From the Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322-3030
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ABSTRACT |
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Xenopus oocytes express several different Ca-activated Cl currents that have different waveforms and biophysical properties. We compared the stimulation of Ca-activated Cl currents measured by two-microelectrode voltage clamp with the Ca transients measured in the same cell by confocal microscopy and Ca-sensitive fluorophores. The purpose was to determine how the amplitude and/or spatio-temporal features of the Ca signal might explain how these different Cl currents were activated by Ca. Because Ca release from stores was voltage independent, whereas Ca influx depended upon the electrochemical driving force, we were able to separately assess the contribution of Ca from these two sources. We were surprised to find that Ca signals measured with a cytosolic Ca-sensitive dye, dextran-conjugated Ca-green-1, correlated poorly with Cl currents. This suggested that Cl channels located at the plasma membrane and the Ca-sensitive dye located in the bulk cytosol were sensing different [Ca]. This was true despite Ca measurement in a confocal slice very close to the plasma membrane. In contrast, a membrane-targeted Ca-sensitive dye (Ca-green-C18) reported a Ca signal that correlated much more closely with the Cl currents. We hypothesize that very local, transient, reversible Ca gradients develop between the subplasmalemmal space and the bulk cytosol. [Ca] is higher near the plasma membrane when Ca is provided by Ca influx, whereas the gradient is reversed when Ca is released from stores, because Ca efflux across the plasma membrane is faster than diffusion of Ca from the bulk cytosol to the subplasmalemmal space. Because dissipation of the gradients is accelerated by inhibition of Ca sequestration into the endoplasmic reticulum with thapsigargin, we conclude that [Ca] in the bulk cytosol declines slowly partly due to futile recycling of Ca through the endoplasmic reticulum.
Key words: store-operated Ca entry; voltage clamp; calcium imaging; Xenopus; oocyte ![]() |
INTRODUCTION |
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Many G protein- and tyrosine kinase-associated receptors stimulate phospholipase C and the production of
inositol 1,4,5-trisphosphate (IP3),1 resulting in a rise in
cytosolic Ca ([Ca]c). The rise in [Ca]c in response to activation of these receptors is typically biphasic, starting
with a transient increase due to IP3-stimulated Ca release from internal endoplasmic reticulum (ER) stores,
followed by a long lasting [Ca]c rise due to entry of extracellular Ca through store-operated Ca channels
(Putney, 1990; Pozzan et al., 1994
; Parekh and Penner,
1997
). Ca entry through this pathway is termed store-operated Ca entry (SOCE), formerly known as capacitative Ca entry (Putney, 1986
). Ca often participates in
multiple signaling pathways that are each controlled by
different extracellular signals in the same cell. For example, Ca controls phototaxis, mating, and deflagellation in the single-celled alga Chlamydomonas (Quarmby and Hartzell, 1994
), mechanosensitivity and afferent
and efferent synaptic transmission in vertebrate hair
cells (Lenzi and Roberts, 1994
), and gene expression
and synaptic transmission in neurons (Ghosh and
Greenberg, 1995
; Finkbeiner and Greenberg, 1996
;
Bito et al., 1997
). A fundamental problem in Ca signal
transduction involves understanding how these different pathways are kept separate and how different effector systems discriminate between Ca signals. There is
growing evidence that spatial, temporal, and amplitude factors are involved in the process of Ca signal discrimination. For example, certain Ca signals may be specifically localized to microdomains that contain the appropriate effector molecules (Thorn, 1996
; Rios and Stern,
1997; Landolfi et al., 1998
; Neher, 1998
). Spatial separation of Ca signals is often achieved by localization of
the Ca sources and sinks, which include Ca buffers that
restrict the diffusion of Ca. Specificity of Ca signals is
also encoded in how effectors respond most efficiently
to Ca. Certain effectors respond optimally to frequency-modulated Ca oscillations, whereas others respond to steady state Ca elevations (Hajnoczky et al., 1995
; Thomas et al., 1996
; Berridge, 1997b
; Bito et al.,
1997
; Dolmetsch et al., 1997
; Deisseroth et al., 1998
; De
Koninck and Schulman, 1998
).
Xenopus oocytes express several endogenous Ca-activated Cl currents that have different waveforms and
biophysical properties (Miledi and Parker, 1984; Parker
et al., 1985
; Gillo et al., 1987
; Dascal, 1987
; Parker and
Miledi, 1987a
,b; Snyder et al., 1988
; Berridge, 1988
;
Boton et al., 1989
; Parker and Ivorra, 1991
; Hartzell, 1996
). We have characterized three Ca-activated Cl currents: a noninactivating outward Cl current (ICl1-S) that
is activated by Ca released from stores, and a slow inward Cl current (ICl2) and a transient outward Cl current (ICl1-T) that are activated by Ca influx through
store-operated Ca channels (Hartzell, 1996
). The presence of multiple currents with distinct properties could be explained by the presence of several types of channels or by a single channel whose properties depend
upon the concentration and/or spatio-temporal features of the Ca signal that activates it (Kuruma and
Hartzell, 1998).
The Cl currents in Xenopus oocytes offer an excellent opportunity to understand how different Ca signals ultimately result in physiologically different responses. Our objective here was to determine how Ca-activated Cl currents were related to changes in cytosolic Ca stimulated by IP3 injection into the oocyte. We were surprised to find that the correlation between Ca signals measured using a cytosolic Ca-sensitive dye, dextran-conjugated Ca-green-1, and Cl currents was quite poor. Analysis of these results suggested that Cl channels, which were located at the plasma membrane, were sensing different Ca than was detected by the Ca-sensitive dye, which was located in the bulk cytosol. This was true even though we were measuring Ca using confocal microscopy in a slice of oocyte very close to the plasma membrane. We demonstrated the presence of transient Ca gradients between bulk cytosol and plasmalemma by showing that a membrane-targeted Ca-sensitive dye (Ca-green-C18) reported a Ca signal that correlated much more closely with the Cl currents. We hypothesize that these transient gradients arise because Ca efflux across the plasma membrane is faster than diffusion of Ca from the bulk cytosol to the subplasmalemmal space. This slow diffusion is partly due to the gigantic size of the oocyte (~1 mm diameter). In addition, we find that slow Ca clearance from the cytoplasm plays a role in maintaining these transient gradients. This slow decline of cytosolic Ca levels is due to futile recycling of Ca through the endoplasmic reticulum.
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MATERIALS AND METHODS |
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Isolation of Xenopus Oocytes
Stage V-VI oocytes were harvested from adult albino (or normal)
Xenopus laevis females (Xenopus I) as described by Dascal (1987).
Xenopus were anesthetized by immersion in Tricaine (1.5 g/liter). Ovarian follicles were removed and digested in normal Ringer with no added calcium, containing 2 mg/ml collagenase
type IA (Sigma Chemical Co.), for 2 h at room temperature. The
oocytes were extensively rinsed with normal Ringer, placed in
L-15 medium (GIBCO BRL), and stored at 18°C. Oocytes were
usually used between 1 and 5 d after isolation.
Imaging and Electrophysiological Methods
Methods are described in detail in Hartzell (1996) and Machaca
and Hartzell (1998)
. In brief, Xenopus oocytes were injected with
23 nl Ca-green-1 coupled to 70 kD dextran (333 µM) for a final
calculated oocyte concentration of ~7.6 µM, or with 13 nl Ca-green-C18 (333 µM) and voltage-clamped with two microelectrodes as described in Fig. 1. Confocal images were acquired using a Zeiss LSM 410 confocal box fitted to a Zeiss Axiovert 100TV
inverted microscope using a Zeiss 10× objective (0.5 NA). For
Ca-green-dextran, the pinhole was fully open, resulting in a focal
section of 1,275 × 1,275 × 35 µm illustrated by the hatched box
in Fig. 1 a. For Ca-green-C18, the focal section was 200 × 1,275 × 4 µm. Image data was analyzed using NIH image 1.60 on a Mac
IIfx and voltage-clamp data was analyzed on a Pentium PC using
Origin 5.0 (Microcal Software). For plots of Ca fluorescence, the
fluorescence intensity of the entire confocal section was averaged
and expressed as a ratio of the background fluorescence taken either before IP3 injection or extracellular Ca addition, depending
on the experimental design. Experiments were performed at
room temperature (22-26°C). The extracellular solutions used
were (mM): normal Ringer, 123 NaCl, 2.5 KCl, 1.8 CaCl2, 1.8 MgCl2, 10 HEPES, pH 7.4; and Ca-free Ringer solution, which
was the same except that CaCl2 was omitted, MgCl2 was increased
to 5 mM, and 0.1 mM EGTA was added.
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Endogenous Ca activated Cl currents, referred to as ICl1-S, ICl2,
and ICl1-T were measured as described in Fig. 1 b. ICl1-S (current at
the end of the first +40-mV pulse, Fig. 1 b) is a sustained outward
current at depolarizing potentials that is activated quickly (~10 s)
after IP3 injection by Ca released from intracellular stores and
has an outwardly rectifying steady state current-voltage relationship. ICl2 (maximum current during the 140-mV pulse, Fig. 1 b)
is an inward current that is activated by Ca entry through store-operated Ca channels (SOCs) driven by the negative membrane potential and has an inwardly rectifying steady state current-voltage relationship. ICl1-T is a transient outward current (peak outward current during the second +40 mV pulse, Fig. 1 b) that was
activated by a depolarizing pulse preceded by a hyperpolarizing
pulse to stimulate Ca influx. For a more detailed characterization
of the Cl currents, see Hartzell (1996)
, Machaca and Hartzell
(1998)
, and Kuruma and Hartzell (1999)
.
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RESULTS |
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Voltage Dependence of Ca Entry
The first goal was to characterize the Ca signal which was
caused by Ca influx through SOCs. Ca influx through
SOCs was measured by imaging Ca in oocytes injected
with dextran-conjugated Ca-green-1 using confocal scanning microscopy while voltage clamping the oocyte with a
two-microelectrode voltage clamp. The confocal fluorescence image reported cytoplasmic Ca primarily because
the injected 70-kD-coupled Ca-green-1 is too large to partition into organelles (Luby-Phelps, 1989; Girard and
Clapham, 1993
; Camacho and Lechleiter, 1993
; Jouaville
et al., 1995
). We also used Ca-green-5N, which has a lower
Ca affinity, and obtained identical results. The oocyte was
voltage clamped at a holding potential of 0 mV and
pulsed to potentials between
140 and +120 mV for 1 s
at 30-s intervals. Before IP3 injection, the Ca fluorescence (FCa/F0) was very small and was independent of
voltage (not shown). However, if we allowed enough
time after IP3 injection for store depletion and activation of SOCE (~10 min), the Ca signal increased significantly with membrane hyperpolarization (Fig. 2 a,
).
We believe that the voltage-dependent Ca fluorescence
reflected the accumulation of Ca in the oocyte due to Ca
influx through SOCs because the Ca fluorescence was
dependent on extracellular Ca (Fig. 2 a,
) and the voltage dependence of the Ca fluorescence corresponded
closely to the voltage dependence of the store-operated
Ca current (ISOC) we have previously characterized electrophysiologically (Hartzell, 1996
; Yao and Tsien, 1997
;
Kuruma and Hartzell, 1999
; Fig. 2 b). ISOC resembled the
prototypic SOC current, ICRAC: it strongly inwardly rectified with a positive reversal potential (Hoth and Penner,
1993
; Yao and Tsien, 1997
).
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The voltage-dependent Ca fluorescence was also
stimulated by depleting intracellular Ca stores independently of IP3. Treatment with ionomycin (Fig. 2 a, ), a
Ca ionophore that selectively releases Ca from ER Ca
stores (Morgan and Jacob, 1994
), or prolonged treatment with thapsigargin, which blocks the ER Ca-ATPase
and depletes Ca stores passively by unopposed leak of
Ca out of the stores (Bird et al., 1992
), stimulated voltage-dependent Ca fluorescence.
Ca Release from Stores Is Voltage Independent
If Ca release from stores is voltage independent, then
the voltage dependence of the Ca signal caused by Ca
influx provides a means to separate the fluorescence
signals due to Ca influx and Ca release from stores. We
therefore tested whether IP3-induced Ca release from
stores was voltage independent by comparing the Ca
fluorescence at +40 and 140 mV when the oocyte was
bathed in Ca-free solution so that there could be no Ca
influx. Fig. 3 a shows pairs of images taken during the
+40- and
140-mV pulses at different times during the
experiment and a thresholded image (
) that shows
the difference between the images at
140 and +40 mV. IP3 injection (at 1 min) released Ca in a wave that
swept through the entire oocyte (2.5 min) as described
by Lechleiter and Clapham (1992)
. The Ca wave eventually subsided (10 min). The amplitudes of the Ca fluorescence at +40 and at
140 mV during the wave of
Ca release were not significantly different (Fig. 3 b).
This shows that Ca release from stores was independent
of voltage. Voltage-dependent Ca fluorescence (Fig. 3
c) was shown to require Ca entry because addition of
Ca to the bath (14.5 min) resulted in an abrupt increase in Ca fluorescence at
140 mV, whereas the fluorescence at +40 mV changed only slightly (Fig. 3 b).
Operationally, store-operated Ca entry has been defined as the increase in Ca fluorescence observed upon
addition of Ca to the bathing solution after internal
stores have been depleted in Ca-free solution (Putney,
1990
; Meldolesi et al., 1991
). On the basis of this definition, the voltage-dependent Ca fluorescence we observe in Fig. 3 is store-operated Ca entry. However,
clearly, Ca fluorescence does not measure Ca influx directly, but rather measures the accumulation of Ca,
which is the final result of influx, efflux, equilibration
with Ca buffers, Ca-induced Ca release from stores, and
sequestration.
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Ca-induced Ca Release Contributes Negligibly to the Voltage-dependent Ca Fluorescence
Ca influx can trigger Ca release from stores in Xenopus
oocytes (Yao and Parker, 1993, 1994
) as a result of the
synergistic interaction of IP3 and Ca on the IP3 receptor
(Iino, 1990
; Parker and Ivorra, 1990
; Bezprozvanny et al.,
1991
; Finch et al., 1991
). Thus, the voltage-dependent
Ca fluorescence could be a combination of Ca entry
and subsequent release of additional Ca from stores not completely depleted by the IP3 injection. Contribution of Ca-induced Ca release (CICR) to the voltage-
dependent Ca signal was assessed in Fig. 3 by adding
ionomycin to release any Ca remaining in stores after
IP3 injection. Ionomycin had no effect on fluorescence at either +40 or
140 mV or the voltage-dependent Ca
signal (Fig. 3, a-c). Thus, CICR contributes negligibly
to the voltage-dependent Ca fluorescence after IP3 injection because IP3 virtually depletes the stores.
Correlation between Ca Fluorescence and Cl Currents
Xenopus oocytes possess several Ca-activated Cl currents
that have been used as real-time detectors of Ca at the
plasma membrane (Miledi and Parker, 1984; Parker et al.,
1985
; Gillo et al., 1987
; Dascal, 1987
; Parker and
Miledi, 1987a
,b; Berridge, 1988
; Snyder et al., 1988
;
Boton et al., 1989
; Parker and Ivorra, 1991
; Hartzell, 1996
). The development of these currents and their relationship to cytosolic Ca after IP3 injection are shown
in Fig. 4. The oocyte was initially bathed in Ca-free solution and voltage clamped from a holding potential of 0 mV with a three-step episode consisting of steps to +40
mV for 1 s,
140 mV for 2 s, and +40 mV for 1 s with
an interpulse interval of 26 s. The first +40 mV step is labeled +40 mV[1], and the second +40 mV[2]. See
MATERIALS AND METHODS and Fig. 1 for details of Cl
currents measurement. Immediately after IP3 injection,
ICl1-S was activated during both +40 mV steps of the episode (Fig. 4, a and b, trace a) and the Ca-green fluorescence (Fig. 4 c) increased in a voltage-independent
manner at all potentials. The activation of ICl1-S preceded the increase in Ca fluorescence (Fig. 4 d). This
was at least partly due to the fact that Cl currents were
measured from the entire cell, whereas the confocal
image was obtained from a superficial optical slice at
the pole opposite to the site of IP3 injection (see Fig.
1). Therefore, the Ca fluorescence reached its maximum more slowly than ICl1-S due to the time required for the Ca wave to travel from the injection site to the
confocal plane (Machaca and Hartzell, 1998
). More
surprisingly, t1/2 of decay of Ca fluorescence was approximately seven times slower (P < 0.005; n = 7) than
the t1/2 of decay of ICl1-S (3.78 ± 0.68 vs. 0.52 ± 0.05 min) (Fig. 4 d, inset). Addition of Ca to the bath increased the voltage-dependent Ca fluorescence, which
was stable for at least 15 min (Fig. 4 e). The amplitude
of the voltage-dependent Ca fluorescence (Ca entry)
was quite small compared with the voltage-independent Ca fluorescence (Ca release). In contrast, the ICl1-T
current activated by Ca entry was larger than the current activated by Ca release (ICl1-S).
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Although the voltage-dependent Ca fluorescence
correlated relatively well with both ICl1-T and ICl2 when
Ca was added after the SOCE mechanism was fully activated (Fig. 4 f), the correlation between ICl2 and Ca fluorescence was poor when one examined their temporal
development after IP3 injection (Fig. 5). Fig. 5 shows the results of an experiment in which the oocyte was
bathed in normal Ca-containing solution from the onset of the experiment. Injection of IP3 rapidly stimulated ICl1-S (Fig. 5, a and b). As ICl1-S returned to baseline, ICl1-T and ICl2 developed (Fig. 5, a and b). Concomitant with the injection of IP3, there were large
increases in Ca fluorescence during both +40- and
140-mV pulses (Fig. 5 c) that closely resembled the
wave of Ca release seen in oocytes bathed in Ca-free solution (Fig. 4 c). The amplitudes of the Ca fluorescence
during the +40- and
140-mV pulses were identical
during the wave of Ca release, but shortly after the peak
of the Ca release wave, the
140-mV Ca fluorescence
became greater than the +40-mV[1] Ca fluorescence
(Fig. 5 c) as SOCE developed (Fig. 5 d). Voltage-dependent Ca fluorescence was apparent immediately after
Ca release from stores and increased gradually over a
period of ~5 min to reach a plateau that remained stable for at least 20 min (Fig. 5 d). Both voltage-dependent Ca fluorescence and the Cl currents were abolished when the cell was switched to Ca-free solution
(Fig. 5, a-d). The time course of development of ICl1-T
and voltage-dependent Ca fluorescence were very similar (half-times 3.12 ± 0.25 and 3.99 ± 0.56 min, respectively), but ICl2 development was significantly (P < 0.0055) slower (t1/2 = 7.2 ± 0.7 min) (Fig. 5, e and f).
These data agree with our other results showing that
ICl1-T development parallels the development of ISOC,
but that ICl2 develops more slowly (Kuruma and Hartzell, 1999
). As was the case when the cell was bathed in
Ca-free solution (Fig. 4 d), the fluorescence Ca signal
decayed significantly slower (P < 0.00004) than ICl1-S
(Fig. 5 f).
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From the data in Figs. 4 and 5, we conclude that in general the correlation between Cl currents and Ca fluorescence is rather poor. Although there are several possible explanations for these data (see DISCUSSION), we believe that the data are most simply explained if the Ca fluorescence and the Cl channels report different Ca.
Fast Dynamics of Ca
We then compared Ca fluorescence and Cl currents on
a faster time scale. We acquired 15 × 512 pixel images
every 100 ms during the voltage-clamp episodes ~10
min after IP3 injection when SOCE had developed. Fig.
6 a shows the time course of the Ca fluorescence during a standard three-step voltage clamp episode every
30 s from a holding potential of 0 mV. The Ca fluorescence remained stable until the membrane was hyperpolarized to 140 mV, at which time the Ca fluorescence increased as Ca entered the cell. Upon returning
to +40 mV[2], the Ca fluorescence began to decline. The decline continued at the same rate when the cell
was returned to the 0-mV holding potential. Ca fluorescence returned to baseline ~10 s after the end of the
140-mV pulse. In Fig. 6 b, Ca fluorescence is superimposed with the current traces on a faster time scale to illustrate the correspondence between Ca fluorescence
and Cl currents. Ca fluorescence did not correlate well
with the Cl current waveforms. For example, although
Ca increased steadily during the
140-mV pulse, ICl2 inactivated partly during the same time period. Furthermore, during the +40 mV[2] pulse, Ca fluorescence declined quite slowly, but ICl1-T had completely inactivated.
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Fig. 6 c shows 3-s-duration Ca traces (as in Fig. 6 b)
taken at 75-s intervals during the time course of an entire experiment before and after IP3 injection. Before
IP3 injection, the Ca trace was flat at all voltages (first
trace). Upon injection of IP3, the fluorescence increased slightly at all voltages (second trace) and,
within 2.5 min (third trace), the Ca fluorescence had
increased significantly at all voltages with the fluorescence being slightly greater during the 140 mV and
+40 mV[2] pulses. As the Ca fluorescence during the
+40 mV[1] step returned toward baseline, the difference in Ca fluorescence between the
140 mV and
+40 mV[1] steps became progressively greater as store-operated Ca entry developed. These data confirm our
previous conclusions that SOCE began to develop very
early after release of Ca from stores, but that its full development required several minutes.
Ca Fluorescence and Cl Currents Report Spatially Different Ca Concentrations
Figs. 4-6 show that Cl currents and Ca fluorescence do not correlate well. Because Cl channels are at the plasma membrane, they necessarily respond to Ca at the plasmalemma. In contrast, Ca fluorescence is collected from an optical section that includes cytoplasm that may be physiologically remote from the plasmalemma. If the Cl channels and the Ca-sensitive dye are reporting Ca signals that are spatially different, this suggests the presence of Ca gradients between the plasma membrane and cytosol. We performed the following experiments to test the hypothesis that after release of Ca from stores, Ca levels in a layer immediately below the plasma membrane become lower than in the adjacent cytosol because of rapid Ca efflux.
Slowing the inactivation of ICl1-S by La.
We predicted that
if Ca levels immediately below the plasma membrane
are lower than in the cytosol because of Ca efflux, inhibition of the plasma membrane efflux pathways would
keep subplasmalemmal Ca levels elevated and the decay of ICl1-S after release of Ca from stores should be
slowed. We tested this prediction by studying the decay
of ICl1-S in oocytes bathed in control or La3+-containing
solutions (1 mM) to inhibit both the plasma membrane Ca-ATPase (Sarkadi et al., 1977) and the Na-Ca exchanger (Kimura et al., 1986
) (Fig. 7). La3+ at 1 mM
has been shown to inhibit ~70% of total Ca efflux
(Brommundt and Kavaler, 1987
). In control cells, ICl1-S
inactivated in <2 min, whereas, when Ca efflux was inhibited by La3+, ICl1-S required >10 min to return to
baseline levels (Fig. 7 a). The t1/2 of decay of ICl1-S in
control (0.55 ± 0.08 min) and La3+-treated (3.62 ± 0.49 min) cells were significantly different (P < 0.000036) (Fig. 7 b). Not only did ICl1-S last longer in La3+-containing solutions, but its amplitude was ~1.5×
greater than that of ICl1-S under control conditions.
These data are consistent with the hypothesis that inhibition of plasma membrane Ca efflux significantly increased the amplitude and duration of the Ca transient below the plasma membrane.
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Ca imaging with lipophilic Ca-sensitive dyes.
A direct test
of the hypothesis that there are Ca gradients between
the bulk cytosol and subplasmalemma would be to image subplasmalemmal Ca. This was done by injecting
the lipophilic Ca-sensitive dye Ca-green-C18, which incorporates into membranes with high affinity and has
been used to measure Ca in the immediate vicinity of
the plasma membrane. We imaged Ca in the plane of the plasma membrane using the smallest scanning pinhole, permitting a z-axis resolution of ~4 µm. Under
these conditions, we found that the Ca fluorescence increased and decreased much more rapidly than with
Ca-green-dextran (Fig. 8 a). The half-time of decay of
the Ca fluorescence after the 140 mV pulse was 3.6 ± 0.4 s with Ca-green-dextran and 1.25 ± 0.1 s with Ca-green-C18 (Fig. 8 b). Furthermore, the waveform of the
Ca fluorescence measured with Ca-green-C18 much
more closely approximated the waveform of the Cl currents (Fig. 8 c) than the fluorescence measured with Ca-green-dextran (Fig. 6 b). These results support the
hypothesis that Ca in the bulk cytosol is different than
Ca immediately under the membrane.
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Recycling of Ca through the ER
The finding that Ca under the plasma membrane is different than in the bulk cytosol raises questions about
the mechanisms responsible for establishing this gradient. It is well known that Ca diffusion in the cytosol is
much slower than diffusion in simple aqueous solution
(Allbritton et al., 1992). If IP3 injection into the cytoplasm stimulates "instantaneous" release of a bolus of Ca from stores, a wave of Ca will diffuse from the site of
injection radially. If efflux of Ca from the cell is faster
than the diffusional flux of Ca to the plasmalemma,
this would create a Ca gradient with the Ca concentration higher deeper in the cytosol. Diffusional flux of Ca
from the cytosol to the subplasmalemmal space could be slowed significantly simply by immobile Ca buffers
in the cytosol. However, the possibility existed that uptake of Ca into the endoplasmic reticulum by sarcoplasmic-ER Ca ATPases (SERCAs) also contributed to the
slow Ca clearance. To test this hypothesis, we measured the effect of thapsigargin on the rate of decline of Ca
fluorescence after IP3-induced Ca release from stores.
We predicted that if SERCAs contribute to the retention of Ca in the cytosol, thapsigargin should accelerate
the decay of Ca fluorescence. Fig. 10 shows that the
half-time of decline of the Ca fluorescence in control
oocytes (t1/2 = 3.78 ± 0.68 min, n = 7) was 2.4× longer
(P < 0.009) than in thapsigargin-treated oocytes (t1/2 = 1.48 ± 0.24 min, n = 7). This result showed that SERCAs play an important role in maintaining elevated Ca
levels in the cytosol for prolonged periods of time.
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This may seem counterintuitive. Normally, one would expect that SERCA-mediated uptake of Ca into stores would accelerate clearance of Ca from the cytosol, but here SERCA-mediated uptake slows clearance. The reason is that, because of the large IP3 injection, a large fraction of the IP3 receptors are open. Nevertheless, Ca will be pumped into the ER by the SERCAs, only to be released into the cytosol through the open IP3 receptors (see DISCUSSION). The uptake of Ca into the ER under these conditions is futile in refilling stores, but clearance of Ca from the cytosol will be greatly slowed because a single Ca molecule may be taken up multiple times in its path towards the plasma membrane, and transient binding to Ca binding proteins in the ER lumen may prolong its residence there.
To test further the idea that the Ca gradients are due
to Ca recycling by the ER, we performed the experiments shown in Fig. 11. The rationale of these experiments was to compare Ca fluorescence and Ca-activated Cl currents in control oocytes, oocytes injected with heparin to block Ca release by the IP3 receptor,
and oocytes treated with thapsigargin to block Ca uptake into the ER. Ca stores were depleted by placing
the oocytes in Ca-free solution and injecting IP3 1-2 h
before voltage clamping. The oocytes were voltage
clamped and the extracellular solution was changed to
one containing Ca at the time indicated. Switching control oocytes to Ca-containing solution quickly activated
ICl1-T and ICl2 (Fig. 11 a) and produced a biphasic increase in Ca fluorescence at all potentials (Fig. 11 b).
The initial abrupt increase was voltage dependent, being larger at 140 mV, and thus related to Ca entry.
The abrupt increase was followed by a slower increase
that was voltage independent, having approximately
the same slope at
140 and +40 mV and which continued to increase for >20 min. The voltage-dependent
Ca fluorescence increased abruptly upon Ca addition,
and then declined slightly over the next 20 min (Fig. 11
c). The decline in Ca entry and in the Cl currents was
probably due to partial refilling of ER Ca stores by influxed Ca.
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When the oocytes were treated with thapsigargin for
>3.5 h to block SERCA-dependent Ca uptake into the
ER, a very different profile was observed (Fig. 11, d-f).
Addition of Ca to the bath produced an immediate and
stable increase in Ca fluorescence during the 140 mV
and +40 mV[2] steps. Ca fluorescence during the +40 mV[1] step increased abruptly by a small amount, but
then remained steady for ~20 min, as did voltage-
dependent Ca entry and ICl1-T and ICl2. This is in marked
contrast to the control oocytes, where the Ca fluorescence increased steadily over time. Ca fluorescence traces at +40 mV[1] and
140 mV from thapsigargin-treated oocytes (Fig. 11 e) were superimposed on control Ca fluorescence traces in Fig. 11 b to illustrate the
differences in Ca fluorescence changes over time. It is
clear that the steady increase in Ca fluorescence observed in control oocytes was eliminated by thapsigargin
treatment. The time-dependent increase of the voltage-independent Ca fluorescence (FCa+40[1]) was significantly
(P < 0.047) greater in control oocytes than in oocytes
treated with thapsigargin or heparin. These data show
that when SERCAs are active, the level of cytosolic Ca becomes greater with time. These data can be explained if
one assumes that transient uptake of Ca into the ER via
SERCAs facilitates the retention of Ca in the cytosol.
Both the Cl currents (ICl1-T and ICl2) and SOCE activated
immediately after Ca addition and remained at steady
levels for at least 20 min (Fig. 11, d and f) because inhibition of the SERCAs prohibited Ca uptake into stores.
When oocytes were injected with heparin to block
the IP3 receptor after the stores were depleted, the converse picture was observed (Fig. 11, g-i). Addition of Ca
resulted in a transient increase in the Ca fluorescence
during the 140 mV and +40 mV[2] steps, but Ca returned to control levels in ~10 min. The transient nature of Ca entry was reflected by the time course of ICl1-T and ICl2 (Fig. 11 g). We interpret the transient nature of
the Ca fluorescence as being caused by refilling of the
Ca stores upon readdition of Ca to the bath. Because
the IP3 receptor was blocked and could not release Ca,
the ER was capable of accumulating Ca, and SOCE entry was inactivated. The negative slope in the voltage-dependent fluorescence in control and heparin-treated oocytes reflects partial refilling of the stores during the
course of the experiment. Refilling is more significant
in heparin-treated oocytes because the stores cannot release sequestered Ca.
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DISCUSSION |
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Reversible Ca Gradients Develop between the Plasma Membrane and Cytosol
We conclude that in Xenopus oocytes gradients of Ca exist between the cytoplasm and the plasma membrane.
When Ca is released from internal stores by injection of
IP3, Ca diffuses from the injection site and activates Ca-activated Cl channels at the plasma membrane. Clearance of Ca from the cytosol is probably mediated under
our conditions largely by uptake into mitochondria
and efflux into the extracellular space, because accumulation of Ca in the ER is circumvented by the open
IP3 receptors. In our studies, large amounts of IP3 were
injected (initial calculated oocyte concentrations were
~50 µM), and the half-life of IP3 in Xenopus oocytes has
been estimated to be between 1 and 10 min, depending on IP3 and Ca concentration (Shapira et al., 1992; Sims
and Allbritton, 1998
). In addition, IP4, which is one of
the metabolic products of IP3, has a lifetime of ~30
min (Sims and Allbritton, 1998
) and is also capable of
releasing Ca from stores (Parker and Ivorra, 1990
; Ivorra et al., 1991
; DeLisle et al., 1995
). For these reasons, it seems reasonable to presume that IP3 receptors remain open for at least tens of minutes under our conditions.
Using Ca-green-dextran and a confocal section including ~35 µm of cytosol below the plasma membrane, we found that the Ca fluorescence after IP3 injection had a half-time of decay of 1.5 min when Ca uptake into ER stores was blocked with thapsigargin. When SERCAs were not inhibited, the decay was 2.4× slower (Fig. 10 a). This shows that the clearance of Ca from the cytosol was slowed by uptake into the ER. In contrast, the half-time of the Ca signal measured at the plasma membrane with Ca-green-C18 was only 0.7 min (Fig. 9 b). This showed clearly that Ca was cleared from the subplasmalemmal space more quickly than from the cytosol further away from the surface. We believe that this gradient arose at least partly because the diffusional supply of Ca released from stores to the plasma membrane was slower than Ca efflux across the plasma membrane. This conclusion was supported by the observation that inhibition of Ca efflux by La, which blocks the plasma membrane Ca-ATPase and Na-Ca exchange, prolongs the rate of decay of the Ca-activated Cl current approximately sevenfold (Fig. 7).
The suggestion that a gradient of Ca exists between
the cytosol and the subplasmalemmal space seems reasonable based on known rates of Ca diffusion and the
distribution of IP3 receptors in Xenopus oocytes. ER Ca
stores, measured by IP3-receptor immunostaining, are
more concentrated close to the plasma membrane in
Xenopus oocytes (Parys et al., 1992; Kume et al., 1993
;
Callamara and Parker, 1994
). Allbritton et al. (1992)
have measured the diffusion coefficient (D) of free Ca
in Xenopus oocyte cytoplasm as 220 µm2/s when all the
Ca buffers are saturated and uptake into organelles is
inhibited. Thus, the average time required for a free
Ca ion to diffuse to the plasma membrane from a site
50-µm deep in the cytoplasm would be ~2 s (t = r 2/
6D). If Ca were heavily buffered so that only 1% of the
Ca were free at any one time (Neher and Augustine,
1992
), the diffusion times would be several minutes
(Dobs = D/k, where k is the ratio of bound/free Ca;
Zhou and Neher, 1993
). This estimate of a 1-2-min diffusion time corresponds qualitatively with the 1.5-min
half-time of Ca clearance we have measured in the presence of thapsigargin. Given this slow diffusion of Ca
from the release sites to the plasma membrane, a relatively low density of Ca efflux pathways in the plasma
membrane (plasma membrane Ca-ATPase and Na-Ca
exchanger) would theoretically be capable of generating a subplasmalemmal Ca gradient. 1 µm2 of membrane containing 25 Ca transporters, each transporting 250 Ca ions/s could clear ~10
20 mol/s. This square
micrometer of membrane could reduce the Ca concentration to a depth of 1 µm (1 µm3 = 10
15 L) at a rate
of 10 µM/s. If Ca release from stores raised cytosolic Ca
to 100 µM, this density of transporters could lower the
Ca concentration within 1 µm of the membrane to
basal levels in ~10 s, if there were no diffusion of Ca
into this space from more distant sites. Because the
peak concentration of an ion diffusing in an infinite
volume decreases proportionally with the cube of the
distance (Berg, 1993
), Ca released more than several
micrometers away from the plasma membrane will not
contribute significantly to the Ca concentration in the
immediate subplasmalemmal space.
We have clearly demonstrated the existence of a gradient of Ca that develops after release of Ca from stores
in which the Ca concentration is lower at the plasma
membrane. Our data also support the idea that this
gradient changes direction when Ca influx occurs. Our
reasons for concluding this are as follows. We have previously shown that ICl2 is less sensitive to Ca than ICl1-S
(Kuruma and Hartzell, 1999), but the Ca fluorescence
reported by cytosolic Ca-green-dextran is less when ICl2
is activated than when ICl1-S is activated (Figs. 4 c and 5 c).
Furthermore, ICl1-T is consistently larger than ICl1-S. If
these currents are mediated by the same type of channel, this would also suggest that Ca concentration is
higher at the plasma membrane during Ca entry than
occurs in response to Ca release from stores.
An important consideration in our studies of the
temporal patterns of Ca distribution was the contribution of the added fluorescent Ca dyes that act as mobile
Ca buffers. Neher and Augustine (1992), Zhou and Neher (1993)
, and Xu et al. (1997)
have shown that even small amounts of added mobile Ca buffers dramatically
alter the spatio-temporal features of Ca dynamics. We
hoped to minimize the contribution of the Ca indicator to the measured Ca dynamics by using relatively immobile Ca indicators, 70-kD-coupled Ca-green-1 and
Ca-green-C18, at low concentration. The fact that the kinetics of the Ca-activated Cl currents were very similar
between uninjected oocytes and oocytes loaded with
the dyes argues that the dyes did not dramatically perturb
the normal patterns of Ca distribution or buffering.
Alternative Explanations
The discrepancy between the Ca-green-dextran fluorescence signal and the Cl current could be explained in
several ways. Although we prefer the interpretation that
the dextran-coupled dye and ICl1-S report different Ca
signals, there are other possibilities. For example,
Parker and co-workers (Parker and Ivorra, 1992, 1993
; Parker and Yao, 1994
) have suggested that the more
rapid turn-off of ICl1-S relative to the Ca fluorescence is
due to inactivation of the Cl channel. However, we have
shown that the turn off of this current is not caused by
an intrinsic inactivation of the channel (Kuruma and
Hartzell, 1999
). Similarly, one could explain the observation that ICl2 does not turn on in response to Ca release from stores and also turns on more slowly than
ICl1-T by supposing that this current requires a slow step,
such as phosphorylation, for activation. We have shown,
however, that this current can be activated very quickly
by Ca influx through exogenously expressed iGluR3
channels and have explained its slow activation entirely by its lower sensitivity to Ca than ICl1-S and ICl1-T (Kuruma and Hartzell, 1999
).
We should mention that the data in Fig. 6 a differ
somewhat from those reported by Yao and Parker
(1993). Yao and Parker (1993)
reported that the Ca
transient continued to rise after repolarization to 0 mV
after a hyperpolarizing step, whereas we found that the Ca signal began to decline immediately upon repolarization. They attribute the increase to Ca-induced Ca
release. The difference between their experiments and
ours is most likely explained by the fact that we injected
~100× more IP3. We found that large IP3 injections
produced a sweeping wave of Ca release that quickly depleted Ca stores, whereas injections similar to those
of Yao and Parker (1993)
produced regenerative spiral
and circular waves of Ca release. With these small injections, Ca stores appeared to refill with Ca shortly after
the wave passed, because within seconds Ca could be
released from the same locale as another wave passed.
Thus, Ca influx under conditions of spiral/circular
wave generation would be expected to stimulate release
by Ca-induced Ca release. In contrast, when the stores
were completely depleted under our conditions, Ca-
induced Ca release would be minimal.
Relationship to Other Studies and Significance
Recently, it has become recognized that Ca signals can
produce different responses depending on both their
amplitude and their frequency (Thomas et al., 1996;
Berridge, 1997a
). Ca signals in many cells occur as oscillations whose frequency is modulated by agonist concentration. The frequency of the oscillations is related
to the magnitude of the cellular response such as secretion or stimulation of enzyme activity (Berridge and
Rapp, 1979
; Osipchuk et al., 1990
; Thorn et al., 1993
).
Moreover, different regions of the cell, particularly
neuronal soma and processes, may exhibit different frequencies of Ca oscillations that elicit qualitatively different responses (Gu and Spitzer, 1995
). Certain effectors, such as mitochondrial dehydrogenases, respond
best to certain Ca oscillation frequencies because mitochondria behave as high-pass filters for the Ca signals
(Hajnoczky et al., 1995
; Rutter et al., 1996
), but other
effectors respond to different Ca oscillation frequencies by mechanisms that remain poorly understood
(Dolmetsch et al., 1998
). Furthermore, different effectors discriminate between different Ca signals by virtue
of differences in their spatial location and their sensitivity to Ca. Probably the best understood examples come
from studies of how Ca dynamics can differentially activate transcription. In AtT20 and hippocampal cells,
spatially distinct Ca signals in the cytoplasm and nucleoplasm can differentially activate transcription from
the serum or cAMP response elements (Bito et al.,
1997
; Hardingham et al., 1997
). In B lymphocytes, high
amplitude transient Ca signals are sufficient to produce translocation of the nuclear factor (NF) kB transcription factor into the nucleus, whereas low amplitude sustained Ca signals are required for persistent translocation of NFAT (Dolmetsch et al., 1997
).
Our present studies provide a novel example of how
Ca signals with different spatial and amplitude characteristics can differentially alter effector (ICl1 and ICl2) activity. Cytoplasmic Ca gradients have been shown to be
physiologically important in a variety of other systems,
including unidirectional fluid secretion in pancreatic acinar cells (Kasai and Augustine, 1990), the activation
of the transcription factor NFAT (Dolmetsch et al.,
1997
), regulation of cytoskeleton in migrating cells
(Brundage et al., 1991
), regulation of ion channel activity (Hoth et al., 1997
; De Koninck and Schulman, 1998
), and vesicular neurotransmitter release (Neher,
1998
). In the case of the Xenopus oocyte, these gradients exhibit several particularly interesting features.
First, the gradient arises partly because of slowed Ca
clearance due to recycling of Ca through the ER stores.
Second, the gradients are rapidly reversible depending on the membrane potential. Third, because inward
and outward Cl currents have different sensitivity to Ca,
these changing gradients produce complex Cl current
waveforms. Although the oocyte is an atypically large
cell, the gradients that we have described are occurring
on the micrometer scale and are likely to also exist in
smaller mammalian cells.
The Ca-activated Cl currents in Xenopus oocytes are
important physiologically: they are responsible for the
depolarizing fertilization potential that provides a fast
block to polyspermy in Xenopus eggs (Jaffe and Cross,
1986). Sperm-egg fusion stimulates IP3 production and
Ca release from stores (Snow et al., 1996
), followed by
activation of Cl currents that closely resemble ICl1-S and ICl2 (D. Glahn and R. Nuccitelli, personal communication). The resting potential of Xenopus eggs is usually
near
60 mV. Since amphibian eggs in the wild are fertilized in fresh water having relatively low [Cl
], ECl is
positive, and activation of Cl currents will depolarize the egg. However, Kuruma and Hartzell (1999)
have
shown that when cytosolic Ca is elevated only moderately, the resulting Cl current is strongly outwardly rectifying (ICl1-S) such that at
60 mV there would be relatively little Cl current. With larger increases in cytosolic
Ca, inward current (ICl2) is also stimulated, which would depolarize the oocyte. This may be an important
mechanism to prevent the oocyte from undergoing the
fast block to polyspermy prematurely. Small elevations
in cytosolic Ca would not have a significant influence
on inward Cl currents at the resting potential, but large
Ca rises would stimulate Cl current at all potentials, effectively voltage clamping the membrane of the oocyte
at ECl and preventing polyspermy.
![]() |
FOOTNOTES |
---|
Address correspondence to H. Criss Hartzell, Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322-3030. Fax: 404-727-6256; E-mail: criss{at}cellbio.emory.edu
Original version received 6 October 1998 and accepted version received 2 November 1998.
We thank Akinori Kuruma for the ISOC current-voltage data in Fig. 2 b, David Glahn and Rich Nuccitelli for access to not yet published material, and David Clapham, Anant Parekh, Reinhold Penner, Lynne Quarmby, Julio Hernandez, Thomas Fisher, and Akinori Kuruma for helpful comments at various stages during these studies.
This work was supported by National Institutes of Health grants GM-55276 and HL-21195.
![]() |
Abbreviations used in this paper |
---|
ER, endoplasmic reticulum; IP3, inositol 1,4,5-trisphosphate; SERCA, sarcoplasmic-ER Ca ATPase; SOC, store-operated Ca channel; SOCE, store-operated Ca entry.
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