homologs of hCLCA3
and mCLCA1/2. The bCLCA1 cDNA codes for 903 amino
acids, giving a predicted product of 100 kDa which shifts
to 140 kDa upon glycosylation (39). Motif analysis predicts 15 PKC acceptor sites, 10 Ca2⫹/calmodulin-dependent protein kinase sites, and 3 tyrosine kinase sites. Four
transmembrane domains and an NH2-terminal signal sequence are predicted from hydropathy plots. Polyclonal
antibodies generated against an NH2-terminal fragment of
the bCLCA1 clone recognized a reduced 36/38-kDa bovine
tracheal lysate product similar to the antigen identified by
the original antibodies that were used to clone bCLCA1
(39, 155). The original purified protein of 140 kDa was
thought to consist of four identical 38-kDa subunits linked
together by disulfide bonds (155). However, the sequence
data and the identification of the 100-kDa unglycosylated
form of bCLCA1 indicate that the 38-kDa protein is a
posttranslational cleavage product that remains associated with its larger counterpart (39). This model has now
been promoted for the other cloned isoforms and orthologs.
2. bCLCA2
The cDNA sequence codes for a predicted hydrophobic NH2-terminal signal sequence and cleavage site for
membrane insertion. In vitro translation produced a 101kDa protein that increased to 120 kDa with glycosylation.
Expression of bCLCA2 in HEK293 cells produced similar
90- and 38-kDa products. Native expression in bovine
endothelial cells produced 130- and 32-kDa glycoforms
(50). As for bCLCA1, a hydropathy plot predicted four
transmembrane domains. Deglycosylation of native
bCLCA2 with N-glycosidase F from endothelial cells reduced the 38- and 32-kDa polypeptides to 22 kDa, and the
90-kDa band to 77 kDa, giving the predicted deglycosylated size of bCLCA2 (50). There is good evidence that the
primary 101-kDa protein is cleaved into two nonidentical
proteins (50), and earlier suggestions of a homotetrameric
structure for this protein (155) are incorrect.
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The pCLCA1 protein is the porcine homolog of
hCLCA1 and mCLCA3. The cDNA sequence contains
3,079 bp and a 2,751-base ORF that is predicted to encode
a 917-amino acid protein with a molecular mass of 100.7
kDa (68). It shares 78% amino acid sequence identity with
hCLCA1, its closest ortholog. Similarities of amino acid
sequence and hydrophobicity between pCLCA1 and
hCLCA1 suggested a similar transmembrane topology for
the two proteins. Modeling this structure according to the
suggestions of Gruber et al. (80) would give an extracellular NH2 terminus followed by four transmembrane domains and a hydrophobic COOH terminus (Fig. 2) (68).
The predicted pCLCA1 protein shares a signal sequence for membrane targeting and potential proteolytic
cleavage sites with hCLCA1. There is an amidation cleavage site at residue 140 and monobasic proteolytic cleavage sites at R662 and K720. This could account for the
multiple sizes of protein (⬃130, 90, and 60 kDa) detected by Western blotting or immunoprecipitation of
brush-border vesicle protein with an inhibitory antibody
(63, 154).
Expression of exogenous pCLCA1 in transfected
Caco-2 cells produced a ⬃60-kDa product upon Western
blotting with polyclonal antibody raised to an NH2-terminal peptide (C250-K266) of pCLCA1. The processing of
CLCA isoforms is known to vary with expression in different tissues (60, 146). Examples include an ⬃60-kDa
bCLCA1 fragment and ⬃60-kDa pCLCA1 product expressed in epithelial cells, as well as a preliminary report
of a ⬃60-kDa protein contributing to anion conductance
in the rabbit ileum (147, 155).
Similar to other CLCA clones, pCLCA1 amino acid
sequence from V307 to Q462 has extensive homology with
sequences that form a VWA domain (Fig. 3). There is a
perfect noncontiguous consensus sequence for a MIDAS
motif (D313-x-S315-x-S317. . . T384. . . D416) within the
putative pCLCA1 VWA domain, a motif shared with
hCLCA1. Structural modeling of the pCLCA1 MIDAS site
reveals that appropriate folding is permitted, although the
-SH of C387 replaces -OH of T384 as one of the divalent
cation ligands (Fig. 4). Loewen et al. (113) have presented
evidence for a direct regulatory role of calcium in the
modulation of chloride conductance by pCLCA1, but
there is no evidence connecting regulation of pCLCA1 to
Ca2⫹ binding to the MIDAS motif.
Four of the C kinase consensus sequences found
within the predicted cytosolic loop between transmembrane domains 3 and 4 are shared with hCLCA1, while
pCLCA1 has a fifth unique site in this same loop. Also
found in this loop is a strong A-kinase consensus sequence that is unique to pCLCA1 (68). The effect of mutagenesis of this highly phosphorylated loop in the mod-
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1. bCLCA1
D. Porcine pCLCA1
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MATTHEW E. LOEWEN AND GEORGE W. FORSYTH
ulation of chloride conductance by CLCA proteins has not
been reported.
IV. ENDOGENOUS TISSUE EXPRESSION SITES
Combining information about sites of tissue expression and structurally related groups of CLCA proteins
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(Fig. 1) could help to clarify CLCA protein function, and
differences in function for each structural group within
the CLCA gene family. However, it is important to note
that some early studies of tissue localization may have
been less than ortholog-specific. The accuracy of specific
CLCA mRNA or protein localization depends on probe
specificity and the stringency of the reaction conditions.
Current studies addressing temporal ortholog expression
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FIG. 4. Predicted folding of CLCA amino acid
sequence with homology to the von Willebrand A
domain including the metal ion dependent adhesion
site (MIDAS) of the ␣2-integrin collagen receptor.
Predicted folding of pCLCA1 (A) and hCLCA1 (B),
based on the high-resolution crystal structure of the
von Willebrand A domain of the human ␣2-integrin
collagen receptor I domain (C), designated 1DZIA
(51, 52). Structures were created in Swiss Model by
replacing amino acids 5 to 133 of the 185-amino
acid sequence of 1DZIA by amino acids 310 to 428
of pCLCA1 or hCLCA1. 1DZIA sequence added to
CLCA protein was removed after modeling so that
A and B show only CLCA polypeptide backbone
folding. Note the classical Rossmann fold with the
open twisted ␤-sheet surrounded by ␣-helices on
both sides. The surface metal ion coordination site
defines the MIDAS motif. Water molecules which
may contribute to divalent cation binding were excluded in the modeling for this figure. Replacement
of threonine with cysteine in the pCLCA1 MIDAS
motif indicates a higher divalent cation binding potential affinity for this isoform.
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are dealing more carefully with issues of probe specificity
as awareness of CLCA isoforms increases.
A. Human CLCAs
1. hCLCA1
2. hCLCA2
By Northern blotting the highest expression of
hCLCA2 mRNA occurs in the trachea and mammary gland
(80). This result was confirmed independently, and the
confirming group also reported expression in the testis,
prostate, and uterus (4). It has also been found in nasal
epithelium (122). Strong expression occurs in most basal
cells in stratified epithelia (35). Although isolated from a
lung cDNA library, hCLCA2 was not detected in the lung
by Northern blot hybridization, and only a weak RT-PCR
signal for hCLCA2 was obtained from the lung. Others
could not confirm expression in the lung (4).
The hCLCA2 mRNA was highly expressed in a nonmalignant transformed human mammary epithelial cell
line MCF10A using both Northern blotting and RT-PCR.
However, hCLCA2 expression could not be detected by
Northern blotting in tumorigenic cell lines MDA-MB-231,
MDA-MD-468, and MCF7. These findings were in agreement with an in situ hybridization staining of acini and
small ducts in normal mammary tissue, but there was no
staining in breast cancer samples (80).
3. hCLCA3
The third human ortholog, hCLCA3, was originally
cloned from spleen. The mRNA transcript is expressed in
the lung, trachea, mammary gland, and thymus (78). Its
presence has also been reported in nasal epithelium (122).
4. hCLCA4
Unlike the other human orthologs, hCLCA4 is
highly expressed in neural tissue (4). On Northern blot
analysis it is found in the amygdala, caudate nucleus,
Physiol Rev • VOL
B. Murine CLCAs
1. mCLCA1
The strongest expression of mCLCA1 was detected in
the lung, aorta, spleen, and bone marrow using real-time
RT-PCR quantitation normalized against expression of
elongation factor 1a (110). High levels were also reported
in the kidney and skin by Romio et al. (160). Lower levels
occur in many tissues, but expression was not detected in
the prostate or in the pregnant, lactating, and involuting
mammary gland. However, mCLCA1 expression was
found in virgin, pregnant, and lactating gland in an independent study (49). There is agreement that mCLCA1
expression is lost during mammary gland involution.
There is a dynamic change in mCLCA1 expression over
gestation (49, 110) pointing to an undefined role of
mCLCA1 in cell maturation and tissue differentiation.
mCLCA1 is the only CLCA reported in the cerebellum
and brain stem (110). This differs significantly from
hCLCA4, which is expressed extensively in other areas of
the nervous system (4). In the same study, mCLCA2 was
undetectable in cerebellum and brain stem (77). These
reports of temporal and tissue-specific expression patterns within the same physiological system may be hints
of diverse roles for CLCA proteins in tissue development
and functional differentiation.
Given the importance of a calcium-activated chloride
conductance in renal epithelium, it was expected that
CLCA cDNAs related to mCLCA1 and mCLCA2 would be
expressed in a mouse inner medullary collecting tubule
cell line (19). RT-PCR of mouse nephron RNA found
mCLCA1 mRNA in the glomeruli and thick ascending
limb, but not in the proximal tubule and cortical collecting duct (19). Interestingly, a study using a nonspecific
mCLCA1/ mCLCA2 probe identified CLCA in the tubular
epithelial cells but not in the glomeruli. The proximal
tubule had the most intense staining with weaker staining
in the loop of Henle and distal tubuli. The collecting duct
was negative (77). These contradictory findings may reflect probe specificity and primers used in the studies, as
much as they show a difference in the CLCA form expressed at different areas within the kidney.
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The strongest expression of hCLCA1 occurs in mucus-secreting cells of large and small intestine (75). With
Northern blotting the highest expression was in the small
intestine, appendix, and colon, with much lower expression levels in the uterus, stomach, testis, and kidney (4).
Another extensive study using highly stringent in situ
hybridization conditions only detected expression in the
small intestine and colon (75). The majority of the mRNA
signal was in cells at the base of the crypts, with the
goblet cells having the highest expression. Induction of
hCLCA1 expression in the airways has been reported
more recently under pathophysiological conditions (83,
88, 135, 181, 209).
cerebral cortex, frontal lobe, hippocampus, medulla
oblongata, occipital lobe, putamen, substantia nigra,
temporal lobe, thalamus, and accumbens expressed
hCLCA4. The cerebellum and spinal cord did not show
any evidence of hCLCA4 expression. The strongest signal from hCLCA4 came from the colon, with twice the
intensity found in neural tissue. Expression also occurred in the bladder, uterus, prostate, stomach, testis,
salivary gland, mammary gland, small intestine, appendix, and trachea (4).
1070
MATTHEW E. LOEWEN AND GEORGE W. FORSYTH
2. mCLCA2
3. mCLCA3
FIG. 5. Differential expression of CLCA proteins along the villus
crypt axis. Areas of higher expression of each isoform are indicated.
cells often had an intensely stained apical membrane,
with obvious labeling around the mucin granules. An
electron microscopic study localized mCLCA3 antigen to
the peripheral membrane of mucin granules in positively
staining cells. Antigen was not found in the center of the
granule, cytosol, nucleus, other organelles, or along the
basolateral membrane of goblet cells (109). These findings support a connection between mCLCA3 expression
and mucin production.
4. mCLCA4
Mucus-secreting cells are the primary site for expression of mCLCA3, the mouse homolog of hCLCA1 and
pCLCA1 (103, 109). The mCLCA3 was originally cloned
from, and found to have highest expression in, the crypts
of the large intestine (103). An extensive immunohistochemical investigation of mCLCA3 showed exclusive expression in the digestive and respiratory tracts and uterus
(109). The mCLCA3 antigen was not found in the gallbladder, kidney, pancreas, sublingual salivary glands, oviduct,
mammary gland, or prostate (109). Concurrent staining
for mucin and immunohistochemical localization of
mCLCA3 antigen indicated that mCLCA3 was expressed
only in mucin-producing cells, but not in all mucin-producing cells.
A dynamic expression pattern was seen in the crypt
goblet cells in both the large and small intestine, with
expression only in approximately the upper two-thirds of
the crypt, whereas weak, or no, staining was observed in
the basal third (109). It is interesting that the distribution
pattern for gastrointestinal mCLCA3 correlates with the
crypt-villus axis (Fig. 5). CLCA proteins have been promoted as proapoptotic (49), but it is the cell group directed toward apoptotic removal at the base of the crypts
that loses mCLCA3 expression. However, mCLCA1/2 expression may increase as mCLCA3 levels decline and play
some role in apoptosis at the base of the crypts.
The development of a suitable antibody allowed intracellular localization of the mCLCA3 antigen (109). In
simple paraffin-embedded tissue sections, the cytosol was
stained positive with a diffuse granular pattern. Goblet
Physiol Rev • VOL
Cloned from smooth muscle, mCLCA4 was detected
in the gastrointestinal tract, uterus, lung, and heart in a
multiple cDNA array using gene-specific primers. A large
component of mCLCA4 cDNA expression was detected
by RT-PCR in the smooth muscle of the dissected tunica
muscularis of the bladder and stomach (49). In situ hybridization with a mCLCA4-specific probe was performed
to determine cell-specific expression in these mixed organs. This probe produced a very strong signal in the
pulmonary vein, aorta, and atrioventricular bundle with a
much lower level in cardiac muscle, coronary artery, and
endothelium. Both the bronchioles and blood vessels
were labeled in the lung. In the gastrointestinal tract,
labeling was associated more with the mucosa than the
muscularis tunica. This mucosal labeling was most intense towards the villus tip (48). It should be noted that
this is the first CLCA with a reported increase in expression towards the villus tip. mCLCA1 and possibly
mCLCA2 are thought to be primarily expressed in the
crypt (77), and mCLCA3 is associated with upper crypt
and midshaft of the villus (109). These differing expression patterns of each isoform within a tissue apparently associate with major differences in cell physiology. Adipose and connective tissue were consistently negative (48).
5. mCLCA5 and mCLCA6
Mouse homologs of hCLCA2 and hCLCA4 have been
designated as mCLCA5 and mCLCA6 (54). Like its human
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mCLCA1 and mCLCA2 may be splice variants of the
same gene locus (158). High mCLCA2 expression was
reported from pregnancy to involution as determined in
two studies by RT-PCR (49, 115), but the strongest
mCLCA2 expression was detected in the involuting mammary gland (48, 107, 110). mCLCA1/2 expression was
detected in both alveolar and ductal epithelial cells by in
situ hybridization in normal murine mammary tissue (77).
Lower mCLCA2 levels were detected in most tissues
tested except for the brain stem, cerebellum, and prostate
(110). In a real time quantitative RT-PCR study, the involuting mammary gland seemed to express purely
mCLCA2. This was the only tissue screen with absolutely
no mCLCA1 signal (110). The dynamic interplay between
the mCLCA1 and mCLCA2 isoforms suggests that they are
involved in different physiological roles. It is interesting
to note that the highest expression of mCLCA2 seems to
be in those tissues with the high rate of cell division and
cell death (49). A possible connection to cell cycle control
or an apoptotic role for mCLCA2 could be considered for
these tissues.
1071
CLCA PROTEINS
counterpart, mCLCA5 is widely expressed, with eye,
spleen, and lactating mammary tissue notable for strong
expression (14, 54). However, mCLCA6 is expressed in
intestine and stomach, but not in whole brain. In this
respect the human and mouse homologs appear to be
functionally distinct.
C. Bovine CLCAs
1. bCLCA1
2. bCLCA2
The bCLCA2 ortholog is reported to be predominantly a luminal membrane protein of the venular endothelia of the lungs and the spleen. However, antibodies to
bCLCA2 located a strong antigen signal in endothelia of
small to medium size venules as well as in the respiratory
epithelial of the bronchi and trachea (50).
D. Porcine pCLCA1
Immunohistochemistry with the IgM monoclonal antibody used to clone pCLCA1 identified antigen on the
enterocyte border of the villi. Labeling intensity was distributed over the mucosal surface, with the most intense
staining in the mid to upper crypt region (154). In the
trachea, pCLCA1 mRNA expression was localized to surface epithelium and the underlying submucosal glands,
with the most intense staining found in a subset of the
submucosal glands (68). These tissues were also positive
for pCLCA1 expression when tested by RT-PCR. pCLCA1
expression was not detected in the colon. RT-PCR identified pCLCA1 mRNA in the parotid, sublingual, and submandibular salivary glands (68). Other tissues that express a variety of chloride channels including the exocrine pancreas, cardiac and skeletal muscle, liver, and
kidney had no detectable pCLCA1 mRNA.
E. Other CLCAs
Antibodies against a pCLCA1 peptide were used in
immunohistochemistry to identify a CLCA epitope in canine retinal pigment epithelial cells (117). Intense staining
was seen on the apical secretory side of the epithelium. The
Muller cells, which are reported to maintain appropriate
extracellular environment for retinal neurons, had significant cCLCA1 expression. CLCA epitope was also prominent
Physiol Rev • VOL
F. Correlating Tissue Expression Sites
to CLCA Groups
Figure 1 illustrates a connection between two structurally related CLCA groups. The first “group” contains
the cross species homologs, hCLCA1, mCLCA3, and
pCLCA1. The divergent connecting branch contains
hCLCA2 and hCLCA4 and now also mCLCA5 and -6 corresponding to these human forms (14, 54). The second
group contains two subgroups. The first of these includes
mCLCA1/2, which may be alternate splice products from
a single locus (158) and mCLCA4. The second subgroup
contains hCLCA3, bCLCA1, and bCLCA2. Functional similarities within these structural subgroups are probable,
and similarities in cross-species tissue expression sites
within CLCA groups support this hypothesis.
The hCLCA1, mCLCA3, and pCLCA1 forms have a
similar distribution pattern in the three species (Table 1),
concentrated in mucus-producing epithelium in the gastrointestinal and respiratory tracts. This group of CLCA
proteins may participate in mucin secretion and modulation of chloride conductance in secretory epithelial tissues. The members are conspicuously absent from pancreas, smooth muscle, and endothelial cells.
The intermediate hCLCA2 and hCLCA4 forms and
mCLCA5 are more widely distributed than the first group.
The extensive expression of hCLCA4 in neural tissue,
with the exception of the cerebellum, is noteworthy. The
absence of expression in the mucosal surfaces of the
small intestine is another prominent difference between
this and the hCLCA1, mCLCA3, pCLCA1 group. In this
respect, mCLCA6 is more like its mCLCA3 counterpart.
Functions related to tumor cell metastasis and chloride
channel activity have been proposed.
The most striking property of the second cluster of
structures, including mCLCA1/2, mCLCA4, hCLCA3,
bCLCA1, and bCLCA2, is the wide distribution of all of
these forms. Their presence has been reported in most
tissues where their expression has been investigated, although there is limited data for hCLCA3, bCLCA1, and
bCLCA2. Disparate functional roles for this third group
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The bCLCA1 ortholog is primarily expressed on the
brush border of ciliated tracheal epithelial cells (50), the
tissue from which it was originally cloned (39). This expression site is similar to that found for mCLCA1/2 (77).
The expression in other tissues has not been reported.
in the corneal epithelium and in the ciliary body (M. E.
Loewen, B. H. Grahn, and G. W. Forsyth, unpublished data).
Antibodies generated to rCLCA1 reacted extensively
in the rat pancreas. The subcellular location of the antigen
was mainly on the zymogen granules (179). In an interesting aside, hCLCA1 expression was not detected by
RT-PCR in a human pancreatic duct adenocarcinoma cell
line that had an active calcium-dependent chloride conductance (56). This negative finding is consistent with
observations by others that CLCA expression does not
always correlate with the presence of calcium-activated
chloride currents (114).
1072
TABLE
MATTHEW E. LOEWEN AND GEORGE W. FORSYTH
1.
Tissue sites of expression of related CLCA proteins
CLCA Species
Tissue
m3
p1
h2/m5
h4/m6
(4)
(75)
(75)
(75)
-
-
(154)
(154)
(154)
-
-/-/(54)
-/(14)
-/(54)
-/(14)
(4)/(14)
(4)/(54)
-/(54)
(4)
-/(54)
-/-
-
-/(54)
-
-/-
(154)
-
(4)
-/(54)
(122)
(4) (80)
-/-
-/(54)
-/-
(4)/(14)
/(14)
(4)/(14)
(4)
(4)
(80)
-//(54)
(4)/-//-
b1
b2
(50)
(50)
(50)
(50)
(109)
(109)
(103)
-
⫺(83, 88)
-
(109)
-
(4)
(4)
-
(109)
-
(83, 88)
(4)
(4)
(4)
CLCA Species
Tissue
GI tract
Salivary
Stomach
Pancreas
Jejunum
Ileum
Colon
Liver
Kidney
Muscle
Cardiac
Smooth
Lung
Nares
Trachea
Alveoli
Spleen
Mammary gland
Lactating
Involuting
Uterus
Testes
Prostate
Neural
Cerebellum
Other
m1/2
(77, 110)
(77, 110)
(77, 110)
(77, 110)
(77, 110)
(77, 110, 160)
(77, 110, 160)
(110)
(77, 110)
(77, 110)
⫺109
(77, 110)
m4
h3
(48)
(48)
(48)
(48)
(48)
(48)
(48)
m1 (74)
m2 (48)
(77, 110)
⫺, (77, 110)
(122)
(78)
(78)
(78)
(78)
⫺108
⫺76
Reference numbers in parentheses identify the source of the positive expression data. Negative data (unattributed) are designated with a
hyphen. Tests not performed or reported are indicated with unmarked cells.
Physiol Rev • VOL
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GI tract
Salivary gland
Stomach
Pancreas
Jejunum
Ileum
Colon
Liver
Kidney
Muscle
Cardiac
Smooth
Lung
Nares
Trachea
Alveoli
Spleen
Endothelium
Mammary gland
Lactating
Involuting
Uterus
Testes
Prostate
Neural
Other
Cerebellum
RPE
h1
1073
CLCA PROTEINS
include suggestions of smooth muscle chloride channel
modulation (mCLCA4, and by extrapolation, the human
isoform hCLCA3), and possible endothelial adhesion factors bCLCA2, hCLCA2, and mCLCA1. A third function is
suggested by the surface epithelial expression of endothelial adhesion CLCAs in the gastrointestinal tract.
V. FUNCTIONAL RESPONSES TO EXPRESSION
OF CLCA PROTEINS
A. Human CLCAs
Transient expression of hCLCA1 in HEK293 human
embryonic kidney cells increased calcium-activated
whole cell currents from 1.57 ⫾ 0.72 pA/pF in control cells
to 11.06 pA/pF in transfected cells, producing a timeindependent outwardly rectifying current (75). This current was said to be “electrically isolated” for chloride,
based on the blockage of cation current using large bulky
cations, but the chloride dependence of the current was
not determined. These putative chloride currents were
inhibited by DIDS, dithiothreitol (DTT), and niflumic acid,
although DIDS is the only one of these compounds known
to be a specific blocker of chloride channels.
This study also presented the only single-channel
patch-clamp study of the CLCA proteins. Single channels
of hCLCA1-transfected cells had a slope conductance of
13.4 pS in cell-attached conformation after the addition of
ionomycin in the presence of 1 mM Ca2⫹ (75). There was
no mention in the report of endogenous HEK293 channels
responsible for the background of nonselective and anion
currents previously reported in these cells (206, 215).
2. hCLCA2
Transient transfection with hCLCA2 produced a calcium-activated, non time-dependent, outwardly rectifying
increase in whole cell current in HEK293 cells. The whole
cell current increased from 1.52 ⫾ 1.83 pA/pF in control to
10.77 ⫾ 3.8 pA/pF in transfected cells. Once again, neither
the background current in untransfected cells nor the
chloride dependence of the current produced in transfected cells were reported. However, the “electrically isolated” anion current was blocked by DIDS, DTT, niflumic
acid, and tamoxifen (80).
3. hCLCA3 and hCLCA4
There are no current reports of functional ion transport data for hCLCA3 and hCLCA4.
Physiol Rev • VOL
1. mCLCA1
mCLCA1 expression in HEK293 cells caused the appearance of a calcium-activated chloride current. The
current was outwardly rectifying and not time dependent
(67). The current increased from 2.05 ⫾ 1.09 to 10.23
pA/pF upon addition of ionomycin. Background chloride
currents were apparently insignificant in this study, but
the anionic dependence of the current was not determined. Niflumic acid, DTT, and DIDS inhibited the inferred chloride current.
Romio et al. (160) concurrently cloned and expressed
mCLCA1 in Xenopus oocytes. Injection of mCLCA1 cRNA
into oocytes caused a significant increase in current without added calcium ionophore. At ⫺80 mV, the current in
mCLCA1 mRNA-injected oocytes was ⫺398 ⫾ 136 nA
compared with ⫺97 ⫾ 12 nA for water-injected oocytes.
The current in the mCLCA1-injected oocytes, but not in
the water-injected oocytes, was chloride dependent. The
lack of a reversal potential (Erev) shift when bath solution
was changed to low NaCl indicated that the background
oocyte current was a combination of both anion and
cation currents. DIDS and niflumic acid inhibited chloride
current in mCLCA1-injected oocytes and in water-injected
controls. The application of a Ca2⫹ ionophore (ionomycin) caused a transient, statistically insignificant increase
in current to 1,805 ⫾ 473 nA in mCLCA1-expressing oocytes compared with 1,109 ⫾ 389 nA in control oocytes
(160). It was difficult to determine any effect of Ca2⫹ on
the chloride conductance associated with CLCA expression due to the large background currents in Xenopus
oocytes.
Generally mCLCA1 expression in HEK293 cells resulted in a mildly outwardly rectifed, time-independent
current (25, 73). This current requires 2 mM Ca2⫹ in the
pipette and does not increase above control values with
500 nM Ca2⫹. The stimulated mCLCA1 current was most
permeable to SCN⫺ ⬎ Cl⫺ ⬎ isethionate (25), as were the
native Ca2⫹-activated chloride channels in smooth muscle
from which mCLCA1 was cloned. The anion dependence
of the whole cell current in control HEK293 cells was not
assessed.
The mCLCA1-dependent calcium-stimulated current
changed significantly from time independent to time dependent, as well as increasing the total whole cell conductance when coexpressed with a potassium channel
␤-subunit (73). Coexpression also resulted in a greater
sensitivity to activation by calcium. Addition of 500 nM
Ca2⫹ in the pipette solution could stimulate the anion
current in ␤-subunit mCLCA1 expressing cells. This difference in current and agonist sensitivity was shown by a
mammalian two-hybrid system to be the result of a direct
interaction between the potassium channel ␤-subunit and
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1. hCLCA1
B. Mouse CLCAs
1074
MATTHEW E. LOEWEN AND GEORGE W. FORSYTH
mCLCA1. Unfortunately, the anionic dependence of the
current was only assessed through the permeability ratios
for SCN⫺ and Cl⫺, which were consistent with those
found in smooth muscle. Again, as with several other
CLCA studies, the background currents were not reported
(73). This study by Greenwood et al. was the first report
suggesting that a CLCA protein requires coexpression
with other proteins to produce functional chloride channels and that the currents induced on CLCA1 expression
could be modified by an accessory protein.
consistent with other reports that are difficult to reconcile
with channel regulation at intracellular calcium concentrations. In contrast to normal mammary epithelial cells,
metastatic mammary tumor cells express little or no
mCLCA5 upon starvation or detachment. Transfection of
a mammary tumor cell line with mCLCA5 caused a dramatic reduction in colony formation by transfected tumor
cells (14).
C. Bovine CLCAs
2. mCLCA2
3. mCLCA3
The effects of mCLCA3 or Gob 5 expression have
been characterized briefly in HEK293 cells (203). mCLCA3
expression increased an outwardly rectifying current
without agonist addition. Current densities at ⫹60 mV
increased from 59 ⫾ 17 to 230 ⫾ 47 pA/pF. Addition of 10
mM EGTA to the pipette solution significantly decreased
these currents (203).
4. mCLCA4
Treating transient mCLCA4-transfected HEK293 cells
held at an undefined controlled voltage with ionomycin or
methacholine increased intracellular calcium and evoked
transient inward currents (48). This effect would be consistent with the inward movement of cations or the outward movement of anions. In this case, a strong anionic
dependence to the stimulated current was shown by a
shift in the current-voltage relationship to the equilibrium
potential of chloride when bath solution was switched to
low chloride after ionomycin addition. Unlike previously
characterized isoforms, the current was found to be nonrectifying. The transient nature of the current was thought
to be analogous to the Ca2⫹-independent inactivation of
the smooth muscle Ca2⫹-activated chloride channel. This
inward current was found to be spontaneous, similar to
those in smooth muscle cells that are induced by a “calcium spark” and are involved in buffering the membrane
potential, relaxation, and spontaneous rhythmic contraction of smooth muscle (92, 136). Current-voltage relationships in nontransfected cells were not examined, as voltage-clamped inward currents were not seen (48).
5. mCLCA5 and 6
Expression of these isoforms caused an ionomycindependent increase in whole cell current in HEK293 cells
(54). Chloride involvement was verified by reversal potential measurements. A requirement for 2 mM calcium in the
pipette to induce these increases in chloride current was
Physiol Rev • VOL
1. bCLCA1
Oocytes transfected with bCLCA1 cRNA had a significantly larger current than water-injected control oocytes
(39). bCLCA1 expression apparently increased an endogenous outwardly rectifying current. This current was sensitive to DIDS and DTT but insensitive to niflumic acid.
Addition of 1 ␮M ionomycin to oocytes resulted in the
activation of endogenous chloride channels which were
inhibited by niflumic acid. Unfortunately, data showing
the effect of the ionophore on bCLCA1-injected oocytes
were not presented or discussed (39).
A nonrectifying, Ca2⫹-activated, DTT-inhibitable current was produced when bCLCA1 was expressed in COS-7
cells (39). This linear current observed after bCLCA1
expression was unlike the outwardly rectified currents
seen with expression of most of the other CLCA family
members. However, no other family members have been
characterized using COS-7 cells. It is interesting to note
that the currents induced in the oocytes had a both timedependent and rectifying quality, whereas those induced
in the COS-7 cells had neither (39).
Lipid bilayers constructed of membranes from oocytes that had been injected with bCLCA1 cRNA had a
unit channel conductance of 21 pS (39). The open probability increased from 0.41 ⫾ 0.07 to 0.60 ⫾ 0.08 in the
presence of Ca2⫹ added only to the side opposite to that
where DIDS was added. The channel was inhibited by
DIDS and DTT, but insensitive to niflumic acid. The channel had an 8:1 anion to cation selectivity ratio and 3:1
selectivity for iodide over chloride under bionic conditions. The biophysical properties were similar to those
found for a purified chloride channel from bovine trachea.
However, some bilayers had a Ca2⫹-activated chloride
conductance that was inhibited by niflumic acid. This was
said to be the endogenous Ca2⫹-activated chloride channel of the oocyte. Others using bCLCA1 as a control for
mCLCA1 characterization found the bCLCA1-induced current was inhibited by 78% with niflumic acid. Subsequent
addition of DIDS (100 ␮M) caused a further 33% inhibition
of the anion current (160). The basis for these differences
is not clear.
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There are no published electrophysiological data on
expressed mCLCA2.
1075
CLCA PROTEINS
sion system influences the properties of the chloride
channels observed upon CLCA expression.
D. Porcine pCLCA1
Physiol Rev • VOL
E. Other CLCAs
A rCLCA1 isoform indirectly implicated in bicarbonate transport and vesicle exocytosis in rat pancreas and a
canine cCLCA1 isoform (117) produced in secretory retinal pigment epithelium have not been fully cloned or
functionally characterized.
VI. PATHOPHYSIOLOGICAL CONNECTIONS
TO CLCA EXPRESSION
A. CLCA in Asthma
Orthologs of the CLCA gene family are overexpressed in bronchial allergic asthmatic responses that
overproduce mucus (88, 135, 181). There is significant
evidence that the CLCA proteins have a causal role in this
condition, suggesting that these proteins may have some
interest as targets for pharmacological intervention in
allergic asthma.
1. Asthma and the genesis of its mediators
Bronchial allergic asthma is a serious inflammatory
condition of the airways resulting in bronchial narrowing,
constriction, and overproduction of mucus (27, 181). Simplistically, the uncontrolled inflammatory response, accompanied by the overexpression of CLCA protein, is
thought to occur through a dysregulation between type 1
helper T (Th1) and type 2 helper T (Th2) cell immune
responses to allergens (10, 27, 47, 90, 134, 159, 192). The
Th1-immune response involving interleukin (IL)-2 and interferon (IFN)-␥ cytokines is seen as a cell-mediated response associated with disease in situations of poor hygiene or infection with Mycobacterium tuberculosis, measles virus, or hepatitis A virus (21, 127, 130, 167, 171). The
Th2 disease response involving a different set of cytokines (IL-4, -5, -6, -9, and -13) is considered to be humoral,
involving the stimulation of B cells and the production of
IgE. Released IgE binds to IgE receptors on mast cells,
lymphocytes, eosinophils, platelets, and macrophages.
Binding of allergen to the IgE results in the release of
inflammatory mediators from mast cells and activation
and potentiation of an inflammatory response (27).
Proper priming of the immune system to different
immunological challenges is essential to develop a normal
Th1 response. The Th1 response then has a role in modulating the Th2 response. The asthma disease state is
dominated by an uncontrolled Th2 response, and blockade of the Th2 cytokine pathways significantly dampens
the asthmatic response (26, 57).
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pCLCA1 expression in NIH/3T3 cells increased a calcium-activated 36Cl⫺ efflux (68, 115, 117). Agonists for
calcium-dependent protein kinase (PKC) or cAMP-dependent protein kinase (PKA) had no effect. However, application of 10 ␮M ionomycin significantly increased
36 ⫺
Cl efflux from transfected cells. Inhibition of this effect
by membrane-permeable Ca2⫹ chelator, combined with
the lack of inhibition by the CaMKII inhibitor KN-93,
suggested that Ca2⫹ may directly regulate a pCLCA1 effect on chloride transport. The pCLCA1 effect on chloride
efflux was inhibited by 5-nitro-2-(3-phenylpropyl-amino)benzoate (NPPB), glibenclamide, diphenylamine carboxylate (DPC), and ␣-phenylcinnamate (␣-PC). NPPB and
DPC inhibited the Ca2⫹-activated chloride efflux at much
lower concentrations than were required for inhibition of
CFTR. NPPB was effective at a concentration similar to
that which was required to inhibit Ca2⫹-activated chloride
conductance in Xenopus oocytes (115).
In whole cell patch clamp, pCLCA1 increased an
endogenous Ca2⫹-activated chloride conductance, making the current more anion dependent (115). The pCLCA1
chloride current was outwardly rectifying and time dependent. This time dependence of chloride currents in
NIH/3T3 fibroblasts has not been reported for other CLCA
proteins, suggesting contributions by the expression system to the chloride current. As with 36Cl⫺ efflux, DIDS did
not inhibit whole cell chloride current, but NPPB, DPC,
and ␣-PC were inhibitory (115). Both DTT and DIDS were
reported to inhibit chloride current activity in previous
studies with different CLCA proteins and different expression systems (39, 67, 75), but did not inhibit effects of
pCLCA1 in 3T3 cells. In contrast to the fibroblast expression system (113), transfection of an epithelial Caco-2
human colon carcinoma cells with pCLCA1 produced a
cAMP-activated chloride conductance, possibly through
effects on CFTR (113). The pCLCA1 and PKA-dependent
currents were anion dependent, time independent, and
nonrectifying. The effects of pCLCA1 expression on PKAdependent chloride conductance were evident in freshly
passaged and in differentiated epithelial cells, both of
which have endogenous CFTR-mediated chloride conductance. This modulation of chloride conductance by
pCLCA1 expression was also seen in nonepithelial NIH/
3T3 fibroblasts coexpressing CFTR and pCLCA1 (117).
Expression of pCLCA1 changed the chloride conductance activated by calcium ionophore or PKA agonists in
Caco-2 epithelial cells to a time-dependent outwardly rectifying current (114). However, the endogenous Ca2⫹activated chloride conductance is lost as Caco-2 cells
mature, and the ability of pCLCA1 to activate Ca2⫹-dependent chloride conductance also disappears (114). Again,
the endogenous chloride channel activity in the expres-
1076
MATTHEW E. LOEWEN AND GEORGE W. FORSYTH
2. Th2 cytokines mediate CLCA expression
FIG. 6. Hypothetical roles for CLCA protein in the asthmatic airway. Increased expression of CLCA is presumably cytokine-driven. CLCA could
then 1) increase the transcription or translation of MUC genes, 2) increase the transport of mucin to the plasma membrane by increasing the
condensation (packaging process) by acidification of the mucin granule, 3) take part in the membrane fusing process by interaction with the SNARE
apparatus, and 4) facilitate the ion transport component of the exocytosis process. CLCA protein may also have a role in increasing net ion/fluid
transport (increasing chloride conductance and decreasing sodium absorption) to the inflamed epithelium to increase mucus transport up the
mucociliary ladder.
Physiol Rev • VOL
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The Th2 response and the associated cytokines IL-9,
IL-4 and IL-13 appear to be directly linked to CLCA overexpression in the asthmatic patient, and in mouse models
of asthma (209). However, instilled Th1 cytokine INF-␥
did not induce expression of mCLCA3. mCLCA3 synthesis
was induced in the lung and bronchi in an allergic asthma
mouse model of ovalbumin sensitization, and transfection
with an adenoviral mCLCA3 antisense construct prevented airway hyperresponsiveness (AHR) and mucus
production after ovalbumin challenge (135). Application
of a sense mCLCA3 mRNA construct to this same model
produced severe AHR and mucus secretion (135). In vitro
overexpression of mCLCA3 or its closest human ortholog
hCLCA1 in human mucoepidermoid cells (NCI-H292) increased mucus secretion and overexpression of the major
secretory mucin gene of asthma, MUC5AC (88). An increase in hCLCA1 correlated with an increase in mucus
and IL-9 receptor in asthmatic patients (Fig. 6). This relationship suggests that hCLCA1 might be a therapeutic
target in asthma patients.
Exposure of human bronchial epithelial cells to Th2
cytokines, IL-13 or -4, greatly increases the magnitude and
duration of activated Ca2⫹-dependent chloride conductance (12, 40). This group of cytokines tends to switch
epithelial transport from net absorption to a secretory
phenotype by decreasing sodium absorption and increasing apical chloride conductance. Inflammation may trigger this phenotypic switch to help flush particulates and
secreted mucus out of the airway. Pseudohypoaldosteronism is an excellent model for this strategy, where increasing net secretion by decreasing absorption significantly
increases the rate of mucociliary clearance (18, 40, 97).
Epithelial Ca2⫹-activated chloride conductance increases in AHR. The pharmacological sensitivity of this
induced chloride conductance is similar to that reported
for CLCA-stimulated conductance (11, 40). There is no
evidence that CLCA induction is responsible for the Th2
cytokine-mediated increase in Ca2⫹-activated chloride
conductance, but CLCA could have a dual role to connect
and coordinate fluid and mucus secretion.
CLCA proteins increase the activity of Ca2⫹-dependent chloride channels, and inhibiting CLCA expression
inhibits mucus production (135), but it is not clear how
these proteins increase mucus secretion. The identification of mCLCA3 expression in mucin granule membranes
of the respiratory and gastrointestinal tract implicates this
protein in mucin storage or release (109). It was suggested that mCLCA3 is needed for mucin maturation.
Negatively charged mucus glycoproteins require strong
acidification for mucin condensation in the granules.
Granule acidification could occur via a H⫹-ATPase with
electroneutrality maintained by a parallel CLCA-modulated chloride conductance (53, 109, 178, 190). Potassium
release and Ca2⫹ entry into the vesicles may also be
important in the condensation process (53, 178).
CLCA proteins could also participate in mucus secretion by promoting rehydration of condensed mucins or by
1077
CLCA PROTEINS
B. CLCA Function in Cystic Fibrosis
Cystic fibrosis (CF) disease arising from problems
with the CFTR protein leads to dysregulation of epithelial
ion transport affecting most secretory epithelial tissues
in the body (128, 129). CLCA expression in secretory
epithelial cells and its function as a modulator of chloride conductance makes it a secondary focal point,
after CFTR, in investigating possible compensatory
mechanisms for defective epithelial ion transport (68,
113–115, 117).
electrochemical gradient created by the 3Na⫹-2K⫹ATPase, making the epithelium absorptive. Chloride then
follows the absorptive sodium movement across the cell
(200, 201).
D. CLCA in CF Epithelium
The CLCA orthologs have a modulatory role on the
normal transepithelial sodium chloride transport, which
is defective in CF disease (113–117, 128, 129). The genetic
abnormality in transport is caused by mutations in the
CFTR. CFTR is the major apical chloride transporting
channel of epithelium (Fig. 7), and it downregulates the
activity of the apical absorptive sodium channel in epithelium (ENaC) (105, 172). The most common mutation in
CFTR, the deletion of phenylalanine at position 508
(F508) (96, 157), can function normally, but ⌬F508 CFTR
transport through the endoplasmic reticulum (ER) and
Golgi to the cytoplasmic membrane is interrupted (30).
Fewer CFTR molecules at the apical membrane of secretory tracheal epithelial cells decrease fluid secretion and
also indirectly increase salt and fluid absorption via the
loss of normal negative effects of CFTR on sodium absorption by ENaC. The resulting reductions in the airway
surface liquid volume (37, 129) increase the viscosity of
airway mucus, decrease mucociliary clearance, and favor
bacterial colonization of the airway surface (119, 150).
C. CLCA in the Normal Epithelium
Ion gradients produced by the basolateral 3Na⫹-2K⫹ATPase provide the electrical driving force for the chloride transport that is modulated by CLCA proteins (101,
102, 111). The inwardly directed Na⫹ gradient produced
by this vectoral cation transport drives the coupled electroneutral entry of Na⫹, K⫹, and Cl⫺ into the cell through
the basolaterally located Na⫹-K⫹-2Cl⫺ cotransporter
(141, 142). The charge separation provided by the stoichiometry of the 3Na⫹-2K⫹-ATPase and the basolateral K⫹
conductance lowers the resting membrane potential, creating a driving force for chloride to exit the epithelial cell
through CLCA-modulated anion conductance channels in
the apical membrane (36, 111, 113–117). Sodium exit
through the tight junctions maintains electrical neutrality,
and water follows, resulting in a secretory epithelium.
However, a decrease in apical chloride conductance can
cause sodium to be drawn back into the cell by the
Physiol Rev • VOL
FIG. 7. Possible roles by which a CLCA protein (pCLCA1) could
contribute to epithelial ion transport in the cystic fibrosis (CF) patient.
Modulation of apical chloride conductance by CLCA protein interactions
may have beneficial effects on ion transport in the CF patient. Beneficial
mechanisms would occur through increasing the activity of CaCC or
promoting proper CF transmembrane conductance regulator trafficking
or function.
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connecting synaptobrevin mucin granule docking proteins with the T-SNARE (syntaxin and SNAP-25) plasma
membrane proteins of exocytosis (131, 145). SNARE machinery permits fusing granules to form a pore through
the cytoplasmic membrane. Rehydration of stored mucus
by influx of extracellular solution through the pore, as
well as by controlled ion and fluid flux through the granule membrane from the cytosol, is required for mucin
release through this pore (178). CLCA protein could control the ion transport required for mucin rehydration
through the vesicle membrane during exocytosis (Fig. 6).
Putative inhibitors of chloride conductance associated with CLCA protein expression can inhibit mucus
secretion (210). However, on closer examination, niflumic
acid inhibited Ca2⫹ entry into the epithelial cells, independent of a chloride channel blockage effect (16). Calcium ion entry is central to the process of mucin exocytosis, triggering activation of the SNARE complex (16).
The role of CLCA protein in controlling mucin secretion
remains obscure, but answers may come from a better
understanding of chloride channel modulation by CLCA
proteins.
1078
MATTHEW E. LOEWEN AND GEORGE W. FORSYTH
for intravascular tumor growth followed by metastatic
tissue invasion (1–3, 211–213).
1. Effects of CLCA overexpression on the cell cycle
Aberrant morphology of cells overexpressing mCLCA2
has been interpreted as evidence for modulation of the
cell cycle by CLCA. This morphologic effect is reported to
require relatively high levels of mCLCA2 expression (49).
Inappropriate amounts or timing of CLCA expression affecting chloride channel activity were assumed to disrupt the intricate depolarization and polarization accompanying the cell cycle. The overexpressing cells tended
to be larger and multinucleated, as a probable consequence of abnormalities in cell cycle control (49).
Blockage or partial arrest at various points in the cell
cycle can disrupt cellular pathways leading to the production of multinuclear giant cells (133). However, specific cell cycle studies with mCLCA2 expression have
not been reported.
2. CLCA suppression of tumor cell cycling
There is a growing body of evidence showing that the
activity of anion channels that are modulated by CLCA, as
well as independent cation channels, have a significant
impact on cell cycle control (98, 99, 113, 115, 121, 204).
Blockage of potassium conductance reduces cellular proliferation and causes synchronization at G0/G1 of the cell
cycle in several cell types (43, 121, 139, 193, 204, 205).
Decreased chloride conductance causes the opposite effect, increasing cell proliferation and protecting cells
from apoptotic death (42, 99, 202, 204). Cell cycle-specific
chloride currents are highest during G1 and lowest during
S phase (188, 191). mCLCA2 could contribute to this
current by stimulating chloride conductance. Then downregulation or loss of mCLCA2 expression or activity could
be a feature of tumorigenic cell lines, and restoring
mCLCA2 expression or activity could be a novel strategic
approach to suppressing the growth of transformed cells
(49, 79, 98, 99).
VII. CLCA PROTEINS IN ONCOLOGY
3. Apoptosis in CLCA tumor suppression
A. Cell Cycle Control
Orthologs of the CLCA gene family apparently play
two seemingly contradictory roles in cell division and
metastasis. CLCA functions as a tumor suppressor within
the transformed cell, possibly by modulating ion transport. Expression of CLCA protein is frequently lost in
tumor cell lines (49, 79, 112). However, tumor cells that
have lost CLCA expression can bind endothelial-expressed CLCA via a novel endothelial CLCA/␤4-integrin
interaction. This tumor cell adhesion to endothelium can
lead to intravascular arrest, promoting a mitogenic signal
Physiol Rev • VOL
The mechanism for these proposed tumor suppressive effects of CLCA proteins is not understood, but it has
been suggested that mCLCA2 could suppress tumor
growth through proapoptotic effects (79). Transfection of
the mammary epithelial cell line HC11 with mCLCA2 significantly increased the rate of apoptosis in cultures subjected to serum starvation compared with serum-starved
control cells (49). Selection for resistance to detachmentinduced apoptosis (anoikis) in the normally nontumorigenic HC11 cell line was accompanied by the loss of
mCLCA1 message and mCLCA2 was assumed to be aberrantly spliced and not functional. These selected cells
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The activity of alternative chloride channels in the
apical membrane of CF epithelium could compensate for
the lack of CFTR. CLCA could increase the activity of
such an alternative channel given its effects on both
cAMP- and Ca2⫹-activated chloride conductance (75, 113–
115, 117). This situation may occur in the CFTR knockout
mouse where upregulation of a Ca2⫹-activated chloride
channel appears to protect against lung pathology (62, 74,
169, 177, 180). Human CF patients also have this Ca2⫹activated chloride channel, but it is apparently unable to
adapt sufficiently to maintain normal airway surface layer
(ASL) hydration and prevent disease (9, 20, 199). Activation of this channel through purinergic receptors has been
explored in CF patients (34, 207). There may be a degree
of natural compensation for defective CFTR, as both
hCLCA1 (83, 84) and Ca2⫹-activated chloride conductance (9, 20, 34, 199) increase in CF airway. CLCA proteins have been shown to increase both endogenous
Ca2⫹-activated and cAMP-activated chloride conductance
(113, 114), although the cAMP-dependent response may
require functional CFTR (Fig. 7). However, activation of a
Ca2⫹-dependent chloride conductance does not replace
the inhibitory effects of CFTR on ENaC, and persistent
sodium absorption aggravates ASL dehydration (37, 129).
The involvement of hCLCA1 in mucus secretion is a
concern when considering attempts to upregulate CLCA
activity in CF patients. Elevations in hCLCA1 expression
in CF patients could contribute to mucus overproduction
and airway obstruction (84). Parallels have been drawn
between elevated levels of hCLCA1 expression in AHR in
asthmatic patients and significantly increased hCLCA1
levels associated with the inflammatory processes of CF
(83). Agents such as niflumic acid may be clinically beneficial through reduction of mucus production (94), although these effects of niflumic acid may not be to inhibit
the chloride conductance of hCLCA1. Future efforts could
be directed toward differentiating beneficial CLCA effects
on net fluid secretion from less desirable stimulation of
mucus secretion (16).
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CLCA PROTEINS
4. A mechanism for CLCA proapoptotic effects
The preceding information suggests that CLCA protein expression can slow cell division through effects on
anion conduction. A number of reports have shown that
blockage of chloride conductance increases cell proliferation and confers protection from apoptotic death (71, 99,
174, 204). Generally, there is an influx of calcium in the
initial stages of apoptosis which activates both potassium
and chloride channels (143). One possible apoptotic scenario would have increasing potassium conductance hyperpolarizing the cell, increasing the driving force for
anion conductance through the already open Ca2⫹-activated chloride channel. Then intracellular pH could be
lowered by bicarbonate exiting through the anion-conducting channel. A reduction in the intracellular pH would
increase the activity of caspase 3, cause destruction of
DNA, and trigger an apoptotic cascade (165). Expression
of CLCA could activate anion conductance to stimulate
the exit of bicarbonate or chloride, resulting in a larger
number of cells entering the apoptosis cascade (Fig. 7).
5. Loss of CLCA in tumorigenic cell lines
Expression of mCLCA2, hCLCA2, or its mouse counterpart mCLCA5 has a significant impact on in vivo and in
vitro growth of tumor cells (14, 49, 79). Nontumorigenic
breast epithelial cells (MCF10 A and MDA-MB-453) express hCLCA2, but transformed tumorigenic cells lines
(MDA-MB-231, MDA-MB-435, MDA-MB-468, and MCF7)
do not (79). Hypermethylation of the hCLCA2 promoter
may cause this transcriptional downregulation in tumoriPhysiol Rev • VOL
genic cell lines, inviting speculation that hCLCA2 is the
1p31 breast cancer tumor suppressor gene (112).
mCLCA1 is also normally expressed in mammary tissue,
and mCLCA1 transcripts were not found in tumor cells,
suggesting disrupted expression at the promoter level in
the tumor (49, 112). Abnormal RNA processing may also
disrupt CLCA2 expression (79, 112). Intron/exon junction
information in mCLCA2 transcripts identified PCR products of mCLCA2 cDNA from JC and CSML-0 adenocarcinoma cell lines as splice variants (79).
Transfecting tumorigenic MDA-MB-435 and MDAMB-231 cells with CLCA2 under the control of a foreign
promotor resistant to inhibitory hypermethylation did not
change growth rate or anchorage-independent growth of
these cells in soft and hard agar. Potential invasiveness,
measured as in vitro cell migration through 8-␮m pore
polycarbonate membranes or Matrigel films, was reduced
upon CLCA2 transfection of tumorigenic MDA-MB-435
and MDA-MB-231 cells (79). In either subcutaneous or
intravenous injections into nude mice, mCLCA2-transfected MDA-MB1–231 cells produced fewer tumors, reduced tumor size, or no lung tumors compared with control MDA-MB-231 inoculates (79). Without changes in
growth rate or anchorage-independent growth, these intriguing in vitro decreases in invasiveness and migration
do not totally account for the loss of tumorigenicity in this
study. Undetermined phenotypic effects of CLCA in relation of cell differentiation remain to be defined.
The relationship between loss of hCLCA2 expression
and tumorigenicity has been confirmed, providing evidence that hCLCA2 is the product of the hitherto unidentified 1p31 breast cancer tumor suppressor gene (112).
The importance of the 1p31 locus is illustrated by the
loss of heterozygosity in this region in 60% of breast
tumors (87).
B. Cell Adhesion and Tumor Metastases
1. CLCA and cell adhesion
The bCLCA2 (Lu-ECAM-1) ortholog was cloned using
antibody that blocked the adhesion-receptor/ligand pair
that mediates binding of lung metastatic melanoma cells
to bovine aortic endothelial cells (50, 70, 212, 214). The
origins of the clone identify bCLCA2 as the endothelial
adhesion molecule that binds hematogeneous tumor cells,
causing vascular arrest before tumor growth and invasion. hCLCA2 and mCLCA5 may be counterparts to the
original bCLCA2 (Lu-ECAM-1) clone (2). Like bCLCA2,
hCLCA2 is expressed by endothelial cells from different
lung vascular compartments (2, 213). This expression is
critical for the establishment and colonization of human
breast cancer cell lines (2).
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were then tumorigenic when injected into mice (49). Apparently mCLCA2 expression does not change growth
rate or anchorage-independent growth in culture conditions where the cellular environment does not favor apoptosis, but the presence of hCLCA2 may create a permissive environment or tip the balance towards apoptosis in
cells receiving an apoptotic signal.
Stable mCLCA1 or mCLCA2 transfectants selected
and maintained with G418 grew normally (79), but transfected cells selected by resistance to Zeocin grew slowly,
and died within a 4-wk period (49). This difference may be
due to the distinct mechanisms of the selective drugs.
Geneticin (G418) binds to the 80S ribosome and prevents
protein synthesis in eukaryotic cells without directly inducing apoptosis. Zeocin causes cell death by intercalating into, and cleaving, DNA (69, 132). This type of stress
activates the p53 apoptotic pathway and, at higher doses,
independent pathways. This means that Zeocin-selected
but not G418-selected cells are always fighting apoptosis.
Combining this stress with proapoptotic effects of
hCLCA2 could account for poor growth and increased
apoptosis seen with Zeocin and not G418.
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MATTHEW E. LOEWEN AND GEORGE W. FORSYTH
2. CLCA binds lung colonizing cell lines in vitro
3. ␤4-Integrin expression, CLCA binding,
and tumor metastasis
A coimmunoprecipitation strategy followed by specific antibody screening identified ␤4-integrin from the
MDA-MB-231 cells as the protein that was coprecipitated
with hCLCA2 (2). There was a strong correlation between
the level of ␤4-integrin expression and the adhesion of
HEK293 cells expressing hCLCA2 in studies involving
additional cancer cells lines expressing different levels of
␤4-integrin. A similar correlation was observed in vivo
between ␤4-integrin expression in tumorigenic cell lines
and the number of lung metastases observed in nude mice
given the different cell lines.
Overexpression of ␤4-integrin in a Kirsten-Ras-transformed Balb/3T3 oncogenic cell line caused an increase in
metastatic performance in syngeneic mice that paralleled
an increase in adhesion of these cells to mCLCA1 (2). The
locations of the metastases were consistent with reported
sites of mCLCA1 expression in the mouse. In addition,
cDNA arrays have shown a definitive increase in ␤4-integrin with increased metastatic potential (2, 182). These
findings support a role for ␤4-integrin binding to CLCA
protein in hematogeneous metastasis.
Antibodies to either ␤4-integrin or hCLCA2 were able
to inhibit binding of the hCLCA2-expressing HEK293 cells
to the MDA-MB-231 cell line. Antibodies directed to integrins other than ␤4 did not reduce binding, but specific
cleavage of cell surface ␤4-integrin with matrilysin greatly
reduced binding. Concurrent injection of nude mice with
antibodies to hCLCA2 and ␤4-integrin along with MDAMB-231 cells reduced lung metastases by 84 and 100% (2).
Nontumorigenic cell lines selected for ␤4 expression
or transfected to add ␤4 expression had increased binding
to CLCA but did not increase lung colonization. The metastatically incompetent MDA-MB-468 cell line has a relatively normal growth phenotype in vitro, and slow adenomatous growth in vivo. Moderate levels of ␤4 overexpression in this cell line did not correlate with a lack of in vivo
metastases for these cells. Apparently, ␤4-integrin effects
Physiol Rev • VOL
4. CLCA binding domains
Specific motifs in both the 90- and 35-kDa fragments
of most CLCA family members are involved in the association with ␤4-integrin (2). These binding motifs belong
to the addressins, which are distinct vascular addresses
used by both immune and blood-borne cancer cells to
colonize specific organs (162). It is believed that cell
binding in lung metastatic cancer is particularly mediated through binding of the ␤4-integrin to the CLCA
addressin (1).
Antibodies that blocked ␤4/CLCA adhesion identified
a candidate binding area that would be in the proposed
second extracellular domain of hCLCA2, bCLCA2, and
mCLCA1 (67, 80). Amino acids 479 – 488 and 740 –749 that
could account for the binding activity in hCLCA2 were
identified in a computer-aided motif search (1). Motifs
with a consensus sequence F(S/N)R(I/L/V)(S/T)S occur in
bCLCA2, mCLCA1 (1), and mCLCA5 (54). When expressed and purified, the motifs in the 90-kDa and the
35-kDa fragments bound to ␤4-integrin, but not to ␤1, ␤3,
fibronectin, or BSA. Adhesion increased with concentration of ligand and was Mn2⫹ but not Mg2⫹ or Ca2⫹ dependent. These purified binding motifs also bound to lung
metastatic MDA-MB-231 cancer cells. Incubation of tumorigenic cells with purified binding motifs before intravenous injection of these cells into SCID/beige mice prevented binding and blocked lung colonization (1).
The ␤4-integrin binding domain in hCLCA2 is present
in the hCLCA1 35-kDa segment, but is disrupted by the
substitution of amino acids G473 A474 for SR in the
hCLCA1 90-kDa segment. The FGALSS sequence in
hCLCA1 and pCLCA1 or FGALAS in mCLCA6 lacks ␤4-
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Only hematogeneous lung colonizing tumorigenic
cells were able to bind to hCLCA2-expressing HEK293
cell lines (2). The MDA-MB-231 cell line, which colonizes
the lungs of nude mice following intravenous injection,
bound to a hCLCA2 expressing monolayer of HEK293
cells at a 75% efficiency. Cell lines that form metastases
after orthotopic tumor xenografts but are unable to form
metastasis on intravenous injection (MDA-MB-435) or
nontumorigenic MCF7 cells only bound at ⬃4 and 7%,
respectively, to hCLCA2-expressing HEK293 monolayers
(2). This evidence suggests that binding of MDA-MB-231
cells to endothelial CLCA is necessary for hematogeneous
metastasis and lung colonization.
on metastasis are somewhat cell type dependent, and
␤4-integrin expression leads to lung metatasis only in
those cancer cells that have the genotype of overexpression combined with altered expression of the appropriate
transforming genes (2).
Overexpressed ␤4-integrin in the nontumorigenic cell
line may be interacting with an unidentified membrane
protein, as ␤4- and ␣6-integrins coimmunoprecipited from
the metastatic MDA-MB-231 cell line but not from the
nonmetastatic MDA-MB-435 cells. When this cell line was
transfected with, and overexpressed ␤4-integrin (MDAMB-435 ␤4), it had an increased adherence to hCLCA2 in
vivo, but was unable to produce metastatic lung colonies.
Failure to coimmunoprecipitate ␤4- and ␣6-integrins may
be due to ␤4-integrin interacting with something other
than its normal ␣6 counterpart in the MDA-MB-435 ␤4cells. The unidentified protein could have a dominant
negative effect on metastasis. It appears that the ␤4-integrin can cause endothelial binding, but that binding to
some target of the ␣6- and ␤4-interaction pair promotes
metastasis (2).
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CLCA PROTEINS
5. Novel ␤4-integrin binding domain
The binding of CLCA to the ␤4-integrin is mediated
through a novel domain in the ␤4-integrin (1, 2). Binding
to the specificity-determining loop (SDL) was predicted,
as this is a nonconserved loop sequence that has been
associated before with ligand binding (151, 175). A short
purified fusion protein of ␤4 segments corresponding to
the predicted loop of ␤1- and ␤3-integrins, found previously to be important in integrin binding, was constructed
and tested to determine the binding domain (175). An area
in the ␤4 SDL sequence (amino acids 184 –203) bound to
CLCA in ELISA and pull-down assays. The same sequence
from the ␤1-integrin did not bind. The relevance of the
binding to hCLCA2 was confirmed by using the synthetic
peptide sequence of the ␤4 loop to block metastatic cell
line adhesion of CLCA protein (1).
The binding of the ␤4-integrin to laminin-5 has been
attributed to a binding site at the NH2 terminus of the SDL
loop as determined by point mutations K177A and Q182L
(186). However, as described above, the ␤4-integrin to
CLCA binding appears to involve the COOH-terminal part
of the SDL (1). Hence, it is likely that laminin-5 and
hCLCA2 interact with different SDL binding motifs. This
difference in interaction may explain the difference in the
mitogenic pathways activated by ␤4-integrin when binding
to CLCA or laminin.
6. Response to CLCA/␤4-integrin binding
As previously discussed, the binding of ␤4-integrin to
hCLCA2 is important for cell adhesion in the beginning
stages of lung metastasis (2, 211). Once adhered, the
tumor cell can either extravasate early, resulting in immediate tissue invasion, or it can undergo intravascular
growth. Extensive intravascular growth leads to occupation of the entire circumference of the vessel lumen by
tumor cells, disintegration of the vessel wall, and invasion
Physiol Rev • VOL
of the adjacent tissue by the tumor (106, 189). It is the
␤4-integrin/CLCA binding that provides the mitogenic
stimulation important for intravascular growth (2, 3).
7. A novel ␤4 mitogenic signaling pathway
A model for the role of ␤4-integrin in a mitogenic
pathway has evolved over the past decade. Initial experiments revealed that laminin or antibody-induced ligation
of the ␣6␤4 complex resulted in the adaptor protein Shc
associating with a phosphotyrosine-containing ␤4-subunit
(Fig. 8). The Shc is then phosphorylated, recruiting Grb2
and potentially activating the ras mitogen-activated protein (MAP) kinase pathway, through the recruitment of
SOS (120, 186). The binding of Shc to ␤4 activates phosphoinositide-3-OH kinase (PI3K) through an undefined
mechanism, which in turn activates Akt kinase resulting
in phosphorylation of Bad, activation of oncogenic transcription factors, and cell survival (33, 66, 166, 176, 182).
Mutagenic studies of the ␤4 cytoplasmic tail show that
Shc preferentially bound to tyrosines 1440 and 1456 (41).
The initial phosphorylation/activation of the ␤4-integrin
for Shc binding is thought to be mediated in part through
the Met tyrosine kinase of the receptor for the hepatocyte
growth factor (HGF) (182). Similarly, the ErbB-2 receptor
oncogene mediates its effects through binding with the
cytoplasmic tail of the ␤4-integrin and activation of the
PI3K (66) (Fig. 8). Interestingly, neither of these oncogenic effects required the extracellular domains of ␤4subunit. Thus the ␤4-subunit is acting more as a modulator of oncogenic signals. The events accompanying the
initial phosphorylation during laminin or antibody binding
of ␤4, and release of its own mitogenic cascade, is still
uncertain.
In comparison with the established activation pathway described above, the binding of CLCA to the ␤4subunit is thought to initiate a novel signal transduction
phosphorylation sequence (Fig. 9). The CLCA-initiated
cascade is described as Met, ErbB-2 independent, and not
requiring phosphorylation of the cytoplasmic tail or Shc
binding domain of the ␤4-subunit for downstream signaling (3). In addition, there is no activation of PI3K. Instead,
the CLCA pathway proceeds from CLCA binding with
␤4-integrin to the aggregation of focal adhesion kinase
(FAK) with the ␤4 cytoplasmic tail through a yet undetermined mechanism. When ␤4-expressing B16-F10 cells
were serum deprived and plated onto mCLCA1 coated
dishes, only FAK was significantly activated. ␤4-Integrin
ligation to mCLCA1 was required to coimmunoprecipitate
FAK. Complex formation between FAK and ␤4-integrin
was assumed to lead to the autophosphorylation of FAK
at tyrosine-397, causing the binding of Src protein tyrosine kinase which then phosphorylates FAK at tyrosine925 (3) (Fig. 9). Phosphorylated FAK binds Grb2 and then
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integrin binding activity (1). Both NH2- and COOH-terminal ␤4-integrin binding motifs are disrupted in pCLCA1,
mCLCA3, and mCLCA6. The COOH-terminal 35-kDa
change is to FSRTAS in both pCLCA1 and mCLCA6. The
mCLCA3 90-kDa ␤4-integrin binding domain region is
changed to (FAALSS) and the 35-kDa sequence is [FSRT(deletion)SS]. The loss of these binding domains from the
same CLCA homolog in mouse, pig, and human parallels
other indications of structural and functional divergence
within the CLCA protein family (Table 1, Fig. 1).
The ␤4-integrin binding motif on both the 90-kDa and
the 35-kDa fragments of the CLCA proteins would have
extracellular locations in either of the proposed topologic
models for membrane insertion (Fig. 2). Hence, the functional data arising from studies of CLCA protein binding
to the ␤4-integrin do not resolve conflicting topology prediction issues.
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MATTHEW E. LOEWEN AND GEORGE W. FORSYTH
recruits Son of Sevenless (SOS), leading to the activation
of the Ras-MAP kinase pathway involving extracellular
signal-regulated kinase (ERK) (3). A similar pathway has
been described in astrocytoma cells and the transforming
growth factor-␤-inducible gene h3 (100).
In addition to FAK, downstream Src kinase and ERK
were also found to be activated (3). ERK reached maximum activation slightly after FAK. However, Src was
constitutively activated in the tumor cell line. Antibodies
that blocked the adhesion of mCLCA1 to ␤4-integrin also
blocked FAK and ERK activation.
The activation of FAK by the mCLCA1/␤4-integrin
complex was somewhat unique, as this activation was not
seen when cells were grown on laminin which binds
␤4-integrin. Transfection of the cell line with the dominant
negative FAK (FANK) (a nonkinase FAK) blocked activation of ERK. Transfection with the wild-type FAK significantly increased ERK activation upon ligation with
mCLCA1 (3).
The FAK-mediated activation of ERK was found to be
Src dependent (3) as the dominant negative FAKY397F
did not allow phosphorylation and reduced complex formation between Src and FAK. Impairing Src binding also
impaired Src phosphorylation of FAK for growth factor
receptor-bound protein (Grb) binding. Antibodies diPhysiol Rev • VOL
rected against ␤-Grb coimmunoprecipitated FAK but not
FAKY397F, indicating that the activated Src must bind
autophosphorylated FAK at 397 and phosphorylate Y925
to allow Grb2 binding, which then allows activation of
ERK (3).
Briefly, the binding of Grb2 allows the translocation
of SOS to the membrane to increase the guanine nucleotide exchange of Ras (45, 161). GTP Ras has increased
activity and activates the downstream MAP kinase pathways or Raf/MEK/ERK (5, 168). In the GTP-bound state,
Ras interacts with and activates Raf, which phosphorylates MEK, which in turn phosphorylates ERK, which
goes on to phosphorylate a number of transcription factors, activating the cell cycle and causing proliferation.
It is interesting to note that both dominant negative
cell lines of B16-F10 melanoma cell produced as described above had greatly reduced lung metastatic potential (3). This is evidence for the importance of CLCA
contributions to this pathway of tumor metastasis. In
addition, this CLCA-activated pathway may also be important in those metastases that undergo extravasation before growth because the ERK activation can also induce
the production of invasive products such as metalloproteinase, and even increase drug resistance (5).
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FIG. 8. Accepted mitogenic signal transduction pathway from ␤4 binding to laminin. Note the initial dependence of Met kinase and the direct
phosphorylation of the ␤4 or the Erb-B2 activation of the cascade. Also note the activation of the phosphoinositide-3-OH kinase (PI3K)/Akt pathway
for cell survival. The presence of Shc and the absence of Src and FAK should also be noted in the activation of the Ras-mitogen-activated protein
kinase pathway.
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CLCA PROTEINS
8. Tumor invasion
Despite the activation of a cell proliferation cascade,
there is little invasion into the actual organ until the vessel
becomes filled with mammary tumor cells (3, 106, 189).
There almost seems to be a need to restrict some of the
blood supply before tumor cells can pass through the
walls of the blood vessels into the tissue. It is also possible that previously discussed ␤4/mCLCA1 interactions
may activate an endothelial chloride conductance that
promotes endothelial cell apoptosis. Tumor cell invasion
could start by adhesion to endothelial cells, proliferation
to fill the vessel and restrict blood supply to the endothelium, which in turn becomes slightly hypoxic, dropping
the intracellular pH. The ␤4/mCLCA1 could then participate in opening a channel to allow chloride entry to
electrically balance ischemic hydrogen ion production
Physiol Rev • VOL
and facilitate a rapid drop in intracellular pH (Fig. 6). The
proposed pH drop would activate caspases and the apoptotic cascade (22, 165). The endothelial cells would die,
and the tumor would invade.
VIII. CLCA, VASCULAR TONE,
AND HYPERTENSION
Orthologs of the CLCA gene family mCLCA1,
mCLCA2, mCLCA4, and, by inference, hCLCA3 are expressed in vascular smooth muscle (25, 48, 110). CLCA
proteins are known to modulate Ca2⫹-activated chloride conductance as well as to interact with the subunits of the Ca2⫹-activated potassium (BK) channel (73,
114, 115). Ca2⫹-activated chloride conductance and potassium conductance control vascular tone, making a
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FIG. 9. Mitogenic signal cascade induced by endothelial CLCA binding to ␤4-integrin on metastatic cell. Note the absence of phosphorylation
of the ␤4 cytoplasmic tail and the dependence on FAK and Src for initiation of the signal transduction cascade. In addition, note the absence of PI3K
pathway activation.
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MATTHEW E. LOEWEN AND GEORGE W. FORSYTH
modulating protein such as CLCA an interesting target
for intervention in conditions such as hypertension and
erectile dysfunction.
A. Hypertension
Physiol Rev • VOL
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Hypertension or an increased vascular tone is defined as the mean arterial blood pressure in humans being
greater than 110 mmHg. This usually results from the
diastolic pressure greater than 90 mmHg and the systolic
pressure greater than 140 mmHg (32, 55, 198). Sustained
increases in vascular tone can be very detrimental. Increased blood pressure contributes to the development of
congestive heart failure by increasing the work load on
the heart, and to chronic renal failure through small renal
hemorrhages and scarring. Hypertension also predisposes
to coronary artery disease and stroke (32, 156).
Hypertension can result from, or be modulated
through, changes to the renin-angiotensin system (32).
The regulation of blood pressure begins with the rate of
renal glomerular sodium clearance. Sodium clearance determines the amount of chloride presented to the macula
densa, which determines the rate of reabsorption from
the filtrate via the Na⫹-K⫹-2Cl⫺ cotransporter (163). Upon
a decrease in the rate of the Na⫹-K⫹-2Cl⫺ cotransporter,
the macula densa signals the juxtaglomerular cells, via a
cyclooxygenase-2 inhibited process, to release renin (24,
29, 72). Renin is converted to angiotensin I and angiotensin II in the lung. Angiotensin II causes vasoconstriction
and increases blood pressure through contraction of the
smooth muscle of the vasculature, increasing glomerular
sodium chloride clearance, and activity of the Na⫹-K⫹2Cl⫺ cotransporter. These effects combine to cause a
decrease in renin release (82). The release of aldosterone
by angiotensin II also increases blood pressure by volume
intake in the distal collecting duct of the kidney, which
also increases glomerular sodium chloride clearance and
decreases renin release. This system permits modulation
of vascular tone, and its malfunction results in hypertension, but the actual etiology of most chronic hypertension
is unknown (195).
Current therapy for hypertension often targets the
renin-angiotensin system, either by blocking angiotensin receptors or by preventing the conversion of renin
to angiotensin (32). Direct modulation of vasoconstriction by reducing vascular smooth muscle tone could be
an alternative to inhibiting agonists of hypertension.
Smooth muscle tone might be reduced by manipulating
the interplay of potassium, chloride, as in the case of
CLCA, and calcium channels that regulate the membrane potential to control calcium influx into the
smooth muscle cell (91).
In smooth muscle, the equilibrium potential for chloride is more positive than the resting membrane potential,
while the potassium equilibrium is more negative (31).
The net result is that negative charges leave the cell upon
chloride channel activation, resulting in membrane depolarization from approximately ⫺60 mV to a chloride equilibrium potential of approximately ⫺30 mV (91). This
depolarization results in the activation of L-type calcium
channels, permitting an influx of extracellular Ca2⫹ that
causes muscle contraction (89, 138). Simplistically, it is
believed that the Ca2⫹ entering the cell bind to calmodulin, resulting in a complex which activates myosin lightchain kinase (149). The resulting phosphorylation of myosin allows myosin/actin cross-bridging and initiates the
“ratchet theory” of contraction through the hydrolysis of
ATP (93, 187). Then the Ca2⫹ which activated contraction
also activates Ca2⫹-dependent potassium channels, resulting in potassium release, cell hyperpolarization, closing of L-type Ca2⫹ channels, and an end to the contraction
cycle (31, 91, 138).
Thus agonist-induced increases in potassium conductance and decreases in chloride conductance in smooth
muscle will result in muscle relaxation, vasodilation, and
hypotension. Conversely, an agonist such as angiotensin
or norepinephrine that decreases potassium conductance
or increases chloride channel conductance will cause
muscle contraction, vasoconstriction, and hypertension
(Fig. 10). Smooth muscle contraction is significantly reduced by the inhibition of norepinephrine or angiotensin
II-induced chloride conductance (81, 194). The presence
of CLCA protein in smooth muscle, the effects of CLCA on
Ca2⫹-activated chloride currents, and CLCA interaction
with subunits of the BK potassium channel make it likely
that this gene family is directly involved in agonist-induced smooth muscle contraction (25, 48).
The molecular mechanism of basal vascular tone is
just beginning to be understood. The basal vascular tone
is thought to be maintained by “calcium sparks” (92, 136)
which originate from a ryanodine-sensitive Ca2⫹ release
from the sarcoplasmic reticulum (92). This Ca2⫹ release is
local and does not cause contraction. However, it does
activate the large-conductance Ca2⫹-activated K⫹ (BK)
channels. This BK channel is made up of a pore-forming
␣-subunit and the ␤1-subunit that regulates its Ca2⫹-sensitive activity (7). Rodent hypertensive models illustrate
the importance of this regulatory subunit. ␤1-Knockout
mice were hypertensive (23), and the ␤1-subunit expression is downregulated in genetically hypertensive rats (8).
In addition, rats made hypertensive by prolonged exposure to angiotensin II were found to downregulate the
␤1-subunit (8). This downregulation correlated with in
vivo whole cell and single-channel studies reporting a
decrease in the channel sensitivity to Ca2⫹.
The CLCA gene family has been implicated in vascular tone and in calcium sparks. CLCA expression has been
identified in the vascular smooth muscle of the mouse (25,
48). When mCLCA4 was expressed in HEK293 cells
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CLCA PROTEINS
(which do not express endogenous BK potassium channel
␤1 regulatory subunit) they produced a nonrectifying and
time-independent Ca2⫹-activated inward current, or outward movement of chloride depolarizing the cell (48).
Similarly, expression of mCLCA1 from murine portal vein
produced a time-independent and only slightly rectifying
anion current upon stimulation by Ca2⫹ (73). These biophysical properties observed upon mCLCA1 or mCLCA4
expression in HEK293 cells are distinctly different from
smooth muscle cell recordings, which show time-dependent currents with strong rectification. However, on cotransfection with the ␤1-subunit of the BK potassium
channel the current became very similar to the smooth
muscle recordings (73). The effect of ␤1-subunit on CLCAassociated conductance was shown by a mammalian twohybrid system to involve a direct protein-protein interaction. Unfortunately, these authors did not report the effect
of cotransfection on the inward-induced current. The inward current (outward movement of chloride) would
likely have been reduced in the dual transfection, given
the strong rectification induced by the ␤1-subunit. An
interaction of CLCA with ␤1-subunit in smooth muscle
would suppress chloride conductance, reduce the depolarization of the membrane, and prevent contraction.
In smooth muscle, the “calcium spark” may occur
through the ␤1 BK subunit interactions that reduce the
stimulatory effects of CLCA proteins on chloride conductance. This makes endogenous chloride channels more
rectifying and stops chloride release. In contrast to the
“spark,” there is larger Ca2⫹ release on agonist addition.
At higher Ca2⫹ concentration, there is a significantly
larger inward current or (movement of chloride out of the
cell) in the dual CLCA ␤1-transfected cells (73). Thus, at
lower [Ca2⫹] during sparks, the outward flux of chloride
may be inhibited by the ␤1-subunit interactions with a
CLCA protein. At higher [Ca2⫹] this inhibitory effect
is lost. Thus, altering CLCA activity in smooth muscle
Physiol Rev • VOL
cells could modulate an endogenous chloride conductance with a significant therapeutic benefit to hypertensive subjects.
IX. CLCA, BESTROPHINS, AND RETINOPATHY
Bestrophin proteins are associated with chloride
transport in the retinal pigment epithelium. Mutant
forms of these proteins cause the inherited disorder
named Best’s vitelliform macular dystrophy. Bestrophins are of particular interest as candidates for the
molecular identity of Ca2⫹-activated chloride conductance (152). However, bestrophins may be only one
component of a hetero-oligomeric calcium-dependent
chloride conductor. Because one major function of
CLCA proteins may be to modulate chloride conductor
activity, it is important to examine the connection between CLCA and bestrophin proteins to get more insight into the disease condition as well as into the
molecular identity of the components that make up the
Ca2⫹-activated chloride conductance.
Best’s vitelliform macular dystrophy (BMD) is an
autosomal dominant inherited disorder of the eye, with
juvenile onset. The disease is characterized by a vitelliform (“egg yolk” or “vitelline”) macular (discolored spots)
lesion easily visible on examining the fundus (15). The
vitelliform lesion is due to deposition of lipofuscin (fatty
pigment)-like material within and below the retinal pigment epithelium (58, 196). This often results in degeneration of the retinal pigment epithelial cells (140). However, the most defining clinical feature of the disease is a
light peak to dark trough ratio in the electrooculogram
(EOG) of ⬍1.5, without abnormalities in the electroretinogram (ERG) (38). The EOG measures the late response
of the eye to light. It is the result of depolarization of the
basal plasma membrane of the retinal pigment epithelium
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FIG. 10. Physiological consequences of smooth muscle chloride channel activation or inactivation. Chloride channel activation results in
membrane depolarization and smooth muscle contraction, whereas inactivation of chloride channels causes membrane hyperpolarization.
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MATTHEW E. LOEWEN AND GEORGE W. FORSYTH
Physiol Rev • VOL
Electrophysiological experiments with expressed
Best1 and -2 as well as C. elegans RFP proteins in HEK293
cells generated chloride currents that increased with micromolar intracellular Ca2⫹ concentration (173, 185).
However, hBest1 was unable to produce a detectable
chloride current when expressed in Xenopus oocytes
(185). In a subsequent study, Xenopus bestrophin produced a Ca2⫹-activated chloride current when expressed
in HEK293 cells (153). Apparently HEK293 cells contain
an accessory subunit that may be required for bestrophin
to function. Introduction of a nonconducting mutation
produces a dominate negative effect in wild-type oocyte
channels (153). It seems likely that bestrophins may have
a direct impact on Ca2⫹-activated chloride conductance,
but that these proteins are not working alone. The
hCLCA1 protein is a good candidate to associate with and
affect the activity of a bestrophin chloride channel. CLCA
proteins have been localized to the same RPE membrane
as Best1, adjacent to the choriocapillaris (117) where
Ca2⫹-activated chloride conductance occurs (65).
X. SUMMARY
The CLCA gene family clusters together on a small
portion of the chordate genome and codes for the production of a structurally similar, but functionally diverse,
group of proteins. The functions associated with members of this gene family implicate the CLCA proteins in
several interesting physiological and pathological processes. They have intriguing roles in cell cycle control and
in apoptosis, which may be associated with tumor suppressor activity. Expression of CLCA protein in different
systems produces major differences in calcium-dependent whole cell chloride currents. cAMP-dependent chloride currents are modulated by CLCA expression. CLCAinduced currents are altered significantly by coexpression
with a potassium channel ␤1-subunit. With the exception
of function as cell-surface adhesion molecules that can
anchor metastasizing tumor cells, these other processes
associated with CLCA expression all have some connection to chloride channel activity. However, the underlying
question of whether CLCA proteins function directly as
chloride channels, or somehow interact with and affect
the conductance of other ion channels has not been resolved.
There may be some convergence in connecting CLCA
proteins to bestrophins, and to Ca2⫹-activated chloride
conductance, as neither protein appears to be a fully
functional chloride conductor without expression in a
proper complimentary background. It is an intriguing possibility that some combination of CLCA and bestrophin
proteins may constitute the molecular identity of a Ca2⫹activated chloride channel. However, the upregulation of
CLCA proteins in the inflammatory process, and the di-
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(RPE) (64, 65). This depolarization is normally caused by
a Ca2⫹-sensitive chloride channel, which is activated by
some yet undefined signal from the stimulated retina (64).
The net result of the depolarization is transport of fluid
and salt away from the retinal rods and cones and into the
choriocapillaris vascular drainage from the eye.
The general ion and fluid transport mechanisms of
the RPE are similar to those in other secretory epithelia,
except that there is no obvious lumen to identify an apical
surface of the epithelium. The Na⫹-K⫹-ATPase located in
the membrane adjacent to the retinal rod and cone photoreceptors generates a large sodium gradient across the
cell membrane (17, 183, 184). This sodium gradient is
coupled to the uptake of Cl⫺ and K⫹ across the membrane by the usual Na⫹-K⫹-2Cl⫺ cotransporter. The vectoral arrangement of ion transport proteins includes a
chloride conductance on the opposite side of the polarized epithelium, adjacent to the choriocapillaris venous
drainage. Normal coordinated operation of these ion
channels drives net ion and fluid movement from the
vitreous to the choroid (17). Chloride conductance is
generally under the control of Ca2⫹ (117), and the rate of
chloride release increases in the light-evoked EOG (65).
These facts lead to the hypothesis that a defect in basolateral Ca2⫹-activated chloride conductance is part of the
pathology of BMD.
The mutation responsible for BMD was found in the
gene coding for a 68-kDa protein. This protein, now
named bestrophin, has become a molecular candidate for
the Ca2⫹-activated chloride channel (148, 152) and a target for activity modulation by CLCA proteins. At least 79
different bestrophin mutations have been found in BMD
patients (125) (summarized at the VMD2 mutation data
base, www.uni-wuerzburg.de/humangenetics/vmd2.html).
These include missense, single amino acid deletions, as
well as splice site and frameshift mutations (6, 13, 28, 46,
104, 118, 123, 126, 144, 148). Bestrophins share homology
with the Caenorhabditis elegans RFP gene family, named
for the presence of a conserved RFP amino acid sequence
motif which is found in 26 transmembrane proteins with
related sequences grouped in worm family eight (170).
Four bestrophin isoforms are known in humans (126,
148, 185). hBest1 or VMD2, the mutated gene in BMD, is
expressed in RPE, retina, and testes. hBest2 or VMD2L1
protein is found in RPE, colon, and testes; hBest3 or
VMD2L3 is expressed in skeletal muscle, brain, spinal
cord, bone marrow, retina, thymus, and testes; and hBest4
or VMD2L2 was found predominantly in the colon and
weakly in fetal brain, spinal cord, retina, lung, trachea,
testes, and placenta (170, 185). It is interesting that
hBest1, hBest2, and hBest4 are highly expressed in the
RPE and colon, two tissues with very active ion and fluid
transport (185). The membrane localization of hBest1 in
the RPE indicates that bestrophins are properly situated
to be calcium-activated chloride channels (124).
1087
CLCA PROTEINS
verse interactions of CLCA proteins with other chloride
channels and with other ion conductances invite speculation that CLCA proteins may have a broader involvement in determining the balance between epithelial absorption and secretion.
17.
18.
Address for reprint requests and other correspondence:
G. W. Forsyth, 52 Campus Dr., Saskatoon, Saskatchewan, Canada S7N 5B4 (E-mail: [email protected]).
19.
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