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From the
Received for publication, March 25, 2002, and in revised form, April 18, 2002
ClC-2 is localized to the apical membranes of
secretory epithelia where it has been hypothesized to play a role in
fluid secretion. Although ClC-2 is clearly the inwardly rectifying
anion channel in several tissues, the molecular identity of the
hyperpolarization-activated Cl Molecular and functional studies have lead to the proposal that
the inwardly rectifying Cl The physiological importance of some epithelial chloride channels has
been revealed by gene mutation-inducing diseases such as cystic
fibrosis (10), Bartter's syndrome (11), and nephrogenic diabetes
insipidus (12). In mice lacking ClC-2, degeneration of the retina and
testis occurs, indicating that this chloride channel is required for
the survival of cells that depend on epithelia forming blood-organ
barriers (1). It is unclear whether this barrier function is related to
the regulation of ClC-2 activity by extracellular pH (13, 14) or cell
swelling (15). The apical location of the ClC-2 channel in rat small
intestine, renal, and airway epithelia further suggests that ClC-2
plays a role in regulating fluid and electrolyte movement in these
tissues (16, 17). Indeed, antisense ClC-2 cDNA reduced native
chloride current in the human intestinal cell line Caco-2 and
significantly reduced Cl Genetic analysis has provided important and sometimes surprising
insights into the function of several chloride channels. Nevertheless,
a clear understanding of the physiological significance of ClC-2 and
other chloride channels in most epithelia remains to be determined.
Functional analysis is complicated in native epithelial cells, because
multiple types of chloride channels are typically present. Salivary
gland acinar cells are no exception, expressing at least five distinct
chloride conductances (18, 19). The first of these to be characterized
(20) is activated by an increase in intracellular free
[Ca2+]i (21). It is likely that the
Ca2+-dependent Cl Therefore, we disrupted the Clcn2 gene to: 1) elucidate the
molecular nature of the inwardly rectifying Cl Generation of the Clcn2
The targeted ES cell clone was injected into C57BL/6 blastocytes to
generate chimeras that were backcrossed against the C57BL/6 strain.
Germline transmission was assessed by Southern blotting, and
heterozygous offspring were crossed to create the F2 animals used in
the present study. In all cases, littermates were paired for each set
of experiments. The general phenotype of our
Clcn2 Electrophysiology--
Whole cell currents were recorded at room
temperature from freshly isolated single parotid acinar cells using the
patch clamp technique (28). An Axopatch 200 B amplifier (Axon
Instruments Corp., Foster City, CA) was used to voltage clamp and
record the resulting chloride currents. Voltage clamp protocols to
activate channels were generated by pClamp 8 software (Axon Instruments Corp.). Chloride currents were filtered at 1 kHz using a low pass Bessel filter and digitized at 2 kHz. A glass pipette had a 2- to
4-M Acinar Cell Preparation--
Parotid acinar cell clumps from
adult (8-10 weeks old) Clcn2+/+ and
Clcn2
Single cell preparations for electrophysiology utilized an initial
10-min digestion of the minced parotid tissue in 12.5 ml of trypsin
digestion media (minimal essential medium, Spinner modification (SMEM),
Biofluids, Inc.) containing 0.01% trypsin, 0.5 mM EDTA,
and 1% BSA under 95%O2 + 5%CO2 gassing and
while shaking (60 cycles/min). The cells were pelleted at 210 × g for 15 s then washed with 10 ml of trypsin inhibitor
solution (SMEM containing 0.2% trypsin inhibitor and 1% BSA). The
cells were spun again and incubated in collagenase digestion solution
as described above. Single cells were rinsed with BSA-free basal medium
Eagle, selected by filtration through 53-µm nylon mesh, and attached
to circular 5-mm polylysine-coated glass coverslides in a 37 °C
incubator containing 95%O2 + 5%CO2.
Cell Volume Determinations--
Cell volume was estimated using
a Nikon Diaphot 200 microscope interfaced with an Axon Imaging
Workbench System (Novato, CA). The dispersed acinar cells were loaded
with the fluoroprobe calcein by incubation for 15 min at room
temperature with 100% O2 in 2 µM calcein-AM
(Molecular Probes, Eugene, OR). Dye-loaded cells were exposed to 490-nm
light, and emitted fluorescence was measured at 530 nm. Changes in cell
volume were monitored by measuring the fluorescence intensity of
calcein within a delimited intracellular volume. Cell volume was
expressed in arbitrary units as 1/normalized calcein fluorescence.
Hyposmotic Shock and the Subsequent Regulatory Volume
Decrease--
Calcein-loaded acinar cell clumps were equilibrated in
an isotonic physiological solution containing (in millimolar): 135 NaCl, 5.4 KCl, 0.4 KH2PO4, 0.33 NaH2PO4, 20 Hepes, 10 glucose, 0.8 MgSO4, and 1.2 CaCl2, pH 7.4. Hypotonic
challenge was induced by switching the perfusate to the above solution
after diluting by 30% with water. Cell volume change was measured as
described above. Regulatory volume decrease (RVD) was followed over the course of ~300 s while the cells remained in the hypotonic solution, and the rate of volume recovery was calculated by determining the slope
of the best-fit line following the switch to hypotonic media and
maximum cell swelling. Some experiments used clotrimazole (Sigma
Chemical Co.) at a final concentration of 1 µM present in
all of the solutions, and others used zinc at a final concentration of
50 µM.
Stimulated Flow Rates and Saliva Composition--
Adult
littermates Clcn2+/+ and
Clcn2 Generation of a Mouse Strain Lacking ClC-2--
A lambda genomic
DNA library derived from 129/SVJ mice was screened using a probe
specific for the Clcn2 gene. Genomic fragments from the
resulting lambda clone were used to flank a positive (PGKneo) selection
cassette in a vector designed to target the Clcn2 gene for
disruption (Fig. 1A). The
construct was intended to replace a genomic segment that includes a
portion of the Clcn2 promoter and 5'-UTR, as well as all of
exon 1 and most of exon 2, with PGKneo. This strategy was expected to
result in the inability to initiate transcription from the defunct
Clcn2 promoter in the transgenic strain, causing an absence
of functional protein, thereby avoiding the possibility of a dominant
negative effect caused by expression of a truncated protein. During the
gene-targeting procedure, a construct with the upstream arm in the
wrong orientation was mistakenly utilized. Electroporation of this
construct into embryonic stem cells resulted in a single targeted cell
line (out of ~900 neomycin-resistant clones; Fig. 1C)
resulting from a hybrid homologous recombination/insertion event, as
described below. After the mistake was recognized we learned that the
promoter of another gene, encoding the RPB-17 protein, overlaps that of Clcn2 in rat (30), as well as in
mouse.2 Because the correct
construct would disrupt both genes, we proceeded to analyze the
embryonic stem cell clone that we had identified.
Analysis using both inside and outside probes as well as genomic PCR
(data not shown) demonstrated that homologous recombination occurred
between the 3'-arm of the targeting vector and the Clcn2 gene. This was followed by a non-homologous insertion event in the
upstream (backwardly oriented) arm (Fig. 1B). The junction between the genomic and vector DNA was not mapped at the single nucleotide level due to a stretch of over 1500 nucleotides of up to
85% GC content, which precluded genomic PCR across that region. The
final homozygous knockout strain was shown to lack ClC-2 protein by
Western analysis (Fig. 1D). In addition, RT-PCR with primers
to the 5'- and 3'-ends of the transcript confirmed that ClC-2 message
was not present in the knockout strain, whereas Northern analysis
indicated that the RPB-17 mRNA, whose promoter overlaps that of
Clcn2, but in the antisense orientation, was present at
normal levels in the Clcn2 Retinal and Testicular Degeneration in ClC-2-deficient
Mice--
The phenotype of the Clcn2 Characterization of Chloride Currents from Parotid Acinar
Cells--
The homozygous knockout strain was used to assess the
molecular nature of the inwardly rectifying Cl
The chloride currents recorded from wild-type parotid acinar cells
using the whole cell configuration (Fig. 4, upper left trace) displayed inward rectification and time dependence, as has
been observed previously (2, 19). To clearly monitor the
hyperpolarization-activated chloride currents, it was necessary to
eliminate the Ca2+-dependent and the
volume-sensitive currents. This was accomplished using an internal
pipette solution containing the calcium chelator EGTA and a hypertonic
bath solution (see "Experimental Procedures"). Relative to acini
from wild-type mice, currents for the acini of
Clcn2
Because the opening of the Ca2+-activated Cl ClC-2 Does Not Function in Regulatory Volume Decrease in Parotid
Acinar Cells--
Mammalian cells undergo a regulatory volume decrease
(RVD) upon exposure to a hypotonic medium. This process allows the
efflux of electrolytes, which are then followed osmotically by water, resulting in cell shrinkage to a normal resting volume. ClC-2 has been
shown to be up-regulated by cell swelling and may play a role in RVD
(15, 33-35), although some evidence exists that ClC-2 can be inhibited
by hypotonicity as well, in the presence of protein phosphatase
inhibitors (36). Fig. 6 shows that under hypertonic conditions ClC-2 Cl
To ascertain whether ClC-2 contributes functionally to volume
regulation in parotid acinar cells, cell volume changes were monitored
following swelling in hypotonic solution (Fig. 7A) and the
initial rates of RVD were determined.
Parotid acini from Clcn2 Loss of Inwardly Rectifying ClC-2 Chloride Channels Does Not Change
the Composition or Flow Rate of Whole Saliva--
The functional
consequences of disrupting expression of the inwardly rectifying ClC-2
Cl
As previously observed (39), the average flow rates became slower as
time progressed (Fig. 8A), but the concentrations of the
electrolytes sodium, chloride, and potassium were qualitatively similar
at both higher and lower flow rates in both genotypes (Fig.
8B). This point is important as it is during transit of the
primary secretions through the water-impermeable ductal network that
electrolytes (primarily Na+ and Cl ClC-2 is a broadly expressed plasma membrane chloride channel that
is active at negative membrane potentials (41). Although the function
of other members of the ClC gene family have become clear following the
identification of disease phenotypes associated with their mutation,
the role of ClC-2 remains an enigma. The distribution of ClC-2 and its
overlap with that of the cystic fibrosis transmembrane conductance
regulator (CFTR), the CF gene product, suggests an important function
for ClC-2 in maintaining chloride homeostasis as well as the potential
to serve a compensatory role in alleviating the severity of the CF
phenotype. ClC-2 has been shown to be present in the developing fetal
lung (42, 43), as well as in the small intestinal epithelium (27), and
contributes to chloride secretion from an intestinal cell line (16).
However, mice deficient in ClC-2 displayed no gross phenotypic deficits in intestinal or lung function.
The focus of the present study was to test three hypotheses in mice
deficient in the expression of ClC-2. 1) Is ClC-2 the hyperpolarization-activated Cl In contrast to the studies of choroid plexus epithelial cells by Speake
et al. (9), we found that targeted disruption of the
Clcn2 gene resulted in loss of the inwardly rectifying
Cl The movement of the primary secretions through duct cells in
salivary glands allows reabsorption of electrolytes, including chloride
and sodium, and results in a hypotonic NaCl-poor final secretion. The
molecular mechanism by which these electrolytes are reabsorbed is still
not understood, but most likely involves the epithelial sodium channel
(48-51), with chloride moving either paracellularly or through a
chloride channel located on the apical membrane of the ducts. Normal
levels of sodium, chloride, and potassium were found in the saliva of
Clcn2 In summary, we have shown that ablation of the Clcn2 gene
does not result in notable deficits in either the production or modification of saliva following stimulation with a cholinergic agonist
in mice, despite the loss of inward-rectifying Cl We thank Dr. Christine Bear for reagents and
technical assistance in assessing the level of ClC-2 protein in the
knockout strain.
*
This work was supported in part by National Institutes of
Health Grants DE09692 and DE13539 (to J. E. M.) and DK50594 (to G. E. S.).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.
Published, JBC Papers in Press, April 25, 2002, DOI 10.1074/jbc.M202900200
2
K. Nehrke, unpublished observations.
The abbreviations used are:
UTR, untranslated
repeat;
RT-PCR, reverse transcription-PCR;
CFTR, cystic fibrosis
transmembrane conductance regulator;
BSA, bovine serum albumin;
SMEM, Spinner-modified minimal essential medium, RVD, regulatory volume
decrease;
NMDG, N-methyl-D-glucamine;
TEA, tetraethylammonium.
Loss of Hyperpolarization-activated Cl
Current in
Salivary Acinar Cells from Clcn2 Knockout Mice*
§,¶,§,,,
,, and§
Center for Oral Biology, Aab Institute of
Biomedical Sciences, the § Eastman Department of
Dentistry, the ** Department of Laboratory Animal
Medicine, and the ¶ Department of Pharmacology and
Physiology, University of Rochester Medical Center, Rochester, New
York 14642 and the Department of Molecular Genetics,
Biochemistry, and Microbiology, University of Cincinnati College of
Medicine, Cincinnati, Ohio 45267
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
current in other
organs, including the salivary gland, is currently unknown. To
determine the nature of the hyperpolarization-activated Cl current and to examine the role of ClC-2 in salivary
gland function, a mouse line containing a targeted disruption of the
Clcn2 gene was generated. The resulting homozygous
Clcn2/ mice lacked detectable
hyperpolarization-activated chloride currents in parotid acinar cells
and, as described previously, displayed postnatal degeneration of the
retina and testis. The magnitude and biophysical characteristics of the
volume- and calcium-activated chloride currents in these cells were
unaffected by the absence of ClC-2. Although ClC-2 appears to
contribute to fluid secretion in some cell types, both the initial and
sustained salivary flow rates were normal in
Clcn2/ mice following in vivo
stimulation with pilocarpine, a cholinergic agonist. In addition, the
electrolytes and protein contents of the mature secretions were normal.
Because ClC-2 has been postulated to contribute to cell volume control,
we also examined regulatory volume decrease following cell swelling.
However, parotid acinar cells from Clcn2/
mice recovered volume with similar efficiency to wild-type littermates. These data demonstrate that ClC-2 is the hyperpolarization-activated Cl channel in salivary acinar cells but is not essential
for maximum chloride flux during stimulated secretion of saliva or
acinar cell volume regulation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
channel in most, if not all,
mammalian cells is ClC-2. Indeed, a null mutation in the
Clcn2 gene resulted in the loss of
hyperpolarization-activated anion currents in Leydig and Sertoli cells
(1). Inwardly rectifying Cl currents have qualitatively
similar properties in numerous tissues, nevertheless, unique activation
kinetics are often observed in different cell types and in heterologous
ClC-2 expression systems. For example, under identical experimental
conditions, the chloride current generated by recombinant rat ClC-2 in
HEK293 cells activates with a faster time course than the current in
rat salivary acinar cells (2). Moreover, cAMP is an important regulator
of recombinant human ClC-2 channel activity (3) and of
hyperpolarization-activated Cl currents in both choroid
plexus (4) and human T84 colon cells (5); in contrast, cAMP sensitivity
is not seen in salivary acinar cells (6). One interpretation of these
contrary results is differential expression of a regulatory subunit
that modulates channel kinetics. Alternatively, splice variants of
ClC-2 may alter the activation properties of this channel (7, 8). However, analysis of the currents in choroid plexus epithelial cells
from ClC-2 knockout animals failed to reveal a loss of the hyperpolarization-activated Cl conductance (9). These
later results demonstrate that another novel gene encodes the inwardly
rectifying Cl current present in choroid plexus cells and
raises the possibility that the hyperpolarization-activated
Cl channel in salivary gland cells and other cell types
is not ClC-2.
-dependent secretion
(16).
channel is
targeted to the apical membrane in parotid acinar cells as has been
shown in pancreatic acinar cells (22). Because salivation is
Ca2+-dependent (23-25), the
Ca2+-gated Cl channel has been predicted to
be the primary Cl channel activated during stimulated
secretion. Additional Cl channels found in salivary gland
cells include those that are volume-sensitive (26),
cAMP-dependent (18), hyperpolarization-activated (2, 19),
and channels with properties like ClC-0 (18). The complexity created by
the expression of multiple chloride channels in acinar cells indicates
that gene knockout model systems will likely be required to
unequivocally assign function to an individual channel.
current in
salivary acinar cells; 2) determine the role of ClC-2 in saliva
secretion; and 3) examine whether ClC-2 is critical for cell volume
regulation. Patch-clamp analysis of chloride currents in salivary gland
acinar cells demonstrated the loss of inwardly rectifying current in
Clcn2/ mice. In contrast, no changes were
observed in the calcium- or volume-activated chloride conductances.
Despite suggestions that ClC-2 may be involved in volume regulation,
acinar cells from Clcn2/ mice recovered cell
volume following swelling by hypotonic shock as well as those from
wild-type littermates. Furthermore, we show that the flow-rate of
saliva secreted during in vivo stimulation, as well as the
protein and electrolyte concentrations of the saliva, were comparable
in wild-type and Clcn2/ mice. These data
unequivocally identity Clcn2 as the gene that encodes for
the inwardly rectifying Cl channel in salivary acinar
cells and demonstrate that the Cl currents required for
stimulated secretion of saliva are mediated by other channels such as
the Ca2+- and/or volume-activated Cl channels.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/ Mouse Strain--
A
clone isolated from a 129/SVJ mouse genomic lambda library was used to
construct a targeting vector with a neomycin-resistance (neo) gene as a
positive selection marker and thymidine kinase gene as a negative
selection marker. A 3.07-kb high fidelity PCR product obtained from a
sub-cloned SstI fragment from the Clcn2 gene was
inserted 3' of the neo cassette, and a 2.3-kb
EcoRI-BamHI fragment was inserted 5' of the neo
cassette (see Fig. 1A). The neo cassette was designed to
replace 1 kb of promoter and ~500 nucleotides of
5'-UTR1 as well as exon 1 and
most of exon 2 of the Clcn2 gene. However, after blunt-end
cloning the 5'-fragment, a clone containing the wrong orientation of
the 5'-arm was mistakenly identified and subsequently linearized and
electroporated into KG ES cells. Southern blotting of
EcoRI-digested genomic DNA from targeted ES cell clones using a 1.3-kb outside probe (3' of the sequence used to create the
targeting vector, as indicated in Fig. 1A) led to the
identification of a recombinant clonal isolate exhibiting the predicted
11- to 7-kb shift in size (Fig. 1C). Further
characterization of this clone indicated that homologous recombination
had taken place between the 3'-arm of the targeting vector and the
genomic DNA, as well as between an unidentified segment of the 5'-arm
of the targeting vector and a region slightly downstream of the 3'-end of the genomic copy of the 5'-arm (possibly due to the presence of long
stretches of repetitive sequence in this area). Although this clone did
not delete the promoter of the Clcn2 gene, the replacement
of most of exons 1 and 2 with PGKneo was expected to and did, in fact,
result in the loss of both the ClC-2 transcript and protein in the
final homozygous animal, as determined by RT-PCR and Western analysis
(Fig. 1D). The generation of the anti-ClC2 antibody has been
described previously (27) and is a kind gift of C. Bear (The Hospital
for Sick Children, Toronto). Amino acids 16-35 of rat ClC-2 contain
the target sequence to which the antibody was raised.
/ strain was essentially
indistinguishable from that reported recently by the Jentsch laboratory
(1). For histological examination, adult
Clcn2+/+ and Clcn2/
age- and sex-matched littermates (7-8 weeks of age) were anesthetized with 300 mg of chloral hydrate/kg of body weight (intraperitoneally) and the tissues were fixed by perfusion with 10% neutral buffered formalin, sectioned at 5 µm, and stained with hematoxylin and eosin.
resistance when filled with the internal solutions. To record
hyperpolarization-activated chloride currents, cells were dialyzed with
an internal solution containing (millimolar): TEA-Cl 140, EGTA 20, HEPES 20, pH 7.3 with TEA-OH. Calcium-dependent chloride
channel currents were recorded from cells dialyzed with an internal
solution containing (millimolar): NMDG-glutamate 80, NMDG-EGTA 50, CaCl2 30, HEPES 20, pH 7.3 with NMDG. The free calcium concentration of this solution was estimated to be 250 nM
(WinMax 2, Stanford CA). Cells were bathed in an external hypertonic
solution containing (millimolar): TEA-Cl 140, CaCl2 0.5, D-mannitol 100, HEPES 20, pH 7.3 with TEA-OH;
volume-sensitive currents were activated by diluting this solution 20%
with water and using the same internal solution as described above for
recording hyperpolarization-activated chloride currents. Square pulses
of 5 or 3 s were delivered every 7 s from a holding potential
of 0 (hyperpolarization-activated and volume-sensitive currents) or
50 mV (calcium-dependent currents). Membrane potential
was changed between 120 to +120 mV in 20-mV steps, and the resulting
currents were recorded after 10 (hyperpolarization-activated) and 5 (volume-sensitive and calcium-dependent currents) min of dialysis. Junction potentials (4.5 mV) and leak currents were not
corrected. Current-voltage relationships were constructed by plotting
the absolute magnitude of the currents at the end of the pulse against
the membrane potential.
/ littermates were prepared by
collagenase digestion as previously described (29). Briefly, mice were
killed by exsanguination following exposure to CO2 gas. The
parotid glands were quickly removed, trimmed of connective tissues, and
finely minced in 7.5 ml of collagenase digestion medium (Eagle's
minimal essential medium, Biofluids, Inc., Rockville, MD) containing
0.04 mg/ml collagenase P and 1% BSA. The minced glands were incubated
at 37 °C in a shaker with continuous agitation (100 cycles/min) and under gas (95%O2 + 5%CO2). After the first
20-min interval the minced glands were dispersed by gentle pipetting
(10 times) and centrifuged (210 × g for 15 s).
The supernatant was discarded, and the pellet was resuspended in 7.5 ml
of collagenase digestion medium for an additional 40 min with pipetting
at 20-min intervals. The cells were then rinsed and harvested by centrifugation.
/ (7-8 weeks of age) were anesthetized
with 300 mg of chloral hydrate/kg of body weight
(intraperitoneally) and then stimulated with 10 mg of
pilocarpine-HCl/kg of body weight (intraperitoneally). Whole saliva,
primarily representing a combination of parotid and submandibular
secretions, with a very minor component from sublingual and minor
salivary, nasal, and tracheal glands, was collected from the lower
cheek pouch by a suction device at intervals of 5, 10, and 15 min. The
protein concentration of saliva was determined using the Bradford
method. Total sodium and potassium contents in saliva samples were
determined by atomic absorption using a PerkinElmer Life Sciences 3030 spectrophotometer. Saliva osmolality was measured using a Wescor 5500 vapor pressure osmometer, and chloride activity was determined using an
Orion EA 940 expandable ion analyzer.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Clcn2 gene targeting. A,
schematic of the design used to create the Clcn2 gene
targeting construct. The genomic DNA is represented by the upper
drawing, whereas the targeting construct is represented by the
lower drawing. Approximate sites where replacement of the
genomic DNA with the targeting construct occurred are indicated by
lines joining the upper and lower
drawings. Homologous arms 1 and 2 correspond to the promoter
region of Clcn2 and from exon 2 through exon 11 of the
coding region, respectively, and are indicated by dashed
arrows. An outside probe, denoted in the schematic, was used to
identify recombinant clones based upon Southern analysis of genomic
EcoRI digests. Primers 1 and 2 (P1 and
P2) were used in genomic PCR of the targeted strain, and
sequencing of the PCR product indicated that recombination had occurred
in the 3'-arm as predicted. Primers 3 and 4 (P3 and
P4) failed to produce an RT-PCR product from the targeted
strain, as did a combination of primer 2 (P2) and a primer
antisense to primer 4 (P4), confirming the lack of ClC-2
message. B, schematic of the final targeted clone. An
apparent recombination/insertion event resulted in the inverted
duplication of arm 1, as indicated by the dashed arrows
facing each other. The junction between the inserted vector DNA and the
genomic DNA was not mapped at the nucleotide level, due to a stretch of
nearly 85% GC-rich DNA in the region that precluded genomic PCR.
C, Southern analysis of EcoRI-digested genomic
DNA from a targeted ES cell line, using the outside probe indicated
above. D, Western analysis of ClC-2 protein from brain
homogenates prepared from Clcn2+/+ and
/ strains.
/ animal (data not shown).
/
strain generated in our laboratory is comparable to that reported
recently (1). The mice appeared generally healthy and displayed normal
behavior and body weight, but histological examination of semi-thin
sections revealed abnormalities of the eye and testis. Unlike the
wild-type eye (Fig. 2A), the knockout exhibited post-natal degeneration of the retina, reflected by
a gradual loss of photoreceptor cells and the outer nuclear layer,
which was not present or remained as only a few cells adjacent to the
inner nuclear layer (Fig. 2, B and C). Maturation
of the testes was normal in wild-type mice (Fig.
3A) but was impaired in the
knockout (Fig. 3B). In Clcn2/
mice at the age of sexual maturity, the germ cell layers were missing,
and no mature spermatozoa were present; in addition, there was
hyperplasia of the Leydig cells and abnormal Sertoli cells were
prominent and widespread (Fig. 3B).
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Fig. 2.
Histology of the retina. A,
retina from a Clcn2+/+ female, with
normal architecture, including inner nuclear layer (INL),
outer nuclear layer (ONL), ganglion cell layer
(GCL), and photoreceptors (PhR). B,
retina from a Clcn2 / female, with decreased
outer nuclear layer, and a single row of photoreceptor nuclei remaining
adjacent to the inner nuclear layer. C, retina from a
Clcn2/ male, which has lost all of the
photoreceptor nuclei, leaving only the inner nuclear layer.
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Fig. 3.
Histology of the testis. 5-µm
sections of testis from a Clcn2+/+ male
(A) and Clcn2 / male
(B) obtained 10 weeks after birth. The normal testicular
architecture found in the +/+ mice is compromised in the
Clcn2/ animal, with missing germ cell layers
(double-headed arrow in the +/+ control), abnormal Sertoli
cells (small arrows) and a relative hyperplasia of the
Leydig cells (large arrows).
current
and to determine the role of ClC-2 in saliva gland function and fluid
secretion from salivary acinar cells. The production of saliva is
initiated by an increase in intracellular Ca2+ that opens
Ca2+-dependent chloride channels on the apical
membranes of the acinar cells. Anion fluxes in parotid acinar cells are
mediated by at least five distinct chloride currents, namely,
volume-sensitive, calcium-dependent, cAMP-activated,
ClC-0-like, and hyperpolarization-activated channels (18, 19). RT-PCR
has demonstrated the presence of ClC-2 in parotid acini, and the
characteristics of the hyperpolarization-activated chloride current in
this cell type are quantitatively similar to that of the cloned ClC-2
channel (2). To unambiguously determine the molecular identity of the
channel mediating this current, we performed patch clamp analysis on
single parotid acinar cells isolated from wild-type and
Clcn2/ mice.
/ mice decreased more than 10-fold in
magnitude at the most negative potentials and exhibited no
rectification (Fig. 4, upper right trace). The lower panels show the current-voltage (IV)
relations of chloride currents for parotid acinar cells derived from
multiple Clcn2+/+ (left, n = 6)
and Clcn2/ (right, n = 8)
mice. A similar analysis of heterozygous
Clcn2+/ mice revealed similar
hyperpolarization-activated currents as present in wild-type acinar
cells, suggesting that there is no dominant negative effect (data not
shown). These results confirm that the ClC-2 channel is, in fact,
responsible for the hyperpolarization-activated Cl
current in parotid acinar cells.
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Fig. 4.
Chloride currents in
Clcn2+/+ and
Clcn2 / mice. Upper
panels, whole cell chloride currents recorded from single acinar
cells isolated from wild-type Clcn2+/+
(left) and Clcn2/
(right) mice. Lower panels, current voltage
relationships from Clcn2+/+ (left;
n = 6) and Clcn2/
(right; n = 8) mice, respectively. The
internal solution contained (millimolar): TEA-Cl 140, EGTA 20, HEPES
20, pH 7.3, with TEA-OH, whereas the external solution contained
(millimolar): TEA-Cl 140, CaCl2 0.5, D-mannitol
100, HEPES 20, pH 7.3, with TEA-OH.
channel on the apical cell membrane is thought to be the primary means
through which chloride exits the cell following stimulation, we
examined Ca2+-dependent chloride currents in
the Clcn2/ mice as well. Although it is
unlikely that ClC-2 contributes to the Ca2+-activated
chloride current directly, oftentimes gene ablations lead to
compensatory mechanisms in overlapping or redundant processes (31, 32).
Fig. 5 (upper panels) shows
whole cell Ca2+-dependent Cl
current obtained from wild-type (left) and
Clcn2/ (right) mice; the
lower panels show the corresponding average current-voltage
relationships. Thus, the Ca2+-dependent
Cl currents from the Clcn2/
mice resembled those from Clcn2+/+ mice,
although with greater variability in magnitude.
View larger version (23K):
[in a new window]
Fig. 5.
Calcium-dependent chloride
currents in Clcn2+/+ and
Clcn2 / mice. The upper
panels display calcium-dependent currents recorded
from single parotid acinar cells isolated from
Clcn2+/+ (left) and Clcn2
/ (right) mice, respectively. The
lower panels are the respective corresponding averaged
current voltage relationships (Clcn2+/+,
n = 3; Clcn2 /,
n = 4). The internal solution contained (millimolar):
NMDG-glutamate 80, NMDG-EGTA 50, CaCl2 30, HEPES 20, pH
7.3, with NMDG, whereas the external solution contained (millimolar):
TEA-Cl 140, CaCl2 0.5, D-mannitol 100, HEPES
20, pH 7.3, with TEA-OH.
currents were present in
acinar cells from wild-type (upper left) but not
Clcn2/ (upper right) mice. In
contrast, upon exposing the same cells to a hypotonic solution, large,
outwardly rectifying chloride currents similar to those previously
reported (26) were recorded in acinar cells from both genotypes (Fig.
6). The currents activated under hypotonic conditions did not resemble
those of ClC-2, nor were they altered in
Clcn2/ acinar cells (Fig. 6, middle
right trace). Also the magnitude of the time-dependent
current due to ClC-2 activation at 100 mV in wild-type acinar cells
was not altered by cell swelling. The IV curves derived from multiple
Clcn2+/+ and Clcn2/
mice indicated that there was no significant change in the
swelling-activated chloride currents (Fig. 6, lower
panels).
View larger version (32K):
[in a new window]
Fig. 6.
Volume-stimulated chloride currents in
Clcn2+/+ and
Clcn2 / mice. The left
and right columns display data obtained from
Clcn2+/+ and Clcn2/
mice, respectively. Upper panel, control currents obtained
from 100 to + 100 mV after 6-min dialysis. Note that the scale is
reduced in comparison to the middle panel to observe the
currents more clearly. Middle panel, chloride currents
obtained from the same cells depicted in the upper row after
4 min in a 20% hypotonic medium. Lower panel, average
current-voltage relationships obtained from cells under control
conditions (squares) and after 4 min in a hypotonic solution
(circles). (Clcn2+/+,
n = 4; Clcn2/,
n = 3). The internal solution contained (millimolar):
TEA-Cl 140, EGTA 20, HEPES 20, pH 7.3, with TEA-OH, whereas the
external solution contained (millimolar): TEA-Cl 140, CaCl2
0.5, D-mannitol 100, HEPES 20, pH 7.3, with TEA-OH, which
was diluted by 20% with water to activate the currents.
/ mice underwent RVD
following swelling at a similar initial rate as their wild-type
littermates (Fig. 7B). In addition, the divalent cation
Zn2+ is known to inhibit ClC-2 chloride currents expressed
in Xenopus oocytes (37) and mouse parotid acinar cells (13),
but had no effect on the initial rate of RVD in wild-type parotid
acinar cells (Fig. 7B). On the other hand, clotrimazole, a
relatively specific inhibitor of IK1 Ca2+-activated
K+ channels (38), reduced the initial rate of RVD by 50%
(Fig. 7B). Together, these data suggest that ClC-2 is not a
major regulator of cell volume homeostasis in parotid acinar cells.
View larger version (34K):
[in a new window]
Fig. 7.
Regulatory volume decrease in parotid acinar
cells from mice lacking ClC-2 expression. The role of ClC-2 in the
RVD response was examined in parotid acini loaded with calcein as
described under "Experimental Procedures." A, parotid
acini isolated from Clcn2+/+ (black
squares) and Clcn2 / (gray
circles) mice were perfused in an isosmotic solution, and then
swelling was induced by switching the perfusate to a hypotonic medium
(30% dilution with water). Changes in cell volume were expressed as
1/calcein Fn, and the initial rate of RVD was
calculated (Clcn2+/+, n = 7 and
Clcn2/, n = 11; representing
four separate acinar cell preparations). B, the initial rate
of RVD was calculated for Clcn2+/+
(n = 21) and Clcn2/
(n = 23) acini and for wild-type acini treated with
either 50 µM zinc (n = 19) to inhibit
ClC-2 channels or with 1 µM clotrimazole
(n = 20) to inhibit the Ca2+-activated
K+ channel mIK1.
channel in salivary glands was first examined by
determining the amount of saliva secreted following stimulation with
the cholinergic agonist pilocarpine. Whole saliva, an indicator of
overall salivary gland function (38), was collected from age- and
sex-matched littermates at 5-min intervals over a 15-min period. The
saliva collected here primarily represents contributions from the
parotid and submandibular salivary glands, as well as a smaller
component from sublingual and minor salivary glands, and nasal
secretions. ClC-2 is present in both the parotid and submandibular
glands (2, 18). Changes in the ability of these glands to secrete fluid
are reflected in the accumulation of whole saliva in the oral cavity.
However, a comparison between wild-type and
Clcn2/ mice failed to reveal significant
differences for either the average flow rates at any point during the
collection period (Fig. 8A) or
the total volume of saliva secreted (Table
I). Over the course of the 15-min
collection period, male Clcn2+/+ and
Clcn2/ mice secreted 13.6 ± 3.4 and
15.4 ± 3.6 µl of saliva per gram of body weight, respectively,
while female wild-type and Clcn2/ mice
secreted 10.1 ± 1.0 and 11.4 ± 2.6 µl per gram of body
weight, respectively.
View larger version (24K):
[in a new window]
Fig. 8.
Saliva flow rates and flow rate dependence of
salivary electrolyte concentrations in wild-type and ClC-2 knockout
mice. A, whole saliva was collected at 5-min intervals
over a 15-min period from mice stimulated to secrete with the
cholinergic agonist pilocarpine (10 mg/kg intraperitoneally). The
volume of saliva collected was normalized to the body weight of the
mice. Standard deviations are presented for each of the four groups
analyzed: Clcn2+/+ and
Clcn2 / males (n = 11 each)
and Clcn2+/+ and
Clcn2/ females (n = 5 each).
More males than females were analyzed due to higher variability among
male animals. B, individual salivary flow rates were
calculated for each 5-min sample taken from every animal as indicated
above then compared with the concentrations of the electrolytes
chloride, sodium, and potassium in the mature saliva (
,
Clcn2+/+; 
,
Clcn2/).
Volume and composition of saliva from Clcn2+/+ and
Clcn2/ mice
) are
reabsorbed, leading to a NaCl-poor, hypotonic mature secretion, and
ClC-2-like Cl currents have been described in salivary
gland duct cells (40). We note here that we are using pilocarpine,
which is a mixed cholinergic agonist that activates both sympathetic
and parasympathetic components, via nicotinic and muscarinic receptors,
respectively, to stimulate salivation, and that salivary gland duct
cells are also responsive to beta-adrenergic agonists; thus, although
the data strongly suggest that ClC-2 is not involved in electrolyte
conservation in salivary glands under these conditions, the data do not
completely rule out a role for ClC-2 in duct function under all
conditions. Finally, we demonstrated that the total amount of protein
present in the saliva was not significantly different in knockout mice compared with their sex-matched wild-type littermates and showed that
the weights of the salivary glands were similar in both wild-type and
Clcn2/ mice (Table I). Although our results
suggest that ClC-2 is involved in neither secretion from salivary
acinar cells following cholinergic stimulation nor electrolyte
conservation in the salivary ductal network, it remains a possibility
that unknown compensatory mechanisms are at work.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
channel in salivary acinar
cells? 2) Does ClC-2 contribute to saliva secretion? 3) Is ClC-2
involved in cell volume regulation? In agreement with a previous report
(1), we found that the only apparent global phenotypic deficits
associated with the lack of ClC-2 include postnatal degeneration of the
retina, including loss of the outer nuclear layer, which results in
blindness, and incomplete maturation of the seminiferous tubules and
abnormal Sertoli cells in the testes, leading to azoospermatic males
that are infertile. A common theme among these phenotypes is the
dependence of the retina and seminiferous tubules on close cell-cell
interactions, as noted by Bösl and colleagues (2001). Briefly,
both affected organs are protected by a blood-organ barrier, and
degeneration occurs in cells that depend upon the barrier-forming
epithelium (for a more detailed discussion, see Ref. 1). Interestingly, a Caenorhabditis elegans homolog of ClC-2, termed CLH-3, has
recently been characterized (44). Although CLH-3 can be activated by cell swelling, the physiological trigger for activation is the induction of oocyte meiotic maturation. In animals exhibiting a CLH-3
loss-of-function, the contractile activity of gonadal sheath cells is
initiated prematurely. Thus, the function of this channel is to couple
two processes that occur between adjacent cells. How this is
accomplished is not known at the present time. However, in mice, ClC-2
has been localized to the tight junction complex between adjacent
intestinal epithelial cells (27) and phenotypically, the
Clcn2/ mice exhibit deficits in male germs
cells and photoreceptor cells, both dependent upon close cell-cell
interactions. It is possible that this member of the ClC family acts in
barrier function and cell-cell communication, raising the question of
whether these processes may be codependent or coupled in some fashion.
Further study of exactly how the loss of ClC-2 leads to these
phenotypes will undoubtedly shed light on its physiological role.
current in salivary acinar cells. Based upon its
location in other polarized cell types, ClC-2 could act at the apical
acinar cell surface to potentiate Cl efflux into the
lumen of the gland during stimulation by acting in concert with other
Cl channels. The Ca2+-dependent
Cl channel is targeted to the apical membrane, however,
down-regulation of this channel is frequently observed (45, 46). This
suggests that an additional Cl channel might also be
activated in response to sustained stimulation, possibly by a
non-Ca2+-dependent mechanism. It is doubtful
that the volume-sensitive Cl channel fills this role,
because cell shrinkage, which occurs during stimulation (47),
down-regulates this channel (26). Moreover, the
cAMP-dependent channel, almost certainly encoded by the
Cftr gene (18), is not significantly involved in salivation. Functionally, due to the strong hyperpolarization required to gate
ClC-2, it is unclear whether ClC-2 would be very active under physiological conditions. In fact, we found that normal levels of
secretion occur in the Clcn2/ mice. Moreover, ClC-2
played little, if any, role in cell volume regulation in salivary
acinar cells. Although our data suggest that ClC-2 is involved in
neither fluid secretion nor cell volume regulation in salivary glands,
we cannot exclude the possibility that ClC-2 may function in such roles
in other epithelial tissues or that yet unknown compensatory mechanisms
alleviate the loss of ClC-2 in the salivary cells.
/ mice; this, combined with the normal
osmolarity of the saliva, suggests that ClC-2 is not a major pathway
for regulating electrolyte reabsorption in salivary glands
following cholinergic stimulation. However, duct cells are also
responsive to beta-adrenergic stimulation and several types of
Cl currents (52, 53), including ClC-2-like currents (40), have been previously described in these cells. Thus, other
Cl channels such as CFTR could compensate for and reduce
the phenotypic severity of ClC-2 loss under the appropriate conditions.
current. Moreover, the phenotypic defects observed in the
Clcn2/ mice indicate that the ClC-2 chloride
channel is involved in the continued viability of both retinal and
testicular cells; this may reflect a role in cell-cell communication,
as is the case with CLH-3, the C. elegans ClC-2 ortholog.
ACKNOWLEDGEMENT
FOOTNOTES
To whom correspondence should be addressed: Center for Oral
Biology, Aab Institute of Biomedical Sciences, University of Rochester Medical Center, Box 611, 601 Elmwood Ave., Rochester, NY
14642. Tel.: 585-275-3444; Fax: 585-506-0190; E-mail:
james_melvin@urmc.rochester.edu.
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 D. Heitzmann and R. Warth Physiology and Pathophysiology of Potassium Channels in Gastrointestinal Epithelia Physiol Rev, July 1, 2008; 88(3): 1119 - 1182. [Abstract] [Full Text] [PDF]
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 X. Liu, K. T. Cheng, B. C. Bandyopadhyay, B. Pani, A. Dietrich, B. C. Paria, W. D. Swaim, D. Beech, E. Yildrim, B. B. Singh, et al. Attenuation of store-operated Ca2+ current impairs salivary gland fluid secretion in TRPC1( / ) mice PNAS, October 30, 2007; 104(44): 17542 - 17547. [Abstract] [Full Text] [PDF]
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O. A. Itani, F. S. Lamb, J. E. Melvin, and M. J. Welsh Basolateral chloride current in human airway epithelia Am J Physiol Lung Cell Mol Physiol, October 1, 2007; 293(4): L991 - L999. [Abstract] [Full Text] [PDF]
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 J. Blanz, M. Schweizer, M. Auberson, H. Maier, A. Muenscher, C. A. Hubner, and T. J. Jentsch Leukoencephalopathy upon Disruption of the Chloride Channel ClC-2 J. Neurosci., June 13, 2007; 27(24): 6581 - 6589. [Abstract] [Full Text] [PDF]
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 G. Cheng, M.-J. Kim, G. Jia, and D. K. Agrawal Involvement of chloride channels in IGF-I-induced proliferation of porcine arterial smooth muscle cells Cardiovasc Res, January 1, 2007; 73(1): 198 - 207. [Abstract] [Full Text] [PDF]
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 Y. R. Yusef, L. Zuniga, M. Catalan, M. I. Niemeyer, L. P. Cid, and F. V. Sepulveda Removal of gating in voltage-dependent ClC-2 chloride channel by point mutations affecting the pore and C-terminus CBS-2 domain J. Physiol., April 1, 2006; 572(1): 173 - 181. [Abstract] [Full Text] [PDF]
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 J. A. de Santiago, K. Nehrke, and J. Arreola Quantitative Analysis of the Voltage-dependent Gating of Mouse Parotid ClC-2 Chloride Channel J. Gen. Physiol., November 28, 2005; 126(6): 591 - 603. [Abstract] [Full Text] [PDF]
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 G. Pena-Munzenmayer, M. Catalan, I. Cornejo, C. D. Figueroa, J. E. Melvin, M. I. Niemeyer, L. P. Cid, and F. V. Sepulveda Basolateral localization of native ClC-2 chloride channels in absorptive intestinal epithelial cells and basolateral sorting encoded by a CBS-2 domain di-leucine motif J. Cell Sci., September 15, 2005; 118(18): 4243 - 4252. [Abstract] [Full Text] [PDF]
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W.-Z. Lan, H. Abbas, H. D. Lam, A.-M. Lemay, and C. E. Hill Contribution of a time-dependent and hyperpolarization-activated chloride conductance to currents of resting and hypotonically shocked rat hepatocytes Am J Physiol Gastrointest Liver Physiol, February 1, 2005; 288(2): G221 - G229. [Abstract] [Full Text] [PDF]
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 S. M. Huber, C. Duranton, G. Henke, C. van de Sand, V. Heussler, E. Shumilina, C. D. Sandu, V. Tanneur, V. Brand, R. S. Kasinathan, et al. Plasmodium Induces Swelling-activated ClC-2 Anion Channels in the Host Erythrocyte J. Biol. Chem., October 1, 2004; 279(40): 41444 - 41452. [Abstract] [Full Text] [PDF]
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A. A. Zdebik, J. E. Cuffe, M. Bertog, C. Korbmacher, and T. J. Jentsch Additional Disruption of the ClC-2 Cl- Channel Does Not Exacerbate the Cystic Fibrosis Phenotype of Cystic Fibrosis Transmembrane Conductance Regulator Mouse Models J. Biol. Chem., May 21, 2004; 279(21): 22276 - 22283. [Abstract] [Full Text] [PDF]
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 M. Hayashi, C. Kunii, T. Takahata, and T. Ishikawa ATP-dependent regulation of SK4/IK1-like currents in rat submandibular acinar cells: possible role of cAMP-dependent protein kinase Am J Physiol Cell Physiol, March 1, 2004; 286(3): C635 - C646. [Abstract] [Full Text]
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N. G. Jin, J. K. Kim, D. K. Yang, S. J. Cho, J. M. Kim, E. J. Koh, H. C. Jung, I. So, and K. W. Kim Fundamental role of ClC-3 in volume-sensitive Cl- channel function and cell volume regulation in AGS cells Am J Physiol Gastrointest Liver Physiol, November 1, 2003; 285(5): G938 - G948. [Abstract] [Full Text] [PDF]
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 K. W. Holmes, R. Hales, S. Chu, M. J. Maxwell, P. J. Mogayzel Jr., and P. L. Zeitlin Modulation of Sp1 and Sp3 in Lung Epithelial Cells Regulates ClC-2 Chloride Channel Expression Am. J. Respir. Cell Mol. Biol., October 1, 2003; 29(4): 499 - 505. [Abstract] [Full Text] [PDF]
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M. L. Olsen, S. Schade, S. A. Lyons, M. D. Amaral, and H. Sontheimer Expression of Voltage-Gated Chloride Channels in Human Glioma Cells J. Neurosci., July 2, 2003; 23(13): 5572 - 5582. [Abstract] [Full Text] [PDF]
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H C. Hartzell and Z. Qu Chloride currents in acutely isolated Xenopus retinal pigment epithelial cells J. Physiol., June 1, 2003; 549(2): 453 - 469. [Abstract] [Full Text] [PDF]
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S. U. Dhani, R. Mohammad-Panah, N. Ahmed, C. Ackerley, M. Ramjeesingh, and C. E. Bear Evidence for a Functional Interaction between the ClC-2 Chloride Channel and the Retrograde Motor Dynein Complex J. Biol. Chem., April 25, 2003; 278(18): 16262 - 16270. [Abstract] [Full Text] [PDF]
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J. Arreola and J. E Melvin A novel chloride conductance activated by extracellular ATP in mouse parotid acinar cells J. Physiol., February 15, 2003; 547(1): 197 - 208. [Abstract] [Full Text] [PDF]
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J. Arreola, T. Begenisich, K. Nehrke, H.-V. Nguyen, K. Park, L. Richardson, B. Yang, B. C Schutte, F. S Lamb, and J. E Melvin Secretion and cell volume regulation by salivary acinar cells from mice lacking expression of the Clcn3 Cl- channel gene J. Physiol., November 15, 2002; 545(1): 207 - 216. [Abstract] [Full Text] [PDF]
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D. Varela, M. I. Niemeyer, L P. Cid, and F. V Sepulveda Effect of an N-terminus deletion on voltage-dependent gating of the ClC-2 chloride channel J. Physiol., October 15, 2002; 544(2): 363 - 372. [Abstract] [Full Text] [PDF]
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