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From the
Received for publication, January 25, 2002, and in revised form, March 12, 2002
To identify the gene products responsible for the
calcium-activated chloride current in smooth muscle, reverse
transcription-PCR with degenerate primers was performed on mouse
intestine and other organs. A new member of the CLCA gene family was
identified, mCLCA4, that is expressed preferentially in organs
containing a high percentage of smooth muscle cells, including
intestine, stomach, uterus, bladder, and aorta. Reverse
transcription-PCR using template RNA prepared from mouse bladder and
stomach smooth muscle layers dissected free of mucosa yielded
mCLCA4-specific bands. In situ hybridization with an
mCLCA4-specific probe confirmed prominent expression in smooth muscle
of major vessels of the heart but not cardiac muscle. High expression
was also detected in the gastrointestinal tract, in bronchioles, and in
aortic and lung endothelial cells. Transient expression of mCLCA4 in
293T cells resulted in the appearance of a prominent calcium-activated
chloride current. Whole-cell currents activated by ionomycin or
methacholine were anion-selective and showed minimal rectification or
voltage-dependent gating. Similar to endogenous currents in
smooth muscle cells, methacholine-induced currents were transient, and
spontaneous transient inward currents were occasionally observed at
resting membrane potentials. These results link calcium-activated
chloride channels in smooth muscle with a gene family whose members
have been implicated in cystic fibrosis, cancer, and asthma.
Calcium-activated chloride currents have been reported in a number
of cell types including exocrine gland (1-3), smooth muscle (4-7),
cardiac muscle (8, 9), epithelium (10-12), and endothelium (13). In
vascular and non-vascular smooth muscle, calcium-activated chloride
channels underlie one component of excitatory postsynaptic potentials
(4-6, 14), and the local gating of Ca2+-activated chloride
channels by unitary Ca2+ release events results in
spontaneous transient inward currents, termed STICs (15-17).
Physiological evidence suggests that activation of
ICl(Ca)1 is an
important component of rhythmic electrical activity (18, 19) and
excitation/contraction coupling (20-22) in smooth muscle. Despite the
prominent role of these channels in evoked and spontaneous electrical
activity of smooth muscle, the molecular identity of the underlying
channel remains unknown.
A family of calcium-activated chloride channels, termed CLCA, has
recently been identified at the molecular level in many epithelial and
endothelial cell types (23-30). CLCA family members have been
implicated in pathological states such as asthma and cancer (30-33),
although their function in normal cell physiology is not well
established. Here we report the cloning and expression of a new member
of the CLCA gene family in mouse. Unlike other CLCA genes, mCLCA4 is
highly expressed in smooth muscle. Transient transfection of HEK293
cells with mCLCA4 results in the expression of calcium-activated
currents with anion selectivity and kinetics similar to those observed
in smooth muscle. These results suggest that mCLCA4 is the gene that
encodes calcium-activated chloride channels in smooth muscle and
broadens the physiological and pathological relevance of the CLCA gene family.
Identification and Isolation of mCLCA4--
RNA was prepared
from mouse large intestine, kidney, and lung by grinding frozen organs
with a mortar and pestle and extracting with Trizol (Invitrogen). 1 µg of RNA was reverse-transcribed (Superscript, Invitrogen) using
random hexamers. cDNA was subjected to PCR (93 °C, 30 s;
55 °C, 30 s; 72 °C, 30 s; 35 cycles) with degenerate
primers based on Lu-ECAM-1 amino acids 36-45
(5'-ATTGCAATTAACCCCAGTGTGCCAGANGA-3') and 165-174
(5'-GCRTAYTCRTCRAANAYNCCCCA-3'). PCR products were subjected to direct
sequencing to rule out Taq PCR error. Distinct sequences
were obtained from intestine versus kidney and lung (which
were identical to mCLCA1) and were consistent across multiple RNA and
cDNA preparations. The full open reading frame of mCLCA4 was
obtained in two steps. Using oligo-dT-primed intestinal cDNA as a
template and primers derived from the mCLCA1 sequence, a product was
obtained comprising the start codon to base pair 2550. 3' RACE was then
employed to obtain the interval from base pair 2359 to the 3' poly(A)
tract. Primers derived from these sequences were used to amplify a
2.7-kb product containing the entire open reading frame. PCR error was
minimized by using high fidelity Herculase DNA polymerase
(Stratagene), by sequencing products directly before cloning, and by
sequencing multiple pGEM-T clones. The final product was transferred as
a SalI/SstII fragment to pIRES2
(XhoI/SstII) for expression in HEK293T cells. One
3' RACE product was obtained that contained an insertion at base pair 2663 (5'-TGGTCTGAGTACCCCCAGCACCCCTCCTGGTCTGAGTACCCCCAGCACCCCTCCTGGTC-3'), resulting in a triple repeat of the amino acid sequence LSTPSTPPG beginning at position 880. The full-length clones all lacked the insertion.
Organ and Tissue Distribution of mCLCA4 by RT-PCR--
Primer
pairs specific for mCLCA4 were designed based on alignment of the four
known mouse CLCA genes and corresponding to the interval base pairs
1123-1443, 5'-catcaatgacagctcctacctagc-3' and
5'-atcaatcaggccattcacgtcttcc-3'. Primers were also designed to bracket
at least one intron to rule out amplification of genomic DNA.
Initially, a mouse multi-tissue cDNA array (RapidScan, Origene) was
probed. To confirm and extend these results, the organs identified in
the array and several others were isolated from a C57BL/6 mouse, and
RNA was extracted and subjected to RT-PCR as before. Smooth muscle
tissue was isolated from mouse bladder and stomach by dissecting away
mucosal and serosal tissue layers under microscopic observation. 18 S
ribosomal RNA was amplified as an internal control for RNA amount and
reverse transcription using a primer:competitive oligomer ratio
of 3:7 as described by Ambion. PCR was varied from 15 to 35 cycles to
determine the range of linear amplification with added template. The
profile at 25 cycles is shown. Total RNA from mouse aortic endothelial
cells was a gift from Bernd Nilius (Leuven, Belgium). Lung
microvascular endothelial cells were isolated from lung tumors as
described (34). In some tissues a smaller product, identified by an
x in Fig. 4, was sometimes obtained in addition to the
expected band. This product was excised, inserted into pGEM-T
(Promega), and determined by sequence analysis of three clones to be an
artifact unrelated to mCLCA4. In addition, a Southern blot of the gel
hybridized with mCLCA4 cDNA detected only the upper band.
In Situ Hybridization of Mouse Tissues--
A subclone
containing base pairs 2670 of the mCLCA4 open reading frame through the
3'-untranslated region was used for in situ hybridization
analysis; this probe avoided regions with high similarity to mCLCA1 and
mCLCA2. In situ hybridization studies were performed using a
modification of procedures described by Wilkinson and Green (35). Mouse
tissues were fixed overnight in freshly prepared ice-cold 4%
paraformaldehyde in phosphate-buffered saline. The embryos were
dehydrated through ethanol into xylene and embedded in paraffin using a
Tissue-Tek V.I.P. automatic processor (Miles, Mishawaka, IN). Sections
(5 µm) were adhered to commercially modified glass slides (Super
Frost Plus, VWR, Rochester, NY), dewaxed in xylene, rehydrated
through graded ethanols, and treated with proteinase K to enhance probe
accessibility and with acetic anhydride to reduce nonspecific
background. Single-stranded RNA probes were prepared by standard
techniques with specific activities of 5 × 109
dpm/mg. Sections were hybridized at Tm Patch Clamp Recording--
The expression of ICl(Ca)
in mCLCA4-transfected and control HEK293T cells was examined by
whole-cell, patch clamp recording using classical and perforated patch
clamp methods as described previously (36, 37). Briefly, HEK293T cells
were transfected with green fluorescent protein-expressing
pIRES2-mCLCA4 or vector alone (LipofectAMINE Plus, Invitrogen),
trypsinized 24 h later, and seeded onto fibronectin-coated glass
coverslips. After 18 h, cells were transferred to a
temperature-controlled chamber maintained at 36 °C (Brook
Industries) and superfused at 1 ml/min with extracellular solution (126 mM NaCl, 1.2 mM MgCl2, 1.8 mM CaCl2, 10 mM HEPES, and 11 mM glucose; the pH was adjusted to 7.4 with NaOH. Anion
shift experiments utilized the above extracellular solution in which
100 mM NaCl was replaced by an equimolar amount of sodium
glutamate, shifting the theoretical chloride equilibrium potential to
24 mV (assuming zero glutamate permeability). Recording pipettes
(resistance, 3-5 megaohms) were filled with 130 mM CsCl, 1.2 mM MgCl2, 10 mM HEPES, 10 mM glucose, 1 mM MgATP, and 0.075 mM EGTA; the pH was adjusted to 7.2 with CsOH. For
perforated patch experiments, pipettes were dipped in pipette solution
for 1-2 s then back-filled with pipette solution containing 200 mg/ml nystatin. After seal formation and establishment of the whole cell
recording configuration, cells were voltage-clamped at Identification and Cloning of a New CLCA Family Member--
To
identify the CLCA isoform present in mouse smooth muscle, RT-PCR was
performed with degenerate primers on template RNA extracted from an
organ rich in smooth muscle, the large intestine. Degenerate primers
for RT-PCR were designed based on published CLCA sequences to amplify
any CLCA cDNA including mCLCA1, mCLCA2, and mCLCA3. Thus, RNA
extracted from established sites of mCLCA1 expression, lung and kidney,
could serve as positive controls for amplification of CLCA mRNA.
RT-PCR of intestinal RNA produced a much stronger band than either lung
or kidney (Fig. 1). Direct sequencing of
the amplification products revealed only the mCLCA1 sequence in lung
and kidney but a new and distinct sequence in intestine, which we have
designated mCLCA4. The electropherogram showed no sign of mCLCA3,
previously reported in intestinal goblet cells, indicating that mCLCA4
is the predominant CLCA family member in large intestine (data not
shown). The full 2.7-kb, 909-amino acid open reading frame of mCLCA4
was obtained using primers derived from the mCLCA1 sequence and 3' RACE
(Fig. 2). Comparison of the mCLCA4 amino
acid and DNA sequences with those of the other mouse CLCA family
members revealed an abrupt divergence at amino acid 873 resulting from
a frameshift mutation (Fig. 2; GenBankTM accession number
AY00827). mCLCA4 retains 79% identity with mCLCA1 and mCLCA2 but only
45% with mCLCA3. All of the general features found in other CLCA
family members, such as the symmetrical cysteine cluster, processing
sites, and glycosylation sites are conserved in mCLCA4 (Fig. 2).
Construction of a phylogenetic tree of known CLCA proteins places
mCLCA4 on Branch A, whose members all bear the sequence RARSPT
(corresponding to amino acids 592-597 of mCLCA4), containing two
adjacent sites for phosphorylation by calcium/calmodulin kinase II and
protein kinase C as well as a site for protein kinase A (Fig.
3). Members on branches B and C lack this
sequence (note that the genetic nomenclature is based on order of
discovery; thus, mCLCA4 is not the ortholog of hCLCA4).
Tissue Distribution of mCLCA4 Expression--
In preliminary
experiments, to identify tissues in which mCLCA4 was expressed, a
multi-organ mouse cDNA array (RapidScan, Origene) was probed by
RT-PCR with primers that recognized mCLCA4 but not other mouse CLCA
family members. Expression was detected in the gastrointestinal tract,
uterus, and heart (data not shown). To confirm and extend these
results, these and other organs were dissected and analyzed from a
C57BL/6 mouse. Expression was highest in large and small intestine and
was also significant in stomach and esophagus but just detectable in
salivary gland (Fig. 4A; lower
band x is an artifact). Outside the gastrointestinal tract, strong expression was detected in uterus and lung, and lower expression was detected in aorta, heart, and skeletal muscle, whereas none was
detectable in kidney or pancreas (Fig. 4A). To determine
whether mCLCA4 expression in mixed organs was associated with
expression in smooth muscle, two mouse organs were chosen in which the
smooth muscle layer could be dissected free of overlying tissue layers. The tunica muscularis was dissected from the mouse bladder and stomach
for RNA preparation and RT-PCR. As shown in Fig. 4B,
isolated smooth muscle from both tissues was strongly positive for
mCLCA4 expression, with stomach more prominent.
Cellular Expression of mCLCA4--
To determine the sites of
expression at higher resolution, we probed tissue sections by in
situ hybridization with an mCLCA4-specific 33P-labeled
cRNA probe derived from the 3'-untranslated region. The best evidence
for expression in smooth muscle was observed in vessels associated with
the heart. A very strong signal was observed in the walls of the aorta
with a much lower signal in adjoining cardiac muscle and little signal
above background in the endothelium of the aortic valve (Fig.
5, a and e). The
pulmonary vein (Fig. 5, b and f) and its branches
were also intensely labeled, whereas the coronary artery was less so
(Fig. 5, c and g). Connective and adipose tissues
were consistently negative. The atrioventricular bundle, consisting of
muscle cells modified to function as nerve fibers, was also positive
(not shown). In lung, the signal was concentrated around bronchioles
and blood vessels (Fig. 6,
a-d). Surprisingly, in the gastrointestinal tract mCLCA4
expression was more strongly associated with the mucosa than with
underlying smooth muscle (Fig. 6, e-h). In small intestine
for example, label was concentrated in villi, whereas smooth muscle
layers were more weakly labeled. In contrast to mCLCA1 and mCLCA3,
mCLCA4 signal was not confined to deep crypt or goblet cells.
The aortic wall in mouse consists mostly of smooth muscle with only a
single layer of endothelial cells lining the vessel interior. Because
endothelial cells are difficult to detect by in situ
hybridization, we obtained endothelial cells that had been
isolated from aorta or lung microvasculature and analyzed them by
RT-PCR. Endothelial cells from either source had high levels of mCLCA4
expression (Fig. 4C).
mCLCA4 Mediates a Calcium-activated Chloride Current--
To
determine whether mCLCA4 encodes a functional chloride channel, the
cDNA was inserted into a vector bearing a green fluorescent protein
marker and transfected into HEK293T cells, and whole cell currents of
fluorescent cells were measured. Recordings were made in extracellular
solution containing Cs+ substituted for Na+ to
block any calcium-activated potassium currents. Under these conditions,
mCLCA4-transfected HEK cells, but not vector-transfected cells,
exhibited a prominent inward current when exposed to ionomycin (Fig.
7A). 16 of 23 cells exposed to
ionomycin displayed prominent inward currents, whereas no current was
observed in 6 cells transfected with vector alone. The evoked currents
were transient in nature even when evoked after exposure to ionomycin,
which results in a sustained increase in [Ca2+]i;
inactivation of the smooth muscle calcium-activated chloride channel
that is independent of Ca2+ has been reported (38).
Methacholine was also used to stimulate calcium release via endogenous
muscarinic receptors. As shown in Fig. 7B, methacholine
activated a transient current of similar time course and smaller
magnitude than ionomycin (Fig. 7B). A second prominent
feature of expression of mCLCA4 was the appearance of spontaneous
transient inward currents (STICs) similar to those observed in
smooth muscle cells from several tissues (17, 39). Spontaneous currents
were observed in 10 of 29 cells transfected with mCLCA4 (Fig.
7C), whereas in 21 cells transfected with a green
fluorescent protein plasmid lacking mCLCA4 no spontaneous methacholine-activated chloride currents were observed.
To obtain the current-voltage relationship of the calcium-activated
inward current, a step depolarization experiment was performed. As
shown in Fig. 8A, the I-V
relationship was linear over the voltage range from The CLCA gene family comprises an expanding group of
calcium-activated chloride channels. To date, the expression of known CLCA genes has been largely restricted to epithelial and endothelial tissue, from which they were first cloned (23, 24). Despite early
reports of prominent calcium-activated chloride currents in
Xenopus oocytes (41), neurons (42, 43), secretory gland (1),
and smooth muscle cells (4), in large measure the genes responsible for
these currents have not been clearly identified, and the extent to
which they represent variants of the described CLCA or ClC (44) gene
families has been uncertain. The identification of a CLCA gene with
prominent expression patterns in smooth muscle significantly expands
the potential importance of this gene family.
In general, calcium-activated chloride channels in various mammalian
tissues are similar in single-channel conductance, anion selectivity,
and drug sensitivity (for review, see Ref. 45). These and other shared
properties led to the proposal that all calcium-activated chloride
channels in mammalian tissues may be mediated by a family of closely
related proteins (20). Although the CLCA family is deeply divergent at
the amino acid level, all share several features. The CLCA precursor is
about 900 amino acids long and contains two proteolytic cleavage sites.
The first cleavage removes the amino-terminal signal sequence,
resulting in a long extracellular amino-terminal tail. The second
cleavage event removes the carboxyl-terminal ~200 amino acids,
yielding products of 90 and 30-40 kDa, both closely associated with
the plasma membrane (24-27). Another signatory feature is a
symmetrical cluster of cysteines,
CX12CX4CX4CX12C,
around amino acid 200. The structure and stoichiometry of the channel
remain to be established.
We report the cloning and functional expression of a novel member of
the CLCA family, termed mCLCA4. The gene product is strongly related to
mCLCA1 and mCLCA2 and shares the potential phosphorylation sites for
calcium/calmodulin kinase II, consistent with regulation by
intracellular calcium. RT-PCR and in situ hybridization
analysis indicates substantial expression of mCLCA4 in smooth muscle
and certain epithelial and endothelial tissues. Expression in smooth muscle was never observed for mCLCA1, mCLCA2, or mCLCA3 (46, 47, 31)
and suggests that mCLCA4 may be associated with chloride currents
recorded in this tissue. Expression studies with mCLCA4 were consistent
with this notion, and transfection of HEK293T cells with mCLCA4
resulted in the expression of a prominent calcium-activated chloride
current. The current-voltage relationship measured in voltage steps
during exposure to ionomycin was consistent with a very weakly
voltage-dependent channel, similar to measurements of
calcium-activated chloride currents in smooth muscle (20) and other
tissues (1, 41). Similarly, despite sustained elevations of
[Ca2+]i obtained with ionomycin, the activated
current decayed with kinetics generally similar to that observed in
smooth muscle (38). The appearance of spontaneous, transient
calcium-activated currents, a prominent feature of smooth muscle
chloride currents (17), also suggests a marked functional similarity
between mCLCA4 and the smooth muscle channel.
In summary, we report the sequence and functional expression of a novel
calcium-activated chloride channel with distinct tissue expression.
Although the definitive determination of the extent to which this
channel underlies specific postsynaptic currents in smooth muscle or
secretory cells will require careful comparison of biophysical
properties of heterologously expressed mCLCA4 with currents recorded in
native cells and gene targeting experiments, our data identify mCLCA4
as a likely candidate for calcium-activated chloride currents in smooth muscle.
We thank Dr. Bernd Nilius for mouse aortic
endothelial cells RNA and the laboratory of Dr. Alan Nixon for
assistance with microscopy.
The dependence of STICs upon unitary
calcium-release events was established by Walsh and co-workers
(Zhu Ge, R., Sims, S. M., Taft, R. A., Fogarty, K. E., and Walsh,
J. V. (1998) J. Physiol. (Lond.) 513, 711-718).
*
This work was supported by United States Army Breast Cancer
Research Fund Grant DAMD17-00-1-0219 (to R. C. E.), NCI, National Institutes of Health (NIH) Public Health Service (PBS) Grant CA47668 (to B. U. P.), PHS Grant DE09692 (to K. N.), and NIH PHS Grants HL45239, HL41084, and DK58795 (to M. I. K.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY00827.
§
To whom correspondence should be addressed. To R. C. E.: Tel.:
607-253-3324; Fax: 607-253-3708; E-mail: rce3@cornell.edu. To
M. I. K.: Tel.: 607-253-3336; Fax: 607-253-3317; E-mail:
mik7@cornell.edu.
Published, JBC Papers in Press, March 14, 2002, DOI 10.1074/jbc.M200829200
The abbreviations used are:
ICl(Ca), calcium-activated chloride current;
RACE, rapid amplification of
cDNA ends;
RT, reverse transcription.
Molecular and Functional Characterization of a Murine
Calcium-activated Chloride Channel Expressed in Smooth Muscle*
§,
,,
Cancer Biology Laboratories and Departments
of Molecular Medicine and ¶ Biomedical Sciences, Cornell
University College of Veterinary Medicine, Ithaca, New York 14853 and
Center for Oral Biology and ** Center for Human
Genetics and Molecular Pediatric Disease, Aab Institute for Biomedical
Research, University of Rochester,
Rochester, New York 14642
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
15 °C,
washed at high stringency (Tm 7 °C) and
treated with RNase A to further diminish nonspecific adherence of
probe. Autoradiography with NBT-2 emulsion (Eastman Kodak Co.) was
performed for 25 days. Slides were developed with D19 (Kodak), and the
tissue was counterstained with hematoxylin.
60 mV
(Axopatch 200 B, Axon Instruments). Records were filtered at 500 Hz and
sampled at 1 kHz. Current reversal potentials were measured either by
step or ramp (60 to 40 mV applied every 30 s) protocols. Cells
were exposed to calcium-mobilizing agents (ionomycin or methacholine)
by means of a puffer pipette connected to a controlled solenoid
(Picospritzer, General Valve Corp.). A microscope equipped with an
ultraviolet source was used to select green fluorescent
protein-positive cells for recording.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Identification of CLCA family members in
mouse organs. RNA was extracted from kidney, large intestine, and
lung and subjected to RT-PCR with degenerate primers based on known
CLCA amino acid sequences.
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Fig. 2.
Comparison of mCLCA4 amino acid sequence with
those of other mouse CLCA family members. Glycosylation sites are
indicated by stars; the cysteine cluster is indicated by
underlines, the phosphorylation hotspot is indicated by a
double underline with closed circles indicating
the phosphorylated residues, and the processing site by an
arrowhead. Alignment was performed using the Megalign
program, Clustal method, version 4.05 of the DNAStar package from
Lasergene.
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[in a new window]
Fig. 3.
Dendrogram showing the relation of mCLCA4
(italics) to the other known family members.
Complete amino acid sequences were used to derive the tree using
DNAStar software. The bar represents 10% sequence
divergence. Note that nomenclature indicates order of discovery rather
than orthology.
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Fig. 4.
Tissue specificity of mCLCA4 expression
determined by RT-PCR. A, whole organs. Lower band
x was sequenced and found to be an unrelated artifact.
B, isolated smooth muscle from bladder and stomach.
C, isolated endothelial cells. MAEC, mouse aortic
endothelial cells. MLEC, mouse lung microvascular
endothelial cells.
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Fig. 5.
Expression of mCLCA4 in vascular tissues
determined by in situ hybridization of tissue
sections. A longitudinal section of the heart was hybridized with
a 33P-labeled mCLCA4 probe, exposed to photo-emulsion, and
examined by UV (ultraviolet autofluorescence, a-c) or
dark-field optics (d-h). Significant hybridization was
observed in aorta (a and e), right pulmonary vein
(b and f), coronary artery (c and
g), and all other vessels in the section (d) with
the antisense RNA probe, whereas the sense control produced no
hybridization above background (h). Specimens were
photographed using a 10× (a and e), 20×
(b, c, f, and g) or 5×
(d and h) objective. aw, aortic wall;
av, aortic valve; cm, cardiac muscle;
ct, connective tissue.
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Fig. 6.
Expression of mCLCA4 in lung and
gastrointestinal tract. Sections of lung (a-d), small
intestine (e and f), and stomach (g
and h) were analyzed as in Fig. 5. Left column,
UV; right column, dark-field microscopy. Insets,
hybridization with sense probe control. Magnification of objective:
a and b, 10×; c and d,
40×; e-h, 20×. Br, bronchiole;
ME, muscularis externa; Muc, mucosa.
View larger version (11K):
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Fig. 7.
Transfection of mCLCA4 results in expression
of a calcium-activated inward current. A, exposure of
HEK293T cells transfected with mCLCA4 to ionomycin (10 µM) activates a transient inward current. No currents
were observed in non-transfected cells. B, exposure of these
cells to methacholine (mACH) evoked a similar transient
current with faster activation kinetics, consistent with a release of
intracellular calcium stores. No inward currents were observed in
vector-transfected control cells. C, in some
mCLCA4-transfected cells spontaneous, transient inward currents were
observed. These spontaneous transient inward currents were not observed
in all cells but were never observed in non-transfected cells.
60 to 40 mV,
indicating little voltage dependence of gating or intrinsic
rectification of mCLCA4 over this voltage range, similar to findings
for mCLCA1 (25) and consistent with the electrophysiological behavior
of these channels in smooth muscle (20). The current was shown to be
chloride-selective by substitution of extracellular Cl
ions with glutamate ions (Fig. 8B). In these experiments,
the current was activated by exposure of cells to ionomycin in the presence of normal extracellular solution or the same solution in which
100 mM NaCl was replaced by 100 mM sodium
glutamate; glutamate ions have a permeability ratio in
calcium-activated chloride channels of approximately 0.05 relative to
Cl ions (1, 40). In the presence of glutamate ions
substituted for chloride ions, the reversal potential of the
calcium-activated inward current shifted from 0 to 18.2 ± 3.6 mV
(n = 6; Fig. 8B), which was quite close to
the theoretical ECl (24 mV, assuming zero
glutamate permeability) and confirmed the anion selectivity of the
channel.
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Fig. 8.
The mCLCA4 current is a calcium-activated
chloride current. A, during exposure of
mCLCA4-transfected HEK293T cells to ionomycin, step depolarization was
imposed to obtain the current/voltage relationship. The magnitudes of
the currents at the end of the step are plotted below. Note that the
current reversal potential is ~0 mV in symmetrical chloride solution.
B, the shift in current reversal potential is shown for a
cell recorded in symmetrical chloride solution and in solution in which
chloride was replaced by glutamate in the extracellular solution. As
shown, the current reversal potential was shifted to close to the
theoretical chloride equilibrium potential (24 mV).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
Note Added in Proof
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1.
Evans, M. G.,
and Marty, A.
(1986)
J. Physiol. (Lond.)
378,
437-460 2.
Smith, P. M.,
and Gallacher, D. V.
(1992)
J. Physiol. (Lond.)
449,
109-120 3.
Arreola, J.,
Melvin, J. E.,
and Begenisich, T.
(1996)
J. Gen. Physiol.
108,
35-47 4.
Byrne, N. G.,
and Large, W. A.
(1987)
J. Physiol. (Lond.)
389,
513-525 5.
Byrne, N. G.,
and Large, W. A.
(1987)
Br. J. Pharmacol.
92,
371-379[Medline]
[Order article via Infotrieve]
6.
Byrne, N. G.,
and Large, W. A.
(1988)
J. Physiol. (Lond.)
404,
557-573 7.
Janssen, L. J.,
and Sims, S. M.
(1992)
J. Physiol. (Lond.)
453,
197-218 8.
Harvey, R. D.,
and Hume, J. R.
(1989)
Science
244,
983-985 9.
Zygmunt, A. C.,
and Gibbons, W. R.
(1991)
Circ. Res.
68,
424-437 10.
Clancy, J. P.,
McCann, J. D., Li, M.,
and Welsh, M. J.
(1990)
Am. J. Physiol.
258,
L25-L32[Medline]
[Order article via Infotrieve]
11.
Anderson, M. P.,
Sheppard, D. N.,
Berger, H. A.,
and Welsh, M. J.
(1992)
Am. J. Physiol. (Lond.)
263,
L1-L14
12.
Morris, A. P.,
and Frizzell, R. A.
(1993)
Am. J. Physiol.
264,
C977-C985[Medline]
[Order article via Infotrieve]
13.
Nilius, B.,
Prenen, J.,
Szucs, G.,
Wei, L.,
Tanzi, F.,
Voets, T.,
and Droogmans, G.
(1997)
J. Physiol. (Lond.)
498,
381-396 14.
Byrne, N. G.,
and Large, W. A.
(1985)
Br. J. Pharmacol.
86,
711-721[Medline]
[Order article via Infotrieve]
15.
Hirst, G. D.,
and Neild, T. O.
(1980)
J. Physiol. (Lond.)
303,
43-60 16.
van Helden, D. F.
(1991)
J. Physiol. (Lond.)
437,
511-541 17.
Wang, Q.,
Hogg, R. C.,
and Large, W. A.
(1992)
J. Physiol. (Lond.)
451,
525-537 18.
Janssen, L. J.,
Hague, C.,
and Nana, R.
(1998)
Am. J. Physiol.
275,
L516-L523[Medline]
[Order article via Infotrieve]
19.
Takano, H.,
Nakahira, Y.,
and Suzuki, H.
(2000)
Jpn. J. Physiol.
50,
597-603[CrossRef][Medline]
[Order article via Infotrieve]
20.
Large, W. A.,
and Wang, Q.
(1996)
Am. J. Physiol.
271,
C435-C454[Medline]
[Order article via Infotrieve]
21.
Criddle, D. N.,
de Moura, R. S.,
Greenwood, I. A.,
and Large, W. A.
(1996)
Br. J. Pharmacol.
118,
1065-1071[Medline]
[Order article via Infotrieve]
22.
Nelson, M. T.,
Conway, M. A.,
Knot, H. J.,
and Brayden, J. E.
(1997)
J. Physiol. (Lond.)
502,
259-264 23.
Cunningham, S. A.,
Awayda, M. S.,
Bubien, J. K.,
Ismailov, I. I.,
Arrate, M. P.,
Berdiev, B. K.,
Benos, D. J.,
and Fuller, C. M.
(1995)
J. Biol. Chem.
270,
31016-31026 24.
Elble, R. C.,
Widom, J.,
Gruber, A. D.,
Abdel-Ghany, M.,
Levine, R.,
Goodwin, A.,
Cheng, H. C.,
and Pauli, B. U.
(1997)
J. Biol. Chem.
272,
27853-27861 25.
Gandhi, R.,
Elble, R. C.,
Gruber, A. D.,
Schreur, K. D., Ji, H. L.,
Fuller, C. M.,
and Pauli, B. U.
(1998)
J. Biol. Chem.
273,
32096-32101 26.
Gruber, A. D.,
Elble, R. C., Ji, H. L.,
Schreur, K. D.,
Fuller, C. M.,
and Pauli, B. U.
(1998)
Genomics
54,
200-214[CrossRef][Medline]
[Order article via Infotrieve]
27.
Gruber, A. D.,
Schreur, K. D., Ji, H. L.,
Fuller, C. M.,
and Pauli, B. U.
(1999)
Am. J. Physiol.
276,
C1261-C1270[Medline]
[Order article via Infotrieve]
28.
Agnel, M.,
Vermat, T.,
and Culouscou, J. M.
(1999)
FEBS Lett.
455,
295-301[CrossRef][Medline]
[Order article via Infotrieve]
29.
Komiya, T.,
Tanigawa, Y.,
and Hirohashi, S.
(1999)
Biochem. Biophys. Res. Commun.
255,
347-351[CrossRef][Medline]
[Order article via Infotrieve]
30.
Elble, R. C.,
and Pauli, B. U.
(2001)
J. Biol. Chem.
276,
40510-40517 31.
Nakanishi, A.,
Morita, S.,
Iwashita, H.,
Sagiya, Y.,
Ashida, Y.,
Shirafuji, H.,
Fujisawa, Y.,
Nishimura, O.,
and Fujino, M.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
5175-5180 32.
Gruber, A. D.,
and Pauli, B. U.
(1999)
Cancer Res.
59,
5488-5491 33.
Abdel-Ghany, M.,
Cheng, H. C.,
Elble, R. C.,
and Pauli, B. U.
(2001)
J. Biol. Chem.
276,
25438-25446 34.
Dong, Q. G.,
Bernasconi, S.,
Lostaglio, S., De,
Calmanovici, R. W.,
Martin-Padura, I.,
Breviario, F.,
Garlanda, C.,
Ramponi, A.,
Mantovani, A.,
and Vecchi, A.
(1997)
Arterioscler. Thromb. Vasc. Biol.
17,
1599-1604 35.
Wilkinson, D. G.,
and Green, J.
(1990)
in
Postimplantation Mammalian Embryos: A Practical Approach
(Copp, A. J.
, and Cockroft, D. L., eds)
, pp. 155-171, Oxford University Press, New York
36.
Fleischmann, B. K.,
Murray, R. K.,
and Kotlikoff, M. I.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
11914-11918 37.
O'Brien, C. D., Ji, G.,
Wang, Y. X.,
Sun, J.,
Krymskaya, V. P.,
Ruberg, F. L.,
Kotlikoff, M. I.,
and Albelda, S. M.
(2001)
FASEB J.
15,
1257-1260 38.
Wang, Y.-X.,
and Kotlikoff, M. I.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
14918-14923 39.
Janssen, L. J.,
and Sims, S. M.
(1994)
Pfluegers Arch. Eur. J. Physiol.
427,
473-480[CrossRef][Medline]
[Order article via Infotrieve]
40.
Amedee, T.,
Large, W. A.,
and Wang, Q.
(1990)
J. Physiol. (Lond.)
428,
501-516 41.
Miledi, R.
(1982)
Proc. R. Soc. Lond. Ser. B Biol. Sci.
215,
491-497[Medline]
[Order article via Infotrieve]
42.
Mayer, M. L.
(1985)
J. Physiol. (Lond.)
364,
217-239 43.
Owen, D. G.,
Segal, M.,
and Barker, J. L.
(1984)
Nature
311,
567-570[CrossRef][Medline]
[Order article via Infotrieve]
44.
Thiemann, A.,
Grunder, S.,
Pusch, M.,
and Jentsch, T. J.
(1992)
Nature
356,
57-60[CrossRef][Medline]
[Order article via Infotrieve]
45.
Gruber, A. D.,
Fuller, C. M.,
Elble, R. C.,
Benos, D. J.,
and Pauli, B. U.
(2000)
Curr. Genomics
1,
201-222[CrossRef]
46.
Gruber, A. D.,
Gandhi, R.,
and Pauli, B. U.
(1998)
Histochem. Cell Biol.
110,
43-49[CrossRef][Medline]
[Order article via Infotrieve]
47.
Leverkoehne, I., and Gruber, A. D. (2002) J. Histochem.
Cytochem., in press
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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