(Received for publication, July 9, 1996, and in revised form, November 19, 1996)
From the Laboratory of Physiology, Catholic
University of Leuven, Campus Gasthuisberg, B-3000 Leuven, Belgium
and the ¶ Laboratory of Toxicology, Catholic University of Leuven,
Van Evenstraat 4, B-3000 Leuven, Belgium
pICln is a protein that induces an
outwardly rectifying, nucleotide-sensitive chloride current
(ICln) when expressed in Xenopus oocytes, but its precise function (plasma-membrane anion channel versus cytosolic regulator of a channel) remains
controversial. We now report that a chloride current identical to
ICln is induced when Xenopus
oocytes are injected with human ClC-6 RNA. Indeed, both the
pICln and the ClC-6 induced current are outwardly
rectifying, they inactivate slowly at positive potentials and have an
anion permeability sequence NO3 > I
> Br
> Cl
> gluconate.
Cyclamate, NPPB, and extracellular cAMP block the induced currents. The
success rate of current expression is significantly increased when the
injected Xenopus oocytes are incubated at a higher
temperature (24 or 37 °C) prior to the analysis. In addition, the
ICln current was detected in 6.2% of
noninjected control Xenopus oocytes. We therefore conclude
that the ICln current in Xenopus oocytes corresponds to an endogenous conductance that can be activated by expression of structurally unrelated proteins. Furthermore, functional, biochemical, and morphological observations did not support
the notion that pICln resides in the plasma membrane either permanently or transiently after cell swelling. Thus, it is unlikely that pICln forms the channel that is responsible for the
ICln current in Xenopus
oocytes.
pICln is a 26-kDa acidic protein that is ubiquitously
expressed not only in various mammalian cell lines and tissues but also in Xenopus oocytes (1-3). Expression of mammalian
pICln in Xenopus oocytes induces an outwardly
rectifying, nucleotide-sensitive Cl current,
ICln, that slowly inactivates at positive
potentials (4). Phenotypical similarities between
ICln and the volume-activated Cl
current, ICl, swell (for a review of
ICl, swell see Ref. 5), have prompted
functional models that describe pICln either as the
volume-activated Cl
channel itself or as a regulator
thereof (2, 6-8). However, using Xenopus oocytes, we have
recently demonstrated that ICln and the
endogenous ICl, swell are two different currents that can be discriminated by biological, biophysical, and
pharmacological criteria (9).
Irrespective of the relation between pICln and
ICl, swell, one major unresolved problem is how
expression of mammalian pICln in Xenopus oocytes
generates a specific Cl current. This was originally
explained by assuming that pICln was a plasma
membrane-spanning protein that constituted the anion channel itself
(4). However, Krapivinsky et al. (2) showed that the
majority of pICln resided in the cytosol, and they
concluded that pICln was a cytosolic regulator of an
endogenous, plasma membrane-located anion channel. Recently, it has
been suggested that pICln, in spite of its cytosolic
location, can still function as a plasma membrane-located anion channel
if one assumes a bimodal distribution for pICln with a
subfraction residing in the plasma membrane (6, 7). Moreover, the ratio
between cytosolic and plasma-membrane pICln could vary,
depending on specific stimuli such as cell swelling (10).
To further characterize the ICln chloride current and to address the role of pICln, we have expressed human pICln in Xenopus oocytes. First of all, we observed that the success rate of ICln expression in Xenopus oocytes injected with pICln RNA markedly depended on the incubation temperature of the oocytes. Furthermore, the ICln current was also triggered in Xenopus oocytes injected with RNA coding for human ClC-6, a protein that is structurally unrelated to pICln. Finally, we also argue against a plasma membrane location for pICln.
A human cDNA clone for pICln
(accession number X91788[GenBank]; see Ref. 1) was PCR-mutagenized by replacing
the 5-untranslated region with an
EcoRI/HindIII/BamHI linker. This
allowed subcloning of human pICln cDNA as a
BamHI fragment in the RNA transcription vector pGEMHE (11),
yielding the pGEMHE/EHBhIClnORF vector. The human ClC-6 clone was
constructed as follows. First we amplified by reverse transcription-PCR
the 5
-end of human ClC-6 (nucleotides 202-1229 of the published open
reading frame; see Ref. 12). Reverse transcription of 1 µg of total
RNA of human K562 cells (ATCC CCL 243) was performed as described (13)
except that random primers were used instead of oligo(dT) primers. The
PCR reaction was carried out with Pfu polymerase
(Stratagene, La Jolla, CA) following the manufacturer's instructions.
The PCR fragment was then digested with BamHI (in forward
primer) and XbaI (internal site at nucleotide 792). The
remainder of the ClC-6 open reading frame (nucleotides 793-2610) and
part of the 3
-untranslated region were isolated as a
XbaI-HindIII fragment from the HA0519 clone. This
is a partial human ClC-6 cDNA clone isolated from a human myeloid
cell line by Miyajima and co-workers (EMBL/GenBankTM
accession number D28475[GenBank]). The
BamHI-XbaI PCR fragment and the
XbaI-HindIII fragment were ligated in a
pBluescript vector digested with BamHI-HindIII.
Nucleotides 1-201 of the open reading frame were then amplified by
reverse transcription-PCR from human K562 RNA and inserted upstream of
the ClC-6 sequence via a NcoI site. This created a
pBluescript vector containing the complete ClC-6 open reading frame.
The ClC-6 sequence was then subcloned in pGEMHE as
BamHI-HindIII fragment.
The pGEMHE/ClC-6 vector was cut with SphI, and the pGEMHE/EHBhIClnORF vector was cut with HindIII. The linearized DNAs were purified (QIAEX desalting and concentration protocol; Qiagen) and in vitro transcribed with T7 RNA polymerase (RiboMAX system, Promega). RNAs were extracted with phenol/chloroform and ethanol-precipitated. Stage V-VI Xenopus oocytes were isolated by partial ovariectomy and defolliculated by collagenase treatment. Between 2 and 4 h after defolliculation, oocytes were injected with 50 nl of 1-100 ng/µl human pICln or ClC-6 RNA. The oocytes were then incubated at 18 °C for 2-4 days in ND-96 solution supplemented with gentamycin sulfate (50 mg/ml). ND-96 contains 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM Hepes, pH 7.5.
Whole-cell currents from oocytes were recorded using the
two-microelectrode voltage clamp technique. Resistances of voltage and
current electrodes filled with 3 M KCl were 0.5-2 M.
Current was sampled at 500- or 2000-µs intervals and filtered at 1 or 0.1 kHz, respectively, using a quadrupole low pass Bessel filter. To
eliminate the effect of voltage drop across the bath-grounding electrode, the bath potential was actively controlled. Linear components of capacity and leak currents were not subtracted. The bath
solution was ND-96 except for the study of the anion permeability
sequence, where NaCl (96 mM) was replaced with various sodium anions (96 mM). These experiments were performed
using an agar bridge. Three voltage protocols were used during the
analysis: (i) in step protocol 1, from a holding potential of
20 mV,
oocytes were clamped for 800 ms from
100 mV to +100 mV spaced 20 mV; (ii) in step protocol 2, oocytes were held at
70 mV, and 800-ms pulses to
40, 0, or +40 mV were applied; and (iii) in the linear voltage ramp protocol, oocytes were held at
20 mV, and a linear voltage ramp from
100 mV to +100 mV (0.4 V/s) was applied. All electrophysiological experiments were performed at room temperature on
Xenopus oocytes that been permanently incubated at 18 °C
or that had been subjected to a temperature elevation (24 °C for 3 h or 37 °C for 30 min) prior to the electrophysiological
analysis.
Permeability ratios (PX/PCl)
for various anions were calculated using the formula,
PX/PCl = (Cle × exp(E × F/RT)
Clrest)/Xe, with
E being
the shift in reversal potential, Cle the extracellular
Cl
concentration in ND-96, Xe the
extracellular anion concentration in anion-substituted ND-96, and
Clrest the remaining Cl
concentration in the
anion-substituted media. Numerical data are represented as mean ± S.E.
Expression of human pICln in Xenopus oocytes was
verified by Western blot analysis using a polyclonal
anti-pICln antiserum (1). Noninjected and pICln
RNA-injected oocytes were lysed in a hypotonic buffer containing 25 mM Tris-HCl, pH 7.5, 20 mM NaCl, 2.5 mM EGTA. The lysate was centrifuged, and the supernatant was stored at 20 °C. 50 µg of Xenopus protein extract
was analyzed on Western blot as described previously (1).
Subcellular fractionation of a human endothelial cell line (EA.hy926 cell line; see Ref. 14) was performed by step centrifugation. Cultured cells were washed with phosphate-buffered saline (PBS) containing 1 mM EDTA, trypsinized, pelleted, and resuspended in 1 ml of isotonic PBS or 1 ml of hypotonic PBS (0.6 ml of PBS, 0.4 ml of H2O). After a 5-min incubation at room temperature or at 37 °C, cells were lysed by sonication. Nuclei plus mitochondria were sedimented by centrifugation for 10 min at 10000 × g. The supernatant was then fractionated into cytosolic and microsomal fractions by ultracentrifugation at 100000 × g for 30 min. The microsomal fraction was resuspended in 20 mM Tris-HCl, pH 7.4, supplemented with 300 mM sucrose.
Cytosolic (50-µg) and microsomal (200-µg) protein fractions were separated on SDS-polyacrylamide gel electrophoresis (10% polyacrylamide) and transferred to polyvinylidene difluoride membranes by semidry electroblotting. Human pICln was detected with a polyclonal anti-pICln antiserum (1:500 dilution; see Ref. 1) using the Amersham ECL method. Secondary antibodies were peroxidase-labeled swine anti-rabbit immunoglobulins (DAKO A/S) diluted 1:1000.
Confocal Immunofluorescence Study of pICln Localization in EA.hy926 CellsAnti-pICln antibodies were first affinity-purified from a polyclonal anti-pICln antiserum using a hexahistidine-tagged human pICln fusion protein (1), which was bound to a polyvinylidene difluoride microporous membrane strip (Immobilon; Millipore). EA.hy926 endothelial cells were grown on gelatin-coated chamber slides (Lab Tek; Nunc Inc.). Prior to fixation in PBS containing 3% paraformaldehyde, cells were incubated for 5 min with isotonic or hypotonic (40% reduction in tonicity) PBS. Cells were permeabilized with 0.5% Triton X-100 and blocked with 10% goat serum in PBS for 1 h. They were then incubated with the affinity-purified polyclonal anti-pICln antibodies for 36 h at 4 °C and thereafter with fluorescein isothiocyanate-conjugated anti-rabbit IgG (Sigma) at room temperature for 1 h. As a control, cells incubated only with the secondary antibodies were analyzed in parallel. Cells were imaged using a Bio-Rad MRC 1000 confocal microscope with a × 40 oil immersion objective and a Kalman filter averaging between 5 and 10 frames. Both confocal (iris confocal aperture, 2 mm), and nonconfocal images (768 × 512 lines) were stored.
Cell Volume MeasurementsEA.hy926 cells were grown on glass coverslips, and cell height was monitored as described by Van Driessche et al. (15).
It has previously been
reported that expression of mammalian pICln in
Xenopus oocytes resulted in an outwardly rectifying chloride
current (ICln) that slowly inactivated at
positive potentials and that could be blocked by extracellular
nucleotides (4, 16, 17). However, our initial attempts to functionally
express human pICln in Xenopus oocytes were
rather unsuccessful; less than 10% of Xenopus oocytes
injected with human pICln RNA displayed currents with an
ICln phenotype. The failure to functionally
express ICln could not be ascribed to the
defolliculation method, since both collagenase-defolliculated and
manually defolliculated Xenopus oocytes gave similar
negative results (data not shown). We then accidentally discovered that
functional expression in some oocyte batches was strongly promoted by
incubating the oocytes at a higher temperature (24 °C for 3 h
or 37 °C for 30 min) prior to the electrophysiological analysis at
room temperature (Fig. 1A). Fig.
1B compares the current amplitude at +100 mV of injected and
noninjected Xenopus oocytes when they were kept at 18 °C
and when the incubation temperature was raised to 24 °C for at least
3 h prior to the electrophysiological analysis at room temperature
(n = 6 for each experimental condition; all oocytes
derived from a single batch). In this experiment, only
Xenopus oocytes that had been injected with human
pICln and that had been subjected to a temperature shift
displayed ICln-type currents (see Fig.
2, A, C, and E, for a
characterization of the current). From then on, we routinely included a
minimum incubation of 3 h at 24 °C in our experimental
protocol.
The absence of ICln-type currents in injected Xenopus oocytes permanently incubated at 18 °C could not be explained by deficient translation of human pICln RNA. Western blot analysis of oocyte extracts confirmed the presence of human pICln protein in Xenopus oocytes that had been injected with human pICln RNA and that had been kept at 18 °C (see Fig. 1C). The overall quality of the Xenopus oocytes within one batch and among different batches was always checked in parallel experiments in which we injected Xenopus oocytes with RNA coding for a Kv1.1 voltage-gated potassium channel (RCK1) (18). Typical K+ currents were observed in all of the injected oocytes independent of the incubation temperature (data not shown).
Human pICln and ClC-6 Induce an Identical ClThe
kinetics, the anion permeability, and the pharmacology of
ICln currents induced by heterologous expression
of mammalian pICln in Xenopus oocytes have been
previously characterized (4, 9). ICln features
are identical in manually defolliculated (4) and
collagenase-defolliculated (9) Xenopus oocytes, and they are
summarized in Fig. 2, A, C, and E.
Briefly, ICln is an outwardly rectifying current
that slowly inactivates at positive membrane potentials (at least +60
mV). It is an anion-selective current with a permeability sequence
NO3 > I
> Br
> Cl
> gluconate. The permeability
ratios PX/PCl calculated from
shifts in reversal potential in anion-substituted media are as follows
(n = 5): 1.35 ± 0.04 (NO3
), 1.19 ± 0.02 (I
), 1.07 ± 0.02 (Br
), and 0.64 ± 0.05 (gluconate). Cyclamate acts as a channel blocker. ICln is blocked by NPPB (83 ± 6% block
with 100 µM at +80 mV; n = 5) and
extracellular cAMP. The cAMP block is clearly
voltage-dependent, since it only affects the outward
current: 5 mM cAMP blocks 42 ± 7% of the current at
+80 mV versus 3.1 ± 4.3% at
80 mV
(n = 5).
ClC-6 is a recently described member of the ClC chloride channel family
(12). The functional characteristics and the physiological role of
ClC-6 are still unknown, since functional expression of ClC-6 in
Xenopus oocytes was reported to be negative (12). When we
injected Xenopus oocytes with human ClC-6 RNA, we initially also obtained negative results. However, when we preincubated the
Xenopus oocytes at a higher temperature prior to the
analysis, we observed an outwardly rectifying current that inactivated
slowly at positive potentials (at least +60 mV; see Fig. 2,
B, D, and F, for a description of the
current). For example, in one batch of Xenopus oocytes, all
oocytes (either injected with ClC-6 RNA or H2O-injected;
n 10 for each condition) remained negative when they
were continuously kept at 18 °C. In contrast, 10 of 14 oocytes
injected with ClC-6 RNA acquired an ICln
phenotype after a temperature shock (either >3 h at 24 °C or 30 min
at 37 °C; data not shown). In this experiment, 1 of 14 H2O-injected control Xenopus oocytes also became
positive after temperature elevation.
In view of the similarities between the ClC-6-induced current and
ICln, we then went on to compare in greater
detail these two currents in Xenopus oocytes (Fig. 2). The
ClC-6-induced currents were outwardly-rectifying, and they inactivated
slowly at positive potentials. We quantified the rectification by
calculating the ratio of the current amplitude at +55 mV to that at
95 mV. These potentials were chosen, since they are approximately
equidistant from the Cl
equilibrium potential in
Xenopus oocytes. In ND-96 the rectification score
(I+55mV/I
95mV) of the
ClC-6-induced current was 6.9 ± 1.5 (n = 20),
which is comparable with the 6.2 ± 0.8 score of the
pICln-induced current. The ClC-6-induced currents reversed
between
25 and
35 mV in ND-96, and they depended on the presence of
extracellular anions, since substitution of extracellular NaCl with
various sodium anion solutions changed the current amplitude and
shifted the reversal potential of the induced current. Based on shifts
in the reversal potential we obtained the following permeability
sequence: NO3
> I
> Br
> Cl
> gluconate.
PX/PCl ratios calculated from
shifts in reversal potential were as follows (n = 5):
1.37 ± 0.04 (NO3
), 1.24 ± 0.03 (I
), 1.12 ± 0.02 (Br
), and
0.46 ± 0.03 (gluconate). The ClC-6-induced current was blocked by
cyclamate. Furthermore, NPPB blocked the ClC-6-induced current to a
similar degree as the pICln-induced current (81 ± 5%
block with 100 µM at +80 mV; n = 5).
Extracellular cAMP only inhibited the outward current (45 ± 8%
block with 5 mM cAMP at +80 mV versus 3.4 ± 1.8% block at
80 mV; n = 5). Thus, expression of
two structurally nonrelated proteins in Xenopus oocytes
induced a chloride current with identical biophysical and
pharmacological characteristics.
A possible explanation
for the observation that pICln and ClC-6 induce an
identical Cl current in Xenopus oocytes is
that these proteins activate, directly or indirectly, a
Cl
conductance that is endogenously present in
Xenopus oocytes (see "Discussion"). One observation in
favor of this interpretation is that an
ICln-type current is occasionally observed in
noninjected or H2O-injected Xenopus oocytes. In
a survey of 81 noninjected or H2O-injected
Xenopus oocytes, we observed in 5 oocytes (6.2%) an
ICln phenotype (outward rectification; slow
inactivation at potentials of at least +60 mV; reversal potential about
30 mV). Similarly, Paulmichl et al. (4) reported that 36 of 943 H2O-injected oocytes (3.8%) displayed an
ICln phenotype.
Expression of mutated pICln proteins
in Xenopus oocytes suggested a close link between
pICln structure and ICln phenotype, since mutations in a putatively extracellular, glycine-rich region led
to mutant currents that could no longer be blocked by extracellular nucleotides such as cAMP (4). However, these mutations also changed the
kinetics and the Ca2+ dependence: in contrast to the wild
type ICln, the mutant current activated slowly
at positive potentials, and reducing extracellular Ca2+
decreased its amplitude. It was therefore concluded that
pICln was a plasma membrane-spanning protein with an
extracellular nucleotide binding site and extracellular
Ca2+ binding sites. However, this interpretation should be
treated cautiously, since the mutant phenotype (slow activation,
dependence on extracellular Ca2+, tail currents) resembles
a conductance that is endogenously present in Xenopus
oocytes (e.g. the steady state current in Ref. 19). We
demonstrated this by eliciting the "mutant phenotype" in
noninjected Xenopus oocytes using the same test protocols as Paulmichl et al. (4). All Xenopus oocytes
possessed a current that slowly activated during an 800-ms voltage step
to +40 mV from a holding potential of 70 mV (Fig. 3).
The time constant of activation at +40 mV was 105 ± 17 ms
(n = 15), which is identical to the time constant
reported by Paulmichl et al. (4) (100 ± 25 ms). The
amplitude of this current at the end of an 800-ms pulse at +40 mV
varied between 0.1 and 2.1 µA (0.62 ± 0.14 µA; n = 15). This current depended on extracellular
Ca2+, since perfusion of the oocytes with ND-96 containing
1 mM EGTA reduced its amplitude to 60 ± 3%
(n = 4). Furthermore, it was not sensitive to 5 mM extracellular cAMP (n = 5). This
observation demonstrates that a current with a phenotype identical to
the mutant ICln is also present in noninjected
control Xenopus oocytes.
Evidence against a Plasma Membrane Location of pICln
Subcellular fractionation of mammalian cells
has revealed that the majority of pICln resides in the
cytosol and that a small part of pICln associates with the
microsomal fraction (2, 10). In addition, it has been reported for
NIH3T3 fibroblasts and LLCPK1 cells that a reduction of the
extracellular osmolarity induces a translocation of pICln
from the cytosolic to the microsomal fraction (10). Although we did not
think that there is a direct link between pICln and
ICl, swell (9), we were still interested in
specific conditions in which pICln could reside in the
plasma membrane. We therefore studied the subcellular distribution of
human pICln in a human endothelial cell line (EA.hy926) in which we had previously documented the expression of the
pICln protein (1). Functional studies also indicated that
EA.hy926 cells were sensitive to changes in extracellular osmolarity
(data not shown). A reduction of the extracellular tonicity with 28% activated a volume-sensitive Cl current that was similar
to ICl, swell of other endothelial cell lines
such as bovine pulmonary artery endothelial cells (CPAE; see Refs. 20
and 21) or human umbilical vein endothelial cells (22). With 140 mM Cs+ in the pipette solution and 5 mM Cs+ in the bath to block the inward
K+ rectifier, ICl, swell in
EA.hy926 cells showed outward rectification, slow inactivation at
positive potentials, and virtually no voltage dependence. Current
densities at
80 mV in isotonic and hypotonic (28% reduction)
conditions were, respectively, 2.8 ± 0.3 pA/picofarad (mean ± S.E.; n = 19) and 49.3 ± 5.2 pA/picofarad (mean ± S.E.; n = 19; p < 0.001). Using the method described by Van Driessche et al.
(15) we directly tested whether EA.hy926 cells swell when the
extracellular tonicity was lowered. A hypotonic stimulus (28 or 50%
reduction) for 5 min induced an increase in cell height to,
respectively, 138 ± 3% (mean ± S.E.; n = 36) or 163 ± 7% (mean ± S.E.; n = 29).
In view of the expression of pICln in EA.hy926 cells and in
view of their sensitivity to changes in extracellular tonicity, we
analyzed the subcellular distribution of pICln in these
cells under isotonic and hypotonic conditions. Subcellular
fractionation followed by Western blot analysis confirmed that the
majority of pICln was recovered in the cytosolic fraction
under isotonic conditions, indicating that pICln is a
soluble and cytosolic protein (Fig. 4). A minor
proportion of pICln was recovered in the microsomal fraction, indicating that the method was sufficiently sensitive to
detect pICln in the membrane fraction under isotonic
conditions. Importantly, the ratio between cytosolic and microsomal
pICln did not change when the cells were subjected to a
hypotonic stimulus (40% reduction in extracellular tonicity for 5 min;
see Fig. 4). Identical results were obtained when the hypotonic
treatment was performed at room temperature or at 37 °C.
The association with the microsomal fraction during subcellular
fractionation does not necessarily mean that pICln is a
plasma membrane protein. Indeed, the microsomal fraction is a mixed
population consisting of plasma membrane and intracellular membrane
vesicles. Moreover, the association can be merely peripheral, for
example mediated by ionic interactions between pICln and
membrane lipids or proteins. To address this problem, we studied the
localization of pICln by immunofluorescence confocal
microscopy in EA.hy926 cells. In this experiment, cells were incubated
at room temperature for 5 min either with isotonic (control cells) or
hypotonic (60% tonicity) PBS prior to fixation. Confocal sections
showed the presence of pICln throughout the cytosol (Fig.
5). Importantly, the periphery of the cell was devoid of
pICln. Furthermore, the intracellular distribution of
pICln was unaffected when the cells were subjected to a
hypotonic stimulus (Fig. 5). Confocal sections revealed an identical
pattern of pICln distribution for control cells and cells
subjected to a hypotonic challenge.
In this study we describe the induction of an identical
Cl current by expression of two structurally unrelated
proteins in Xenopus oocytes. The current induced by human
pICln and ClC-6 corresponds phenotypically to the
ICln current observed after heterologous
expression of other mammalian pICln proteins in
Xenopus oocytes (4, 16, 17). In short,
ICln is an outwardly rectifying anion current
with slow inactivation kinetics at positive potentials. Its
permeability sequence is NO3
> I
> Br
> Cl
> gluconate.
It is blocked by cyclamate, NPPB, and extracellular cAMP. In
Xenopus oocytes, the ICln current can
be clearly discriminated from the volume-activated chloride current
(9). The success rate of ICln expression was
greatly enhanced in some Xenopus oocyte batches by
incubating the Xenopus oocytes injected with
pICln or ClC-6 RNA for
3 h at 24 °C or for 30 min at
37 °C prior to the electrophysiological analysis. Brandt et
al. (12) mention that they incubated ClC-6 injected
Xenopus oocytes at 18 °C for 2 or 3 days before current
measurements. The absence of a temperature elevation may very well
explain why they did not observe a chloride current in ClC-6-injected
Xenopus oocytes. The temperature dependence was specific for
ICln, since functional expression of a
voltage-dependent K+ channel (RCK1) did not
require a preceding temperature shift. In principle,
temperature-dependent processes can affect channel expression by interfering with the biosynthesis (translation, membrane
insertion, and folding), the transport from rough endoplasmic reticulum
to the plasma membrane, and/or the gating mechanism. As for
pICln, we can rule out a temperature effect on translation of pICln RNAs, since we were able to detect human
pICln in extracts of injected Xenopus oocytes
that had only been incubated at 18 °C. A similar effect of
incubation temperature on channel expression in Xenopus
oocytes has been described for epithelial Na+ channels, in
which case insertion into the plasma membrane seems to be the
temperature-dependent step (23).
How can we explain the finding that pICln and ClC-6 induce
an identical Cl current when expressed in
Xenopus oocytes and that a similar current can be observed
in a small minority of control Xenopus oocytes? There are,
in principle, two alternative possibilities. (i) Both pICln
and ClC-6 are anion channels that reside in the plasma membrane and
that mediate the ICln current. However, we then
have to explain how two structurally unrelated proteins can form a
channel with identical biophysical and pharmacological characteristics.
Furthermore, this does also not account for the the occasional
appearance of ICln in control oocytes. Finally, as argued below, there is no firm experimental evidence in favor of a
plasma membrane location of pICln. (ii) The alternative
explanation is that pICln and ClC-6 activate, directly or
indirectly, an identical anion channel that is endogenously present in
Xenopus oocytes. Opening of this channel would then lead to
the ICln current. The sporadic appearance of
ICln in control Xenopus oocytes is
consistent with this explanation. Whether proteins other than
pICln or ClC-6 can induce ICln when
expressed in Xenopus oocytes, remains a possibility that
cannot be discarded at the moment. Finally, the induction of an
endogenous conductance by exogenous pICln or ClC-6 is in line with several other reports that have described the activation of
other endogenous channels after overexpression of foreign proteins in
Xenopus oocytes (24-29).
In principle, one could argue that the heterologously expressed
proteins, i.e. human pICln and ClC-6, interact
directly or indirectly with pICln that is endogenously
present in Xenopus oocytes (1, 2) and that the
Xenopus pICln forms the actual channel. This
interpretation presupposes that pICln is an intrinsic membrane protein that spans the plasma membrane. However, in our opinion there are no compelling arguments in favor of this assertion. (i) The amino acid sequence of pICln contains no
hydrophobic regions that are sufficiently long to traverse the membrane
as an -helix. A structural model has been proposed in which the
transmembrane part of pICln consists of an amphipathic
-sheet (4). However, no experimental evidence in favor of this model
has been presented. (ii) The changes in current phenotype (loss of
block by extracellular nucleotides, sensitivity to extracellular
Ca2+) observed after expression of a mutant
pICln do not necessarily mean that pICln spans
the plasma membrane. As demonstrated, an identical phenotype was
present in noninjected oocytes. (iii) The association of small amounts
of pICln with the membrane fraction after cell
fractionation does not necessarily imply that it is a transmembrane
protein inserted into the plasma membrane. Alternative explanations are
that pICln is a peripheral membrane protein and/or that it
associates with intracellular membrane structures (endoplasmic reticulum, Golgi, endosomes, etc.) rather than with the plasma membrane. (iv) The preferential association with the soluble fraction (our data and Ref. 2) as well as the confocal immunofluorescence data
point to a cytosolic location. (v) In contrast to Paulmichl et
al. (10) we did not observe a shift of pICln to the
plasma membrane upon reducing extracellular osmolarity. (vi)
Krapivinsky et al. (2) have examined the distribution of
endogenous pICln in Xenopus oocytes, and they
were unable to identify pICln in oocyte microsomes. We
therefore conclude that pICln is not a plasma membrane-located channel protein and, consequently, that the
ICln current is not carried by the
pICln protein. As to ClC-6, it has formally not yet been
proven that it resides in the plasma membrane. However, its structural
relationship to well documented plasma membrane Cl
channels such as ClC-0 and ClC-1 and the presence of several hydrophobic segments in the hydropathy analysis strongly suggest that
ClC-6 is a membrane protein (12).
Our interpretation of the present data implies a molecular pathway that links a cytosolic protein, pICln, with a plasma membrane anion channel. Krapivinsky et al. (2) have shown that pICln forms oligomeric complexes with several cytosolic proteins, one of which has been identified as actin. In addition, a small amount of pICln was found to be associated with the membrane cytoskeleton (2). These observations are consistent with pICln forming part of a protein-protein interaction cascade that may be responsible for the activation of a plasma membrane anion channel. Clearly, more experimental work is required to unravel this pathway. Similarly, it is not known how ClC-6 activates the endogenous channel. Since ClC-6 most likely resides in the membrane (see above), it may interact with the anion channel either directly or indirectly via one or more intervening proteins.
At present we do not know whether the activation of
ICln is related to the proper physiological
function of pICln and of ClC-6 or whether it represents a
mere side effect of expressing exogenous proteins in Xenopus
oocytes. Yet, the present data directly impinge on the functional
models for pICln and its proposed physiological role as a
volume-regulated anion channel or as a regulator of ICl, swell (2, 6-8). Collectively, the lack of
experimental evidence that pICln is a plasma membrane anion
channel and our previous observation that ICln
and ICl, swell are two different currents (9)
argue against a role for pICln as a volume-regulated anion
channel or as a regulator thereof. As to the physiological role of
ClC-6, there is as yet no formal evidence that it is a Cl
channel, since expression studies have either yielded negative results
(12) or the ICln phenotype (this study). The
structural relationship of ClC-6 with proven Cl
channels
such as ClC-0, ClC-1, ClC-2, and ClC-5 is compatible with a role in
Cl
or anion transport, but further experiments are
required to clarify this issue.
Finally, although Xenopus oocytes have proven to be a
reliable and powerful tool to analyze ion channels, they may contain specific pitfalls when used to identify or characterize exogenous Cl currents. First of all, at least four distinct
endogenous Cl
currents have been described in
Xenopus oocytes: a Ca2+-activated
Cl
current (30), a hyperpolarization-activated
Cl
current (31), a volume-activated Cl
current (9, 32, 33), and the ICln current.
Moreover, two of them can be activated by the expression of exogenous
proteins. Expression of small integral membrane proteins such as
phospholemman, MAT-8, IsK, SYN-C, and NB activates the
hyperpolarization-activated Cl
current (24-28), whereas
ICln is induced by pICln and
ClC-6.
We thank L. Missiaen (Laboratory of Physiology, Catholic University of Leuven) for help with confocal microscopy, G. Szücs for characterizing ICl, swell in EA.hy926 cells, and W. Van Driessche (Laboratory of Physiology, Catholic University of Leuven) for performing volume measurements on EA.hy926 cells. We thank N. Nomura (Kazusa DNA Research Institute, Japan) for providing us with the HA0519 clone. The EA.hy926 cell line was given to us by C.-S. J. Edgell (University of North Carolina). We acknowledge the technical expertise of D. Hermans, J. Prenen, A. Florizoone, and M. Crabbé.