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* Dipartimento di Fisiologia e Biochimica Generali, Ia Università di Milano, 20133 Milano, Italy;
Centre for Immunology, St. Vincent’s Hospital and University of New South Wales, Sydney, NSW 2010, Australia;
Department of Medicine, and Victor Chang Cardiac Research Institute, University of New South Wales, Sydney NSW 2010, Australia; and
§ Dipartimento di Biologia Cellulare e dello Sviluppo, Università ‘La Sapienza’, 00185 Roma, Italy
1Correspondence: Dipartimento di Biologia Cellulare e dello Sviluppo, P.le Aldo Moro 5, Università ‘La Sapienza’, 00185 Roma, Italy. E-mail michele.mazzanti{at}unimi.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: single-channel currents • NCC27 • ion channel
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4)
. We recently described the molecular cloning and
characterization of a novel nuclear chloride ion channel protein,
NCC27, which localizes principally to the cell nucleus
(5)
. This protein is a member of a new class of organellar
chloride ion channel proteins, the first identified member of which was
the bovine chloride channel, p64 (6)
. Of the several
members of this family now cloned, only NCC27 and the newly described
CLIC3 (7)
are dominantly nuclear in distribution. The
localization of NCC27 to the nuclear membrane, and its presence in
virtually all cells and tissues we have so far examined, suggested to
us that this channel is highly conserved and could play an important
role in the function of all or most cells.
NCC27 differs from other putative nuclear ion channels in several ways.
It is found predominantly in the nucleoplasm (in soluble form), and
only a small proportion of the protein is inserted into the nuclear
envelope (5)
. No other ion channel proteins have been
reported to have this unusual distribution in eukaryotic cells. NCC27
is also a very small protein of 27 kDa. These factors led us to
consider the possibility that it may represent only a subunit or
regulator of an unidentified ion channel rather than a membrane
resident channel in its own right. We thought this to be unlikely based
on our earlier electrophysiological studies (5)
, and were
further reassured by the knowledge that p64, a close relative of NCC27,
has been shown to function as a chloride ion channel in lipid bilayers
(8)
. Nonetheless, we wanted to confirm that NCC27 actually
forms part or the entire chloride ion channel we described in our
earlier report.
The studies we present here were carried out in our model of Chinese
hamster ovary cells (CHO-K1) stably transfected with NCC27. This ion
channel is constitutively expressed in the nucleus and nuclear membrane
of CHO-K1 cells; as we previously reported, when CHO-K1 cells are
transfected with an NCC27 containing eukaryotic expression vector,
synthesized protein spills over into the cytoplasm. Ion channel
activity can then be detected in the plasma as well as the nuclear
membrane of the transfected cells (9)
. We first used
conventional single-channel methods to further define the
electrophysiological properties of the resulting ion channel. We were
able to undertake a detailed comparison of the properties of this
channel in its locations on the nuclear and plasma membranes, something
that has not previously been possible. We then used a technique, novel
to this field, involving the incorporation of epitope tags at both the
amino and carboxyl-terminal ends of the NCC27 protein. This then
allowed us to use a monoclonal antibody to this tag as a uniquely
specific modulator of ion channel properties. In this way we were able
to definitively associate the expressed protein with the ion channel
activity, and define the orientation of the protein in the plasma
membrane.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Eukaryotic expression of NCC27 and NCC27 tagged with the FLAG peptide
(DYKDDDDK) at its carboxyl terminus was performed as described
previously (5)
. A construct incorporating a modified FLAG
peptide (DYKDDDDN) at the amino terminus of NCC27 was directionally
cloned into pRc/CMV vector (Invitrogen, Groningen, The
Netherlands). This construct was transfected into CHO-K1 (80%
confluent) for 24 h in 35 mm2 dishes using 9
µl lipofectamine reagent (Life Technologies, Inc., Milano,
Italy) and 1 µg of DNA, as recommended by the manufacturer. Stable
transfectants were selected with G418 (Boehringer Mannheim, Mannheim,
Germany), followed by subcloning. The subclones were screened by
immunofluorescent staining using anti-FLAG M2 (M2-AB, Sigma, Milano,
Italy) monoclonal antibody (5)
.
Isolation of cell nuclei
Nuclei were isolated from CHO-K1 cells transfected with NCC27
cells by shearing 2 x 106 cells in a
homogenizer with a Teflon pestle of 0.025 µm clearance and
centrifuging the homogenate on a sucrose pad (in mM: 50 Tris HCl, 25
KCl, 5 MgCl2, 250 sucrose) at 4000 rpm in a
Labofuge M (Heraeus, Milano, Italy) at 0–4°C. This procedure results
in a pellet of pure nuclei, which were resuspended in a standard bath
solution (in mM: 120 KCl, 0.1 CaCl2, 2
MgCl2, 1.1 EGTA, 10 HEPES 5 glucose, pH 7.4) at
room temperature .
Patch-clamp recording
The patch electrodes were pulled from hard borosilicate glass on
a Brown-Flaming P-87 puller (Sutter Instruments, Novato, Calif.). The
pipettes were coated with Sylgard (Dow Corning, Seneffe, Belgium) and
fire polished to an external tip diameter of 1–1.5 µm. These
electrodes had resistances of 7–10 M
. We applied standard
cell-attached and nucleus-attached patch-clamp techniques to obtain
seals of 20–50 G in the single-channel recordings. The bath
solution for the CHO-K1 cells contained (in mM): 130 NaCl, 4.8 KCl, 1.2
MgCl2, 1 CaCl2, 10 HEPES,
12.5 glucose, 1.2 NaH2PO4.
For cell-attached and inside-out recordings, the same solution was used
in the micropipettes. For outside-out experiments, the patch electrode
solution contained (in mM): 10 NaCl, 130 KAsp, 2
MgCl2, 1.3 CaCl2, 10 HEPES,
10 EGTA. The antibody (M2-AB, Sigma) was applied at a concentration of
1.5 µg/ml in both inside-out and outside-out experiments, using a
custom-made fast perfusion system. The solution used for patch clamping
of isolated nuclei was (in mM): 120 KCl, 0.1
CaCl2, 2 MgCl2, 1.1 EGTA,
10 HEPES 5 glucose, pH 7.4. The same solution was used to fill the
electrode in nucleus-attached experiments.
Single-channel currents were recorded with an Axon 200B patch-clamp amplifier (Axon Instruments, Novato, Calif.), digitized, and stored on a VCR (Panasonic, Milano, Italy). Data were analyzed on a PC after filtering at 1000 Hz using both pClamp 7 (Axon Instruments) and custom-made programs developed by W. Gooldby, Department of Anatomy and Cell Biology, Emory University, Atlanta, Georgia.
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RESULTS |
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. We found such channel behavior in the plasma membranes of more than
25% of patches in transfected cells (n=50), but in only one
patch out of more than 30 experiments with native CHO-K1 cells. This
observation suggests that the ion channel activity detected after
transfection is related to the functional expression of NCC27 protein.
In our experimental conditions a different channel activity,
characterized by strong voltage dependence, was occasionally recorded
in both transfected and untransfected cells. This ionic pathway is
likely to be an endogenous channel and it was easily distinguished from
NCC27-associated channel activity by its kinetics and its larger
conductance.
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The measured resting membrane potential (Vm) of transfected
CHO-K1 cells was -35 ± 12.4 mV (n=12). The channel
opened outwardly at all potentials and only occasionally conducted
inward current. At very positive membrane voltages (above +40-+50 mV),
the single-channel current became nonlinear, with current amplitude
either becoming constant or in many cases actually declining with
increasing depolarization. This is seen in the single-channel
current/voltage (i/V) relationship of Fig. 1B
as well as on
averaged i/V data shown in the insert. The single-channel conductance
in the example shown (Fig. 1B
), calculated by fitting the
points in the linear part of the i/V plot, was 7.26 ± 0.64 pS and
the average conductance in five experiments was 8.05 ± 0.35 pS
(Fig. 1B
, insert). The channel open probability increased
slightly with increasing membrane potential and fell to zero at
potentials below approximately -60 mV (Fig. 1C
). Calculated
mean open and close time at 0 mV Vm was 8.1 ± 0.4 and
4.3 ± 0.2 ms. The extrapolated reversal potential was -82 ± 18.5 mV. In four experiments we recorded continuously channel
activity at +40 mV membrane potential. No significant opening rundown
was observed even after more that 20 min of recording. In all these
respects, the biophysical properties of the NCC27-associated channels
in this transfection model closely resemble both the chloride
conductance already described (5)
and that reported by
others for the closely related channel, p64 (10)
NCC27 conductance is chloride dependent
We noted a wide range of reversal potentials for this channel and
also considerable variability in single-channel conductance, which
ranged between 6.8 and 9.5 pS in different patches. We hypothesized
that this may reflect variations in intracellular chloride
concentration. To test this hypothesis, we studied NCC27-associated
channels expressed in plasma membrane in the cell-attached mode, before
and after intracellular cell perfusion with a 140 mM chloride solution
and in inside-out configuration. Figure 2A
illustrates the result of a two-electrode experiment in
which a single NCC27-associated channel was observed before (left) and
after (right) dialysis of the cell with 140 mM
Cl-. To dialyze the cell, we used a second
electrode filled with high chloride solution. The two electrodes were
simultaneously placed on the cell in cell-attached mode. After
recording of several single-channel current traces using the electrode
containing bath solution, we changed the configuration of the patch
pipette filled with 140 mM Cl- from
cell-attached to whole cell. We waited 1–2 min after patch break to
collect a single-channel opening in the new configuration. The
current-voltage (i/V) relationships are illustrated in Fig. 2B
; from these we calculated a conductance of 8.8 ±
0.5 pS before and 17.4 ± 0.6 pS after dialysis with 140 mM
chloride. Exposure to internal high chloride concentration in
two-electrode experiments not only increased the single-channel
conductance (16.3±0.85 vs. 8.6±0.7; n=3), but
also removed the nonlinear behavior described above (illustrated in
Fig. 1B
). In addition, the probability of the channel to
conduct inward current increased in high chloride, supporting the
hypothesis that there is an active role of the anion in the current
flow modulation.
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We observed the same chloride dependence of the ionic pathway in
inside-out experiments (n=6). In Fig. 2C
we show
an example of channel recording in which the ‘cytoplasmic’ side of
the inside-out patch was serially exposed to 20, 50, and 140 mM Cl at
Vp ±50 mV. This resulted in measured channel conductance increasing
from 9 ± 0.8 pS at 20 mM Cl- to 11 ±
1.3 pS at 50 mM Cl- and 17 ± 1.4 pS at 140
mM Cl- (Fig. 2D
).
NCC27-associated channels have similar properties whether expressed
on nuclear or plasma membranes
In native cells this protein is predominantly localized in the
nucleus. The ionic channel associated with NCC27 expression was
frequently detected on the nuclear envelope using patch-clamp on
isolated nuclei obtained from transfected CHO-K1 cells. The probability
to find a single channel was higher than in the plasma membrane (43%
vs. 25%; n=45). Furthermore, in the case of the nuclear
envelope as well as plasma membrane, the single-channel current was
recorded much more frequently in NCC27 protein-transfected cells
compared with channel appearance in native cell isolated nuclei (20%;
n=27). We were therefore interested to compare
single-channel properties in nuclear and plasma membranes. Figure 3
(top) depicts current recordings obtained from both cell membrane and
the envelopes of isolated nuclei from NCC27-transfected CHO-K1 cells.
Whereas active channels were much more frequently detected in the
nuclear membrane than in the plasma membrane, most of the biophysical
characteristics of the channel were very similar. In the example shown
in Fig. 3
, the open probabilities at comparable potential differences
were 0.4 ± 0.03 and 0.45 ± 0.08 for NCC27 inserted in the
plasma membrane and in the nuclear membrane, respectively. There were,
however, major differences in single-channel conductance and in the
linearity of the i/V relationship between the two preparations (Fig. 3
,
middle panels). The cell membrane channel had, on average, a
conductance of 8.5 ± 0.6 pS (n=6) and showed a
nonlinear i/V relationship at depolarized membrane potentials (Fig. 3
,
middle left). The i/V for the nuclear channel was quite different (Fig. 3
, middle right). This channel conducts both inward and outward
current, with a reversal potential around 0 mV and conductances of
16.2 ± 1.4 pS (n=5). Furthermore, single-channel
currents recorded from the nuclear envelope increased linearly with
voltage.
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Despite the differences in voltage dependence, most of the channel
parameters show similar behavior. We can already see from the foregoing
text that the open probability of the channel is the same. The bottom
of Fig. 3
shows that the calculated open and close time of the channel
at a comparable potential are also similar.
Electrophysiological characteristics of FLAG-tagged and unmodified
NCC27-associated ionic channel are very similar
These experiments were carried out using two clones in which the
transfected NCC27 protein was tagged at either the amino or carboxyl
terminus with the eight amino acid FLAG epitope. Studies with the
FLAG-tagged, NCC27-transfected CHO-K1 plasma membrane were performed at
the single-channel level in cell-attached configuration. The
electrophysiological characteristics of both FLAG-tagged and unmodified
NCC27-associated channel were very similar. This is demonstrated in
Fig. 4
, which compares recordings made from three cells transfected with
nontagged, amino-terminal-tagged, and carboxyl-terminal-tagged
NCC27. Two examples of single-channel, cell-attached current recordings
(pipette potential = 0 mV) are illustrated at the top of each
column. Channel kinetics, mean open times, and open probabilities are
very similar in the three NCC27 cell lines and characteristic of this
channel (see above). Single-channel conductances calculated from the
linear part of the i/V curves (Fig. 4
, lower panel) are also very
similar. We observed, however, a consistent difference in the voltage
at which the current changes slope at positive potential. In nontagged
channels, the point of inflection was at a membrane potential of
approximately +40 mV, compared to +20 mV for amino FLAG-tagged channel
and 0 mV for carboxyl FLAG-tagged channel. This observation suggests
that modification of the NCC27 proteins directly altered single-channel
current. The eight amino acids positioned either at the amino- or
carboxyl-terminal of NCC27 proteins are highly charged and could
interfere with the ionic flow.
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NCC27 is a transmembrane protein with the amino terminus orientated
outward
The FLAG epitope is recognized by a monoclonal antibody (anti-FLAG
M2) that identifies only the tagged proteins, as demonstrable on
Western blots (5)
. We undertook a series of inhibition
experiments using the FLAG antibody and the plasma membrane of
epitope-tagged, transfected CHO-K1 cells (Fig. 5
). We made recordings in both outside-out and inside-out configurations
from plasma membranes of CHO-K1 cells transfected with either the amino
FLAG-tagged or carboxyl FLAG-tagged NCC27. Exposure of an outside-out
patch of an amino-tagged NCC27 channel to anti-FLAG M2 antibody
resulted in complete silencing of channel activity within 5 min (Fig. 5
, left; n=7). Average time of channel blockade was 4.8 ± 1.24 min. By contrast, in six similar experiments using the
inside-out configuration (n=5), anti-FLAG M2 antibody had no
effect on channel activity during 15 min of exposure (not illustrated).
Conversely, when six similar experiments were undertaken with the
carboxyl FLAG-tagged NCC27 construct, the anti-FLAG M2 antibody blocked
channel activity in the inside-out configuration (Fig. 5
, right), but
had no effect in studies using the outside-out configuration
(n=5; not illustrated). The time to stop the single-current
flow was higher than outside-out amino FLAG experiments. On average,
the current was completely canceled in 6.8 ± 2.16 min.
Furthermore, we were unable to detect any effect on single-channel
currents in either inside-out or outside-out configurations using
either nonspecific antibodies (IgG, UBI; 
-tubulin, Sigma) or
anti-FLAG M2 antibody already saturated with the FLAG peptide
(n=3 and n=4, respectively).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Little is known about
the role of anion channels present in intracellular compartments, but
presumably they serve the same functions (4)
. Because of
its localization, NCC27 could play an important role in nuclear
physiology. Most of the protein is concentrated and soluble in
the nucleoplasm; only a small percentage is present in the nuclear
membranes in association with an ionic channel (12)
. The
possibility for the protein to become inserted in the membranes of the
envelope suggests a transient modulatory effect of NCC27. The
mechanisms promoting the protein insertion in lipid bilayers are still
unclear; the increment of intracellular protein concentration certainly
must be one of them. In an overexpression system represented by the
NCC27-transfected CHO-K1 cells, not only there is an increase of the
proteins in the nuclear envelope, but the probability of finding it in
the cytoplasm and cell membrane is also much higher (5)
.
We have previously reported the possibility that NCC27 proteins form a
chloride ion channel that localizes to the nuclear envelope. NCC27 is
present in a wide variety of cells and cell lines of human origin, the
murine cell line RAW 264.7, and untransfected (hamster) CHO-K1 cells;
it is also highly conserved across species (5)
. In the
present study we have shown that after transfection of NCC27 protein in
CHO-K1 cells, the likelihood of finding chloride channels with the
patch-clamp technique in both the cell and the nuclear membrane is
increased by many fold. This could suggest that the functional
expression of NCC27 is represented by chloride selective ion channel or
that the protein is closely related to it.
The NCC27-associated channel is sensitive to intracellular changes of
chloride concentrations. Cytosolic chloride in CHO-K1 cells is on
average quite low: ~12 mM (13)
. We observed a relatively
high variability in single-channel conductances and current reversal
potentials in our studies of transfected CHO-K1 cells consistent with a
range of intracellular chloride concentration in these cells from 4–20
mM. CHO-K1 cell population are actively dividing; not only is it
possible to recognize different cell shapes, but by measuring the
resting potential it is possible to find values ranging from -30 to
-50 mV. We believe this variability could be caused by and at the same
time could influence intracellular chloride concentrations that
correspond to the presence in culture of cells at various stages of the
cell cycle.
Examining Fig. 2D
, it is clear that the NCC27-associated
channel is very sensitive to the chloride concentration changes,
particularly at low values. Conductance and reversal potential are not
the only parameters modified by the increase of
[Cl-]in. According to
the data reported in Fig. 2
, the same single channel exposed to 130 mM
intracellular chloride increases the open time and the probability of
opening for the inward current. Even the nonlinear part of the i/V
curve present in cell-attached experiments at a membrane potential
above 40 mV is completely abolished in high chloride solutions.
The study of single-channel chloride dependence could be useful in
interpreting the data in which we compared cell membrane and nuclear
single ionic pathways (Fig. 3)
. Searching for similarities, the first
observation is the marked increase of channel appearance in both plasma
and nuclear membranes in CHO-K1-transfected cells: 25% vs. 5% and
43% vs. 25%, respectively. In the kinetics parameters, there is good
agreement in the open probability (0.4 for the cell membrane channel
and 0.45 for the nuclear envelope conductance) calculated at each
potential. Measuring the open and close time at potential in which the
single-channel current shows comparable amplitude (Fig. 3
, bottom), we
obtained analogous values. The big difference is visible in the lower
panels of Fig. 3
and concerns the voltage dependence of the current.
The i/V relationships of the nuclear envelope and cell membrane channel
show different conductances and linearity. However, if we compare
nuclear channel i/V with the current/voltage relationship obtained from
a single channel recorded in high cytoplasmic chloride solution (Fig. 2B
), we have an almost perfect match. Not only are the two
curves fully linear, but the measured channel conductances of 16.3 and
16.2 pS are almost identical. Taking this into account, we suggest that
the nuclear channels recorded in cell-free configuration share the same
high chloride ionic solution in the inner membrane side. A previous
work has already discussed that during a nuclear isolation procedure
there is a disruption of the contacts with the endoplasmic reticulum
(14)
. Since the solution in which the nuclei are isolated
contains 140 mM KCl, it is reasonable to think that there is a washout
of the contents of the nuclear cisterns. Eventually the nuclear
envelope regains integrity in vitro after repair of the
external membrane of the envelope. However, the solution inside the
nuclear cavities is the same high chloride solution used to preserve
isolated nuclei.
The mechanism by which intracellular chloride modulates channel activity is unclear, but an intriguing possibility is that it may promote aggregation of channels into multimeric units. We frequently observed multiple conductance states when the inner face of the membrane under study was exposed to a high chloride concentration. At times during these studies it became difficult to distinguish individual single-channel openings, and some of our recordings suggest that single proteins may be combining to form a larger conductance channel. As we reported previously, conductance was not the only property modified by intracellular chloride. The nonlinearity of the voltage dependence of the channel was altered, suggesting that chloride also modifies the gating properties of the channel protein. This observation and our finding that the addition of a small peptide ‘tag’ to the amino or carboxyl terminus of the NCC27 protein also alters the rectification properties of the channel both support our hypothesis that the NCC27 protein forms either all or a structural component of the ion channel.
The most direct evidence for the latter conclusion is provided by the antibody studies. The monoclonal antibody directed at the FLAG epitope tag rapidly inhibits the chloride conductance, but only in situations where it has access to the FLAG epitope (i.e., only if the tag is facing outward into the buffer containing the antibody). We conclude from this observation that NCC27 must be a transmembrane protein that directly forms part of the ion channel.
Furthermore, based on our observations of the various combinations of
amino-tagged and carboxyl-tagged proteins using inside-out and
outside-out configurations, we conclude that the amino terminus
projects ‘extracellularly’ and the carboxyl terminus is directed
inwardly, at least when NCC27 channels are expressed on the plasma
membrane. If we assume an alpha-helical structure for the NCC27 ion
channel, the only putative transmembrane segment is that lying between
about amino acids 25–46 (see ref (5)
. This would leave
only a short region of ~24 amino acids lying exterior to the membrane
and the bulk of the protein projecting intracellularly (or into the
nucleus, as the case may be). By analogy with other well-described ion
channel proteins, the small size and the presence of only a single
transmembrane domain on NCC27 suggest that it normally coassembles as
multimers to form functional ion channels. Our ability to label NCC27
with a peptide to which a monoclonal antibody is available has provided
us with a channel ‘blocker’ of far greater specificity than existing
chloride channel-blocking agents. We are unaware of previous reports of
the use of this methodology in the elucidation of transfected ion
channel behavior.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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