Plant Physiol. (1993) 102: 1139-1146
Calcium Retrieval from Vacuolar Pools'
Characterization of a Vacuolar Calcium Channel
Angie Celli and Eduardo Blumwald*
Centre for Plant Biotechnology, Department of Botany, University of Toronto, 25 Willcocks Street,
Toronto, Ontario M5S 3B2, Canada
Voltage patch-clamp experiments at the whole-vacuole and single-channel levels were employed to study the retrieval of CaZ+
from vacuoles into the cytoplasm in sugar beet cell (Beta vulgaris
1.) suspension cultures. Channels allowing the movement of Ca2+
out of the vacuole were identified at physiological conditions of
pH, vacuolar membrane potential, and vacuole/cytoplasm Caz+
concentrations. The operation of the channel was voltage dependent and inositol-l,4,5-triphosphate insensitive and displayed high
selectivity for CaZ+ ions. These channels bear similarities to the
dihydropyridine-sensitive i-type CaZ+channels from animal cells.
Bay K-8644, an agonist, increased the frequency of channel openings, whereas nifedipine, an antagonist, reduced the channel activity. Both effects were elicited only from the vacuolar side of the
channel. Channel activities were also inhibited by verapamil, La3+,
and cytoplasmic Caz+ concentrations higher than 1 x 10-6 M. The
modulation of the channel currents by cytoplasmic CaZ+ would
suggest the role of these channels in triggering the initiation of
signal transduction processes in plant cells.
The vacuole of a mature plant cell is the main storage
compartment of intracellular Ca2+.Release of vacuolar Ca2+
induces an elevation of free cytoplasmic Caz+ concentration
from steady-state levels of 150 nM to greater than 300 nM
(Gehring et al., 1990). These fluctuations in cytoplasmic CaZ+
levels are thought to mediate severa1 physiological processes
in plant cells, such as phototropism and geotropism (Gehring
et al., 1990) and hormone action (Felle, 1988).
In animal cells, cytoplasmic free Ca2+ is known to play a
role as a second messenger in signal-transduction processes
(Carafoli, 1987). The influx of Ca2+into the cytoplasm from
intracellular stores, other than mitochondria, is mediated by
two different Caz+-selectivechannels. One of these channels,
localized in the ER (Ross et al., 1989), is activated by Ins-P3
and is heparin sensitive. The second type is a Ca2+-induced
Ca2+release channel isolated from the SR (Lai et al., 1988).
The SR channel is activated by submicromolar to micromolar
Ca2+concentrations and is heparin insensitive. Also, the SR
Ca2+-releasechannel will open in the presence of caffeine at
micromolar levels of cytoplasmic Ca2+,but this effect has not
' This research was supported by an operating grant of the Natural
Sciences and Engineering Research Council of Canada to E.B.
* Corresponding author; fax 1-416-978-5878.
been observed on the Ins-P3-activated channel (Wakui et al.,
1990).
In plant cells, patch-clamp studies have demonstrated two
different types of Ca2+-sensitive channels that serve as a
pathway for the release of vacuolar Ca2+.Alexandre et al.
(1990) have shown a Ca2+-selective channel that is active
only in the presence of Ins-P3. This channel, identified only
in excised patches of tonoplast from Beta vulgauis, is partially
inhibited by verapamil (a Ca2+ channel antagonist) and has
a conductance of 30 picosiemens with 5 mM CaZ+on the
vacuolar side of the tonoplast. Ins-P3has also been shown to
release Ca2+ from tonoplast vesicles (Schumaker and Sze,
1987) and intact vacuoles (Ranjeva et al., 1988).
A second type of Ca'+-selective, voltage-dependent, and
Ins-P3-insensitive channel has been reported in excised
patches of tonoplast by Johannes et al. (1992a). The unitary
current of these channels saturated at vacuolar membrane
potentials of -40 mV. Channel activity was sensitive only to
the lanthanide Gd3+, and changes in cytoplasmic Ca2+concentrations (from nM to mM) did not affect channel activity.
Increased levels of vacuolar Ca2+ increased the open-state
probability of the channel and shifted the voltage activation
to more positive potentials.
It should be noted that in this report vacuolar membrane
potentials are considered according to the recent proposed
convention for electrical measurements in endomembranes
(Bertl et al., 1992). According to this convention, the vacuolar
lumen is considered to be equivalent to the extracellular
space. Thus, for plant vacuoles, the tonoplast voltage is
considered to be the difference between the voltage at the
cytosolic side and the voltage at the vacuolar side. Therefore,
when positive voltage differences are set across the tonoplast,
these voltage differences will be considered negative tonoplast potentials, making the tonoplast electrically equivalent
to the cell plasma membrane.
The presence of these two different Ca2+-selectivechannels
in the vacuolar membrane may provide two potential pathways for the release of vacuolar Ca2+ when elevation of
cytoplasmic Ca2+is required by the cell for signal transduction
mechanisms. In this report, we present evidence for a Ca2+selective channel in the tonoplast of sugar beet cells that is
voltage dependent, allowing the movement of CaZ+ions from
Abbreviations: DHP, dihydropyridine; E,, reversal potential; InsP3, inositol- 1,4,5-triphosphate; SR, sarcoplasmic reticulum.
1139
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Gelli and Blumwald
1140
the vacuole into the cytoplasm at physiological tonoplast
membrane potentials. This channel is differentially sensitive
to 1,4-DHPs, being inhibited by nifedipine (a DHP antagonist) and activated by Bay-K 8644 (a DHP agonist). The
characteristics of this channel differ from those reported by
Johannes et al. (1992a) in that no saturation of single-channel
current was observed with decreasing tonoplast membrane
potentials and cytoplasmic Ca2+concentrations greater than
1 X 10-4 M blocked the release of Caz+from the vacuole. The
Ca2+-selectivechannel reported here bears similarities to the
L-type Ca2+channels of plasma membrane from animal cells.
MATERIALS AND METHODS
Plant Material
Cell suspensions of sugar beet (Befa vulgaris L.) were grown
as described previously (Blumwald and Poole, 1987). Vacuoles were obtained by osmotic lysis of protoplasts as described
by Pantoja et al. (1989).
Measurements of Whole-Vacuole and Single-Channel
Currents
Patch-clamp techniques were used to record ionic currents
from intact vacuoles and isolated outside-out patches (Hamil
et al., 1981). The vacuole-attached configuration was initially
obtained by applying gentle suction to the pipette, resulting
in seals with resistances of 6 to 10 GO. AppIying a pulse of
1 V for 30 ms broke the small area of vacuolar membrane
within the pipette tip, thus resulting in the whole-vacuole
configuration. Liquid junction potentials were calculated as
described by Barry and Lynch (1991) and clamp voltages
were adjusted accordingly. Currents moving from the vacuole
into the cytoplasm are presented as inward currents and
defined as inward-rectifying currents, according to the recently proposed convention for the electrical measurements
in endomembranes by Bertl et al. (1992). Also, the tonoplast
voltage is considered to be the difference between the voltage
at the cytosolic side and the voltage at the vacuolar side.
Therefore, when positive voltage differences are set across
the tonoplast, these voltage differences will be considered
negative tonoplast potentials.
Pipettes were pulled from borosilicate glass capillaries
(Rochester Scientific Co., Rochester, NY) in two stages with
a vertical pipette puller (Narishige Co., Tokyo, Japan). Pipette
tips with resistances of 3 to 5 MO were coated with a layer
of silicone (Sigmacote, Sigma) and heat-polished for 3 to 4
min .
Whole-vacuole currents were obtained by holding the
membrane potential at O mV and applying alternate pulses
of +10 mV every 10 s with increments of 10 mV from -100
to 100 mV or as described in the legends to the figures.
Currents were recorded with the pCLAMP software program
(Axon Instruments, Foster City, CA), and data were stored in
a PCII-386 computer operating on-line.
Selectivity of the whole-vacuole currents was determined
by tail current experiments. Currents were activated by -100
mV pulses for 5 s followed by a step up in voltage to -80
mV. This protocol was repeated 15 times, and the voltage
was stepped up by 10 mV for each successive pulse.
Plant Physiol. Vol. 102, 1993
Outside-out patches were obtained from the whole-vacuole configuration by withdrawing the pipette from the vacuole. Single-channel currents were measured in exciseci outside-out patches of tonoplast by continuously polarizing the
isolated patch to potentials between -100 to 100 mV.
Voltage clannp measurements of whole-cell and singlechannel currents were performed at 2OoC to 23OC writh a
Dagan 3900 patch-clamp amplifier (Dagan Corp., Minneapolis, MN) and filtered at 500 Hz. Single-channel recordings
were low-pass filtered at 200 Hz with a four-pole Bessel filter
contained in the patch-clamp amplifier. The signal from the
patch-clamp system was digitized at 44 kHz by a pulse-code
modulator and stored on videotape (DAS 900, Dagan Corp).
Single-channel data were digitized and processed with the
pCLAMP program.
Solutions
Vacuoles were held in a recording chamber (cytoplasmic
side of vacuole) in 100 mM KC1 along with 2 mM ME;C12, 5
mM Tris/Mes, pH 7.5, 1 m CaC12, and 1.1 mM [bis-(oaminophenoxy)-ethane-hJ,N,N’,N’,-tetraacetic
acid] trrtrasodium salt to give a final concentration of 1 x 1 0 P M free
Ca2+.The pipette-filling solution (vacuolar interior) consisted
of either BaCl,, SrC12,or CaC12at the concentrations inclicated
in the legends to the figures. Also included in the pipette
solution were 2 mM MgC12 and 5 mM Tris/Mes, pH 6.0 or as
indicated. Both the bathing and the pipette solutionij were
adjusted to 450 milliosmolar with D-sorbitol.
The mean single-channel open probabilities were calculated by using amplitude distribution histograms constructed
from single-channel records obtained at -60 mV as derxribed
by Pantoja et al. (1992a). A modification of the GoldmanHodgkin-Katz equation was used to calculate the permeability ratio between Ba2+and K+ (Pantoja et al., 1992b).
RESULTS
Calcium Release from lsolated Vacuoles
Whole vacuolar currents were recorded with BaC12, SrC12,
or CaC12 (10 mM) in the pipette (inside the vacuole) and 100
mM KC1 in the recording chamber (cytoplasm) while maintaining cytoplasmic calcium (1 X 10-6 M)and the pH gradient
(1.5 units) at physiological levels. Figure 1 shows the activation of currents from the vacuole into the cytoplasm (inward
currents) in response to voltage pulses applied to the membrane ranging from a holding potential of O mV to f I00 mV
in increments of 20 mV. Negative voltage pulses elicited large
time-dependent currents in the presence of BaC12 (Fig. lA),
whereas positive voltage pulses resulted in small instantaneous currents. The same protocol was applied to vacuoles
exposed to SrC1, (Fig. 1B) and CaCl, (Fig. 1C) in the pipette.
Again, negative voltage pulses elicited outward currents;
however, these currents were of smaller magnitude than
those recorded in the presence of BaC12.
The currents recorded with the various divalent ions were
plotted against their respective voltages (Fig. 1D). l’he current-voltage relationship demonstrated that these (currents
were voltage dependent, and of the three divalent ions used,
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Copyright © 1993 American Society of Plant Biologists. All rights reserved.
Vacuolar Calcium Channels
1141
Ba2+induced currents with the largest magnitude, followed
by SrZ+and Ca2+.
An estimate of the selectivity of the channels was obtained
by tail current experiments (Fig. 2). Inward currents reversed
at 30 mV with vacuoles exposed to 10 mM BaZ+in the pipette
and 100 mM KC1 in the recording chamber, indicating a
of 20. It should be noted that
permeability ratio, PBaZ+/PK+,
during whole-vacuole measurements a run-down of the
channel activity was observed. This phenomenon has also
been reported in a comparable study by Johannes et al.
(1992a). Because of the time-dependent inactivation in the
whole-vacuole configuration, isolated patches of tonoplast
were used to characterize further the Ca'+-selective channels.
C
Single-Channel Recordings
D
L
500 ms
E
m e m b r a n e potential (mV)
-120
-80
'
o
0
O
l0
O
-40
O
9 3
ea
o
1400
I
Single-channel activity was recorded in isolated outsideout patches (vacuolar side exposed to the pipette) with 10
m~ BaCI2,SrCI,, CaClz in the pipette (vacuolar side) and 100
mM KCl in the recording chamber (cytoplasmic side) while
maintaining cytoplasmic concentration of free Ca2+ at 1 x
10-6 M. Figures 4A and 5A show a record of single-channel
currents obtained at -80 mV with 10 mM BaClZ.
The single-channel currents were plotted against their respective voltages (Fig. 3). The current-voltage plot demonstrated that the current levels increased as the membrane
potential was made more negative and that the currents
varied linearly with decreasing membrane potentials. A single-channel conductance of 14, 10, and 6 picosiemens was
obtained with 10 mM BaZ+,Srz+, and Ca2+ as the charge
carrier, respectively. Currents recorded in the presence of
Ba2+ were of much larger magnitude than those recorded
T
A
A
Figure 1. Voltage-dependent currents from whole vacuoles of sugar
beet cells exposed to BaCI,, SrCI,, and CaCI,. A, Voltage clamp
recordings were obtained by applying voltage pulses of +100 to
-100 mV in steps of 20 mV from a holding potential of O mV. B,
With t h e vacuolar side of t h e tonoplast (pipette)exposed to 10 mM
BaCI,, negative voltage pulses (V, = V, - V,) induced time-dependent currents that reached a maximum current leve1 in 3 s. The timedependent currents were not activated at positive membrane potentials. C, Time-dependent currents were induced by negative
membrane potentials in the presence of SrCI,; however, current
levels were smaller in magnitude. Again, positive potentials did not
elicit time-dependent currents. D, In the presence of CaCI,, negative membrane potentials induced currents of a much smaller
magnitude. E, Current levels measured at 4.8 s from the onset ol
the voltage pulses (O to -100 mV) were plotted against their
respective voltages. Current levels were obtained from experiments
performed in the presence of BaCI, (A), SrCI, (O),and CaCI, (O).
Experimental data are the mean k S E of nine vacuoles.
B
i
L
1s
recordings determine t h e reversal potential
of the time-dependent currents. An estimate of current specificity
was obtained through tail current experiments. A, Currents were
activated by voltage pulses of -100 mV for 6.5 s and deactivated
by stepping up the voltage to -80 mV. This protocol was repeated
15 times with a subsequent increase in the deactivating pulse of 10
mV. Tail currents reversed direction (Erev) between 20 and 30 mV.
B, Negative voltage pulses of -1 O 0 mV induced time-dependent
inward currents. These currents were subsequently deactivated as
the voltage pulses were stepped up to less negative potentials in
increments of 10 mV. These experiments were performed with
whole vacuoles exposed to 10 m M BaCl, (vacuolar side of tonoplast)
and 100 mM KCI (cytoplasmic side of tonoplast).Similar results were
observed in at least five vacuoles.
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Copyright © 1993 American Society of Plant Biologists. All rights reserved.
Figure 2. Tail current
Gelli and Blumwald
1142
A
with S?+ or Ca2+. The current-voltage graph also revealed
-90
-60
the inward rectification of the currents at negative poteritials,
m e m b r a n e potential (mv)
corresponding to the release of the divalent cation frorn the
-30
O
30
60
90
4
+
vacuole.
11 t
A
A
-2.0
‘ s c (PA)
B
3.0 T
O
10
20
Cation activity
C
Plant Physiol. Vol. 102, 1993
30
(mM)
40
To.1o
-90
-60
-30
O
m e m b r a n e potential (mV)
Figure 3. The current-voltagerelationship of single-channel records
with Ba2+, Sr2+, and Ca2+as the charge carrier. lsolated patches of
tonoplast (outside-out)were exposed to 100 mM KCI and 10-6 M
free Ca2+ (cytoplasmic side) with 10 mM BaCI, (A), SrCb (O), and
CaC12 (O)(vacuolar side). Single-channel activity was recorded and
plotted against the respective voltage. The linear relationship between the single-channel current and the voltage is evident. Current
levels were largest with BaZ+as t h e charge carrier, as was observed
in the whole-vacuole configuration. Least-squares analysis gave
zero-current potentials (!Erev)of 40, 45, and 55 mV (arrows)for 10
mM CaCI,, SrCI,, and BaCI,, respectively. B, The dependence of
single-channel currents on the activity of Ba2+,Sr2+,and Ca2+.Singlechannel currents (Isc)recorded at -60 mV were plotted against the
activity of the different divalent cations. Channel currents saturated
with increasing concentrations of t h e cations. Inset, An EadieHofstee plot of the experimental data gave K d values of 13.3, 18.1,
and 24.3 mM and single-channel maximum currents of 3.7, 3.4, and
3.1 pA for Baz+, Si-’+, and Ca2+,respectively. C, The open probability
The selectivity of these currents was determined from
single-channel records obtained under the same conditions
as explained above. From the current-voltage graph, the zerocurrent potentials (arrows on Fig. 3A) appeared at +40, +45,
and +55 mV for Ca2+, Sr2+,and Ba2+,respectively. By substituting these values into a modified version of the GoldmanHodgkin-Katz equation (Pantoja et al., 1992b) permeability
of 20 to 23 were obtained for Ca2+,Srz+,
ratios, Pcatlon2+/PK+,
and Ba2+.Single-channel currents were also recorded in the
presence of increasing concentrations of Ba2+,Sr‘+, ancL Ca2+
to observe any saturation in the magnitudes of currents.
Currents were recorded with concentrations of 10, 20, 40,
and 80 mM BaZf, Sr2+, and CaZ+and plotted against the
activity of the divalent cations. The currents displayed an
apparent saturation with increasing activity of Ba2+,Sr2+,and
Ca2+(Fig. 3B). These current values were used to generate an
Eadie-Hofstee plot (Fig. 38, inset) from which Kd values of
13.3, 18.1, and 24.3 mM and maximum currents of 3.7, 3.4,
and 3.1 pA for Ba2+, SrZ+, and Ca2+, respectively, were
obtained. To calculate the open probability of the chamnels,
amplitude histograms were generated from the single-channel current levels (Fig. 3C). The histograms revealed that the
single-channel open probability was dependent on the vacuolar membrane potential and reached a maximum at -70
to -80 mV.
Pharmacological Characterization of the Vacuolar
CaZ+Channels
The effects of the Ca2+ channel antagonists nifedipine (a
DHP) and verapamil (a phenylalkylamine) and the c hannel
agonist Bay K 8644 (a DHP) on the single-channel ciirrents
were studied. In animal cells, verapamil and nifedipine have
been reported to inhibit voltage-gated Ca2+channels by two
distinct mechanisms (Catteral and Striessnig, 1992). F’henylalkylamines such as verapamil are effective inhibitors of the
Ca2+channel only when applied to the intracellular surface
of the channel (cytoplasmic side), where they bind to the
transmembrane pore of the channel and occlude it. DHPs
bind to their receptor from the extracellular surface of the
channel (vacuolar side) and modulate the channel actjvity by
favoring distinct modes of channel gating. With 1 X 10-4 M
verapamil present in the bath, the vacuolar calcium currents
were almost totally inhibited (not shown). Currents rtxorded
from an outside-out patch in the presence and abs’ence of
nifedipine are shown in Figure 4. These currents were recorded at a membrane potential of -80 mV with 10 m M Ba2+
in the pipette to maximize current levels. Prior to the ahddition
of nifedipine, the single-channel current recordings from
controls displayed large channel activity with at least three
of the channels is voltage dependent. Analysis of amplitude histograms constructed from single-channel records demonstrated that
the open-channel probability increased between O and -60 mV
and reached a maximum at -70 to -80 mV. Experimental data are
the mean k SE for five vacuoles.
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Vacuolar Calcium Channels
A
-C
-O,
-O,
-0,
1800
1440
2
P
1080 o,
m
720
8
$
360
O
l?
400ms
C
D
n1
T
L12
Amplitude (pA)
Figure 4. A reduction in single-channel activity by the 1,4-DHP
antagonist nifedipine. A, An original record of single-channel currents from an outside-out patch of tonoplast exposed to 10 m M
BaCl, (vacuolar side) and 100 m M KCI and 10-6 M free Ca2+ (cytoplasmic side). This trace obtained at -80 mV shows t h e activation
of at least three channels in the patch of tonoplast, as indicated by
the different current levels (O,, O,, and 03).
Similar results were
observed in at least eight vacuoles. 6, Amplitude distribution histogram from single-channel records similar to that shown above.
The largest peak of the histogram occurs at O pA and represents all
channels in the closed state. The additional peaks correspond to
one (approximately -2.0 pA) and two (approximately -4.0 pA)
channels opening simultaneously. C, A reduction in single-channel
activity by nifedipine. A single-channel record was obtained at -80
mV from an outside-out patch with the vacuolar side of the tonoplast exposed to 100 PM nifedipine. Channel activity is inhibited as
demonstrated by the reduced number of events and the shortened
open times. Similar results were observed in at least four vacuoles.
D, Amplitude distribution histogram from single-channel records
similar to that shown in C. lnhibition of channel activity is demonstrated by t h e absence of the peaks representing the simultaneous
opening of one and two channels.
1143
channels in the isolated patches (Fig. 4A). After the addition
of 100 PM nifedipine to the pipette (vacuolar side of the
tonoplast), the currents were inhibited, as shown by the
shortened open times and the reduced number of channel
openings (Fig. 4C). The inhibition of channel activity is
further demonstrated in the amplitude histogram obtained in
the absence (Fig. 4B) and presence (Fig. 4D) of nifedipine.
The absence of the peaks corresponding to the simultaneous
opening of one and two channels is evident when comparing
the two amplitude histograms.
Figure 5 shows the effect of an agonist, Bay K 8644, on the
vacuolar Ca2+ currents. Current recordings obtained in the
presence of 1 X 10-4 M Bay K 8644 in the pipette demonstrated an increase in the frequency of channel opening while
the current levels remained unchanged (Fig. 5A). Dwell time
distribution histograms were generated from single-channel
recordings obtained in the absence (Fig. 5A) and presence of
Bay K 8644 (Fig. 5C). The values for the mean open and
closed times of these channels were calculated as described
by Pantoja et al. (1992b). The similarities in the channel mean
open time (23.6 f 3.5 versus 25.2 f 4.4 ms) and mean closed
time (1388 +_ 25 versus 1212 +_ 32 ms) before and after the
addition of the agonist suggest that Bay K 8644 did not
significantly affect the time that the channel spent in the
open and closed states. The histograms of the open and
closed states were fitted with one exponential, suggesting
that only one conductive and one nonconductive state were
detected in the absence of the agonist. However, two exponentials were required when fitting the histograms of currents
recorded in the presence of Bay K 8644. This suggests that
the agonist induced the activation of another conducting and
nonconducting state of the channel. These results would
suggest that the increase in the frequency of channel openings
elicitated by Bay K 8644 was due to a mechanism other than
the prolongation of the open times.
DISCUSSION
An elevation of the cytoplasmic free Ca2+concentration to
levels above 1 X 10-6M may lead to Ca2+-activatedresponses.
Fluctuations in Ca2+concentration are the result of the Ca2+
release from intracellular Ca2+ pools such as those from
vacuoles where free Ca2+concentration is between 1 and 10
mM. The DHP-sensitive channel described in this paper provides a pathway for the release of vacuolar Ca2+ into the
cytoplasm. Other divalent cations (Ba2+and Sr2+)also moved
through these channels, suggesting that any Ca2+-mediated
response is related to a somewhat general quality of the
cations and might not be due to the action of these cations
as specific second messengers (Kauss, 1987).
The release of vacuolar Ca2+into the cytoplasm has been
shown here to be active at physiological pH values (pH =
7.5 in the cytoplasm; pH = 6.0 in the vacuole) and Ca2+
concentrations (10 mM in the vacuole; 1 X 10-6 M in the
cytoplasm). Also, the channels were active at physiological
vacuolar membrane potentials, showing voltage dependency
with an inward rectification at negative tonoplast potentials.
The probability of opening was strongly voltage dependent
and reached a maximum at higher negative potentials. These
channels are more than 20 times more selective for Caz+than
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Copyright © 1993 American Society of Plant Biologists. All rights reserved.
Gelli and Blumwald
1144
A
-
O2
r
400 mr
B
500
400
n
200
c
zo
,
O0
240
,,
,n
480
O
2400
,
4800
Time (ms)
yr
D
500
for K+. It should be made clear that in this work vacuolar
membrane potentials are considered according to a rccent
proposed convention (Bertl et al., 1992) where the vacuolar
lumen is considered to be equivalent to the extracellular
space. Therefore, the tonoplast voltage is considered to be
the difference between the voltage at the cytosolic side and
the voltage at the vacuolar side.
Measurement of unitary channel current revealed that
slope conductance was largest and the zero-current potential
was slightly more positive when Ba2+was the charge carrier.
The zero-current potential and the Kd value suggest tha,t the
channel has a higher affinity for Ba2+and Ba2+has a higher
mobility (larger conductance) through the channel compared
to Ca2+ and Sr2+.However, further information is required
before a mechanism of selection at the channel pore ciln be
proposed. For the r-type Ca2+channel in animal cells, Tsien
et al. (1987) and Hess et al. (1986) have argued that seloction
occurs through affinity for a binding site inside the channel
pore rather than rejection from a sieve. A similar process of
selectivity has been suggested for the Ca2+-selectivechannel
in the tonoplast (Johannes et al., 1992b). For excised peitches
of membrane under bi-ionic conditions (K+ on cytoplasmic
side; Ca2+on vacuolar side), the authors reported a large K+
conductance (out of the cytoplasm) and a small Ca2+ conductance (into the cytoplasm) with 15 to 20 times higher
selectivity for Ca2+over K+ where the Ca2+currents saturated
at higher membrane potentials. They suggested that there
was ionic competition for an intrapore binding site so that
permeant ions do not move through the channel independently (Johannes et al., 1992b). However, experiments reported here were also carried out under similar bi-ionic
conditions with physiological levels of free cytoplasmic Ca2+,
and the results are in contrast to those reported above, in
that the current-voltage relationship does not show a K+
conductance, nor does it show any Ca2+ current saturation
with increasing membrane potentials.
The channel reported in this paper is similar to the DHPsensitive, L-type Ca2+ channel from animal cells. In the Ltype Ca2+channels, both Ba2+ and Sr2+can replace Ca2+as
the charge carrier. L-Type Ca2+channels are inhibited by La3+
and verapamil (when applied to the cytoplasmic side of the
membrane). These channels are also sensitive to DHPs, being
inhibited by nifedipine (when applied to the extracellular side
of the membrane). The agonistic effects of Bay K 8644 on the
extracellular side of the L-type channel has been well documented in animal cells. Reports have suggested that Bay K
8644 enhanced Ca2+channel currents by increasing tlne duration of time that the channel spent in the open state (Hess
et al., 1984). Experiments where the vacuolar side (extracellular side of the channel) of the tonoplast was expc'sed to
Bay K 8644 demonstrated that the mean open time of the
channel remained essentially unchanged while the frequency
of opening increased. This was reflected by the difference
between the mean closed times of the channel (before and
after the addition of Bay K 8644). In addition, Bay IC 8644
activated a different conducting state of the channel ias well
as an additional closed state.
A similar type of response was reported by Tohse et al.
(1991) in Ca2+channels of embryonic chick heart cells, where
Bay K 8644 increased the open probability of the channel but
:
:
:kn,",
oL,
. Closed times
350
I Open times
1
0 O-
220
L
440
,
f Closed times
OO
Time (ms)
L 2400L
4800
.
Figure 5 . The effects of Bay K 8644 (a 1,4-DHP agonist) on the
single-channel kinetics. A, A single-channel record obtained from
an outside-out patch of tonopiast exposed to 10 mM BaCiz(vacuolar
side) and 100 mM KCI and 10-6 M free Caz+ (cytoplasmic side).
Channel activity was recorded at a membrane potential of -80 mV.
The trace shows the presence of at least two channels in the patch
(O1,Oz).B, The distribution of dwell times for the channel in the
open and closed state from single-channel records similar to that
shown above. The histograms for the open and the closed times
were best fit with a single exponential. Rate constants for the open
and closed times are 32.14 ms-' and 588.24 ms-', respectively. The
mean open and mean closed times are 23.6 -C 3.5 ms and 1388 f
25 ms, respectively. C, Single-channel record obtained from an
outside-out patch of tonoplast voltage clamped at -80 mV and
exposed to 1O 0 p~ Bay K 8644 (vacuolar side). Bay K 8644 increased
the frequency of channel opening and induced a second conducting state of the channel as demonstrated in the single-channel
records. D, Open-close kinetics of the channels in the presence of
100 FM Bay K 8644. Dwell time distribution histograms were constructed from single-channel records similar to that shown in C.
Histograms for both the open and closed times were best fit with
two exponentials with rate constants of 6.43 ms-' and 32.14 ms-'
(open times) and 8.22 ms-' and 428.74 ms-' (closed times). The
mean open and closed times are 25.2 k 4.4 ms and 1217 k 32 ms,
respectively.
Plant Physiol. Vol. 102, li 993
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Copyright © 1993 American Society of Plant Biologists. All rights reserved.
Vacuolar Calcium Channels
failed to prolong channel opening. These results suggested
that Bay K 8644 may be activating nonfunctional channels
or that it may be enhancing the availability of each Ca2+
channel. Thus, the agonistic action of Bay K 8644 on Ca2+
channels may include multiple mechanisms.
The sensitivity of this channel to DHPs was further supported by experiments performed with nifedipine. Experiments revealed an inhibition of channel activity when nifedipine was present on the vacuolar side of the tonoplast. The
single-channel current was not completely blocked, because
some brief openings were still observed. This suggests that
nifedipine does not block the pore of the channel but rather
may bind near the outer surface of the channel. A similar
response was observed when the cytoplasmic side of the
tonoplast was exposed to the phenyalkylamine verapamil.
Inorganic inhibitors such as La3+eliminated channel activity
almost completely. These channels bear similarities to the
L-type Ca2+channels from animal cells in their sensitivity to
DHPs; however, their mode of action, as in the case of Bay
K 8644, may involve different mechanisms.
Another similarity between the inward rectifying Ca2+
channel reported here and the L-type channel is the rundown
in channel activity observed in excised patches. Channel
rundown of the L-type channel has been reported mostly in
isolated patches; however, we observed this both in the
whole vacuole and in excised patches of tonoplast. Johannes
et al. (1992a) report a similar channel rundown when vacuolar pH was lowered, and they suggest that cytoplasmic
factors might be involved in channel activity. The pH difference, acidic inside, that was maintained across the tonoplast
in this work could have contributed to the observed rundown
in channel activity.
The evidence provided in this paper suggests that the
currents recorded in both the whole vacuole and the isolated
patch moved through voltage-dependent Ca2+ channels.
Moreover, these channels were active at physiological vacuolar membrane potentials, cytoplasmic and vacuolar pH values, and reported resting levels of cytoplasmic and vacuolar
Ca2+ concentrations. The elevation of cytoplasmic Ca2+ to
values higher than 1 X 10-6 M reduced channel activity, and
values of 1 X 10-4 M resulted in total inhibition of channel
currents (data not shown). These results contradict the findings by Johannes et al. (1992a) in which channel activity was
unaffected by relatively high cytoplasmic CaZ+concentrations. Thus, our results would suggest that the inward-rectifying vacuolar Ca2+channels reported here can play a central
role in the release of Ca2* from the vacuolar pool into the
cytoplasm that is needed to initiate various signal transduction processes in higher plants.
An alternative explanation to our results could be that the
currents characterized in this paper were due to a Caz+activated chloride channel that would facilitate the movements of chloride out of the cytoplasm into the vacuole. This
hypothesis can be ruled out due to the following observations: (a) the replacement of divalent cations with KCI in the
vacuole did not induce a significant anion current into the
vacuole (results not shown). With 100 mM KCl in the cytoplasmic side and 10 mM KCl with 1 mM CaC12in the vacuole,
ion currents reversed at a E,,, approximately -30 mV, indicating that the currents were predominantly due to the move-
1145
ment of K+ ions out of the cytoplasm into the vacuole: (b)
recent experiments in our laboratory revealed instantaneous
vacuolar chloride currents (time independent) that would
facilitate the transport of chloride ions out of the vacuole into
the cytoplasm (our unpublished results).
A second pathway for the intravacuolar Ca2+ release
through a channel activated by IP3 has been reported in
excised tonoplast patches (Alexandre et al., 1990) and in
plant microsomes (Brosnan and Sanders, 1990). We have
tried to confirm these results through the application of the
patch-clamp technique in both excised patches and whole
vacuoles under the conditions reported by the authors and
the different conditions reported in this paper. Nevertheless,
all our attempts to confirm these findings failed.
In conclusion, this study provides further evidence of a
mechanism for the elevation of cytoplasmic Ca2+. These
channels are active at physiological Ca2+concentrations, pH,
and vacuolar membrane potentials. The increase in cytosolic
Ca2+ could act as a positive feedback mechanism for Ca2+activated Ca2+release from other intracellular storage pools
(Schroeder and Thuleau, 1991) that could trigger the initiation of signal transduction processes. Moreover, the efflux of
Ca2+from the vacuole into the cytoplasm will depolarize the
tonoplast potential toward the Ca2+ equilibrium potential
(50-100 mV). The depolarization of the tonoplast could serve
as a negative feedback mechanism to attenuate the signal
transduced by Ca2+. Thus, depolarization of the tonoplast
together with the increase in Ca2+concentration in the vicinity of the vacuole will allow the influx of K+ into the vacuole
through Ca2+-activated K+ channels (SV-type channels)
(Hedrich and Neher, 1987) and the influx of Ca2+through
Ca2+channels activated at positive tonoplast potentials (Pantoja et al., 1992b). The influx of positively charged ions into
the vacuole will result in the polarization of the vacuolar
potential to resting levels. Return of the remaining cytoplasmic Ca2+to steady-state levels would be achieved by the
plasma membrane Ca2+-ATPase(Rasi-Caldogno et al., 1987)
and the vacuolar Ca2+/nH+antiport (Blumwald and Poole,
1986; Schumaker and Sze, 1987; Blackford et al., 1990).
ACKNOWLEDCMENT
The authors thank Dr. Omar Pantoja, Oxford University, UK, for
his assistance and helpful discussions.
Received February 5, 1993; accepted April 13, 1993.
Copyright Clearance Center: 0032-0889/93/l02/1139/08.
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