From the Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557-0046
In many mammalian cells, ClC-3 volume-regulated chloride channels maintain a variety of normal
cellular functions during osmotic perturbation. The molecular mechanisms of channel regulation by cell volume,
however, are unknown. Since a number of recent studies point to the involvement of protein phosphorylation/dephosphorylation in the control of volume-regulated ionic transport systems, we studied the relationship between
channel phosphorylation and volume regulation of ClC-3 channels using site-directed mutagenesis and patch-clamp techniques. In native cardiac cells and when overexpressed in NIH/3T3 cells, ClC-3 channels were opened
by cell swelling or inhibition of endogenous PKC, but closed by PKC activation, phosphatase inhibition, or elevation of intracellular Ca2+. Site-specific mutational studies indicate that a serine residue (serine51) within a consensus PKC-phosphorylation site in the intracellular amino terminus of the ClC-3 channel protein represents an important volume sensor of the channel. These results provide direct molecular and pharmacological evidence indicating that channel phosphorylation/dephosphorylation plays a crucial role in the regulation of volume sensitivity
of recombinant ClC-3 channels and their native counterpart, ICl.vol.
Key words:
 |
INTRODUCTION |
To avoid excessive alterations of cell volume that may
jeopardize structural integrity and a variety of cellular
functions, mammalian cells are able to precisely maintain their size in the face of osmotic perturbations
through the regulated loss or gain of intracellular ions
or other osmolytes (Nilius et al., 1996
; Strange et al.,
1996
; Okada, 1997
; Lang et al., 1998
). Even under isotonic conditions, volume constancy of any mammalian
cell is challenged by the transport of osmotically active
substances across the cell membrane and alterations in
cellular osmolarity by metabolism (Lang et al., 1998
).
Thus, the continued operation of cell volume regulatory mechanisms, such as volume-regulated chloride
(Cl
) currents (ICl.vol), is required for cell volume homeostasis in many mammalian cells (Nilius et al., 1996
;
Strange et al., 1996
; Okada, 1997
). We have recently
provided evidence that the channel protein responsible for ICl.vol in the heart and many other mammalian
cells is encoded by the ClC-3 gene (Duan et al., 1997b
;
Yamazaki et al., 1998
). ClC-3 belongs to the large gene family of ClC Cl
channels that are comprised of 12 putative transmembrane-spanning domains (Kawasaki et al.,
1994
; Kawasaki et al., 1995
; Jentsch, 1996
; Schmidt-Rose and Jentsch, 1997
). Expressed ClC-3 Cl
channels
in oocytes and mammalian cells are strongly inhibited by activation of PKC (Kawasaki et al., 1994
, 1995
; Duan
et al., 1997b
) and hypertonic cell shrinkage while they
are activated by hypotonic cell swelling (Duan et al.,
1997b
). Little is currently known, however, about the
molecular mechanisms of regulation of ICl.vol by cell volume (Okada, 1997
; Strange, 1998
; Clapham, 1998
).
Alternations of cell volume during extra- and intracellular osmotic perturbation trigger a multitude of intracellular signaling events, including various second
message cascades, phosphorylation or dephosphorylation of target proteins, as well as altered gene expression (Waldegger et al., 1997a
,b; Lang et al., 1998
). Cell swelling has been shown to induce protein dephosphorylation, which in turn activates K-Cl cotransport (Jennings and al-Rohil, 1990
; Jennings and Schulz, 1991
;
Bize and Dunham, 1994
; Starke and Jennings, 1993
)
and inhibits Na-K-2Cl cotransport (Klein et al., 1993
;
Haas et al., 1995
; Lytle, 1998
). This swelling-induced
protein dephosphorylation may be due to decreased kinase activity (Jennings and al-Rohil, 1990
; Bize and
Dunham, 1994
; Gibson and Hall, 1995
) and/or increased activities of serine/threonine protein phosphatases (PPs, probably PP1 and PP2A)1 (Jennings and
Schulz, 1991
; Starke and Jennings, 1993
; Lytle, 1998
).
On the other hand, cell shrinkage has been shown to
cause protein phosphorylation, which in turn inhibits
K-Cl cotransport (Jennings and al-Rohil, 1990
; Jennings
and Schulz, 1991
; Bize and Dunham, 1994
; Starke and
Jennings, 1993
) and activates Na-K-2Cl cotransport
(Klein et al., 1993
; Haas et al., 1995
; Lytle, 1998
). In
Ehrlich mouse ascites tumour cells, cell shrinkage
causes a rapid increase (174% within 1 min) in PKC activity in the membrane fraction that appears to be involved in the activation of the Na-K-2Cl cotransport after cell shrinkage (Larsen et al., 1994
). PKC-dependent
phosphorylation is also involved in the cell shrinkage activation of a nonselective conductance in Caco-2 cells
(Nelson et al., 1996
). In fact, cell swelling and shrinkage have been shown to induce protein dephosphorylation and phosphorylation, respectively, in a variety of
cell systems (Grinstein et al., 1992
; Palfrey, 1994
), including epithelial (Haas et al., 1995
) and endothelial
(Santell et al., 1993
; Klein et al., 1993
) cells, erythrocytes (Jennings and al-Rohil, 1990
; Jennings and Schulz, 1991
; Starke and Jennings, 1993
; Lytle, 1998
), Ehrlich
mouse ascites tumour cells (Larsen et al., 1994
; Krarup
et al., 1998
), and cardiac myocytes (Hall et al., 1995
).
Therefore, these studies all suggest that phosphorylation/dephosphorylation of proteins (such as ionic channels and transportors) due to altered protein kinase and/or phosphatase activities may be a common
process linking changes in cell volume to protein functions.
It is noteworthy that native ICl.vol in heart and many
other tissues is also strongly regulated by phosphorylation/dephosphorylation. Activation of intracellular
PKC (Duan et al., 1995
; Hardy et al., 1995
; Coca-Prados
et al., 1996
; Bond et al., 1998
; Dick et al., 1998
) and inhibition of PPs (Hall et al., 1995
; Doroshenko, 1998
) both strongly inhibit ICl.vol even under hypotonic conditions, while inhibition of PKC can activate ICl.vol under
isotonic conditions (Coca-Prados et al., 1995
, 1996
;
Dick et al., 1998
). To study the volume-sensing mechanism of ClC-3 channels and its potential linkage to
channel phosphorylation, we used a variety of approaches from the tight-seal whole-cell voltage-clamp
study of pharmacological agents on native and cloned
channels to analysis of the functional expression of
wild-type and site-directed mutants of ClC-3 channels in NIH/3T3 cells. All of the results in this study are
consistent with the identification of the volume sensor
as a distinct serine residue in a consensus PKC-phosphorylation site in the intracellular amino terminus of
ClC-3 chloride channel. These results thus provide a
new structural link between protein function (channel
activity) and alterations in phosphorylation-dephosphorylation in response to changes in cell volume during osmotic perturbations.
 |
MATERIALS AND METHODS |
Site-directed Mutation and Functional Expression of
Guinea-Pig Cardiac ClC-3
The serine at position 51 and/or 362 was altered by a S51, S362,
and S51 + S362 to an alanine site-specific mutation introduced into gpClC-3 cDNA (Deng and Nickoloff, 1992
). The mutation
was confirmed by nucleotide sequencing of both strands of the
mutated cDNA. NIH/3T3 cells were transiently transfected by
electroporation as previously described (Duan et al., 1997b
).
Each dish was transfected with appropriate combinations of CD8
(a lymphocyte cell surface antigen) in the
H3-CD8 plasmid construct as a marker for transfection (4 µg) and wtClC-3, S51AClC-3,
S362AClC-3, or S51A + S362A ClC-3 in the pZeoSV vector (20 µg). Transfected cells were identified by their binding to CD8-coated beads (M-450 CD8; Dyna-beads). Cells were subcultured
on glass coverslips for electrophysiological recording.
Electrophysiological Recordings
Currents were measured from isolated NIH/3T3 cells or guinea-pig atrial and ventricular myocytes at room temperature (22- 24°C) by the tight-seal whole-cell voltage-clamp technique as described (Hamill et al., 1981
; Duan et al., 1997a
,b). To obtain whole-cell current-voltage relations, cells were held at
40 mV and test potentials were applied from
100 to +120 mV for 400 ms in +20-mV increments at an interval of 5 s (voltage-clamp
protocol is shown in Fig. 1, top). Current amplitudes were measured at 8 ms after the corresponding voltage step relative to 0 current level and normalized to cell capacitance (pA pF
1). To
obtain time-dependent changes in current amplitude before and
after different interventions, cells were clamped from a holding potential of
40 mV to hyperpolarizing potential of
100 mV
for 100 ms, back to
40 mV for 10 ms, and then to a depolarizing
potential of +100 mV for 100 ms (voltage-clamp protocol is
shown in Fig. 3, top). The same hyperpolarizing and depolarizing pulses were imposed every 30 s. All results are expressed as
mean ± SEM. Statistical comparisons were performed by Student's t test and a two-tailed probability of <5% was taken to indicate statistical significance.

View larger version (47K):
[in this window]
[in a new window]
|
Fig. 1.
Relations between
PKC activity and volume regulation of wtClC-3 in NIH/3T3
cells. (A) PKC activation inhibited both basally active and hypotonic cell-swelling-induced IClC-3.
When overexpressed in NIH/
3T3 cells, only a small portion of
the ClC-3 channels were active
under isotonic conditions (a).
Subsequent exposure of the
same cell to hypotonic solutions
caused a further increase in current amplitude (b). Activation of
PKC by PDBu (100 nM) under
hypotonic conditions caused a
closure of most channels (c).
Mean current-voltage (I-V) curves
from six cells under isotonic ( ),
hypotonic ( ), and hypotonic
PDBu 100 nM ( ) conditions
are shown in d. (B) Downregulation of endogenous PKC by
exposure of the wtClC-3-transfected NIH/3T3 cells to PDBu
1 µM for >24 h not only abolished the inhibition of IClC-3 by
acute application of PDBu (100 nM), but also changed the sensitivity to changes in osmolality.
(a) Representative current traces
recorded from the PDBu-pretreated ClC-3 stably transfected
cells under isotonic conditions.
A larger IClC-3.b was elicited under
the same isotonic conditions. (b)
Subsequent hypotonic cell swelling failed to further increase the
current amplitude of these cells.
(c) Acute application of PDBu
(100 nM) under hypotonic conditions no longer inhibited the
currents. (d) Mean I-V curves
from four different cells under
isotonic ( ), hypotonic ( ), and
hypotonic PDBu 100 nM ( )
conditions. (C) Inhibition of endogenous PKC by BIM (100 nM) activated ClC-3 channels in isotonic solutions, and subsequent hypotonic
cell swelling failed to further increase the current density. (a) IClC-3.b under isotonic conditions. (b) Exposure of the same cell to BIM (100 nM) under isotonic condition increased the current. (c) Subsequent exposure of the cell to hypotonic solution caused no further increase
in current amplitude. (d) Mean I-V curves from five different cells under isotonic ( ), isotonic BIM 100 nM ( ), and hypotonic BIM 100 nM ( ) conditions.
|
|

View larger version (45K):
[in this window]
[in a new window]
|
Fig. 3.
Inhibition of endogenous PKC by BIM (100 nM) activated large outwardly rectifying
Cl currents under isotonic conditions in guinea-pig atrial (A)
and ventricular (B) myocytes.
Subsequent hypotonic cell swelling failed to significantly increase
the current densities in these
cells. (a) Whole-cell current recordings under isotonic conditions. (b) Exposure of the same
cell to BIM (100 nM) under isotonic conditions caused a significant increase in the membrane
current with similar biophysical
properties as ICl.vol (see A). (c)
Subsequent exposure of the cell
to hypotonic solution caused no
significant increase in current
amplitude. (d) Mean I-V curves
from six (C and D) different cells
under isotonic ( ), isotonic BIM
100 nM ( ), and hypotonic BIM
100 nM ( ) conditions. (C) Pharmacology of BIM-induced ICl.vol
in atrial myocytes. a and b compare the effects of DIDS (100 µM) on the swelling-induced
ICl.vol (a) and the BIM-induced
ICl.vol (b) in atrial myocytes. DIDS
inhibited these currents in an
identical voltage-dependent manner. Extracellular ATP (10 mM)
also inhibited BIM-induced ICl.vol
in a characteristic voltage-dependent manner (c) identical to its
inhibitory effect on the wild-type
gpClC-3 channels (Duan et al.,
1997b ) and ICl.vol in many other
tissues and species (Strange et al.,
1996 ).
|
|
Solutions and Drugs
Bath and pipette solutions were chosen to facilitate Cl
current
recording. The hypotonic (250 mOsm/kg H2O, measured by
freezing point depression, Osmomette; Precision Systems Inc.)
bath solutions for recording in NIH/3T3 cells contained (mM):
125 NaCl, 2.5 MgCl2, 2.5 CaCl2, 10 HEPES, pH 7.2, [Cl]o = 135 mM. The isotonic and hypertonic bath solutions were the same
as the hypotonic solution except that the osmolarity was adjusted
to 300 and 350 mOsm/kg H2O, respectively, with mannitol.
When experiments were performed with decreased [Cl]o, iodide
(I
) or aspartate (Asp
) was used to replace Cl
at equimolar
concentration (110 mM). The pipette (internal) solution for recordings in NIH/3T3 contained (mM): 135 N-methyl-D-glucamine chloride (NMDG-Cl), 2 EGTA, 5 Mg-ATP, 10 HEPES, pH
7.2, [Cl]i = 135 mM, 300 mOsm/kg H2O using mannitol). The
hypotonic (220 mOsm/kg H2O) bath solutions for recording in
cardiac myocytes contained (mM): 90 NaCl, 0.8 MgCl2, 1.0 CaCl2,
0.2 CdCl2, 2.0 BaCl2, 0.33 NaH2PO4, 10 tetraethylammonium-Cl,
10 HEPES, 5.5 glucose, pH 7.4, [Cl]o = 108 mM. The isotonic
bath solutions were the same as the hypotonic solution except
that the osmolarity was adjusted to 300 mOsm/kg H2O, respectively, with mannitol. When low [Cl]o was needed, NaI or Na-aspartate was used to replace NaCl at equimolar concentration
(90 mM). The pipette (internal) solution for recordings in cardiac myocytes contained (mM): 108 NMDG-Cl, 2 EGTA, 5 Mg-ATP, 10 HEPES, pH 7.4, [Cl]i = 108 mM, 290 mOsm/kg H2O using mannitol. In some experiments, cell diameters were measured continuously using a video edge detector (Crescent Electronics) and cell volumes were calculated assuming a simple
spherical geometry. All chemicals were from Sigma Chemical Co.
Phorbol 12,13-dibutyrate (PDBu), bisindolylmaleimide I-HCl
(BIM), okadaic acid, and calyculin A were obtained from Calbiochem Corp. and prepared as stock solutions of 1 or 10 mM in
dimethyl sulfoxide and added to known volume of superfusion
solutions to produce the desired concentrations.
 |
RESULTS |
Under isotonic (300 mOsm/kg H2O) symmetrical Cl
(135 mM) conditions, wtClC-3-transfected cells generated basally active outwardly rectifying whole-cell currents (Fig. 1 A, a) with a mean current density of 442 ± 38 pA pF
1 at +80 mV and
253 ± 27 pA pF
1 at
80
mV and a mean reversal potential of
1.8 ± 0.3 mV (n = 6). Exposure of these cells to hypotonic solutions (250 mOsm/kg H2O, 17% hypotonic) for >2 min caused
significant cell swelling and increased the membrane
current densities to 946 ± 56 pA pF
1 at +80 mV and
593 ± 46 pA pF
1 at
80 mV (n = 6) due to an increase in the number of active channels (Strange et al.,
1996
; Duan et al., 1997a
,b) (Fig. 1 A, b). These results
indicate that under basal isotonic conditions, most expressed ClC-3 channels remain in a closed state that
can be activated by hypotonic cell swelling, suggesting
the possible existence of an endogenous cytosolic inhibitor under isotonic conditions (Krick et al., 1991
;
Kawasaki et al., 1995
). Activation of PKC by PDBu (100 nM) under hypotonic conditions strongly inhibited the
currents (Fig. 1 A, c) in a voltage-independent fashion as previously described (Duan et al., 1997b
), while hypotonic solutions induced a similar increase in cell volume
in control (129 ± 5.4%, n = 8) and PDBu-containing
solutions (122 ± 2.0%, n = 7, P = NS). Downregulation of endogenous PKC by exposure of wtClC-3-transfected NIH/3T3 cells to PDBu (1 µM) for >24 h
(Duan et al., 1995
; Pears and Goode, 1997
) not only
abolished the inhibition of wtClC-3 currents by acute
application of PDBu (Fig. 1 B, c), but also, surprisingly,
changed the volume sensitivity of these channels. In
downregulated cells under isotonic conditions, most
channels were constitutively open with a mean current
density of 1,009 ± 92 pA pF
1 at +80 mV and
678 ± 60 pA pF
1 at
80 mV (n = 4) (Fig. 1 B, a) and subsequent hypotonic cell swelling failed to further significantly increase current densities (Fig. 1 B, b) in these
cells (1,039 ± 91 pA pF
1 at +80 mV and
701 ± 70 pA pF
1 at
80 mV, P = NS). Inhibition of endogenous PKC in wtClC-3-transfected cells by acute application of BIM (100 nM), a highly selective PKC inhibitor
(Toullec et al., 1991
), also dramatically increased membrane Cl
current densities under isotonic conditions
(Fig. 1 C, a and b) and abolished further activation of
expressed channels by cell swelling (Fig. 1 C, c). The
endogenous Cl
currents in NIH/3T3 cells, while also
volume sensitive, contribute very little to the results described above (Duan et al., 1997b
; also see Fig. 6, A and
B). These results strongly suggest that endogenous
PKC in these NIH/3T3 cells is a strong cytosolic inhibitor of ClC-3 channels and that relief of PKC inhibition
may be linked to hypotonic-induced opening of the
channel. To further test this hypothesis, we performed
similar experiments in isolated guinea-pig atrial and
ventricular cells from which the ClC-3 gene was originally cloned (Duan et al., 1997b
). As shown in Fig. 2,
both the basally active and swelling-activated currents
in atrial (Fig. 2 A) and ventricular (Fig. 2 B) myocytes
were also strongly inhibited by PKC activation. Identical to the cloned gpClC-3 channel expressed in NIH/
3T3 cells (Duan et al., 1997b
), these cell swelling-
induced and PKC-sensitive currents in both atrial (Fig.
2 C) and ventricular (Fig. 2 D) myocytes had an anion
selectivity of I
> Cl
>> Asp
. Consistent with our observations in NIH/3T3 cells, BIM-induced inhibition of
endogenous PKC also activated native ICl.vol in both
atrial (Fig. 3 A, b) and ventricular (Fig. 3 B, b) myocytes under isotonic conditions and prevented further
activation by subsequent hypotonic cell swelling (Fig. 3,
A, c and B, c). As shown in Fig. 3 C, the swelling- and
BIM-induced currents were inhibited by extracellular
4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) (100 µM, Fig. 3 C, a and b) and ATP (10 mM, Fig. 3 C, c) in
a characteristic voltage-dependent manner that closely
resembles the inhibition of native ICl.vol by these compounds in a wide variety of cells (Duan et al., 1995
;
Vandenberg et al., 1994
; Nilius et al., 1996
; Strange et
al., 1996
; Okada, 1997
; Yamazaki et al., 1998
) and ClC-3
currents in oocytes and mammalian cells (Kawasaki et al.,
1994
; Duan et al., 1997b
). These results support the idea
that PKC phosphorylation and dephosphorylation of
both the wtClC-3 and native channel protein play a crucial role in channel regulation by changes in cell volume.

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 6.
Comparison of current densities at +80 mV in NIH/
3T3 cells transfected with CD8,
wtClC-3, S51AClC-3, S362AClC-3,
and S51A+ S362AClC-3 under
isotonic (A), hypotonic (B), and
hypotonic PDBu 100 nM (C)
conditions, respectively. Whole-cell currents were recorded from
cells transfected with different
cDNAs that were randomly coded
during experiments. Results were
revealed after all experiments
were completed.
|
|

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 2.
PKC activity and volume-regulation of native ICl.vol in
guinea-pig atrial (A) and ventricular (B) myocytes. (a) Representative whole-cell current traces
recorded under isotonic conditions. (b) Subsequent exposure
of the same cell to hypotonic solutions caused a further increase
in the currents (ICl.swell). (c) Activation of PKC by PDBu (100 nM)
under hypotonic conditions
caused closure of most channels.
(d) Mean I-V relationships from
different atrial (n = 6, in A) or
ventricular (n = 5, in B) cells under isotonic ( ), hypotonic ( ),
and hypotonic PDBu 100 nM
( ) conditions. (C and D) I-V
relationship of whole-cell currents in atrial (C) and ventricular (D) myocytes before and
after [Cl ]o was replaced by
equimolar (90 mM) I or Asp
(ECl = +46 mV). I and Asp
substitution of [Cl ]o shifted the
reversal potential of ICl.vol in atrial
myocytes (C) from 2.0 ± 0.5 to
15.3 ± 1.3 and +39.5 ± 1.7 mV (n = 6), respectively, and
of ICl.vol in ventricular myocytes
(D) from 3.2 ± 0.9 to 13.8 ± 2.4 and +41.2 ± 3.9 mV (n = 5), respectively. Voltage-clamp
protocol is the same as shown in
Fig. 1, top.
|
|
In intact cells, processes that are reversibly controlled
by protein phosphorylation require not only a protein
kinase but also a protein phosphatase (Hunter, 1995
).
The net level of protein phosphorylation depends on
the balance of kinase and phosphatase activities (Cohen, 1992
), and both protein kinases and phosphatases have been reported to be subject to regulation by cell
volume (Jennings and al-Rohil, 1990
; Jennings and
Schulz, 1991
; Starke and Jennings, 1993
; Waldegger et al.,
1997a
; Lang, 1998). In fact, inhibition of serine/threonine protein phosphatases by calyculin A in NIH/3T3
cells causes not only a marked increase in protein phosphorylation in both cytosolic and insoluble cellular
fractions, but also a reversible cell shape change
(Chartier et al., 1991
). To further test the hypothesis
that a balance between channel protein phosphorylation and dephosphorylation may be the key regulatory event responsible for ClC-3 channel regulation by cell
volume in the face of osmotic perturbation, we studied
the effects of two highly potent serine/threonine protein phosphatase inhibitors, okadaic acid (Cohen et al.,
1990
) and calyculin A (Ishihara et al., 1989
), on wtClC-3 channels expressed in NIH/3T3 cells. As shown
in Fig. 4, both okadaic acid (100 nM, A) and calyculin A (20 nM, B) not only inhibited basally active wtClC-3
channels under isotonic conditions, but also prevented
hypotonic cell-swelling activation of these channels.
Similar results were observed in four (okadaic acid)
and five (calyculin A) different cells in which wtClC-3
were stably or transiently transfected. Both basally and
swelling-activated ClC-3 channels were always strongly
inhibited by either okadaic acid or calyculin A, when
added before or after induction of cell swelling, indicating that PPs are continuously involved in channel
regulation and the balance of PKC-PP activity is constantly regulated by cell volume.

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 4.
Effects of serine/threonine protein phosphatase inhibitors, okadaic acid (A) and calyculin A (B), on wtClC-3 channels. Whole-cell currents were monitored continuously (Voltage-clamp protocol is shown on the top and described in MATERIALS
AND METHODS) when cells transfected with wtClC-3 were consecutively exposed to isotonic solutions for 10 min, hypotonic solutions
until the changes in current amplitudes reached a steady state
(~20 min), isotonic solutions until the changes in current amplitudes reached a steady state (~20 min), isotonic solutions with 100 nM okadaic acid (A, OA) or calyculin A (B, CLA) for 10 min, hypotonic solutions with OA or CLA for 10 min, washout of OA or
CLA with hypotonic solutions until currents reached a steady state,
reapplication of OA or CLA in hypotonic solutions and then washout of OA or CLA. Both OA (A) and CLA (B) not only inhibited
the basally active wtClC-3 channels, but also prevented hypotonic
cell-swelling activation of these channels. Both basally and swelling
activated ClC-3 channels were always strongly inhibited by both
OA and CLA, when added either before or after cell swelling. Similar results were observed in four (OA) and five (CLA) different
cells in which wtClC-3 were stably or transiently transfected.
|
|
The apparent link between PKC phosphorylation-
dephosphorylation and cell swelling-induced activation of ClC-3 channels prompted a consideration of putative protein PKC phosphorylation sites as potential
volume sensors (Kawasaki et al., 1994
; Duan et al., 1997b
; GenBank accession #U83464). One is serine 51 near the amino terminus (Arg-Arg-lle-Asn-Ser51-Lys-Lys-Lys) and the other is serine 362 within the cytoplasmic loop between transmembrane domains D7 and D8
(Arg-Arg-Lys-Ser362-Thr-Lys). We tested the hypothesis
that the inhibition, by PKC activators and phosphatase
inhibitors, and the activation, by hypotonic cell swelling and PKC inhibitors, of ClC-3 channels may be due
to direct phosphorylation-dephosphorylation of S51 or
S362. Serine 51 and/or serine 362 were changed to alanines and three mutants were generated: single mutants S51A, S362A, and double mutant S51A + S362A
(Fig. 5 A). Results of whole-cell patch-clamp recording
from these three ClC-3 mutants transfected into 3T3
cells are shown in Fig. 5, B-D. Cells were exposed
either to isotonic (a), hypotonic (b), or hypotonic
PDBu 100 µM (c) solutions. The double mutant
S51A+S362AClC-3 generated a large constitutively activate IClC-3 with a mean current density of 918 ± 23 pA
pF
1 at +80 mV and
557 ± 48 pA pF
1 at
80 mV
(n = 5) (Fig. 5 B, a) that was not significantly augmented (mean current density of 970 ± 48 pA pF
1 at
+80 mV and
629 ± 85 pA pF
1 at
80 mV, P = NS)
by hypotonic cell swelling (Fig. 5 B, b) or inhibited by
PDBu-induced activation of PKC (Fig. 5 B, c, mean current density of 952 ± 41 pA pF
1 at +80 mV and
606 ± 71 pA pF
1 at
80 mV, P = NS). Similar results were
obtained from S51AClC-3-transfected cells. Most channels were open even under isotonic conditions and
generated a large constitutively active IClC-3 (Fig. 5 C, a,
mean current density of 930 ± 18 pA pF
1 at +80 mV
and
608 ± 22 pA pF
1 at
80 mV, n = 7) that was no
longer responsive to cell swelling (Fig. 5 C, b, mean
current density of 971 ± 27 pA pF
1 at +80 mV and
650 ± 23 pA pF
1 at
80 mV, P = NS, compared with
isotonic conditions) or PDBu (Fig. 5 C, d, mean current density of 907 ± 31 pA pF
1 at +80 mV and
627 ± 31 pA pF
1 at
80 mV, P = NS compared with isotonic
and hypotonic conditions). As shown in Fig. 5 D, however, the S362AClC-3 mutant yielded a channel with an
intermediate responsiveness to cell swelling and PKC.
The mean current densities of S362AClC-3 under isotonic conditions were 610 ± 42 pA pF
1 at +80 mV and
348 ± 31 pA pF
1 at
80 mV (n = 6) that are significantly higher (P < 0.05) than the mean current densities of wtClC-3 under the same isotonic conditions. Exposure of these S362AClC-3-transfected cells to hypotonic solutions caused a further increase in current
densities (912 ± 90 pA pF
1 at +80 mV and
468 ± 37 pA pF
1 at
80 mV, n = 6, P < 0.05 compared with isotonic conditions). Activation of PKC by PDBu under
hypotonic conditions caused significantly less inhibition of S362AClC-3 current than that of wtClC-3 (55 ± 7% inhibition of S362AClC-3 currents [n = 6] vs. 81 ± 3% inhibition of wtClC-3 currents [n = 6] at +80 mV,
P < 0.01). We also performed blinded experiments in
which coded NIH/3T3 cells transiently transfected with
CD8 alone (control), wtClC-3, S51AClC-3, S362AClC-3,
and S51A+S362AClC-3 mutants were used in a randomized fashion to study the response of expressed
currents to hypotonic cell swelling and PKC activation.
Fig. 6 compares the mean current densities of cells
transiently transfected with CD8 alone (control), wtClC-3, S51AClC-3, S362AClC-3, and S51A+S362AClC-3
mutants at +80 mV under isotonic (A), hypotonic (B),
and hypotonic PDBu 100 nM (C) conditions, respectively. While only very small currents could be detected from CD8-transfected cells under isotonic and hypotonic conditions with similar densities as reported before (Duan et al., 1997b
), it is very clear that wtClC-3
and all three ClC-3-mutant transfected cells elicited
significantly larger basally active outwardly rectifying currents under isotonic conditions. Wild-type ClC-3
channels were activated by cell swelling and inhibited
by PKC activation, as previously shown (Fig. 1 A, a).
Both S51AClC-3 and S51A+S362AClC-3 channels were
constitutively opened under isotonic conditions and
thus generated significantly larger "basal" currents than wtClC-3 (Fig. 6 A). These phenotypes exhibited no significant response to changes in cell volume (Fig. 6 B)
or activation of PKC (Fig. 6 C). Basal S362AClC-3 currents were larger than wtClC-3 (P < 0.05) but smaller
than S51AClC-3 and S51A+S362AClC-3 (P < 0.05)
(Fig. 6 A). This phenotype had intermediate response
to cell volume (Fig. 6 B) and PKC (Fig. 6 C). As shown
in Fig. 7 A, a, hypertonic (350 mOsm/kg H2O) cell
shrinkage also failed to inhibit S51AClC-3 channels
(898 ± 32 pA pF
1 under isotonic conditions vs. 869 ± 48 pA pF
1 under hypertonic conditions, n = 5, P = NS). However, extracellular nucleotides (ATPo, 10 mM,
Fig. 7 A, b), DIDS (100 µM, Fig. 7 A, c), and tamoxifen
(10 µM, Fig. 7 A, d) all blocked S51AClC-3 with a characteristic voltage dependence closely resembling their
effects on wtClC-3 (Duan et al., 1997b
; Yamazaki et al.,
1998
). The anion selectivity of these ClC-3 mutants
were examined (Fig. 7 B). Table I summarizes the shifts
in the reversal potentials of native ICl.vol in guinea-pig
atrial and ventricular myocytes and wild-type and mutant ClC-3 channels in NIH/3T3 cells induced by I
or
Asp
and their relative permeabilities. The permeability ratios were calculated from the shifts using the modified Goldman-Hodgkin-Katz equation (Hille, 1992
)
for monovalent anion substitutions. All three mutants
of ClC-3 had a similar permeability ratio to I
or Asp
with respect to Cl
and an identical anion-selectivity of
I
> Cl
>> Asp
as native ICl.vol and wtClC-3.

View larger version (54K):
[in this window]
[in a new window]
|
Fig. 5.
Effects of PKC on
mutant S51A, S362A, and S51A + S362A gpClC-3 transiently expressed in NIH/3T3 cells. (A)
Transmembrane topology model
for ClC Cl channels (Schmidt-Rose and Jentsch, 1997 ) and the
location of mutated residues. (B-
D) Whole-cell currents recorded
from three mutants of ClC-3 under isotonic (a), hypotonic (b),
and hypotonic PDBu 100 nM (c)
conditions, respectively. d show
corresponding mean I-V curves
from different cells transfected
with S51A+S362AClC-3 (B, d, n
= 5), S51AClC-3 (C, d, n = 7),
and S362AClC-3 (D, d, n = 6),
respectively.
|
|

View larger version (50K):
[in this window]
[in a new window]
|
Fig. 7.
Pharmacology and
ion selectivity of ClC-3 mutants in
NIH/3T3 cells. (A) Representative whole-cell recording of S51A
ClC-3 currents under isotonic
conditions and in the presence
of extracellular hypertonicity (a),
ATP (b), DIDS (c), and tamoxifen (d). While hypertonic cell
shrinkage failed to inhibit S51A
ClC-3 current, extracellular ATP
(10 mM), DIDS (100 µM), and
tamoxifen (TMX, 10 µM) all inhibited S51A ClC-3 currents in a
characteristic voltage-dependent
manner identical to their inhibitory effects on the wtClC-3 channels (Duan et al., 1997b ). (B)
I-V relationship of whole-cell
currents in NIH/3T3 cells transfected with S51A + S362A ClC-3
(a), S51A ClC-3 (b), S362A ClC-3
(c under isotonic conditions and
d under hypotonic conditions)
before and after [Cl ]o was replaced by equimolar (110 mM)
I or Asp (ECl = +42.8 mV). I
and Asp substitution of [Cl ]o
shifted the reversal potential of
S51A + S362A ClC-3 current (a)
from 1.0 ± 1.2 to 14.2 ± 1.1 and +43.8 ± 3.2 mV (n = 4), respectively, of S51A ClC-3 current
(b) from 3.8 ± 0.3 to 14.9 ± 2.6 and +37.7 ± 1.6 mV (n = 3),
respectively, of S362A ClC-3 current under isotonic condition (c)
from 2.3 ± 0.8 to 17.0 ± 2.3 and +41.0 ± 2.6 mV (n = 4), respectively, and S362A ClC-3 current under hypotonic condition
(d) from 2.2 ± 1.1 to 14.5 ± 2.1 and +38.1 ± 1.5 mV (n = 3),
respectively. Voltage-clamp protocol is the same as shown on the
top of Fig. 1.
|
|
It has been reported that rat ClC-3 channels may be
inhibited by increases in intracellular Ca2+ when expressed in Chinese hamster ovary cells (Kawasaki et al., 1995
). Similarly, exposure of guinea-pig cardiac wtClC-3-
transfected cells to the Ca2+ ionophore, ionomycin (1 µM), also caused a dramatic inhibition of wtClC-3 currents under hypotonic conditions (Fig. 8 A, c and d).
As shown in Fig. 6 B, however, the inhibition of gpClC-3
currents by ionomycin was prevented by inhibition of
endogenous PKC when cells were pretreated with BIM
(100 nM). These results suggest that inhibition of gpClC-3 channels by an increase in intracellular Ca2+ may
be due to a Ca2+-sensitive PKC-phosphorylation-mediated mechanism. This is different from rat ClC-3 channels (Kawasaki et al., 1995
) in which the Ca2+ inhibition of ClC-3 channels in inside-out membrane patches
from Chinese hamster ovary cells was reported to be
phosphorylation independent (Kawasaki et al., 1995
).
The reason for this discrepancy is unclear. Both Ca2+-sensitive (PKC
) and -insensitive (PKC
and PKC
)
PKC isozymes are abundantly expressed in NIH/3T3
cells (Szallasi et al., 1994
), and BIM inhibits all PKC
,
I,
II,
,
, and
isozymes (Gekeler et al., 1996
; Toullec et al., 1991
). Thus, it is possible that both Ca2+-sensitive and -insensitive PKC isozymes can phosphorylate
the ClC-3 protein and cause similar conformational
changes to close the channel. To further examine
whether the inhibition of gpClC-3 channels by intracellular Ca2+ is dependent on PKC phosphorylation of the
channel, we performed further experiments to study
the response of expressed mutant S51AClC-3 channel to
ionomycin. While the properties of S51AClC-3 channels under isotonic and hypotonic conditions (Fig. 8 C,
a and b) were identical to those shown in Fig. 5 C, a and
b, respectively, ionomycin failed to inhibit S51AClC-3
channels (Fig. 8 C, c and d), confirming the involvement of amino-terminal PKC-phosphorylation in the
Ca2+ inhibition of ClC-3 channels.

View larger version (49K):
[in this window]
[in a new window]
|
Fig. 8.
Effects of increase in
intracellular Ca2+ on wild-type
and mutant S51AClC-3 currents
in NIH/3T3 cells. (A) Ionomycin (1 µM) inhibited wtClC-3
currents under hypotonic conditions in the presence of 2.5 mM
external Ca2+. Representative
whole-cell currents recorded under isotonic, hypotonic, and hypotonic ionomycin 1 µM conditions and the mean I-V curves of
each (n = 4) are shown in a-d,
respectively. (B) Pretreatment of
wtClC-3 transfected NIH/3T3
cell with BIM (100 nM) under
isotonic condition (a) abolished
the upregulation effect by hypotonic cell swelling (b) and inhibitory effect by ionomycin (c). d
shows mean I-V curves from six
different cells. (C) Mutation at
the amino-terminal PKC-phosphorylation site of ClC-3
(S51AClC-3) also abolished the
upregulation effect by hypotonic cell swelling (b) and inhibitory effect by ionomycin (c).
Similar results were obtained
from five different cells and the
average I-V curves under each
condition are shown in d.
|
|
 |
DISCUSSION |
In this study, we examined the molecular mechanism
responsible for the activation of volume-regulated Cl
channels by hypotonic cell swelling using both cloned
guinea-pig cardiac ClC-3 expressed in NIH/3T3 cells
and native ICl.vol currents in guinea-pig atrial and ventricular myocytes. Our functional and mutational studies of the ClC-3 gene product indicate that the activation of ClC-3 channels during hypotonic-induced cell swelling is attributable to relief of endogenous PKC inhibition of these channels caused by cell swelling-
induced dephosphorylation of a serine residue within
the amino terminus of the channel protein. Thus, these
results provide an important new clue into the molecular link between changes in cell volume, protein phosphorylation-dephosphorylation, and channel function.
Protein phosphorylation or dephosphorylation is a
common rapid and reversible means of transducing signals from the extracellular environment to many cellular responses (Witters, 1990
; Hunter, 1995
). It should
not be surprising that cells are able to use these rapid
and reversible means to control their sizes. How
changes in cell volume are linked to changes in activity
of protein kinases or phosphatases is still not clear. One
of the most acute signaling events triggered by osmotic
challenge may be dilution or concentration of cellular
constituents including proteins leading to changes in
intracellular macromolecular crowding and confinement, which may profoundly alter kinase-phosphatase
activities (Fulton, 1982
; Jennings and al-Rohil, 1990
;
Jennings and Schulz, 1991
; Minton et al., 1992
; Starke
and Jennings, 1993
; Garner and Burg, 1994
). Alternatively, slower subacute signaling events caused by cell swelling or shrinkage may be related to changes in the
synthesis of second messengers. For example, exposure
of mammalian and plant cells to acute hyperosmotic
stress stimulates rapid synthesis of phosphatidylinositol-3,5-bisphosphate, a new phosphoinositide second messenger in the phospholipase C-PKC cascade that may
associate with the cytoskeleton (Dove et al., 1994
,
1997
). Finally, the slowest and long-term intracellular
signaling events involved in continuous volume regulation may be due to alterations in gene expression of
second messengers (Waldegger et al., 1997a
; Waldegger and Lang, 1998
). The exact interaction between
cell volume and elements of intracellular signaling and
detailed intermediate processes of how PKC and PP activities may be regulated by cell volume requires further study.
Our data suggests that ClC-3 channels may exist in either a closed phosphorylated state or an active dephosphorylated state. PKC phosphorylation of the NH2 terminus of ClC-3 channel may cause a crucial change in
channel conformation and close the channel pore.
Combined with data from our previous studies and
those of other laboratories (Li et al., 1989
; Jennings
and al-Rohil, 1990
; Witters, 1990
; Garner and Burg,
1994
; Duan et al., 1995
, 1997a
,b; Coca-Prados et al.,
1995
, 1996
; Strange et al., 1996
; Dove et al., 1997
;
Waldegger et al., 1997a
), we propose that ClC-3 channels are continuously controlled by a volume-sensitive phosphorylation-dephosphorylation reaction mediated by PKC (both Ca2+-sensitive and -insensitive
isozymes) and PPs (probably PP1 and PP2A). Under
isotonic conditions, a balance of basal PKC and PP activities usually keeps most ClC-3 channels in a phosphorylated closed state and only few channels are in
the dephosphorylated open state. These few active
channels generate a "basal" current (Duan et al., 1992
,
1995
, 1997a
,b; Liu et al., 1993
; Coca-Prados et al., 1995
;
Voets et al., 1996
; Dick et al., 1998
). Under hypotonic conditions, PKC activity is diminished due possibly to
dilution (Jennings and al-Rohil, 1990
; Garner and
Burg, 1994
), redistribution or alteration in PKC or PP
activity (Jennings and Schulz, 1991
; Starke and Jennings, 1993
; Palfrey, 1994
; Lytle, 1998
); ClC-3 channels
become dephosphorylated and more channels open,
producing a larger macroscopic current (Duan et al.,
1995
, 1997a
,b; Strange et al., 1996
). Under hypertonic
conditions, PKC activity may be increased (Jennings
and al-Rohil, 1990
; Larsen et al., 1994
; Garner and
Burg, 1994
; Nelson et al., 1998; Dove et al., 1997
;
Waldegger et al., 1997a
,b) and PP activity may be diminished (Palfrey, 1994
; Lytle, 1998
), thus more channels become phosphorylated and close. Therefore, it is
proposed that serine 51, a putative PKC phosphorylation site near the NH2 terminus of ClC-3, may represent
an important volume sensor of the channel that directly links channel activity to alterations in intracellular
PKC-PP activity.
PKC activation has been previously reported to inhibit voltage-dependent Cl
conductances in hippocampal pyramidal cells (Madison et al., 1986
), resting
Cl
conductance in vascular smooth muscle cells (Saigusa and Kokubun, 1988
), skeletal muscle cells (Brinkmeier and Jockusch, 1987
), ClC-1 in skeletal muscle
and HEK cell expression system (Rosenbohm et al.,
1995
), and the hyperpolarization-activated ClC-2 Cl
channel that is also regulated by cell volume (Staley,
1994
; Staley et al., 1996
; Fritsch and Edelman, 1996
,
1997
). Consistent with our finding in native guinea-pig
atrial and ventricular myocytes and cloned ClC-3-transfected NIH/3T3 cells, PKC activation inhibits ICl.vol and
outwardly rectifying chloride channel in rabbit atrial myocytes (Duan et al., 1995
), MDR1-transfected 3T3
cells (Hardy et al., 1995
), human airway epithelial cells
(Li et al., 1989
), and canine visceral smooth muscle
cells (Dick et al., 1998
). Inhibition of PPs has also been
shown to inhibit ICl.vol in chick heart cells (Hall et al.,
1995
) and bovine chromaffin cells (Doroshenko,
1998
). Recent studies in human nonpigmented ciliary
epithelial cells and canine colonic smooth muscle cells
have also shown that PKC inhibitors isosmotically upregulated ICl.vol (Coca-Prados et al., 1995
, 1996
; Dick et
al., 1998
). Dephosphorylation and cell swelling also activate a voltage-gated Cl
channel in ascidian embryos
(Villaz et al., 1995
). Inhibition of PKC also activates the
volume-sensitive ClC-2 channel in human intestinal
T84 epithelial cells (Fritsch and Edelman, 1996
). Our results provide direct molecular and pharmacological
evidence indicating that channel phosphorylation-
dephosphorylation plays a crucial role in regulation of
volume sensitivity of ClC-3 channels and native ICl.vol.
Therefore, these data provide further evidence that
protein kinases and phosphatases may be secondary
mediators of a subset of cellular responses to cell volume changes that directly control the function of proteins such as ClC-3 channels.
It has also been reported, however, that ICl.vol in some
tissues is either stimulated by phorbol esters, presumably through activation of PKC, or not regulated by
PKC activators and inhibitors (Jackson and Strange,
1993
; Szücs et al., 1996
; Miwa et al., 1997
; reviewed by
Strange et al., 1996
; Okada, 1997
; and Strange, 1998
).
While most of these studies did not directly measure
PKC activity, Miwa et al. (1997)
reported that phorbol
ester TPA (12-O-tetradecanoylphorbol-13-acetate) still
had no effect on ICl.vol in human epidermoid KB cells
even though activation of PKC by this compound could
be biochemically proven. The reason for these discrepancies is not clear. It is possible, however, that the intrinsic response of cells to phorbol esters in terms of activation of different PKC isozymes may vary from cell to
cell (Nishizuka, 1988
; Hug and Sarre, 1993
) and the phosphorylation of ClC-3 chloride channel may be mediated by specific PKC isozymes. Another possibility is
that the so called "PKC activators" (phorbol esters) and
"PKC inhibitors" may also act on unknown protein kinases other than PKC. It may be possible that the kinase involved in regulation of ClC-3 is not simply PKC
but another serine/threonine kinase acting at the same
S51. Waldegger et al. (1997a)
have recently cloned a
volume-regulated serine/threonine protein kinase designated h-sgk, which is upregulated by hypertonic cell
shrinkage and depressed by hypotonic cell swelling.
h-sgk has 50% homology throughout its catalytic domain with PKC and is widely expressed in many tissues.
However, gene transcription and translation of h-sgk
may be too slow to account for the change in ICl.vol. Simple biochemical experiments or attempts to measure activities or translocations of PKC or PKC isozymes may
not be sufficient to provide a definitive answer to the
complex question whether or not PKC is in fact activated by cell shrinkage and inhibited by swelling. Obviously, more extensive experiments will be needed to
detect rapid but more subtle changes in PKC or other protein kinases or phosphatases near the membrane
during volume alterations. Although our mutation experiments on the consensus PKC phosphorylation sites
provide strong evidence supporting the role of PKC in
the volume regulation of the channel, direct measurement of changes in kinase and/or phosphatase activity
and biochemical evidence for phosphorylation of ClC-3
during alternations of cell volume are needed. It
should also be pointed out that consensus phosphorylation sites of a protein can be promiscuous and mutations can alter the conformation of the protein independent of phosphorylation. In some cells, the mechanism of channel activation by cell swelling may be more
complicated than described here since it is also possible that the volume-regulated Cl
channels in some native cells may actually be composed of heterodimers; e.g., ClC-3 along with other ClC subunits (Lorenz et al.,
1996
). Whether a non-PKC-regulated but volume-sensitive ClC-3 isoform, or heteromultimer, or other members of the ClC-3/ClC-4/ClC-5 subbranch (Jentsch,
1996
) are also involved in cell-volume regulation and account for ICl.vol in other cell types needs further investigation.
Address correspondence to Dr. Joseph R. Hume or Dr. Burton
Horowitz, Department of Physiology and Cell Biology/351, University of Nevada School of Medicine, Reno, NV 89557-0046. Fax: 702-784-4360; E-mail: joeh{at}med.unr.edu or burt{at}physio.unr.edu
Original version received 14 July 1998 and accepted version received 12 November 1998.
D. Duan was supported by an MRC fellowship from the Medical Research Council of Canada. This study was supported by
NationalWe are indebted to N. Horowitz, L. Ye, and T. Truong for their excellent technical assistance. We thank Dr. Bertil Hille for suggesting the experiments in Fig. 4.
BIM, bisindolylmaleimide I-HCl;
DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid;
I-V, current-
voltage;
PDBu, phorbol 12,13-dibutyrate;
PP, protein phosphatase.
1.
|
Bize, I., and
P.B. Dunham.
1994.
Staurosporine, a protein kinase
inhibitor, activates K-Cl cotransport in LK sheep erythrocytes.
Am. J. Physiol.
266:
C759-C770
[Abstract/Free Full Text].
|
2.
|
Bond, T.D.,
M.A. Valverde, and
C.F. Higgins.
1998.
Protein kinase C
phosphorylation disengages human and mouse-1a P-glycoproteins from influencing the rate of activation of swelling- activated
chloride currents.
J. Physiol. (Lond.).
508:
333-340
[Abstract/Free Full Text].
|
3.
|
Brinkmeier, H., and
H. Jockusch.
1987.
Activators of protein kinase C
induce myotonia by lowering chloride conductance in muscle.
Biochem. Biophys. Res. Commun.
148:
1383-1389
[Medline].
|
4.
|
Chartier, L.,
L.L. Rankin,
R.E. Allen,
Y. Kato,
N. Fusetani,
H. Karaki,
S. Watabe, and
D.J. Hartshorne.
1991.
Calyculin-A increases
the level of protein phosphorylation and changes the shape of
3T3 fibroblasts.
Cell Motil. Cytoskelet.
18:
26-40
[Medline].
|
5.
|
Clapham, D.E..
1998.
The list of potential volume-sensitive chloride
currents continues to swell (and shrink).
J. Gen. Physiol.
111:
623-624
[Free Full Text].
|
6.
|
Coca-Prados, M.,
J. Anguita,
M.L. Chalfant, and
M.M. Civan.
1995.
PKC-sensitive Cl channels associated with ciliary epithelial homologue of pICln.
Am. J. Physiol.
268:
C572-C579
[Abstract/Free Full Text].
|
7.
|
Coca-Prados, M.,
J. Sanchez-Torres,
K. Peterson-Yantorno, and
M.M. Civan.
1996.
Association of ClC-3 channel with Cl transport by human nonpigmented ciliary epithelial cells.
J. Membr.
Biol.
150:
197-208
[Medline].
|
8.
|
Cohen, P.,
C.F. Holmes, and
Y. Tsukitani.
1990.
Okadaic acid: a
new probe for the study of cellular regulation.
Trends Biochem.
Sci.
15:
98-102
[Medline].
|
9.
|
Cohen, P..
1992.
Signal integration at the level of protein kinases,
protein phosphatases and their substrates.
Trends Biochem. Sci.
17:
408-413
[Medline].
|
10.
|
Deng, W.P., and
J.A. Nickoloff.
1992.
Site-directed mutagenesis of
virtually any plasmid by eliminating a unique site.
Anal. Biochem.
200:
81-88
[Medline].
|
11.
|
Dick, G.M.,
K.A. Kuenzli,
B. Horowitz,
J.R. Hume, and
K.M. Sanders.
1998.
Functional and molecular identification of a novel
chloride conductance in canine colonic smooth muscle.
Am. J. Physiol.
275:
C940-C950
[Abstract/Free Full Text].
|
12.
|
Doroshenko, P..
1998.
Pervanadate inhibits volume-sensitive chloride current in bovine chromaffin cells.
Pflügers Arch.
435:
303-309
[Medline].
|
13.
|
Dove, S.K.,
C.W. Lloyd, and
B.K. Drobak.
1994.
Identification of a
phosphatidylinositol 3-hydroxy kinase in plant cells: association
with the cytoskeleton.
Biochem. J.
303:
347-350
[Medline].
|
14.
|
Dove, S.K.,
F.T. Cooke,
M.R. Douglas,
L.G. Sayers,
P.J. Parker, and
R.H. Michell.
1997.
Osmotic stress activates phosphatidylinositol-3,5-bisphosphate synthesis.
Nature.
390:
187-192
[Medline].
|
15.
|
Duan, D.,
B. Fermini, and
S. Nattel.
1992.
Sustained outward current observed after Ito1 inactivation in rabbit atrial myocytes is a
novel Cl current.
Am. J. Physiol.
263:
H1967-H1971
[Abstract/Free Full Text].
|
16.
|
Duan, D.,
B. Fermini, and
S. Nattel.
1995.
Alpha-adrenergic control
of volume-regulated Cl currents in rabbit atrial myocytes. Characterization of a novel ionic regulatory mechanism.
Circ. Res.
77:
379-393
[Abstract/Free Full Text].
|
17.
|
Duan, D.,
J.R. Hume, and
S. Nattel.
1997a.
Evidence that outwardly
rectifying Cl channels underlie volume-regulated Cl2 currents in
heart.
Circ. Res.
80:
103-113
[Abstract/Free Full Text].
|
18.
|
Duan, D.,
C. Winter,
S. Cowley,
J.R. Hume, and
B. Horowitz.
1997b.
Molecular identification of a volume-regulated chloride channel.
Nature.
390:
417-421
[Medline].
|
19.
|
Fritsch, J., and
A. Edelman.
1996.
Modulation of the hyperpolarization-activated Cl current in human intestinal T84 epithelial
cells by phosphorylation.
J. Physiol. (Lond.).
490:
115-128
[Abstract].
|
20.
|
Fritsch, J., and
A. Edelman.
1997.
Osmosensitivity of the hyperpolarization-activated chloride current in human intestinal T84
cells.
Am. J. Physiol.
272:
C778-C786
[Abstract/Free Full Text].
|
21.
|
Fulton, A.B..
1982.
How crowded is the cytoplasm?
Cell.
30:
345-347
[Medline].
|
22.
|
Garner, M.M., and
M.B. Burg.
1994.
Macromolecular crowding and
confinement in cells exposed to hypertonicity.
Am. J. Physiol.
266:
C877-C892
[Abstract/Free Full Text].
|
23.
|
Gekeler, V.,
R. Boer,
F. Uberall,
W. Ise,
C. Schubert,
I. Utz,
J. Hofmann,
K.H. Sanders,
C. Schachtele,
K. Klemm, and
H. Grunicke.
1996.
Effects of the selective bisindolylmaleimide protein kinase C
inhibitor GF 109203X on P-glycoprotein-mediated multidrug resistance.
Br. J. Cancer.
74:
897-905
[Medline].
|
24.
|
Gibson, J.S., and
A.C. Hall.
1995.
Stimulation of KCl co-transport in
equine erythrocytes by hydrostatic pressure: effects of kinase/
phosphatase inhibition.
Pflügers Arch.
429:
446-448
[Medline].
|
25.
|
Grinstein, S.,
W. Furuya, and
L. Bianchini.
1992.
Protein kinases,
phosphatases, and the control of cell volume.
NIPS (News Physiol.
Sci.).
7:
232-237
.
[Abstract/Free Full Text] |
26.
|
Haas, M.,
D. McBrayer, and
C. Lytle.
1995.
[Cl-]i-dependent phosphorylation of the Na-K-Cl cotransport protein of dog tracheal
epithelial cells.
J. Biol.Chem.
270:
28955-28961
[Abstract/Free Full Text].
|
27.
|
Hall, S.K.,
J. Zhang, and
M. Lieberman.
1995.
Cyclic AMP prevents
activation of a swelling-induced chloride-sensitive conductance
in chick heart cells.
J. Physiol. (Lond.).
488:
359-369
[Abstract].
|
28.
|
Hamill, O.P.,
A. Marty,
E. Neher,
B. Sakmann, and
F.J. Sigworth.
1981.
Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.
Pflügers Arch.
391:
85-100
[Medline].
|
29.
|
Hardy, S.P.,
H.R. Goodfellow,
M.A. Valverde,
D.R. Gill,
V. Sepulveda, and
C.F. Higgins.
1995.
Protein kinase C-mediated
phosphorylation of the human multidrug resistance P-glycoprotein regulates cell volume-activated chloride channels [published
erratum appears in EMBO J. 1995. 14:1844].
EMBO (Eur. Mol.
Biol. Organ.) J.
14:
68-75
[Abstract].
|
30.
| Hille, B. 1992. Ionic Channels of Excitable Membranes. Sinauer Associates, Inc., Sunderland, MA.
|
31.
|
Hug, H., and
T.F. Sarre.
1993.
Protein kinase C isoenzymes: divergence in signal transduction?
Biochem. J.
291:
329-343
[Medline].
|
32.
|
Hunter, T..
1995.
Protein kinases and phosphatases: the yin and
yang of protein phosphorylation and signaling.
Cell.
80:
225-236
[Medline].
|
33.
|
Ishihara, H.,
B.L. Martin,
D.L. Brautigan,
H. Karaki,
H. Ozaki,
Y. Kato,
N. Fusetani,
S. Watabe,
K. Hashimoto, and
D. Uemura.
1989.
Calyculin A and okadaic acid: inhibitors of protein phosphatase activity.
Biochem. Biophys. Res. Commun.
159:
871-877
[Medline].
|
34.
|
Jackson, P.S., and
K. Strange.
1993.
Volume-sensitive anion channels mediate swelling-activated inositol and taurine efflux.
Am. J. Physiol.
265:
C1489-C1500
[Abstract/Free Full Text].
|
35.
|
Jennings, M.L., and
N. al-Rohil.
1990.
Kinetics of activation and inactivation of swelling-stimulated K+/Cl transport. The volume-sensitive parameter is the rate constant for inactivation.
J. Gen.
Physiol.
95:
1021-1040
[Abstract].
|
36.
|
Jennings, M.L., and
R.K. Schulz.
1991.
Okadaic acid inhibition of
KCl cotransport. Evidence that protein dephosphorylation is
necessary for activation of transport by either cell swelling or
N-ethylmaleimide.
J. Gen. Physiol.
97:
799-817
[Abstract].
|
37.
|
Jentsch, T.J..
1996.
Chloride channels: a molecular perspective.
Curr. Opin. Neurobiol.
6:
303-310
[Medline].
|
38.
|
Kawasaki, M.,
S. Uchida,
T. Monkawa,
A. Miyawaki,
K. Mikoshiba,
F. Marumo, and
S. Sasaki.
1994.
Cloning and expression of a protein kinase C-regulated chloride channel abundantly expressed
in rat brain neuronal cells.
Neuron.
12:
597-604
[Medline].
|
39.
|
Kawasaki, M.,
M. Suzuki,
S. Uchida,
S. Sasaki, and
F. Marumo.
1995.
Stable and functional expression of the CIC-3 chloride
channel in somatic cell lines.
Neuron.
14:
1285-1291
[Medline].
|
40.
|
Klein, J.D.,
P.B. Perry, and
W.C. O'Neill.
1993.
Regulation by cell
volume of Na(+)-K(+)-2Cl cotransport in vascular endothelial
cells: role of protein phosphorylation.
J. Membr. Biol.
132:
243-252
[Medline].
|
41.
|
Krarup, T.,
L.D. Jakobsen,
B.S. Jensen, and
E.K. Hoffmann.
1998.
Na+-K+-2Cl cotransport in Ehrlich cells: regulation by protein
phosphatases and kinases.
Am. J. Physiol.
275:
C239-C250
[Abstract/Free Full Text].
|
42.
|
Krick, W.,
J. Disser,
A. Hazama,
G. Burckhardt, and
E. Fromter.
1991.
Evidence for a cytosolic inhibitor of epithelial chloride
channels.
Pflügers Arch.
418:
491-499
[Medline].
|
43.
|
Lang, F.,
G.L. Busch,
M. Ritter,
H. Volkl,
S. Waldegger,
E. Gulbins, and
D. Haussinger.
1998.
Functional significance of cell volume
regulatory mechanisms.
Physiol. Rev.
78:
247-306
[Abstract/Free Full Text].
|
44.
|
Larsen, A.K.,
B.S. Jensen, and
E.K. Hoffmann.
1994.
Activation of
protein kinase C during cell volume regulation in Ehrlich mouse
ascites tumor cells.
Biochim. Biophys. Acta
1222:
477-482
[Medline].
|
45.
|
Li, M.,
J.D. McCann,
M.P. Anderson,
J.P. Clancy,
C.M. Liedtke,
A.C. Nairn,
P. Greengard, and
M.J. Welsch.
1989.
Regulation of chloride channels by protein kinase C in normal and cystic fibrosis
airway epithelia.
Science.
244:
1353-1356
[Medline].
|
46.
|
Liu, S.,
J.R. Stimers, and
M. Lieberman.
1993.
A novel Cl conductance in cultured chick cardiac myocytes: role of intracellular
Ca2+ and cAMP.
J. Membr. Biol.
141:
59-68
.
|
47.
|
Lorenz, C.,
M. Pusch, and
T.J. Jentsch.
1996.
Heteromultimeric
CLC chloride channels with novel properties.
Proc. Natl. Acad.
Sci. USA.
93:
13362-13366
[Abstract/Free Full Text].
|
48.
|
Lytle, C..
1998.
A volume-sensitive protein kinase regulates the Na-K-2Cl cotransport in duck red blood cells.
Am. J. Physiol.
274:
C1002-C1010
[Abstract/Free Full Text].
|
49.
|
Madison, D.V.,
R.C. Malenka, and
R.A. Nicoll.
1986.
Phorbol esters
block a voltage-sensitive chloride current in hippocampal pyramidal cells.
Nature.
321:
695-697
[Medline].
|
50.
|
Minton, A.P.,
G.C. Colclasure, and
J.C. Parker.
1992.
Model for the
role of macromolecular crowding in regulation of cellular volume.
Proc. Natl. Acad. Sci. USA.
89:
10504-10506
[Abstract].
|
51.
|
Miwa, A.,
K. Ueda, and
Y. Okada.
1997.
Protein kinase C-independent correlation between P-glycoprotein expression and volume
sensitivity of Cl channel.
J. Membr. Biol.
157:
63-69
[Medline].
|
52.
|
Nelson, D.J.,
X.Y. Tien,
W. Xie,
T.A. Brasitus,
M.A. Kaetzel, and
J.R. Dedman.
1996.
Shrinkage activates a nonselective conductance:
involvement of a Walker-motif protein and PKC.
Am. J. Physiol.
270:
C179-C191
[Abstract/Free Full Text].
|
53.
|
Nilius, B.,
J. Eggermont,
T. Voets, and
G. Droogmans.
1996.
Volume-activated Cl channels.
Gen. Pharmacol.
27:
1131-1140
[Medline].
|
54.
|
Nishizuka, Y..
1988.
The molecular heterogeneity of protein kinase C
and its implications for cellular regulation.
Nature.
334:
661-665
[Medline].
|
55.
|
Okada, Y..
1997.
Volume expansion-sensing outward-rectifier Cl
channel: fresh start to the molecular identity and volume sensor.
Am. J. Physiol.
273:
C755-C789
[Abstract/Free Full Text].
|
56.
| Palfrey, H.C. 1994. Protein phosphorylation control in the activity
of volume-sensitive transport systems. In Cellular and Molecular
Physiology of Cell Volume Regulation. K. Strange, editor. CRC
Press Inc., Boca Raton, FL. 201-214.
|
57.
| Pears, C.J., and N.T. Goode. 1997. PKC downregulation: signal or
adaptation? In Protein Kinase C. P.J. Parker and L.V. Dekker, editors. R.G. Landes Company, Austin, TX. 45-56.
|
58.
|
Rosenbohm, A.,
A.L. George,
R. Rudel, and
C. Fahlke.
1995.
Effect
of activators of protein kinase C (PKC) on the muscle chloride
channel, CLC-1, studied in native and heterologous expression
systems.
Pflügers Arch.
429:
R25
.
|
59.
|
Saigusa, A., and
S. Kokubun.
1988.
Protein kinase C may regulate
resting anion conductance in vascular smooth muscle cells.
Biochem. Biophys. Res. Commun.
155:
882-889
[Medline].
|
60.
|
Santell, L.,
R.L. Rubin, and
E.G. Levin.
1993.
Enhanced phosphorylation and dephosphorylation of a histone-like protein in response to hyperosmotic and hypoosmotic conditions.
J. Biol.
Chem
268:
21443-21447
[Abstract/Free Full Text].
|
61.
|
Schmidt-Rose, T., and
T.J. Jentsch.
1997.
Transmembrane topology
of a CLC chloride channel.
Proc. Natl. Acad. Sci. USA.
94:
7633-7638
[Abstract/Free Full Text].
|
62.
|
Staley, K.J..
1994.
The role of an inwardly rectifying chloride conductance in postsynaptic inhibition.
J. Neurophysiol.
72:
273-284
[Abstract/Free Full Text].
|
63.
|
Staley, K.J.,
R. Smith,
J. Schaack,
C. Wilcox, and
T.J. Jentsch.
1996.
Alteration of GABAA receptor function following gene transfer of
the CLC-2 chloride channel.
Neuron.
17:
543-551
[Medline].
|
64.
|
Starke, L.C., and
M.L. Jennings.
1993.
K-Cl cotransport in rabbit
red cells: further evidence for regulation by protein phosphatase
type 1.
Am. J. Physiol.
264:
C118-C124
[Abstract/Free Full Text].
|
65.
|
Strange, K.,
F. Emma, and
P.S. Jackson.
1996.
Cellular and molecular physiology of volume- sensitive anion channels.
Am. J. Physiol.
270:
C711-C730
[Abstract/Free Full Text].
|
66.
|
Strange, K..
1998.
Molecular identity of the outwardly rectifying,
swelling-activated anion channel: time to reevaluate pICln.
J.
Gen. Physiol.
111:
617-622
[Free Full Text].
|
67.
|
Szallasi, Z.,
C.B. Smith,
G.R. Pettit, and
P.M. Blumberg.
1994.
Differential regulation of protein kinase C isozymes by bryostatin 1 and phorbol 12-myristate 13-acetate in NIH 3T3 fibroblasts.
J.
Biol. Chem.
269:
2118-2124
[Abstract/Free Full Text].
|
68.
|
Szücs, G.,
S. Heinke,
C. De Greef,
L. Raeymaekers,
J. Eggermont,
G. Droogmans, and
B. Nilius.
1996.
The volume-activated chloride current in endothelial cells from bovine pulmonary artery is
not modulated by phosphorylation.
Pflügers Arch.
431:
540-548
[Medline].
|
69.
|
Toullec, D.,
P. Pianetti,
H. Coste,
P. Bellevergue,
T. Grand-Perret,
M. Ajakane,
V. Baudet,
P. Boissin,
E. Boursier, and
F. Loriolle.
1991.
The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C.
J. Biol. Chem.
266:
15771-15781
[Abstract/Free Full Text].
|
70.
|
Vandenberg, J.I.,
A. Yoshida,
K. Kirk, and
T. Powell.
1994.
Swelling-activated and isoprenaline-activated chloride currents in guinea
pig cardiac myocytes have distinct electrophysiology and pharmacology.
J. Gen. Physiol.
104:
997-1017
[Abstract].
|
71.
|
Villaz, M.,
J.C. Cinniger, and
W.J. Moody.
1995.
A voltage-gated
chloride channel in ascidian embryos modulated by both the cell
cycle clock and cell volume.
J. Physiol. (Lond.).
488:
689-699
[Abstract].
|
72.
|
Voets, T.,
G. Droogmans, and
B. Nilius.
1996.
Membrane currents
and the resting membrane potential in cultured bovine pulmonary artery endothelial cells.
J. Physiol. (Lond.).
497:
95-107
[Abstract].
|
73.
|
Waldegger, S.,
P. Barth,
G. Raber, and
F. Lang.
1997a.
Cloning and
characterization of a putative human serine/threonine protein
kinase transcriptionally modified during anisotonic and isotonic
alterations of cell volume.
Proc. Natl. Acad. Sci. USA.
94:
4440-4445
[Abstract/Free Full Text].
|
74.
|
Waldegger, S.,
G.L. Busch,
N.K. Kaba,
G. Zempel,
H. Ling,
A. Heidland,
D. Haussinger, and
F. Lang.
1997b.
Effect of cellular
hydration on protein metabolism.
Miner. Electrolyte Metab.
23:
201-205
[Medline].
|
75.
|
Waldegger, S., and
F. Lang.
1998.
Cell volume and gene expression.
J. Membr. Biol.
162:
95-100
[Medline].
|
76.
|
Witters, L.A..
1990.
Protein phosphorylation and dephosphorylation.
Curr. Opin. Cell Biol.
2:
212-220
[Medline].
|
77.
|
Yamazaki, J.,
D. Duan,
R. Janiak,
K. Kuenzli,
B. Horowitz, and
J.R. Hume.
1998.
Functional and molecular expression of volume-regulated chloride channels in canine vascular smooth muscle
cells.
J. Physiol. (Lond.).
507:
729-736
[Abstract/Free Full Text].
|