Max-Planck-Institut für experimentelle Medizin, D-37075 Göttingen, Germany
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Release from arrest in G2 phase of the cell cycle causes profound changes in rat ether-à-go-go (r-eag) K+ channels heterologously expressed in Xenopus oocytes. The most evident consequence of the onset of maturation is the appearance of rectification in the r-eag current. The trigger for these changes is located downstream of the activation of mitosis-promoting factor (MPF). We demonstrate here that the rectification is due to a voltage-dependent block by intracellular Na+ ions. Manipulation of the intracellular Na+ concentration indicates that the site of Na+ block is located ~45% into the electrical distance of the pore and is only present in oocytes undergoing maturation. Since the currents through excised patches from immature oocytes exhibited a fast rundown, we studied CHO-K1 cells permanently transfected with r-eag. These cells displayed currents with a variable degree of block by Na+ and variable permeability to Cs+. Partial synchronization of the cultures in G0/G1 or M phases of the cell cycle greatly reduced the variability. The combined data obtained from mammalian cells and oocytes strongly suggest that the permeability properties of r-eag K+ channels are modulated during cell cycle-related processes.
Key words: cell cycle; CHO cells; electrophysiology; potassium channels; Xenopus laevis ![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE Xenopus oocyte maturation has proven useful
for studies of development and cell cycle. Although
some key processes have been identified (Cork and
Robinson, 1994; Murakami and Vande Woude, 1997), the
exact biochemical pathway leading to maturation remains unknown. The physiological trigger of maturation in amphibians is progesterone, which is secreted by follicular
cells (Fortune et al., 1975
; Schuetz and Glad, 1985
). It acts
on surface receptors and induces the activation of pre-MPF molecules already present in the oocyte. This is
thought to happen through a process that includes the inhibition of protein kinase A and the activation of Mos protein kinase (Sagata, 1997
). This initial activation is amplified by the synthesis of new cyclin B (the regulatory subunit of mitosis-promoting factor [MPF]1) and positive
feedback of MPF on pre-MPF activation. Injection of exogenous MPF is able to induce oocyte maturation by itself. Hence, MPF is regarded as a key molecule in the process
(Nurse, 1994
).
We have recently shown that rat ether-à-go-go (r-eag)
(Ludwig et al., 1994) K+ channels heterologously expressed in Xenopus oocytes undergo profound modification during maturation of the host cell (Brüggemann et al.,
1997
). The modification consists of a voltage-dependent block that causes rectification of the ionic currents. We
also demonstrated that the modification of the channel occurs downstream from the activation of MPF, indicating
that it is a cell cycle-related process and is not due to one
of the collateral pathways that converge on MPF activation. In this paper, we investigate the mechanism underlying the maturation-dependent rectification.
Block of K+ channels by internal Na+ has been known,
particularly in delayed rectifiers from squid axon and in
Ca2+-activated K+ channels (Bezanilla and Armstrong,
1972; French and Wells, 1977
; Marty, 1983
; Yellen, 1984a
;
Howe et al., 1992
). Some cloned channels, such as Kv2.1
(Lopatin and Nichols, 1994
), have been shown to be
blocked by Na+ in a voltage-dependent manner. Na+
seems to act as an open channel blocker since it decreases
the conductance of the channel but not its open probability. Moreover, a K+ current present in human neuroblastoma cells, which shares many properties with eag currents (Meyer and Heinemann, 1998
), had previously been
shown to be blocked by intracellular Na+ (Johansson et
al., 1996
). The intracellular Na+ concentration changes in
response to many cellular events, usually related to the activation of either Na+/Ca2+ or Na+/H+ antiporters (see for
example Harootunian et al., 1989
; Borin and Siffert, 1991;
Johnson et al., 1991
). In this paper, we show that Na+ is
able to block the r-eag channel only after MPF activation in oocytes, implying profound modifications in the ion-conduction pathway during cell cycle-related processes.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
cRNA Preparation
All constructs used in this work are based on the pCDNA3-r-eag DNA,
which was a generous gift from Prof. O. Pongs (Zentrum für Neurobiologie, Hamburg, Germany). It was used to subclone the r-eag cDNA into
the high-efficiency expression vector pSGEM (M. Hollmann, Max-Planck-Institut). RNA encoding r-eag was prepared using a template with
a T7 promoter following a standard protocol (Krieg and Melton, 1987),
and injection into Xenopus oocytes was performed as described previously (Stühmer, 1992
). Only oocytes considered to be in stages V and VI
were injected. The oocytes were incubated in Barth's medium (including
0.33 mM Ca(NO3)2 and 0.41 mM CaCl2) at 18°C.
Cell Culture and Construction of Stably Transfected Cell Lines
CHO-K1 cells (CCL61; American Type Culture Collection, Rockville,
MD) were maintained in 90% Ham's F-12 nutrient mixture and 10% FCS
in a humidified atmosphere at 37°C. Medium was replaced twice a week,
and cells were passed every 7-12 d. Transfection was performed using the
Ca2+ phosphate method of Chen and Okayama (1987). Stable cell lines were selected based on their resistance to G418 encoded in the pCDNA3
vector.
Cell populations in a particular phase of the cell cycle were enriched using arresting media. To inhibit proliferation and increase the percentage
of cells in G0/G1 phase, the serum concentration was reduced to 0.5% for
48 h before the measurement. To obtain an enrichment in M phase cells,
500 nM taxol (Schiff et al., 1979) was added to the medium for 18-24 h,
and only round cells were used for measurements. In a few experiments,
the cells were stained in vivo after 18 h in taxol with 5 µg/ml Hoechst
33342 (HO) for ~2 min. We then recorded from cells displaying mitotic
figures of their chromosomes under UV illumination.
Electrophysiology
Two-microelectrode recordings were performed 1-7 d after cRNA injection (using a Turbo TEC-10CD amplifier; npi Electronics, Tamm, Germany). The intracellular electrodes had resistances of 0.6-1 M when
filled with 2 M KCl. All the records presented were leak subtracted online
using a P/n protocol. Recordings were performed in an external solution
(normal frog Ringer [NFR]) containing (mM) 115 NaCl, 2.5 KCl, 1.8 CaCl2, 10 Hepes/NaOH, pH 7.2. Recombinant human MPF (0.75 U/µl)
was purchased from Promega (Madison, WI). It was injected (~50 nl) into
oocytes during or immediately before electrophysiological measurements.
For some experiments, we took advantage of spontaneous maturation, a
frequent phenomenon in mammalian oocytes that also occurs in amphibian oocytes after detachment of the follicular cell layer (Zelarayan et al.,
1996
).
Patch-clamp (Hamill et al., 1981) experiments in macropatches from
oocytes were carried out using an EPC-9 amplifier (HEKA Electronics,
Lambrecht, Germany). Pipettes were prepared from aluminum-silicate
glass with resistances of 0.7-1.5 M
when filled with the extracellular solution NFR. The bath solution contained (mM) 100 KCl, 10 EGTA, 1 1,2-bis(2-aminophenoxy)ethaneN,N,N'N'-tetraacetic acid (BAPTA), 10 Hepes/
KOH, pH 7.2. NaCl was added to the intracellular solution at the indicated concentrations without changing the KCl concentration to simplify
the analysis. Equivalent increase in ionic strength and osmotic properties
of the solution with KCl did not noticeably alter the current properties.
Single channel measurements were performed in the inside-out configuration of the patch clamp technique, with NFR as pipette solution and an internal solution containing (mM) 100 KCl, 10 EGTA, 1 BAPTA, 10 Hepes/KOH, pH 7.2.
All oocyte experiments were repeated at least three times using oocytes from at least two different donors. The figures show representative experiments with internal controls performed in the same oocyte or patch.
For whole-cell recordings in CHO-K1 cells, pipettes pulled from Kimax
glass (2-3 M) were filled with a solution containing (mM) 140 KCl or
CsCl, 10 BAPTA, 10 Hepes, pH 7.2, with the corresponding hydroxide
(Cs or K). The extracellular medium contained (mM) 140 NaCl, 2.8 KCl, 2 CaCl2, 2 MgCl2, 10 glucose, 10 Hepes/NaOH, pH 7.2. To measure reversal
potentials, the cells were bathed in a solution containing (mM) 140 KCl, 2 CaCl2, 10 Hepes/NaOH, pH 7.2, and the pipette solution contained (mM)
140 CsCl, 10 BAPTA, 10 Hepes, pH 7.2 (CsOH). Unless otherwise stated,
the holding potential for all experiments was
80 mV. Electrophysiological experiments were carried out at a room temperature of 20-22°C.
Acquisition and data analysis was achieved using the Pulse-PulseFit software package (HEKA Electronics). For single-channel measurements, the analysis was performed using TAC software (Bruxton Corp., Seattle, WA). The sampling rate was set in order to obtain five times oversampling at any particular filter setting.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Rectification of r-eag Current in Mature Oocytes Is Compatible with Block by an Intracellular Factor
Fig. 1 A shows a comparison of the current-voltage (I-V)
relationships of an oocyte before and after maturation by
progesterone (see Brüggemann et al., 1997). Before MPF
activation by progesterone, the I-V relationship is fairly
linear at depolarized potentials (open circles). The situation is quite different after MPF activation (filled circles);
the two curves overlap for small depolarizations, but diverge at larger depolarizations (starting at about +10 mV in this particular oocyte). This phenomenon is termed
"rectification" (Hille, 1992
) since the membrane conductance decreases with increasing potentials.
|
Rectification can be caused by at least two different processes (Hille, 1992). In some channels, it is caused by a
block by mobile factors, such as Mg2+ or polyamines. In
others, the rectification is directly caused by voltage-dependent conformational changes of the channel. A member of the eag family, human eag-related gene [HERG],
belongs to the second group (Schönherr and Heinemann,
1996
; Smith et al., 1996
; Spector et al., 1996
). HERG channels undergo inactivation with such fast kinetics that the
activation of the channel is not detectable. Currents can
only be observed upon repolarization while they deactivate. This prompted us to test whether a related mechanism could be responsible for the rectification of r-eag currents after MPF activation. Although no inactivation was
detected in r-eag currents from immature oocytes, this
property could have been conferred to the channels by
MPF activation.
We attempted to evoke inactivation by applying long-lasting depolarizing pulses. Fig. 1 B shows that there is no
decrease in current amplitude during long depolarizations
(up to 20 s) at voltages at which the current shows substantial rectification (+80 mV), indicating that the reduction
on current amplitude reaches its equilibrium before activation is completed. Cumulative inactivation during repetitive stimulation (Baukrowitz and Yellen, 1995) was also
tested. Fig. 1 C shows that the current amplitude did not decrease during trains of stimuli at different frequencies.
(2 and 4 Hz in this case; the strong voltage-dependence of
activation [Terlau et al., 1996
] precludes the use of higher
frequencies.) Moreover, at a given frequency there was no
detectable difference between depolarizations to potentials where there is rectification (+60 mV) or to potentials
where the rectification is minimal, if at all present (0 mV).
Our observations indicate that the inactivation process, if it exists, would have very fast kinetics, and thus we favor the hypothesis of a blocking agent acting from the inside (see below).
Block by Internal Na+ Ions Can Account for the Rectification
An alternate mechanism to account for rectification is block by diffusible factors. This possibility is supported by the fact that cell-attached patches from mature oocytes exhibit rectification properties similar to the ones observed with two-electrode voltage clamp in whole oocytes. Furthermore, current amplitude increases and rectification disappears upon the excision of the patch (data not shown), as would be expected of a block by a diffusible, intracellular factor.
In a previous report (Brüggemann et al., 1997), we had
tested and rejected two potential candidates that could
cause rectification: Ca2+, a potent blocker from the cytoplasmic side (Stansfeld et al., 1996
), and spermidine. Intracellular Ca2+ did not show a voltage-dependent block, and
spermidine failed to block the channel even at high concentrations.
High extracellular K+ or Cs+ concentrations reversibly
remove rectification (not shown). This observation is compatible with either relief of C-type inactivation (López-Barneo et al., 1993; Baukrowitz and Yellen, 1995
) or block
by intracellular Na+ (Yellen, 1984b
). We have already
shown (Fig. 1) that inactivation is unlikely to be responsible for the rectification. Therefore, we tested intracellular
Na+ as a voltage-dependent blocker. Inside-out patches
taken from mature oocytes expressing r-eag recover their
rectification properties in the presence of millimolar concentrations of Na+. Fig. 2, A and B, shows raw current
traces, and Fig. 2 C shows the corresponding I-V relationship of r-eag currents exposed to either 0 or 20 mM NaCl
from the intracellular side. This current-voltage relationship closely resembles those recorded from mature whole oocytes (see Fig. 1 A). Na+ ions are known to induce rectification in various K+ channels (Bezanilla and Armstrong,
1972
; French and Wells, 1977
; Marty, 1983
; Yellen, 1984a
;
Howe et al., 1992
; Lopatin and Nichols, 1994
). Mg2+, commonly a blocker of K+ channels, also causes block of r-eag
currents, but this effect is only slightly voltage dependent
(data not shown). Since the phenomenon described here is
similar to block by Na+, we decided to further characterize
the effects of intracellular Na+.
|
The degree of rectification depends on the Na+ concentration (Fig. 3 A), and the concentration required for the
50% inhibition (IC50) is strongly voltage dependent (Fig. 3
B), ranging from 10 mM at +100 mV to more than 20 mM
at +20 mV. This data can be described according to
Woodhull (1973) by the following equation:
![]() |
(1) |
|
where KNa(V) represents the IC50 for Na+ at a given voltage, KNa(0) is the IC50 at 0 mV, Z is the effective valence of
the ion, and F, R, and T have their usual meaning. The effective valence of the blocking ion is Z = z, where z is the
true valence and
stands for the fraction of the electrical
distance that the blocking ion has to enter to reach its
binding site. A fit of the experimental IC50 to Eq. 1 (Fig. 3
C) gave a value of 0.446 for
, indicating that the Na+ ion
has to enter ~45% of the electrical distance within the
channel to reach the blocking site.
Single-channel records from inside-out patches show that Na+ induces an apparent decrease of the single-channel conductance from 1.5 to 0.9 pA at +80 mV, while at 0 mV the conductance is unaffected (Fig. 4). This behavior is typical for an open channel blocker, which possibly interferes with the K+ flow by binding to a site within the conducting pathway itself.
|
The Sensitivity to Na+ Ions Is Conferred by MPF Activation
Na+ concentration is expected to rise inside the oocyte
during maturation, due to the activity of the Na+/H+ antiporters (Rezai et al., 1994). The rectification of the r-eag current could simply be a consequence of this rise in Na+
concentration. To test this hypothesis, we examined the effect of a rise in Na+ concentration in immature oocytes.
The Na+ increase was achieved by injection of ~50 nl of
2 M Na+ into an oocyte (final concentration > 20 mM)
while recording. Fig. 5, A-C, shows that the current amplitude is decreased after Na+ increase. Once the amplitude
differences were normalized, the shape of the I-V relationship remained unchanged (Fig. 5 C, inset). Similar results
were obtained increasing the Na+ concentration by incubation of the oocytes in ouabain (0.5 or 1.3 mM for 180 min; data not shown).
|
To determine the possible correlation between the intracellular Na+ concentration in a particular oocyte and
the degree of rectification of r-eag currents, we coinjected
cRNAs coding for Na+ channel type II from rat brain
(Noda et al., 1986) and r-eag. The reversal potential for
the Na+ current was then used to estimate the internal
Na+ concentration in the oocyte. The recordings were performed with two-electrode voltage clamp in standard extracellular solution (NFR) containing 1 mM MgCl2 and
stimulating from a holding potential of
120 mV. Both
conditions were designed to slow down the activation of
r-eag current (Terlau et al., 1996
) and therefore minimize the interference of outward K+ currents with Na+ currents. Subsequently, the outward current was measured
from a holding potential of
80 mV in the absence of extracellular Mg2+. In these experiments (Fig. 5 D), the intracellular Na+ concentration was between 5 and 35 mM. We
did not observe correlation with the degree of rectification
assessed as ratio between the steady-state current at +100
mV and at +40 mV. This ratio is ~2 when the current does
not show rectification (see Fig. 1 A). Taken together, these
results demonstrate that an increase in Na+ concentration
by itself is not sufficient to induce rectification of r-eag
currents.
Inside-out patches from immature oocytes show a fast
and strong run-down of the current. This run-down does
not depend on diffusible factors since the current is not recovered after reinserting the membrane patch into the oocyte ("patch-cramming"; Kramer, 1990). This has precluded a detailed characterization of the behavior of the
current in these oocytes. However, in a limited number of
patches (n = 3), we were able to measure currents with
only slight rectification when exposed to 20 mM internal
Na+ (Fig. 6, A-C). In contrast, patches obtained from the
same cell, this time after injection of MPF (Fig. 6 D, filled
squares), showed a strong rectification that closely resembled the one described earlier in this paper (see Fig. 1 A).
We conclude that MPF renders the channels sensitive to
internal Na+.
|
r-eag Currents Expressed in Mammalian Cells Are Blocked by Internal Na+
We checked the ability of internal Na+ ions to block eag
currents in CHO-K1 cells stably expressing r-eag currents.
For this purpose, we measured the currents in the whole-cell configuration, in the presence of 10 mM NaCl in the
internal solution. We detected rectification in 9 out of 12 cells. In six cases, the current amplitude peaked around
+80 mV when the I-V relationship was determined between 60 and +100 mV, and in the other three the peak
current was obtained at a less positive potential (+60 mV).
The I-V relationship resembled the one obtained in oocytes, although the rectification was usually not strong
(Fig. 7).
|
This weak rectification could depend on differences between the oocyte system and mammalian cells, but most surprising were the differences within the mammalian cell line itself. A tempting hypothesis is that the differences from cell to cell are due to cells that are at different stages of the cell cycle since oocytes are arrested at G2 phase of the cell cycle and CHO-K1 cells are not. To test this hypothesis, we performed analogous experiments in cells treated with 500 nM taxol to enrich the population of cells in M phase. Only cells with spherical morphology typical of mitotic cells and/or displaying mitotic figures of chromosomes stained in vivo with HO were selected for recording. None of the cells tested (n = 8) showed rectification, supporting the hypothesis that the variability observed within cells from the same culture may be due to cell cycle-related events.
Since Na+ seems to enter deeply into the pore to block the channel, and the site appears to be only accessible under certain conditions, we addressed the possibility that changes in the conducting pathway of r-eag are occurring during cell cycle-related processes. If this was the case, some of the selectivity properties of r-eag might be altered as well. We decided to study the behavior of the current in the presence of Cs+ as the intracellular charge carrier.
r-eag Channels Are Permeable to Cs+ Ions
Drosophila eag channels are half as permeant to Cs+ as to
K+ (Brüggemann et al., 1993). This is an unexpected property for a K+ channel. In contrast, r-eag channels appeared
to be Cs+ impermeant when measured in whole oocytes
with the two-electrode voltage clamp technique (Ludwig
et al., 1994
). However, we have seen some Cs+ outward
current when Cs+ is the only available monovalent cation
in the inside of patches obtained from immature oocytes.
As previously mentioned, we have not succeeded to measure in patches from immature oocytes long enough to
completely rule out any contamination by K+ in the internal solution. Therefore we decided to characterize the Cs+
permeability in transfected CHO-K1 cells.
Most cells in the whole-cell configuration with pipettes
containing Cs+ as the sole charge carrier gave outward
currents (Fig. 8 A). In symmetrical Cs+, outward and inward tail currents are at equilibrium around 0 mV (Fig. 8
B). When exposed to different external K+ to Cs+ ratios,
the behavior of the tail currents is more complex (Fig. 8
C). Increasing Cs+ concentration results in a reduction of
the tail amplitude, which is almost abolished at 35 mM
Cs+. The amplitude, however, increases again with further
reductions in the K+ concentration (with the concomitant
increase in Cs+ concentration), indicating a close interaction between Cs+ and K+ (Fig. 8, C and D). This "anomalous mole fraction" effect is analogous to the one observed
in Ca2+ channels for Ca2+ and Na+ or for Ca2+ and Ba2+,
and also for the interaction between K+ and Tl+ in the inward rectifier of echinoderm eggs (Hille, 1992). The anomalous mole fraction indicates that there is interaction
between both ions in the conducting pathway, i.e., that
both K+ and Cs+ go through the same pore.
|
The Permeability Ratio Cs+/K+ Changes in Different Phases of the Cell Cycle
Approximately 90% of the cells tested (n > 100) showed Cs+ outward currents, but some were impermeable to Cs+, although they showed K+ inward currents, proving that r-eag channels were indeed expressed. These cell-to-cell differences could be explained in several alternative ways. We show in this paper that r-eag channels are blocked by internal Na+ once MPF is activated, and that Na+ has to enter the pore to almost one half of its electrical distance. We have also shown that most (but not all) of the transfected CHO-K1 cells show a similar behavior, and that this variability is abolished by synchronization of the cells in M phase. This indicates that the r-eag pore properties change during progression through the cell cycle.
The reversal potential measured in cells with Cs+ as internal ion and K+ in the outside was also strongly variable.
Fig. 9 shows some examples of the currents elicited by depolarizing ramps from 100 to +75 mV lasting 1 s. The
currents have been normalized to the maximal amplitude,
and evidently the reversal potential varies strongly from
cell to cell, although other features of the current remain
unaltered. We determined the value of the reversal potential using either voltage ramps, as in the experiments depicted in Fig. 9, or discrete depolarizations lasting 500 ms
to potentials between
60 and +75 mV in 15-mV increments. Both methods gave essentially identical results.
The reversal potential in these cells (Table I) ranged between
28 and +60 mV, and the coefficient of variation
was 0.9. Thus, the Cs+/K+ permeability ratio varied between 0.09 and 2.76 (mean 0.38). In these experiments, we
did not find any correlation between the reversal potential
and cell size, current density, or series resistance.
|
|
To test the possibility that the Cs+/K+ permeability ratio
changes during progression through the cell cycle, we measured the reversal potential of the current under the same
conditions in cultures enriched in G0/G1-phase cells (by
withdrawal of growth factors, i.e., 0.5% FCS in the medium). These cells showed much less variation, ranging between 2 and +34 mV, with a coefficient of variation of
0.6 (Table I).
We also tested cultures enriched in M phase cells. After taxol treatment (18 h at 0.5 µM), cells having a spherical morphology had reversal potential in the range of +16 to +49 mV (Table I). Again, the coefficient of variation (0.3) was much smaller than the one of untreated cells. We think that the narrower interval is due to better recognition of M phase cells.
To further assure the identification of cells in M phase, we stained the DNA of a batch of cells in vivo using HO. Cells with clear mitotic figures had variations in their reversal potential within the limits of the voltage clamp errors (Erev = 37.8 ± 5 mV, n = 5, coefficient of variation 0.1).
To ensure that the level of expression of eag channels was sufficiently high to overcome any endogenous current contribution, we only used cells showing a robust K+ inward tail current. Neglecting cells with small tail currents systematically excludes cells that have very negative reversals caused by increased Cs+ permeability. Therefore, the measured variability in reversal potentials is a lower estimate of the true variance. We never detected Cs+ currents, and K+ currents never exceeded 0.1 nA in untransfected cells, neither in normal cultures (n = 20), nor in cultures containing 0.5% FCS (n = 3) or treated with 500 nM taxol (n = 5).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Modulation of ion channels during cell cycle has been reported (see for example Day et al., 1993; Moody, 1995
;
Kuga et al., 1996
), although the molecular mechanisms underlying such modulation remain unclear. MPF is the
ubiquitous molecule controlling the G2-to-M transition of
the cell cycle, both during meiotic and mitotic divisions
(Nurse, 1994
). Furthermore, somatic cells and oocytes
share many common effects after MPF activation, although usually at different stages of the cell cycle. r-eag
channels heterologously expressed in Xenopus oocytes are
strongly modulated during MPF-dependent maturation.
We studied the modulation of r-eag by MPF as a tool to
characterize cell cycle-related changes in conductance
properties of an ion channel. This modulation might give
further insight into r-eag function in native cells.
As a first conceivable source of rectification, inactivation
was excluded since MPF is unable to induce inactivation in
r-eag channels. The next possible cause of rectification,
block by intracellular agents such as Mg2+ and Na+, was
examined. This was attempted despite technical problems caused by run-down of the currents in oocytes and their
fragility after MPF injection. Although Mg2+ and Ca2+ are
able to block the channel, the block is not voltage dependent. Na+ acts as an open channel blocker of r-eag channels, analogous to other delayed rectifier K+ channels.
During maturation, there is an activation of the Na+/H+
antiporter (Rezai et al., 1994), and consequently the intracellular Na+ concentration will increase. Rectification in
r-eag could therefore be only due to a rise in intracellular
Na+ concentration. However, Na+ block alone does not
explain the rectification since (a) the injection of Na+ does
not induce rectification in immature oocytes; (b) the rise in
Na+ concentration caused by ouabain incubation does not
induce rectification in immature cells, nor does it increase
the rectification present in spontaneously mature cells; (c)
there is no correlation between the Na+ content of an oocyte and the degree of rectification of the current; and (d)
the increase in Na+ concentration happens before MPF
activation, in contrast to the induction of rectification,
which requires active MPF.
From the voltage dependence of the block, we found
that Na+ ions cross 45% of the electrical distance within
the channel. Since no block is observed in immature cells,
either Na+ ions are unable to reach the blocking site, or
they do not block because they can permeate the channel.
Both cases would implicate a change in pore properties of
the channel during maturation. This was tested using
Cs+usually a blocker of K+-channels
as the intracellular permeant ion in mammalian cells permanently transfected with r-eag. We found that Cs+ can permeate the
channel when extracellular K+ is absent. Exposing the cells
to mixtures containing both ions caused a dramatic reduction in current. This anomalous mole fraction effect has
been explained as a competition for binding sites (Hille, 1992
) and proves that the two ions share the same conduction pathway. The presence of a Cs+ outward current allows us to define the channel as permeable to Cs+. This
property of r-eag K+ channels is reminiscent of the lack of
selectivity of Ca2+ channels in the absence of Ca2+ and has
not been described for any other K+ channel.
Strikingly, the channel is not always permeable to Cs+ since not all cells tested gave Cs+ outward currents. Neither were currents blocked in all cells by Na+, and several cells produced measurable Na+ currents (not shown). Together with the oocyte data, there seems to be a population of cells in which the r-eag channels are permeable to Cs+ and are not blocked by Na+. After progression through the cell cycle, these channels become impermeable to Cs+ and blockable by Na+. A direct comparison between the data obtained in oocytes and in CHO cells is not possible at the moment since the effects observed in oocytes correspond to the onset of the M phase, and we have only measured CHO cells in which M phase was already clearly established. At present we cannot define different permeability states at various phases of the cell cycle. We have measured permeability properties in M phase cells based on morphological features and the possibility to stain mitotic chromosomes with vital stains. All of these cells show Cs+ permeability with a ratio PCs/PK of 0.2 (as calculated from reversal potentials) and no Na+ block.
The differences between currents measured in oocytes and in mammalian cells could be explained if some factors, such as accessory subunits, are missing and/or different in one of the cell types. This would, however, produce homogeneous currents in all cells of the same type, and it cannot explain the variability observed between cells of the same type.
Assuming that the permeability properties of the r-eag channel change because of cell cycle-related events, there are two possible mechanisms to explain such modulation. First, it is possible that the channel molecule itself is physically changed (for example, by phosphorylation), or secondly, some other factor (subunit or attached protein) linked to the channel might induce the change. In the latter case, the modifying factor has to interact tightly with the channel since the Na+ block is maintained under cell-free conditions.
Our data demonstrate that one and the same channel
can carry different currents depending on the stage of the
cell cycle, indicating a novel possibility for ion channel
modulation. We have shown that the permeability properties of channels can themselves be modulated during cell
cycle-related processes, independently of changes in expression levels and kinetic properties. A similar variability
of channel properties has been recently described for cardiac Na+ channels that can permeate Ca2+ in a modulated
manner (Santana et al., 1998). Here, we have detected changes in the conducting properties of r-eag channels using a biophysical approach. The precise molecular mechanisms leading to these profound changes remain to be elucidated. However, given that these changes are modulated
by the cell cycle itself, they harbor the potential for being
relevant to cellular biology.
![]() |
Footnotes |
---|
Received for publication 11 May 1998 and in revised form 17 September 1998.
Andrea Brüggemann's present address is Hoechst Marion Roussel, DG
Cardiovascular, H 821, D-65926 Frankfurt, Germany. Tel.: 49-69-305-13547. Fax: 49-69-305-16393.
Address all correspondence to Luis A. Pardo, Max-Planck-Institut für
experimentelle Medizin, Hermann-Rein-Str. 3, D-37075 Göttingen, Germany. Tel.: 49-551-3899-643. Fax: 49-551-389-9644. E-mail: pardo{at}mail.mpiem.gwdg.de
We thank Dr. O. Pongs for the generous gift of the r-eag clone, Dr. M. Stocker for the Na+ channel cRNA, Dr. M. Hollmann for the pSGEM vector, Drs. S. Beckh and G. Busch for critical comments on the manuscript, and V. Díaz-Salamanca, S. Voigt, and B. Scheufler for expert technical assistance.
![]() |
Abbreviations used in this paper |
---|
BAPTA, 1,2-bis(2-aminophenoxy)ethaneN,N,N'N'-tetraacetic acid; HO, Hoechst 33342; IC50, concentration required for 50% inhibition; I-V, current-voltage; MPF, mitosis-promoting factor; NFR, normal frog Ringer; r-eag, rat ether-à-go-go K+ channel.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Baukrowitz, T., and G. Yellen. 1995. Modulation of K+ current by frequency and external [K+]: a tale of two inactivation mechanisms. Neuron. 15: 951-960 |
2. |
Bezanilla, F., and
C.M. Armstrong.
1972.
Negative conductance caused by entry of Na+ and Cs+ ions into the K+ channels of squid axons.
J. Gen. Physiol.
60:
588-608
|
3. |
Borin, M., and
W. Siffert.
1990.
Stimulation by thrombin increases the cytosolic
free Na+ concentration in human platelets. Studies with the novel fluorescent cytosolic Na+ indicator Na+-binding benzofuran isophthalate.
J. Biol.
Chem.
265:
19543-19550
|
4. | Brüggemann, A., L.A. Pardo, W. Stühmer, and O. Pongs. 1993. Ether-à-go-go encodes a voltage-gated channel permeable to K+ and Ca 2+ and modulated by cAMP. Nature. 365: 445-448 |
5. |
Brüggemann, A.,
W. Stühmer, and
L.A. Pardo.
1997.
Mitosis-promoting factor-mediated suppression of a cloned delayed rectifier K+ channel expressed in
Xenopus oocytes.
Proc. Natl. Acad. Sci. USA.
94:
537-542
|
6. | Chen, C., and H. Okayama. 1987. High-efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol 7: 2745-2752 |
7. | Cork, R.J., and K.R. Robinson. 1994. Second messenger signaling during hormone-induced Xenopus oocyte maturation. Zygote. 2: 289-299 |
8. | Day, M.L., S.J. Pickering, M.H. Johnson, and D.I. Cook. 1993. Cell cycle control of a large-conductance K+ channel in mouse early embryos. Nature. 365: 560-562 |
9. | Fortune, J.E., P.W. Concannon, and W. Hansel. 1975. Ovarian progesterone levels during in vitro oocyte maturation and ovulation in Xenopus laevis oocytes and eggs. Biol. Reprod. 13: 561-567 |
10. | French, R.J., and J.B. Wells. 1977. Na+ ions as blocking agents and charge carriers in the K+ channel of the squid giant axon. J. Gen. Physiol. 70: 707-724 [Abstract]. |
11. | 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 |
12. |
Harootunian, A.T.,
J.P. Kao,
B.K. Eckert, and
R.Y. Tsien.
1989.
Fluorescence
ratio imaging of cytosolic free Na+ in individual fibroblasts and lymphocytes.
J. Biol. Chem.
264:
19458-19467
|
13. | Hille, B. 1992. Ionic Channels of Excitable Membranes. Sinauer Associates, Inc., Sunderland, MA. 607 pp. |
14. | Howe, J.R., M. Baker, and J.M. Ritchie. 1992. On the block of outward K+ current in rabbit Schwann cells by internal Na+ ions. Proc. R. Soc. Lond. B Biol. Sci. 249: 309-316 |
15. | Johansson, S., A.K. Sundgren, and U. Kahl. 1996. Potential-dependent block of human delayed rectifier K+ channels by internal Na+. Am. J. Physiol. 39: C1131-C1144 . |
16. |
Johnson, E.M.,
J.M. Theler,
A.M. Capponi, and
M.B. Vallotton.
1991.
Characterization of oscillations in cytosolic free Ca2+ concentration and measurement of cytosolic Na+ concentration changes evoked by angiotensin II and
vasopressin in individual rat aortic smooth muscle cells. Use of microfluorometry and digital imaging.
J. Biol. Chem.
266:
12618-12626
|
17. | Kramer, R.H.. 1990. Patch cramming: monitoring intracellular messengers in intact cells with membrane patches containing detector ion channels. Neuron. 2: 335-341 . |
18. | Krieg, P.A., and D.A. Melton. 1987. In vitro RNA synthesis with SP6 RNA polymerase. Methods Enzymol. 155: 397-415 |
19. |
Kuga, T.,
S. Kobayashi,
Y. Hirakawa,
H. Kanaide, and
A. Takeshita.
1996.
Cell
cycle-dependent expression of L- and T-type Ca2+ currents in rat aortic
smooth muscle cells in primary culture.
Circ. Res.
79:
14-19
|
20. | Lopatin, A.N., and C.G. Nichols. 1994. Internal Na+ and Mg2+ blockade of DRK1 (Kv2.1) K+ channels expressed in Xenopus oocytes. Inward rectification of a delayed rectifier. J. Gen. Physiol. 103: 203-216 [Abstract]. |
21. | López-Barneo, J., T. Hoshi, S.H. Heinemann, and R.W. Aldrich. 1993. Effects of external cations and mutations in the pore region on C-type inactivation of Shaker K+ channels. Recept. Channels. 1: 61-71 |
22. | Ludwig, J., H. Terlau, F. Wunder, A. Brüggemann, L.A. Pardo, A. Marquardt, W. Stühmer, and O. Pongs. 1994. Functional expression of a rat homologue of the voltage gated ether a go-go K+ channel reveals differences in selectivity and activation kinetics between the Drosophila channel and its mammalian counterpart. EMBO (Eur. Mol. Biol. Organ.) J. 13: 4451-4458 [Abstract]. |
23. | Marty, A.. 1983. Blocking of large unitary Ca2+-dependent K+ currents by internal Na+ ions. Pflügers Arch. 396: 179-181 |
24. |
Meyer, R., and
S.H. Heinemann.
1998.
Characterization of an eag-like K+
channel in human neuroblastoma cells.
J. Physiol. (Lond.).
508:
49-56
|
25. | Moody, W.J.. 1995. Critical periods of early development created by the coordinate modulation of ion channel properties. Persp. Dev. Neurobiol 2: 309-315 . |
26. | Murakami, M.S., G.F. Vande, and Woude. 1997. Mechanisms of Xenopus oocyte maturation. Methods Enzymol. 283: 584-600 |
27. | Noda, M., T. Ikeda, H. Suzuki, H. Takeshima, T. Takahashi, M. Kuno, and S. Numa. 1986. Expression of functional Na+ channels from cloned cDNA. Nature. 322: 826-828 |
28. | Nurse, P.. 1994. Ordering S phase and M phase in the cell cycle. Cell 79: 547-550 |
29. |
Rezai, K.,
A. Kulisz, and
W.J. Wasserman.
1994.
Protooncogene product, c-mos
kinase, is involved in upregulating Na+/H+ antiporter in Xenopus oocytes.
Am. J. Physiol.
267:
C1717-C1722
|
30. | Sagata, N.. 1997. What does Mos do in oocytes and somatic cells? Bioessays. 19: 13-21 |
31. |
Santana, L.F.,
A.M. Gomez, and
W.J. Lederer.
1998.
Ca2+ flux through promiscuous cardiac Na+ channels![]() |
32. | Schiff, P.B., J. Fant, and S.B. Horwitz. 1979. Promotion of microtubule assembly in vitro by taxol. Nature. 277: 665-667 |
33. | Schönherr, R., and S.H. Heinemann. 1996. Molecular determinants for activation and inactivation of HERG, a human inward rectifier K+ channel. J. Physiol. (Lond.). 493: 635-642 [Abstract]. |
34. | Schuetz, A.W., and R. Glad. 1985. In vitro production of meiosis inducing substance (MIS) by isolated amphibian (Rana pipiens) follicle cells. Dev. Growth Differ. 27: 201-211 . |
35. | Smith, P.L., T. Baukrowitz, and G. Yellen. 1996. The inward rectification mechanism of the HERG cardiac K+ channel. Nature 379: 833-836 |
36. | Spector, P.S., M.E. Curran, A. Zou, M.T. Keating, and M.C. Sanguinetti. 1996. Fast inactivation causes rectification of the IKr channel. J. Gen. Physiol. 107: 611-619 [Abstract]. |
37. |
Stansfeld, C.E.,
J. Roper,
J. Ludwig,
R.M. Weseloh,
S.J. Marsh,
D.A. Brown, and
O. Pongs.
1996.
Elevation of intracellular Ca2+ by muscarinic receptor
activation induces a block of voltage-activated rat ether-à-go-go channels in
a stably transfected cell line.
Proc. Natl. Acad. Sci. USA.
93:
9910-9914
|
38. | Stühmer, W.. 1992. Electrophysiological recordings from Xenopus oocytes. Methods Enzymol. 207: 319-339 |
39. | Terlau, H., J. Ludwig, R. Steffan, O. Pongs, W. Stühmer, and S.H. Heinemann. 1996. Extracellular Mg2+ regulates activation of rat eag K+ channel. Pflügers Arch. 432: 301-312 |
40. |
Woodhull, A.M..
1973.
Ionic blockage of Na+ channels in nerve.
J. Gen. Physiol.
61:
687-708
|
41. | Yellen, G.. 1984a. Ionic permeation and blockade in Ca2+-activated K+ channels of bovine chromaffin cells. J. Gen. Physiol 84: 157-186 [Abstract]. |
42. | Yellen, G.. 1984b. Relief of Na+ block of Ca2+-activated K+ channels by external cations. J. Gen. Physiol 84: 187-199 [Abstract]. |
43. |
Zelarayan, L.,
J. Oterino, and
M.I. Buhler.
1996.
Spontaneous maturation in
Bufo arenarum oocytes![]() |