1 Department of Physiology and Biophysics and 2 Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas 77555
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ClC-3 is a voltage-gated Cl
channel that is highly conserved and widely expressed, although its
function, localization, and properties remain a matter of considerable
debate. In this study, we have shown that heterologous expression of
ClC-3 in either Chinese hamster ovary (CHO-K1) or human hepatoma
(Huh-7) cells results in the formation of large, acidic vesicular
structures within cells. Vesicle formation is prevented by bafilomycin,
an inhibitor of the vacuolar ATPase, and is not induced by an E224A mutant of ClC-3 with altered channel activity. This demonstrates that
vesicle formation requires both proton pumping and Cl
channel activity. Manipulation of the intracellular Cl
concentration demonstrated that the ClC-3-associated vesicles shrink
and swell consistent with a highly Cl
-permeable membrane.
The ClC-3 vesicles were identified as lysosomes based on their
colocalization with the lysosome-associated proteins lamp-1, lamp-2,
and cathepsin D and on their failure to colocalize with fluorescently
labeled endosomes. We conclude that ClC-3 is an intracellular channel
that conducts Cl
when it is present in intracellular
vesicles. Its overexpression results in its appearance in enlarged
lysosome-like structures where it contributes to acidification by
charge neutralization.
endosomes; bafilomycin; ClC channels
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CL
channels are present in all cells both at the plasma membrane and
in intracellular sites. They are involved in diverse cell functions
including stabilization of membrane potential, transepithelial
transport, maintenance of intracellular pH, and cell volume regulation
(14, 15). Some Cl
channel functions have
been associated with specific channel molecules. For example, ClC-1 is
a myocyte plasma membrane channel that regulates membrane potential
(33, 38), and ClC-5 is an endosomal Cl
channel that participates in acidification and endocytosis (29, 34). However, for many Cl
channels, it has not
been possible to associate molecular identity with function
(19). In hepatocytes, as yet unidentified Cl
channels participate in cell volume regulation (1, 7, 22, 23) and acidification of intracellular organelles (5, 24, 28, 36).
The ClC Cl channel family has proven to be important in
diverse cellular functions (10, 14, 40). In this family,
ClC-3, along with the highly homologous ClC-4 and ClC-5, form one
distinct branch. Each has been functionally expressed, and all possess similar channel properties (8, 20). ClC-5 localizes in
endocytic vesicles in the kidney where it is necessary for
acidification and endocytosis (12, 29). The precise
function of ClC-3 is controversial. Although it was first proposed as a
swelling-activated Cl
channel in plasma membranes
(3), recent studies support a predominantly intracellular
localization (20, 39). In knockout mice, Stobrawa et al.
(35) found that disruption of the ClC-3 gene impaired
acidification of synaptic vesicles in hippocampal neurons. However,
ClC-3 is highly conserved and broadly distributed, and its functions
may not be limited to acidification of synaptic vesicles.
We have previously cloned ClC-3 from rat hepatocytes and have
characterized its channel activity (20, 32). In the
present study, we have expressed ClC-3 in human hepatoma (Huh-7) and
Chinese hamster ovary (CHO-K1) cells and noted that ClC-3 expression
induced formation of large intracellular vesicles. ClC-3 protein
localized abundantly in the vesicular membranes. Further examination
revealed that ClC-3 functions as an intracellular Cl
channel in these vesicles, cooperating with vacuolar
H+-ATPase to achieve H+ and
Cl
flux. Our results provide direct evidence that ClC-3
is a functional intracellular channel and demonstrate the importance of
ClC-3 in organelle acidification.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ClC-3 expression vectors. Four different ClC-3 constructs were used. pClC3sFlag (short form) and pClC3lFlag (long form) were constructed in pcDNA 3.1 as described previously (20, 32). pClC3sGFP was prepared by subcloning the ClC-3 short form open reading frame into pEGFP-N1 (Clontech) between the XhoI and BamH1 sites. This construct produced a fusion protein with the enhanced green fluorescent protein (EGFP) moiety attached to the COOH terminus of ClC-3. A pClC3sFlag E224A mutation was produced by using the QuikChange site-directed mutagenesis kit (Stratagene). All constructs were confirmed by sequencing in the Protein Chemistry Laboratory of the University of Texas Medical Branch.
Cell culture and transfection. CHO-K1 or Huh-7 cells were cultured in DMEM/F-12 medium (Mediatech) and grown on glass coverslips in six-well plates. Cells were transiently transfected at 50-70% confluence with pClC3sFlag, pClC3lFlag, pClC3sGFP, or pClC3sFlag E224A by using Fu-GENE6 (Boehringer Mannheim) or Lipofectamine Plus (Life Technologies) following the manufacturer's protocol. Cells were used for experiments 48-72 h after transfection.
For patch-clamp experiments, cells were transfected with a mixture of either pClC3sFlag or its E224A mutant plus a GFP plasmid (pEGFP; Clontech) at a 30:1 ratio and observed under epifluorescence microscopy 48-72 h after transfection. Fluorescent cells were chosen for patch-clamp analysis.Electrophysiology. Whole cell current recordings of ClC-3-expressing cells were performed by using a patch-clamp system (Axopatch-200 and pCLAMP v6.03, Axon Instruments) at room temperature (23°C) as described previously (20). The bath solution consisted of (in mM): 114 NaCl, 5.4 CsCl, 1 MgSO4, 1.5 CaCl2, 10 HEPES, and 10 glucose. Osmolality (measured with a vapor pressure osmometer, model 5500, Wescor, Logan, UT) was adjusted to 300 mosmol/kg H20 by adding sucrose, and pH was adjusted to 7.4 with NaOH. Pipette solution contained (in mM) 120 CsCl, 3 MgSO4, 1 CaCl2, 11 EGTA, 3 Na2ATP, 10 HEPES, and 10 glucose and was adjusted to pH 7.2 with CsOH. Osmolality was 290 mosmol/kg H20.
Immunofluorescence.
Immunofluorescence was performed as described previously
(32). Transfected cells were grown on glass coverslips.
They were fixed with methanol at 20 °C for 10 min, washed in PBS,
and incubated with the m2 anti-FLAG monoclonal antibody (1:500, Sigma)
in 10% goat serum for 1.5 h. Coverslips were then washed for
2 h in PBS and incubated with Alexa Fluor 488-conjugated goat
anti-mouse IgG (1:500, Molecular Probes) for 1 h followed by
another hour of washing. Similar immunostaining was performed with
primary antibodies against Golgi marker 58k protein (1:100, Abcam),
human cathepsin D (1:100, Upstate Biotechnology), anti-lamp-1
monoclonal antibody H4A3, and anti-lamp-2 monoclonal antibody H4B4
(each at 1:200, Developmental Studies Hybridoma Bank, University of Iowa). Secondary antibodies were Alexa Fluor 594-conjugated goat anti-mouse IgG or Alexa Fluor 594 goat anti-rabbit IgG (1:500, Molecular Probes). Nuclear staining was performed with
4,6-diamidino-2-phenylindole (DAPI) dye as described previously
(18). Cells were observed in a Nikon Eclipse 800 (Melville, NY) epifluorescence microscope with the FITC-filter set
(excitation 465-495 nm, dichroic mirror 505 nm, emission
515-555 nm) for Alexa Fluor 488 conjugates, Texas red filter set
(excitation 540-580 nm, dichroic mirror 595 nm, emission
600-660 nm) for Alexa Fluor 594 conjugates, and the DAPI filter
set (excitation 340-380 nm, dichroic mirror 400 nm, emission 435-485 nm) for the nuclear DAPI dye. Images were acquired by using a Dage-MTI (Michigan City, IN) camera for later processing by
Adobe Photoshop software.
Identification of acidic intracellular compartments. After transfection of pClC3sGFP for 48 h, LysoSensor blue DND-167 (Molecular Probes) was added to the medium to a final concentration of 1 µM and incubated for 30 min. Cells on coverslips were directly observed under the microscope with the FITC filter set for ClC-3 protein or DAPI filter set for LysoSensor. Images were recorded immediately to avoid photobleaching.
Examination of fluid-phase endocytosis. Cells grown on small coverslips were washed with HEPES-buffered saline solution (HBSS, in mM: 150 NaCl, 0.8 MgSO4, 2.2 D-gluconic acid, hemicalcium salt, 4.2 K-gluconic acid, 10 HEPES, and 18 glucose, adjusted to pH 7.4 with NaOH) and incubated with Alexa Fluor 594-conjugated dextran (MW 10,000, Molecular Probe) at 5 mg/ml for 10 min. Dextran was removed by washing three times with HBSS, and the cells were subsequently incubated for variable times in the same solution before fixation with 3% paraformaldehyde. They were then washed in PBS and observed by epifluorescence microscopy.
Live-cell fluorescence microscopy.
Cells transfected with pClC3sGFP were placed in a 0.5-ml thermostatic
chamber (36°C) and perfused continuously at 2 ml/min with
Cl-free or Cl
-containing solutions.
Epifluorescence images were acquired with a Nikon Eclipse TE200
inverted microscope using a ×60, 1.40 N.A. objective and
Metamorph software. Cl
-containing isotonic solution was
HBSS. Cl
-free solution used Na-gluconate to substitute
the NaCl. Hypotonic solutions were prepared by reducing NaCl or
Na-gluconate to 100 mM.
Materials. Bafilomycin A1 and DAPI were purchased from Sigma. Other chemicals were from Sigma or Fisher Scientific unless noted otherwise.
Data analysis.
Assessment of the presence of vesicular structures in cells was
performed by selecting four corner fields and a central field on each
coverslip and scoring each fluorescent cell for the presence or absence
of vesicles as assessed visually by a blinded observer. Transfection
efficiency was also estimated each time. Statistical significance was
tested using the 2 method.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ClC-3 expression induces the formation of intracellular vesicles.
Huh-7 hepatoma cells and CHO-K1 cells were transiently transfected with
pClC3sFlag or pClC3sGFP as described in METHODS. Neither of
these tags at the COOH terminus affected channel properties (20). A consistent observation was that ClC-3 expression
resulted in the formation of large intracellular vesicles, as shown in Fig. 1A. The vesicles
themselves appeared to contain ClC-3 in their membranes. This
phenomenon was observed in both CHO-K1 and Huh-7 cells. Similar
vesicles could be observed with difference-interference contrast optics
after transfection of untagged ClC-3 as well. In nontransfected or GFP
only-transfected cells, the distinctive vesicles were never seen. This
pattern of vesicle formation was specific to the short form of ClC-3
and was not seen with ClC-3 long form (Fig. 1B).
|
|
Vacuolar proton-ATPase is essential for the formation of
ClC-3-associated vesicles.
We hypothesized that abnormally large Cl conductance in
association with proton pumping from the vacuolar proton-ATPase might lead to vesicle enlargement by the accumulation of osmotically active
Cl
ions in the vesicle lumen. We then determined whether
proton pumping was necessary for the formation of the enlarged
structures. Figure 3 demonstrates
that inhibition of the V-type ATPase (V-ATPase) with bafilomycin A1, a
highly specific inhibitor of vacuolar proton-ATPase, nearly abolished
formation of enlarged vesicles. ClC-3 was still abundantly expressed,
but it was now located primarily in punctate spots without a
discernable lumen. This indicates that vesicle enlargement requires
proton pumping.
|
A ClC-3-channel mutation disables vesicle formation.
As previously reported (20), expression of ClC-3 in CHO-K1
cells results in a strongly outward-rectifying Cl
current, presumably because a fraction of the expressed ClC-3 also
appears on the plasma membrane. To determine whether vesicle formation
requires the channel activity of ClC-3, we introduced an E224A mutation
in pClC3sFlag. This mutation corresponds to a highly conserved segment
that lines the permeation pore of ClC channels (6) and
forms part of the selectivity filter (4). The identical
mutation in ClC-4 and ClC-5 dramatically changed channel conductance
and rectification (8). Figure
4 demonstrates that the E224A mutation in
ClC-3 produced the identical effect on ClC-3 voltage dependence as it
did for ClC-4 and ClC-5. The mutant whole cell currents are largely
inward rectifying and have reduced maximal conductance.
|
|
ClC-3-mediated Cl flux across the vesicle
membrane.
To determine whether the ClC-3-associated vesicles possess high
Cl
conductance, we manipulated Cl
gradients
across the vesicle membrane and recorded resulting changes in vesicle
size. Live cells expressing the ClC-3-GFP-fusion protein were observed
by epifluorescence microscopy, superfused with Cl
-free
solution, and then returned to normal Cl
bath solution.
Preliminary studies, however, showed that the plasma membrane was
relatively impermeable to Cl
, and therefore changes in
bath Cl
concentration only slowly affected the gradients
across the membrane of the intracellular vesicles.
|
ClC-3 vesicles are lysosomes.
ClC-3-expressing cells were fixed and immunostained with marker
antibodies as described in METHODS. Figure
7, A-C, demonstrates that
ClC-3 expressed in Huh-7 cells did not colocalize with a Golgi marker
(Golgi 58K protein). However, the lumen of many of the vesicular
structures demonstrated the presence of cathepsin D (Fig. 7,
D-F), a lysosome-specific protease, suggesting that some of these enlarged ClC-3-containing structures may be lysosomes. This was further confirmed by the demonstration that both
lysosome-associated membrane proteins lamp-1 and lamp-2
(9) strongly colocalize with ClC-3 (Fig. 7,
G-L). Similar results were obtained in CHO-K1 cells
(data not shown).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The functions and properties of ClC-3 have been difficult to
determine. ClC-3 has variously been proposed to be a swelling-activated plasma membrane Cl channel, a Ca2+-activated
channel, and an intracellular channel necessary for acidification of
synaptic vesicles (3, 13, 35). We have previously
determined that ClC-3 Cl
currents are different from
swelling-activated Cl
currents and have nearly identical
biophysical properties as ClC-5 (20).
In the present study, we have shown that a single amino acid
substitution at position 224 of ClC-3 produced the same effect on
channel properties as does the corresponding mutation in ClC-4 and
ClC-5 (8). This definitively confirms that the ClC-3
molecule itself mediates the channel activity that we have observed
here and in our previous study (20). Furthermore,
expression of ClC-3 in CHO-K1 or Huh-7 cells results in the development
of large intracellular vesicular structures with the ClC-3 molecules
localized in the vesicular membrane. The vesicles possess high
Cl permeability, and the interior pH of these vesicles
was <5.1 as assessed by Lysosensor blue. Expansion of these
ClC-3-associated vesicles was dependent on both proton pumping and
normal ClC-3 channel activity. These findings strongly support the
conclusion that ClC-3 is an intracellular Cl
channel that
participates in vesicular acidification.
Our data demonstrate that the large ClC-3 vesicular structures have the properties of lysosomes. Although they are larger than normal lysosomes, they contain the lysosomal membrane proteins lamp-1 and lamp-2, as well as the lysosomal protease cathepsin D. In addition, endocytosed dextran colocalizes with them only at times >1 h postinternalization, a time at which endosomal contents have been delivered to lysosomes (31). This pattern shows that the ClC-3 vesicles are not endosomes but rather lysosomes. It is important to note, however, that the distinction between late endosomes and lysosomes may be difficult to establish, and hybrid organelles may also exist (25). In addition, not all expressed ClC-3 is present in these enlarged lysosomal structures, and other sites of localization may occur as well.
Multiple organelles require an acidic environment for proper
functioning. This is achieved by an electrogenic vacuolar
H+-ATPase that actively transports protons from the cytosol
into the vesicle lumen (11, 27, 37). Efficient pumping
requires charge neutralization by Cl flux into the lumen
where the steady-state pH depends on the balance of proton pumping,
Cl
permeability, and proton leakage. This acidification
process may promote enlargement of the luminal size by translocation of Cl
ions and also by the trapping of basic osmolytes
(37). Overexpression of ClC-3 in lysosomes may thus be
responsible for both abnormal enlargement and hyperacidification.
The precise properties of ClC-3 are the subject of dispute, and different laboratories appear to have obtained conflicting results with this channel. We believe that our present results may shed some insight on the reasons for these differences. Our laboratory has previously reported whole cell currents associated with expression of both the short and long form of ClC-3 (32). The mutation results presented in Fig. 4 clearly show that these short form-associated currents are mediated by ClC-3. The current has extreme outward rectification, is insensitive to NPPB [5-nitro-2-(3-phenylpropylamino)benzoic acid] and DIDS, and is never seen in untransfected cells (20). Each of these characteristics is identical to what has been reported for the highly homologous ClC-5 and ClC-4 (8).
In contrast, the currents that we reported to be associated with expression of the long form of ClC-3 (32), as well as the properties of ClC-3 that have been observed by Duan et al. (3), are quite different from those of ClC-4 and ClC-5. They have only weak outward rectification, are sensitive to both NPPB and DIDS, and are identical to endogenous currents that are present in untransfected cells. We thus believe that the currents that we previously observed associated with the long form of ClC-3 (32) were endogenous to the cells and were not mediated by ClC-3 itself. In subsequent experiments in which greater care has been taken to prevent cell swelling, we no longer see plasma membrane currents associated with expression of ClC-3 long form.
It is important to note that we have only been able to observe wild-type ClC-3 currents in transiently transfected cells that possess large intracellular vesicles. We have not been able to observe ClC-3 currents in transfected cells without intracellular vesicles. We suggest that the presence of vesicles indicates an extreme degree of ClC-3 overexpression. Only in this case does enough of the channel appear on the plasma membrane to produce measurable whole cell currents. The long form of ClC-3 is also expressed well in our transient transfection assays, but it does not form vesicles. Our inability to see currents with this molecule may reflect more efficient intracellular retention.
These findings need to be understood in the context of other recent
studies of ClC-3. As discussed in detail previously (20), our results differ markedly from those of Duan et al. (3)
and Kawasaki et al. (16). We find that ClC-3 appears to be
an intracellular channel that contributes to acidification of
intracellular vesicles and is not activated by cell swelling.
However, our results are entirely compatible with the recent studies of
Stobrawa et al. (35), who used knockout mice and
confirmed that ClC-3 is not associated with swelling-activated
currents. These authors also observed abnormalities of acidification in
synaptic vesicles of hippocampal neurons. Synaptic vesicles share some
membrane protein components with lysosome-related organelles such as
melanosomes and platelet-dense granules (2, 26). Thus the
observations of Stobrawa et al. are compatible with a role of ClC-3 in
promoting acidification of lysosomes in other cell types. Our results
are also partially consistent with the recent paper by Weylandt et al.
(39), who confirmed that ClC-3 is primarily intracellular and is not a swelling-activated channel. However, Weylandt et al.
obtained a different voltage dependence than we did, with much more
conduction at negative voltages. The explanation for this difference is
not clear. It could reflect a difference in cell systems with
specific regulatory proteins present only in some cell types.
Huang et al. (13) also studied ClC-3 in stably transfected cells. They observed a Ca2+-dependent
Cl current that was weakly outward rectifying and
sensitive to DIDS. The rectification and inhibitor sensitivity of these
currents are different from what we see for ClC-3, but we have not
examined Ca2+ dependence in our system. Another difference
between our work and that of Huang et al. is that ClC-3 channels were
open constitutively in our experiments but required elevated
Ca2+ to become open in the studies of Huang et al.
Several alternative explanations for our results also need to be considered. The possibility exists that the vesicles result from some effect of overexpressed ClC-3 that is not related to its channel function. For example, this could be a nonspecific effect of membrane protein overexpression, or the introduction of ClC-3 into these cells could alter vacuolar H+-ATPase expression levels. Several facts can be used to argue against these possibilities. First, the E224A mutant is structurally identical to ClC-3 wild type except for the loss of a single-charged residue in the channel pore. It was abundantly expressed in CHO-K1 cells and appeared to have similar intracellular localization, but it did not produce vesicle enlargement. In addition, overexpression of other similar molecules such as ClC-3 long form does not produce these vesicles (Fig. 1). Second, vacuolar H+-ATPase is expressed in multiple intracellular compartments (11). If the primary effect of ClC-3 were to increase its expression, the observed effects would not be selective for lysosomes.
An important issue concerning ClC-3, ClC-4, and ClC-5 is how the
extreme outward rectification (8, 20) is compatible with
channel function in any real cellular compartment, where the cytosolic
potential is always negative. In most cells, cytosol positive
potentials, which would allow ion conduction, never occur in either the
plasma membrane or in any conceivable intracellular organelle. This
problem is so significant as to suggest that the main function of ClC-3
might not involve its ion channel activity. However, our study
demonstrated intracellular Cl fluxes into
ClC-3-containing vesicles (Fig. 6). ClC-3, therefore, does function as
an intracellular channel. One possible explanation is suggested by the
recent determination of the crystal structure of the bacterial ClC
channel by Dutzler et al. (4). This structure reveals that
a COOH-terminal
-helix (the R helix) is oriented with its
NH2 terminus in the pore selectivity filter and its COOH terminus in the cytosol. As suggested by Dutzler et al., binding of an
accessory protein to the cytoplasmic COOH-terminal domain of the
protein could alter the confirmation of the selectivity filter and
modify the voltage dependence of ClC-3. In this scenario, failure to
conduct Cl
at negative voltages at the plasma membrane
could keep the channel closed in the plasma membrane. Because the
vesicles of the secretory and endocytic pathways do indeed fuse with
the plasma membrane, it is inevitable that some ClC-3 will appear on
the plasma membrane. However, the absence of the appropriate accessory
proteins in plasma membrane would effectively close any ClC-3 channel
molecules that appear there, helping to ensure that ClC-3 functions
exclusively as an intracellular channel. This explanation is, of
course, speculative, and the exact purpose of the extreme outward
rectification has yet to be determined. Further experiments are
required to clarify this point.
We therefore conclude that ClC-3 functions as a Cl
channel in lysosome membranes and contributes to vesicular
acidification. This finding is consistent with the demonstrated
function of other members of the ClC family, particularly ClC-5
(12, 29). Mutations in ClC-7 also result in a defect in
acidification by osteoclasts that leads to abnormal bone resporption
(17). We may thus cautiously generalize that this class of
intracellular Cl
channels is involved in a diverse array
of intracellular acidification processes. It cooperates with vacuolar
H+-ATPase to allow acidification and vacuolation to occur.
We believe this is the first demonstration of functional activity of
ClC-3 in non-neuronal cells. Additional expression in other cell lines or observations in native cells is needed to understand further details
of ClC-3 function.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. R. Van Dyke and N. Wills for helpful discussions and Dr. Leoncio Vergara for assistance with microscopy.
![]() |
FOOTNOTES |
---|
This work was supported by National Institute of Diabetes and Digestive and Kidney DiseasesGrant DK-42917.
Address for reprint requests and other correspondence: S. A. Weinman, Dept. of Physiology and Biophysics, Univ. of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0641 (E-mail: sweinman{at}utmb.edu).
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.
First published February 13, 2002;10.1152/ajpcell.00504.2001
Received 22 October 2001; accepted in final form 6 February 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Bodily, K,
Wang Y,
Roman R,
Sostman A,
and
Fitz JG.
Characterization of a swelling-activated anion conductance in homozygous typing cell hepatoma cells.
Hepatology
25:
403-410,
1997[ISI][Medline].
2.
Dell'Angelica, EC,
Mullins C,
Caplan S,
and
Bonifacino JS.
Lysosome-related organelles.
FASEB J
14:
1265-1278,
2000
3.
Duan, D,
Winter C,
Cowley S,
Hume JR,
and
Horowitz B.
Molecular identification of a volume-regulated chloride channel.
Nature
390:
417-421,
1997[ISI][Medline].
4.
Dutzler, R,
Campbell EB,
Cadene M,
Chait BT,
and
MacKinnon R.
X-ray structure of a ClC chloride channel at 3.0 A reveals the molecular basis of anion selectivity.
Nature
415:
287-294,
2002[ISI][Medline].
5.
Eliassi, A,
Garneau L,
Roy G,
and
Sauvé R.
Characterization of a chloride-selective channel from rough endoplasmic reticulum membranes of rat hepatocytes: evidence for a block by phosphate.
J Membr Biol
159:
219-229,
1997[ISI][Medline].
6.
Fahlke, C,
Yu HT,
Beck CL,
Rhodes TH,
and
George AL, Jr.
Pore-forming segments in voltage-gated chloride channels.
Nature
390:
529-532,
1997[ISI][Medline].
7.
Feranchak, AP,
Fitz JG,
and
Roman RM.
Volume-sensitive purinergic signaling in human hepatocytes.
J Hepatol
33:
174-182,
2000[ISI][Medline].
8.
Friedrich, T,
Breiderhoff T,
and
Jentsch TJ.
Mutational analysis demonstrates that ClC-4 and ClC-5 directly mediate plasma membrane currents.
J Biol Chem
274:
896-902,
1999
9.
Fukuda, M.
Lysosomal membrane glycoproteins. Structure, biosynthesis, and intracellular trafficking.
J Biol Chem
266:
21327-21330,
1991
10.
George, AL, Jr,
Bianchi L,
Link EM,
and
Vanoye CG.
From stones to bones: the biology of ClC chloride channels.
Curr Biol
11:
R620-R628,
2001[ISI][Medline].
11.
Grabe, M,
and
Oster G.
Regulation of organelle acidity.
J Gen Physiol
117:
329-344,
2001
12.
Gunther, W,
Luchow A,
Cluzeaud F,
Vandewalle A,
and
Jentsch TJ.
ClC-5, the chloride channel mutated in Dent's disease, colocalizes with the proton pump in endocytotically active kidney cells.
Proc Natl Acad Sci USA
95:
8075-8080,
1998
13.
Huang, P,
Liu J,
Di A,
Robinson NC,
Musch MW,
Kaetzel MA,
and
Nelson DJ.
Regulation of human CLC-3 channels by multifunctional Ca2+/calmodulin-dependent protein kinase.
J Biol Chem
276:
20093-20100,
2001
14.
Jentsch, TJ,
Friedrich T,
Schriever A,
and
Yamada H.
The CLC chloride channel family.
Pflügers Arch
437:
783-795,
1999[ISI][Medline].
15.
Jentsch, TJ,
and
Gunther W.
Chloride channels: an emerging molecular picture.
Bioessays
19:
117-126,
1997[ISI][Medline].
16.
Kawasaki, M,
Suzuki M,
Uchida S,
Sasaki S,
and
Marumo F.
Stable and functional expression of the CIC-3 chloride channel in somatic cell lines.
Neuron
14:
1285-1291,
1995[ISI][Medline].
17.
Kornak, U,
Kasper D,
Bosl MR,
Kaiser E,
Schweizer M,
Schulz A,
Friedrich W,
Delling G,
and
Jentsch TJ.
Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man.
Cell
104:
205-215,
2001[ISI][Medline].
18.
Lerat, H,
Honda M,
Beard MR,
Loesch K,
Sun J,
Yang Y,
Okuda M,
Gosert R,
Xiao SY,
Weinman SA,
and
Lemon SM.
Steatosis and liver cancer in transgenic mice expressing the structural and nonstructural proteins of hepatitis C virus.
Gastroenterology
122:
366-375,
2002[ISI][Medline].
19.
Li, XH,
and
Weinman SA.
Chloride channels and hepatocellular function. Prospects for molecular identification.
Annu Rev Physiol
64 (13):
1-13.25,
2002[ISI][Medline].
20.
Li, X,
Shimada K,
Showalter LA,
and
Weinman SA.
Biophysical properties of ClC-3 differentiate it from swelling-activated chloride channels in CHO-K1 cells.
J Biol Chem
275:
35994-35998,
2000
21.
Lin, HJ,
Herman P,
Kang JS,
and
Lakowicz JR.
Fluorescence lifetime characterization of novel low-pH probes.
Anal Biochem
294:
118-125,
2001[ISI][Medline].
22.
Maglova, LM,
Jackson AM,
Meng XJ,
Carruth MW,
Schteingart CD,
Ton-Nu HT,
Hofmann AF,
and
Weinman SA.
Transport characteristics of three fluorescent conjugated bile acid analogs in isolated rat hepatocytes and couplets.
Hepatology
22:
637-647,
1995[ISI][Medline].
23.
Meng, XJ,
and
Weinman SA.
Cyclic AMP and swelling activated chloride conductance in rat hepatocytes.
Am J Physiol Cell Physiol
271:
C112-C120,
1996
24.
Morier, N,
and
Sauvé R.
Analysis of a novel double-barreled anion channel from rat liver rough endoplasmic reticulum.
Biophys J
67:
590-602,
1994[Abstract].
25.
Mullins, C,
and
Bonifacino JS.
The molecular machinery for lysosome biogenesis.
Bioessays
23:
333-343,
2001[ISI][Medline].
26.
Mullins, C,
Hartnell LM,
and
Bonifacino JS.
Distinct requirements for the AP-3 adaptor complex in pigment granule and synaptic vesicle biogenesis in Drosophila melanogaster.
Mol Gen Genet
263:
1003-1014,
2000[ISI][Medline].
27.
Nelson, N,
and
Harvey WR.
Vacuolar and plasma membrane proton-adenosinetriphosphatases.
Physiol Rev
79:
361-385,
1999
28.
Nordeen, MH,
Jones SM,
Howell KE,
and
Caldwell JH.
GOLAC: an endogenous anion channel of the Golgi complex.
Biophys J
78:
2918-2928,
2000
29.
Piwon, N,
Gunther W,
Schwake M,
Bosl MR,
and
Jentsch TJ.
ClC-5 Cl-channel disruption impairs endocytosis in a mouse model for Dent's disease.
Nature
408:
369-373,
2000[ISI][Medline].
30.
Sanchez-Olea, R,
Fuller C,
Benos D,
and
Pasantes-Morales H.
Volume-associated osmolyte fluxes in cell lines with or without the anion exchanger.
Am J Physiol Cell Physiol
269:
C1280-C1286,
1995
31.
Schmid, SL.
Toward a biochemical definition of the endosomal compartment. Studies using free flow electrophoresis.
Subcell Biochem
19:
1-28,
1993[Medline].
32.
Shimada, K,
Li XH,
Xu GY,
Nowak DE,
Showalter LA,
and
Weinman SA.
Expression and canalicular localization of two isoforms of the ClC-3 chloride channel from rat hepatocytes.
Am J Physiol Gastrointest Liver Physiol
279:
G268-G276,
2000
33.
Steinmeyer, K,
Klocke R,
Ortland C,
Gronemeier M,
Jockusch H,
Grunder S,
and
Jentsch TJ.
Inactivation of muscle chloride channel by transposon insertion in myotonic mice.
Nature
354:
304-308,
1991[ISI][Medline].
34.
Steinmeyer, K,
Schwappach B,
Bens M,
Vandewalle A,
and
Jentsch TJ.
Cloning and functional expression of rat CLC-5, a chloride channel related to kidney disease.
J Biol Chem
270:
31172-31177,
1995
35.
Stobrawa, SM,
Breiderhoff T,
Takamori S,
Engel D,
Schweizer M,
Zdebik AA,
Bosl MR,
Ruether K,
Jahn H,
Draguhn A,
Jahn R,
and
Jentsch TJ.
Disruption of ClC-3, a chloride channel expressed on synaptic vesicles, leads to a loss of the hippocampus.
Neuron
29:
185-196,
2001[ISI][Medline].
36.
Tilly, BC,
Mancini GM,
Bijman J,
van Gageldonk PG,
Beerens CE,
Bridges RJ,
De Jonge HR,
and
Verheijen FW.
Nucleotide-activated chloride channels in lysosomal membranes.
Biochem Biophys Res Commun
187:
254-260,
1992[ISI][Medline].
37.
Van Dyke, RW.
Acidification of lysosomes and endosomes.
Subcell Biochem
27:
331-360,
1996[Medline].
38.
Wagner, S,
Deymeer F,
Kurz LL,
Benz S,
Schleithoff L,
Lehmann-Horn F,
Serdaroglu P,
Ozdemir C,
and
Rudel R.
The dominant chloride channel mutant G200R causing fluctuating myotonia: clinical findings, electrophysiology, and channel pathology.
Muscle Nerve
21:
1122-1128,
1998[ISI][Medline].
39.
Weylandt, KH,
Valverde MA,
Nobles M,
Raguz S,
Amey JS,
Diaz M,
Nastrucci C,
Higgins CF,
and
Sardini A.
Human ClC-3 is not the swelling-activated chloride channel involved in cell volume regulation.
J Biol Chem
276:
17461-17467,
2001
40.
Wills, NK,
and
Fong P.
ClC chloride channels in epithelia: recent progress and remaining puzzles.
News Physiol Sci
16:
161-166,
2001