The Plant Journal (1998) 13(2), 167–175
The action potential in Chara: Ca2F release from internal
stores visualized by Mn2F-induced quenching of furadextran
Christoph Plieth1*, Burkhard Sattelmacher2,
Ulf-Peter Hansen3 and Gerhard Thiel4
1Department of Plant Sciences, University of Oxford,
South Parks Road, Oxford OX1 3RB, UK,
2Institut für Pflanzenernährung und Bodenkunde,
Christian-Albrechts-Universität, Olshausenstr. 40, 24098
Kiel, Germany,
3Institut für Angewandte Physik, Christian-AlbrechtsUniversität, Olshausenstr. 40, 24098 Kiel, Germany, and
4Pflanzenphysiologisches Institut, Universität Göttingen,
Untere Karspüle 2, 37073 Göttingen, Germany
Summary
The action potential (AP) in Chara is associated with a
transient elevation in the concentration of cytoplasmicfree Ca2F ([Ca2F]cyt). The quenching properties of the
fluorescent Ca2F indicator dye fura-dextran, in combination with Mn2F, was used to investigate whether this
[Ca2F]cyt transient is due to Ca2F release from internal
stores or to Ca2F influx across the plasma membrane.
Adding Mn2F to the external medium or pre-injection of
Mn2F into the vacuole caused no perceivable quenching
of the fura fluorescence, during an AP. This makes it
unlikely that Ca2F influx across the plasma membrane or
the tonoplast contributes significantly to the [Ca2F]cyt
transient in an excited cell. When cells were pre-incubated
in external solutions containing Mn2F from 25 to 30 mM
APs evoked a transient quenching of fura fluorescence in
Mn2F-free solutions. Under these conditions, the quenching must be attributed to an AP-associated release of
Mn2F from internal stores. Based on the finding that
exposing cells to millimolar concentrations of Mn2F
caused a progressive quenching of the fura fluorescence
in non-excited cells, it can be assumed that some Mn2F
enters the cells during pre-incubation and is loaded into
internal stores. During excitation, this stored Mn2F is
released together with Ca2F.
Introduction
The plasma membrane of plant cells is excitable, in a
similar manner to that of nerve- or muscle-cells in animals.
Received 28 May 1997; revised 5 September 1997; accepted 19 September 1997.
*For correspondence (e-mail
[email protected]).
© 1998 Blackwell Science Ltd
Electrical, mechanical or other stimuli evoke in plants a
transient rise in the permeability of the membrane to ions
(Andjus and Vucelic, 1990; Knight et al., 1992; Shimmen,
1996; Thiel et al., 1997). This increase in permeability
underlies the transient depolarization of the membrane
voltage known as action potential (AP) (Shimmen et al.,
1994).
In Characean algae, the AP is associated with a shortlasting elevation of the concentration of cytoplasmic-free
Ca21 ([Ca21]cyt) (Kikuyama and Tazawa, 1983; Williamson
and Ashley, 1982). This rise in Ca21 from a resting level of
200 nM to a peak at about 700 nM (Plieth and Hansen, 1996)
is considered as primary step in membrane excitation. It
is believed that the increase in [Ca21]cyt activates Ca21sensitive Cl– channels in the plasma membrane (reviewed
by Tazawa et al., 1987) and in this way triggers the rise in
bulk Cl– conductance (Lunevsky et al., 1983).
As yet there is no clear-cut discrimination between the
possibilities that a movement of Ca21 through channels in
the plasma membrane or the release of Ca21 from internal
stores provides the source of the intracellular [Ca21]cyt
signal. Evaluation of available experimental data provides
support for both hypotheses. The idea of Ca21 influx across
the plasma membrane is largely based on the finding that
excitation is correlated with an increase in the influx of
45Ca21 into the vacuole of Chara (Hayama et al., 1979; Reid
and Tester, 1992). This is also consistent with the finding
that replacement of external Ca21 by Mg21 abolishes
electrically triggered AP as well as the [Ca21]cyt transient
(Williamson and Ashley, 1982). When they were transferred
back to the medium containing Ca21, in the same study, the
cells regained excitability, however the [Ca21]cyt transient
(∆[Ca21]cyt) remained absent or reduced for some 10 min
(Williamson and Ashley, 1982). Such a reduced [Ca21]cyt
transient in the presence of sufficient external Ca21 is
difficult to reconcile with the idea that ∆[Ca21]cyt is simply
the result of Ca21 influx across the plasma membrane.
Another argument for Ca21 influx via plasma membrane
channels is based on the effect of inhibitors. La31, which
is anticipated to block Ca21 channels in Chara (Reid and
Tester, 1992), also abolishes electrical excitation in these
algae. But notably, the time of exposure to La31 required
to abolish excitability is in the range of tens of minutes
(Beilby, 1984; Tsutsui et al., 1987). If La31 was only blocking
Ca21 channels in the plasma membrane, much shorter
times would be expected for channel blockade (Bauer
et al., 1997).
167
168 Christoph Plieth et al.
Finally, evidence in support of a Ca21 influx through
channels in the plasma membrane comes from electrophysiological measurements. The amplitude of the AP was
correlated with the external Ca21 concentration and this
was interpreted as evidence for Ca21 influx during the
downstroke of the AP (Findlay, 1962; Hope, 1961a,b; Staves
and Wayne, 1993). Furthermore, current measurements
under voltage clamp control suggested that one component of the complex excitation current is sensitive to the
external Ca21 concentration (Beilby and Coster, 1979;
Lunevsky et al., 1983; Shiina and Tazawa, 1987). This
rendered the current a probable candidate for the Ca21
inward current anticipated during excitation. The latter
interpretation, however, is challenged by two arguments.
Firstly, the reversal voltage of the Ca21-sensitive excitation
current was well negative of the Ca21 equilibrium voltage,
which casts doubt on the idea that the current carries Ca21
(Beilby, 1984; Thiel et al., 1997). Secondly, analysis of the
putative Ca21 current with respect to the voltage and
time dependency of activation revealed that this current
activates with a delay (Beilby and Coster, 1979). Thus, even
if the channel was carrying Ca21 influx it would be unlikely
that it is responsible for the initial sharp rise in [Ca21]cyt
(Beilby, 1984).
Other experimental data, albeit more indirect, favour the
view for release of Ca21 from internal stores in the course
of excitation. Elevation of inositol-trisphosphate (InsP3) in
the cytoplasm triggered AP in Chara and Nitella (Thiel
et al., 1990). This indicates, by analogy to the situation in
animal cells (Berridge and Irvine, 1989), that internal Ca21
stores are present in the algae and that release of Ca21
from these stores is sufficient to trigger excitation.
Another line of evidence for internal release is related to
(Ca21)cyt measurements in tonoplast-free Chara cells in
which a biphasic Ca21 transient during the AP could be
resolved (Kikuyama and Tazawa, 1983). The suggestion
that the initial transient reflects release of Ca21 from
depletable internal stores was based on the finding that
the initial transient was abolished after multiple AP, even
when sufficient Ca21 was present in the external medium
(Kikuyama and Tazawa, 1983).
A similar model involving Ca21 stores that were refilled
by Ca21 from the external medium was obtained from
patch clamp experiments on the plasma membrane of
Chara. In this case it was found that the Cl– channels,
which form the elementary basis of the excitation Cl–
current, activated without a temporal correlation to the
putative plasma membrane Ca21 influx (Thiel et al., 1993).
One plausible explanation for these data is that the activation of the Cl– channels was due to a release of Ca21 from
internal stores (Thiel et al., 1993, 1997).
In this study, we used fluorescence-ratio imaging to
record Ca21 transients during excitation and attempted to
resolve the origin of Ca21. An important feature of the
Figure 1. Series of action potentials in a fura-dextran loaded Chara
corallina cell.
At t 5 1550 sec, 50 µM Mn21 was added to the perfusion stream. (a)
Membrane potential; (b) cytoplasmic Ca21 ion concentration calculated by
the F340/F380 ratio.
Ca21-indicator fura-2 is that its fluorescence is quenched
by Mn21 ions under all excitation wavelengths (Gilroy and
Jones, 1992; McAinsh et al., 1995; Malhó et al., 1995;
Thomas and Dellaville, 1991; Zottini and Zanoni, 1993).
Mn21 is similar to Ca21 and, hence, able to penetrate
Ca21 channels and other pores (i.e. K1 channels) that are
permeable to Ca21 ions (Fasolato et al., 1993; Piñeros and
Tester, 1995; Striggow and Ehrlich, 1996). In the case
that the external solution contains Mn21, it is possible to
discriminate between Ca21 influx and Ca21 mobilization
from internal stores. Therefore the Ca21-independent
fluorescence of the dye excited by light near the isosbestic
point (i.e. F360 with λex 5 360nm), was measured in addition
to the Ca21 dependent wavelengths for ratio measurements
of [Ca21]cyt (i.e. F340 and F380) and inspected for Mn21
evoked quenching (Merrit et al., 1989).
Results
Measurement of action potentials in Chara cells with
Mn21 ions in the bathing medium
Figure 1 illustrates the simultaneous recording of the membrane voltage and [Ca21]cyt in a fura-dextran loaded
internodal cell of Chara during repetitive excitation in the
absence and in the presence of 50 µM Mn21. As a general
observation it appeared that addition of submillimolar
Mn21 to the bath solution did not abolish excitability over
the period of recording. But the concomitant [Ca21]cyt
transients gradually decayed in amplitude with time of
exposure to Mn21 (Figures 1; 2c,d; 3c,d), while the magnitude of the AP increased (Figures 1; 2 a,b; 3a,b)
Figure 2 shows a close up of the fluorescence signals
© Blackwell Science Ltd, The Plant Journal, (1998), 13, 167–175
Calcium release from internal stores 169
Figure 2. Examples of APs from Figure 1, without Mn21 in the external
bathing medium (a, c, e) and with 50 µM Mn21 in the external bathing
medium (b, d, f);. membrane potential (a, b); cytoplasmic calcium
concentration (c, d); fluorescence signals (e, f): open circles 5 F340; closed
circles 5 F380; line without symbols 5 F360.
recorded during two AP, one without and one with 50 µM
Mn21 in the bath. In the standard experimental medium
(left panel) the AP was associated with a transient antiparallel change of the dye fluorescence in the Ca21sensitive wavelengths 340 nm and 380 nm, respectively
(Figure 2b). The altered fluorescence ratio was converted
to a rise in [Ca21]cyt from a resting level of about 200 nM
to a peak value of 700 nM (Figure 2c), by reference to the
in vitro calibration.
Figure 2(e) shows that the fluorescence measured at 360
nm near the isosbestic point (F360) underwent, at the
resting voltage, random variations that were not reflected
in [Ca21]cyt (see also Figure 4b; 7c).The amplitudes of
variations in F360 decreased as the cytoplasmic streaming
ceased due to AP (data not shown), and could therefore
be assigned to fluctuations in dye concentration generated
by moving particles in the cytoplasm (Plieth and Hansen,
1996).
During the downstroke of the AP the F360 signal generally
increased transiently (Figure 2e,f). The reason for this unexpected transient rise in F360 during excitation is not known.
One possible explanation for this phenomenon could be
that the massive loss of KCl during the AP (Kikuyama,
1986, 1987; Kikuyama et al., 1984) produces a short-lasting
water loss and consequently a rise in cytoplasmic dye
concentration. Another explanation could be that the cytoplasmic environment causes changes in the optical properties of the dye that lead to a shift of the isosbestic point to
longer excitation wavelengths in vivo compared with data
obtained in vitro (Bancel et al. 1992; Blatter and Wier,
1990; Haugland, 1992). Under these conditions the F360
measurements retained a slight amount of Ca21 sensitivity.
The right panel of Figure 2 shows, for comparison, the
fluorescence signals obtained during the AP with Mn21 in
© Blackwell Science Ltd, The Plant Journal, (1998), 13, 167–175
Figure 3. Average of five time series during action potential.
Membrane potential (a, b); [Ca21]c (c, d); fura fluorescence excited under
λex 5 360 nm (F360, e, f). Without Mn21 ions in the bathing medium (a, c,
e) and with 50 µM Mn21 (b, d, f); the difference curve (g) was calculated by
substracting the curve in (e) from that in (f) (compare Figure 6).
the bath. Under these conditions the AP evoked similar
changes in the three fluorescence signals as those obtained
in the absence of Mn21. Compared to the control, only the
magnitudes of changes in [Ca21]c were reduced by the
presence of Mn21 in the bath (Figure 2c,d). Most important,
the F360 signal indicated no obvious quenching of the fura
fluorescence during the AP in the Mn21 containing bath
medium (Figure 2e,f).
To uncover potentially small Mn21-related differences of
the fluorescence signal, the F360 responses during AP with
and without Mn21 from Figure 1 were normalized to the
time of excitation and averaged (Figure 3). In essence, the
mean value of F360 remained, for both treatments, with the
exception of the initial transient fluorescence increase,
constant during AP. The control data (mean F360 in the
absence of Mn21) were further subtracted from the mean
F360 signal recorded in the presence of Mn21. This operation
should abolish the contribution of Mn21-independent background fluctuations in fluorescence intensity and leave
170 Christoph Plieth et al.
Figure 4. Membrane potential (a) and fura fluorescence (λex 5 360 nm) (b)
in a Chara cell during exposure to 30 mM external Mn21 concentration.
only Mn21-related effects on F360. Figure 3(g) shows the
respective F360 difference curve, which revealed no perceivable Mn21-related quenching during the AP.
Neither increasing the concentration of Mn21 up to
150 µM (NExp 5 6) nor using Ni21 as another potential
quenching ion (Haugland, 1992) in the outer medium
(NExp 5 4) produced any significant quenching of F360
during an AP (data not shown).
The lack of quenching of F360 during the AP in Mn21containing solution prompts two alternative explanations.
(i) The transient rise in [Ca21]cyt may be due to Ca21 release
from internal stores without contribution of significant
influx across the plasma membrane. (ii) The quenching
ion Mn21 may, unlike the situation in other organisms,
not permeate Ca21 transporting channels in the plasma
membrane of Chara (Fasolato et al., 1993; Piñeros and
Tester, 1995; Striggow and Ehrlich, 1996).
To test the latter hypothesis, namely that Mn21 is unable
to cross the plasma membrane, fura-dextran loaded Chara
cells were exposed to high concentrations of Mn21. Figure 4
shows the response of the electrical and fluorescence
parameters to an exposure of 30 mM Mn21. Immediately
after adding Mn21 to the perfusion stream, VM depolarized.
The [Ca21]cyt concentration remained unaffected by this
treatment. A scrutiny of F360 revealed that high concentrations of Mn21 caused a slow progressive quenching of the
fluorescence, indicating that Mn21 could enter the cell via
plasma membrane-resident transporters. In comparable
experiments a mean quenching of 41 6 10% (mean 6 SD;
NExp 510) of fura fluorescence occurred with 25–30 mM
Mn21 in the bath.
A typical loading of Chara with fura results in a cytoplasmic dye concentration of about 5 µM (Plieth and
Hansen, 1996). Considering that 40% of the fluorescence
intensity (i.e. 2 µM fura) is quenched during Mn21 incubation, we can, based on the high affinity of Mn21 to fura
Figure 5. Action potentials in Chara before and after incubation with high
Mn21 ion concentrations.
Membrane potential (a); cytoplasmic calcium concentration as calculated
from the F340/F380 fluorescence ratio (b).
(Kd(fura-Mn21) ™ 5 nM) estimate that the cytoplasmic
concentration rose by ca. 2 µM.
In control experiments cells were also exposed to comparable concentrations of Ni(NO3)2, an operation that similarly produced membrane depolarization but no quenching
of F360. Hence, the loss of F360 fluorescence upon exposure
to Mn21 appeared specific for Mn21 influx and was not
due to osmotic effects or due to the concomitant membrane
depolarization.
After pretreatment in Mn21, AP cause fluorescence
quenching
In the presence of millimolar Mn21 concentrations, the
cells were not excitable. However, cells gradually regained
excitability after washing the heavy metal out of the bath
solution (Figure 5). AP elicited after pre-treatment with
Mn21 were somewhat reduced in amplitude and duration.
The recovery of excitability was also associated with progressive reappearance of [Ca21]cyt transients (Figure 5b).
The most important observation with respect to the
fluorescence signals in cells pretreated with 25 mM Mn21
is detailed in Figure 6. Under control conditions, F360
remained largely constant during the AP (Figure 6e). After
pretreatment with high Mn21, the F360 signal decreased in
all cases during the AP, concomitantly with the rise in
[Ca21]cyt (Figure 6f). The amplitude of quenching varied
from 8% to 12% between different AP, with no apparent
decay during continuous excitation. Figure 6(g) shows the
mean quenching of F360 during AP in Mn21 pre-treated cells
(Figure 6f) after subtraction of the background variations
obtained before Mn21 exposure (Figure 6e).
Similar quenching of the F360 signal in parallel with the
elevation of [Ca21]cyt was obtained after Mn21 pretreatment
© Blackwell Science Ltd, The Plant Journal, (1998), 13, 167–175
Calcium release from internal stores 171
Table 1. Time constants (in sec) and amplitude factors (see
equation below) of the F360 quenching during AP in Mn21 pretreated Chara cells (mean 6 SD; nAP 5 8; nCells 5 3). The
fluorescence signals were normalized to 1 (Figure 6h)
τ1
τ2
k0
k1
k2
27.5 6 11.2
266 6 79
1.01 6 0.04
–0.31 6 0.15
0.66 6 0.38
Kinetic analysis
The AP-associated quenching of F360 was fitted by the sum
of two exponentials
t
t
F 5 k0 1 k1·exp –
1 k2·exp –
τ1
τ2
( )
( )
The resulting first time constant of the biphasic F360
response provides a measure for the release kinetics of
Mn21 from the internal stores (an example is given in
Figure 6f). This operation provides a time constant of τ1 5
27.5 sec for the release of Mn21 into the cytoplasm. The
second time constant was found to be τ2 5 266 sec
(Table 1).
Figure 6(h) shows that the noise in the fluorescence
signal increased ca. 30 sec after the AP. This was due to the
inhomogeneous dye distribution in the cell and recovery of
cytoplasmic streaming, which ceased during excitation
(Plieth and Hansen, 1996).
Figure 6. Average of five time series during action potentials.
Membrane potential (a, b); [Ca21]c (c ,d); F360 fluorescence quenching (e,
f); before (a, c, e) and after (b, d, f) incubation in 25 mM Mn21; the difference
curve (g) was calculated by substracting the curve in (e) from that in (f)
(compare Figure 3). An example for the fit of the biphasic fluorescence
response (h), the fitted curve (5 line) was fitted to the scaled data (5
circles) by a sum of two exponentials (equation 1). The time constants
here are τ1 5 25 sec and τ2 5 325 sec; ca. 30 sec after the AP the scatter
increases due to the recovery of cytoplasmic streaming.
during all APs with measurable Ca21 transients (NCells 5 3;
nAP 5 24). Notably, AP-associated quenching was observed
in all cases in the absence of external Mn21, even 30 min
up to 3 h after washing the cell thoroughly with Mn21-free
medium. Hence, it is most unlikely that Mn21 ions from the
external solution provided a source of ions for quenching.
A washout of cell wall-bound Mn21 caused by K1 efflux
during the AP is very unlikely. Firstly, the rate of quenching
is maximal at the peak of the AP (Figure 6b,g), i.e. a time
when the delayed voltage activated K1 channels are still
closed (Thiel, 1995; Thiel et al., 1997). Secondly, the amplitude of fura quenching does not decay during subsequent
AP as would be expected if a pool of Mn21 bound to the
cell wall is mobilized during AP.
Consequently, the quenching of F360 must reflect release
of Mn21 together with Ca21 from internal stores.
© Blackwell Science Ltd, The Plant Journal, (1998), 13, 167–175
Measurement of action potentials in Chara cells with
Mn21-loaded vacuoles
In plant cells the vacuole is believed to be a Ca21 store. It
contains several mM of Ca21 (Okihara and Kiyosawa, 1988),
some of which might be released upon stimulation. We
therefore tested whether the vacuole was the store releasing Ca21 during the AP. In order to investigate potential
vacuolar Ca21 efflux during excitation, the vacuoles of
Chara cells were loaded with 50 µM , [Mn21]v ,100 µM (for
details of vacuolar loading see the Experimental procedures). The AP recorded under these conditions were associated with the typical rise in cytoplasmic [Ca21]cyt. Inspection
of the F360 signal showed no evidence for AP-associated
quenching (Figure 7). The same absence of quenching was
found in all experiments conducted (NCells 5 3, nAPs 5
16). This makes Ca21 release from the vacuole during
excitation unlikely.
Discussion
A key finding of the present work is that the fura fluorescence near the isosbestic point is not quenched during an
AP when Mn21 is present in the external medium at
172 Christoph Plieth et al.
Figure 7. Example of an AP in a Chara cell with Mn21-loaded vacuole.
Membrane potential (a); cytoplasmic calcium concentration (b);
fluorescence signals (c): open circles 5 F340; closed circles 5 F380, line
without symbols 5 F360.
concentrations of up to 150 µM. The conclusion, therefore,
is that the bulk of the Ca21 responsible for the transient
rise in [Ca21]cyt during the AP must be due to release from
internal stores. Ca21 influx across the plasma membrane
(PM) does not add significantly to the rise in [Ca21]cyt. The
idea of Ca21 release from internal stores during excitation
is further supported by the finding that APs were associated
with a quenching of the fura fluorescence after prolonged
pretreatment of the cells with high Mn21 concentrationcontaining solution. In this case quenching was detectable
even after thorough washing of the heavy metal out of the
bath solution. Under these conditions only release of Mn21
from internal stores can possibly provide a source for the
fluorescence quenching.
So in summary, the data are consistent with a model
that Chara contains cytoplasmic stores that are slowly filled
under resting conditions with divalent ions. In the course
of an AP, these stores discharge Ca21, which is responsible
for the transient rise of [Ca21]cyt during excitation. If the
stores contain Mn21, discharge during the AP leads to a
quenching of the fluorescence signal.
It is widely believed that the vacuole serves in plant cells
as a site of internal Ca21 release via InsP3-dependent or
-independent mechanisms (Allen et al., 1995; Ward et al.,
1995). Loading the vacuole in Chara with Mn21 prior to
excitation caused no detectable quenching of the fura
fluorescence during an AP in the present study (Figure 7).
Therefore the vacuole can be excluded as relevant source of
Ca21 discharge during excitation, and the Ca21 transporters
activated upon excitation do not appear to reside in the
tonoplast. Hence, Chara cells must have Ca21 stores other
than the vacuole. A possible candidate is the endoplasmic
reticulum (ER), which in animal cells contains InsP3 receptors with Mn21 permeability (Striggow and Ehrlich, 1996).
With these properties, the stores could, as in the present
case, be loaded with Ca21 or Mn21 and discharge their
content upon stimulation.
Previous luminescent measurements have explained the
rise in [Ca21]cyt during the AP as the result of Ca21 influx
across the PM or a combination of internal release and
influx (Kikuyama and Tazawa, 1983; Williamson and Ashley,
1982). In contrast to the present study, the latter measurements of [Ca21]cyt in tonoplast-free cells detected not one
but two separable Ca21 peaks during excitation (Kikuyama
and Tazawa, 1983). The first of the two peaks appeared to
reflect Ca21 release from internal stores, like the Ca21
transient in the present study. Similar to the present data,
these internal stores seemed to require refilling with Ca21
from the external medium. The second Ca21 transient
detected by Kikuyama and Tazawa (1983) was not observed
in the present study. Also, luminescent Ca21 measurements
in intact Chara cells only observed a single Ca21 transient
(Williamson and Ashley, 1982). Therefore, it seems likely
that the second long-lasting [Ca21]cyt transient, which is
probably due to Ca21 influx across the plasma membrane
(Kikuyama and Tazawa, 1983), is related to specific properties of the tonoplast-free cell system.
In most experiments it was noted that after quenching
during the AP the F360 fluorescence signal slowly reversed
back near the resting level, which lead to the biphasic
response (see τ2, Table 1).
It is of course tempting to interpret the second (slow)
time constant as an indirect measure for the refilling of
the Ca21 stores. However, Mn21 binds fura with a roughly
50 times higher affinity than Ca21 (dissociation constants:
Kd(Mn21-fura) ™ 5nM; Kd(Ca21-fura) ™ 250nM). Therefore,
fura binding to Mn21 must be considered quasi irreversible
in the cytoplasmic environment. This renders retrieval of
fura-bound Mn21 into the stores unlikely. This is supported
by the finding that the fluorescence quenched after the
influence of high Mn21 concentrations (Figure 4) never
recovered its previous magnitude.
An alternative explanation for the slow time constant
could be that electrical stimulation does not cause excitation of the entire membrane and hence increases [Ca21]c
only in parts of the cell (Homann and Thiel, 1994). Thus,
during recovery of Ca21-sensitive cytoplasmic streaming
after an AP, cytoplasm with elevated [Ca21]c in the window
of recording is mixed with the part of cytoplasm that has
been left unaffected. Such a dilution of Mn21 quenched
with non-quenched fura could explain the recovery of the
F360 signal. On this background the mean time of τ2 5 266
sec (Table 1) could be seen as the time of streaming
recovery.
In the past it has been observed that AP-associated
Ca21 transients in Chara cells gradually reappeared after
replacing external Mg21 with Ca21 (Williamson and Ashley,
1982). This was interpreted as a role of Ca21 influx in the
© Blackwell Science Ltd, The Plant Journal, (1998), 13, 167–175
Calcium release from internal stores 173
immediate process of excitation. In the context of the
present data, it can be argued that sufficient filling of
internal stores with Ca21 from the external medium is
necessary for transient elevation of [Ca21]cyt. Such a temporal uncoupling of events would explain the observation
that regeneration of Ca21 transients does not occur immediately after transfer of cells from a Mg21- to a Ca21-containing medium but only gradually over several tens of
minutes (Williamson and Ashley, 1982).
The same arguments hold true for the effect of the
putative Ca21 channel blocker La31. The unusually long
exposure of cells to the blocker required to abolish excitability (Beilby, 1984; Tsutsui et al., 1986, 1987) and the lack
of recovery from La31 treatment (Beilby, 1984; Kourie,
1994) are better understood in terms of a reduced loading
of internal Ca21 stores than in terms of a direct block of
plasma membrane Ca21 channel activity during the AP. In
the same context, the enhanced influx of 45Ca21 into excited
cells (Hayama et al., 1979; Reid and Tester, 1992) could
therefore be due to a stimulated tracer uptake between AP
into stores rather than influx during the AP.
In essence, the present evidence for Ca21 release from
internal stores during excitation is in line with recent
electrophysiological data. In these studies it was found that
elevation of the cytoplasmic InsP3 concentration triggered
excitation (Thiel et al., 1990). In combination with the
present data it underlines that cytoplasmic Ca21 stores are
present in Chara and that triggered release of Ca21 is
associated with excitation.
Furthermore, in patch clamp investigations it was found
that the activation of excitatory Cl– channels was not
correlated in time with the activity of a putative Ca21
permeable channel (Thiel et al., 1993, 1997). It was therefore
proposed that for activation of Ca21-sensitive Cl– channels
internal stores must be filled with Ca21 from the external
solution. Upon stimulation, the stores discharge their content and transiently activate Cl– channels. In this sense
both fluorescence optic measurements and the electrophysiological data support the same model for the rise of
[Ca21]cyt in membrane excitation in Chara. At this stage we
cannot directly interpret the measured [Ca21]cyt transients
as those anticipated from electrophysiological measurements. Thus, it is not clear whether the stores discharging
Ca21 during the AP are indeed the same ones that are
InsP3-sensitive. Furthermore, the anticipated kinetics of
[Ca21]cyt transients underlying Cl– channel activation is
significantly faster than the global Ca21 changes recorded
with Ca21 dyes (Thiel et al., 1997). However, in analogy to
studies in oocytes (Yao et al., 1995), it could be assumed
that single short-lived release events initiate global longer
lasting Ca21 waves by increasing the frequency of Ca21
release from individual release sites. In this sense the
measured [Ca21]cyt signal would reflect the statistical superposition of many single events.
© Blackwell Science Ltd, The Plant Journal, (1998), 13, 167–175
The absence of an apparent Ca21 influx in the present
studies raises the question of how an electrical trigger
(depolarization) initiates the release of Ca21 from internal
stores. It may be suggested that a tiny Ca21 influx reaches
stores adjacent to the membrane, causes a calcium-induced
release (Berridge 1990) and starts a propagating wave of
Ca21 release throughout the cytosol. The detection of this
putative influx would be difficult. In the studies here,
quenching of the fluorescence near some membrane-adjacent stores would not cause a detectable change of the
overall signal. In the case of patch clamp studies, the
suggested restriction to parts of the cell (Homann and
Thiel, 1994) may prevent the detection of depolarizationinduced Ca21 influx. However, if the reason for not finding
the depolarization-induced Ca21 influx is its non-existence,
it has to be questioned whether the concept of depolarization-induced ion fluxes as known from animal cells (Hille,
1992) can be transferred to excitable plant cells (Beilby
and Coster, 1979). An alternative approach is offered by
integrins. These membrane-bound proteins are connected
to the cytoskeleton and are known to be involved in the
transduction of mechanical signals into a biochemical
response (Ingber, 1991). Wayne et al., 1992) assume the
involvement of integrins in gravisensing in characean cells.
Integrins bearing electrical charges could be moved by
membrane potential similar to the S4 helices of Shakertype K1 channels (Durell and Guy, 1992) and open internal
Ca21 stores in the ER by mechanical signal transduction
without involvement of voltage-sensitive ion channels.
Experimental procedures
Plant material
Chara corallina was grown in the laboratory in plastic basins filled
with APW (0.1 mM KCl, 1.0 mM NaCl, 0.5 mM CaCl2) as described
in Plieth and Hansen (1992).
Loading procedures
In order to prevent dye sequestration into the vacuole and other
cell compartments, the dextran derivative of fura-2 was used (i.e.
fura-dextran; MW 5 10 kDa; F-3029; Molecular Probes, Eugene,
OR; for emission and excitation spectra see Plieth et al., 1997). A
solution of 1 mM fura-dextran was injected into the cytoplasm of
the cells by manual micropressure injection or by loading via
neighbouring cells, as described in detail by Plieth and Hansen,
1996).
In some experiments Mn21 ions were also loaded into the
vacuole by means of micropressure injection. A mixture of 440 µM
BCECF–dextran, 20 mM Mn(NO3)2 and 100 mM KCl was injected
into the vacuole under the control of fluorescence imaging (λex 5
490nm, λem 5 530 nm, using a DM510 dichroic and BA520–560
bandpass filter from Nikon, Düsseldorf, Germany) in order to get
an estimate of the amount of the [Mn21]v. Loading was stopped
when the vacuole showed a BCECF fluorescence corresponding
174 Christoph Plieth et al.
to about 1.5 µM. Thus, the Mn21 ion concentration in the vacuole
([Mn21]v) was 50–100 µM.
Forschungsgemeinschaft. G. Thiel was supported by SFB 523.
C. Plieth is funded by PL253/1–1.
Preparations of cells and excitation
References
Individual dye-loaded Chara whorl cells of 5–15 mm length were
dissected and transferred to a perfusion chamber (i.e. Petri dish
with an outlet as described in Plieth, 1995). The cells were pressed
to the bottom with needles sticking in silicon rubber at the bottom
of the Petri dish, as previously described (Plieth and Hansen, 1992).
All experiments were carried out in standard medium (SM 5
0.1 mM KNO3, 0.1 mM MgCl2, 0.1 mM CaCl2, 2.0 mM Mes adjusted
with NaOH to pH 5 5.5). This medium was found to be best suited
for eliciting AP with large amplitudes. The perfusion flux was
adjusted to 10 ml min–1. Solutions of different ionic composition
(i.e. SM with and without Mn21 ions) could be switched to the
perfusion stream by a group of three-way valves (Plieth, 1995).
Two bath electrodes (glass tubes with an inner diameter of
3 mm, filled with 1 M KCl in 3% agar) were positioned near both
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3 V/cm) was sufficient to elicit an AP. In order to avoid incomplete
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Fluorescence ratio imaging
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by Mühling et al. (1995). Fluorescence values were obtained by
averaging the grey levels in a region of interest (i.e. an area of
about 100 µm 3 200µm). The ratio of F340/F380 was used as
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the sample rate was increased to 4 ratios per 10 sec. The F360
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Calibration
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Probes; Haugland, 1992) containing 3 µM fura-dextran (Plieth, 1995,
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needs a comment: fura-dextran has a higher dissociation constant
(Kd ™ 350 nM, varying from lot to lot; Haugland, 1992) than the
pure pentapotassium salt fura-K5 (Kd ™ 140 nM; Lattanzio, 1991).
Thus it is not surprising that fura-dextran saturates above 1 µM
Ca21 (i.e. pCa , 5).
Acknowledgements
We are grateful to Dr Mark Fricker (Oxford) for critically reading
the manuscript and to Dr Jack Dainty (Montpellier) for helpful
discussion. This study was partly supported by the Deutsche
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