From the Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709
Received for publication, September 12, 2000, and in revised form, October 18, 2000
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ABSTRACT |
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The regulation of store-operated,
calcium-selective channels in the plasma membrane of rat basophilic
leukemia cells (RBL-2H3 m1), an immortalized mucosal mast cell line,
was studied at the single-channel level with the patch clamp technique
by removing divalent cations from both sides of the membrane. The
activity of the single channels in excised patches could be modulated
by Ca2+, Mg2+, and pH. The maximal
activation of these channels by divalent cation-free conditions
occurred independently of depletion of intracellular Ca2+
stores, whether in excised patches or in whole cell mode. Yet, a number
of points of evidence establish these single-channel openings as
amplified store-operated channel events. Specifically, (i) the single
channels are exquisitely sensitive to inhibition by intracellular
Ca2+, and (ii) both the store-operated current and the
single-channel openings are completely blocked by the capacitative
calcium entry blocker, 2-aminoethoxydiphenyl borane. In addition, in
Jurkat T cells single-channel openings with lower open probability have been observed in the whole cell mode with intracellular
Mg2+ present (Kerschbaum, H. H., and Cahalan, M. D. (1999) Science 283, 836-839), and in RBL-2H3 m1 cells a
current with similar properties is activated by store depletion.
In a variety of cells, binding of ligands such as
neurotransmitters, hormones, or growth factors to receptors on the cell surface generates intracellular calcium signals. Receptor-mediated release of Ca2+ from
IP31-sensitive
intracellular stores triggers an influx of Ca2+ into the
cell via store-operated channels (SOCs) in the plasma membrane, a
process termed "capacitative calcium entry" (1, 2). The resulting
increase in intracellular Ca2+ concentration regulates
important processes, ranging from cell growth and differentiation to
apoptosis and cell death. Molecular candidates for SOCs are the
mammalian homologues of the Drosophila TRP (transient
receptor potential) protein (3). Expression of the related genes,
however, has resulted in varying patterns of response, including
constitutive activity, augmentation of capacitative calcium entry,
apparent direct activation by IP3, and activation by
diacylglycerol (4). Therefore, the mechanism by which emptying of
intracellular Ca2+ stores activates SOCs remains elusive.
Recent findings have favored a "direct coupling" model, whereby
IP3 receptors in the store membranes sense the
Ca2+-filling status of the stores and transfer this
information via direct coupling to subunits of SOCs in the plasma
membrane (5-8).
For the present, the best knowledge of SOCs comes from
electrophysiological studies of a specific current associated with SOCs, known as the Ca2+ release-activated calcium current
(Icrac) (9). Despite extensive characterization
of the macroscopic CRAC currents, the single-channel signature is
poorly understood. This is because the channels are presumed to have a
minute single-channel conductance, too low to be resolved at the
single-channel level (10). During whole cell measurements in Jurkat
T-lymphocytes, recently, Kerschbaum and Cahalan (11) were able to
detect for the first time single-channel events which they attributed
to CRAC channels. Their approach was to measure currents in the
complete absence of intra- and extracellular divalent cations. They
reasoned that under these conditions CRAC channels, like voltage-gated
Ca2+ channels should pass monovalent cations
indiscriminately and with enhanced single-channel conductance (12-14).
Indeed, in the absence of divalent cations large nonselective cation
currents were observed, and during the initial stage of activation,
single-channel conductances of 36 to 40 pS were measured in whole cell
mode (11). However, the relationship of these large nonselective cation
currents to the process of store depletion, and thus their identity as CRAC channels, has not been definitively established.
Here we report similar single-channel activities measured for the first
time in excised plasma membrane patches, using a mast cell line
(RBL-2H3 m1) and divalent cation-free solutions. Our whole cell data
are similar in some respects to those from Jurkat T-lymphocytes. In
addition, because of direct access to the cytoplasmic surface of the
channels, we have elucidated kinetic and regulatory properties of the
channels. In particular, we have sought to determine whether or not
these cation channels truly represent CRAC channels. In divalent
cation-free solutions excised channels of 25 to 39 pS conductance
showed stable activation, independent of IP3 or IP3 receptors, and were blocked by 2-APB, a relatively
specific inhibitor of SOC activation (15, 16). The effects of 2-APB, as
well as the modulation of channel activity by Ca2+ provide
evidence that the Na+-conducting channels are in fact
single CRAC channels. Hence, this report offers new insight into the
possible regulation mechanism of native SOCs and provides a novel
approach to investigate these channels under low divalent conditions.
Cell Culture--
Rat basophilic leukemia cells (RBL-2H3 m1), an
immortalized mucosal mast cell line expressing m1 muscarinic receptors,
were obtained from Dr. M. Beaven, National Institutes of Health (17). The cells were cultured in Earle's minimal essential medium with Earle's salts, 10% fetal bovine serum, 4 mM
L-glutamine, 50 units/ml penicillin, and 50 mg/ml
streptomycin (37 °C, 5% CO2). For experiments, cells
were plated onto glass coverslips and used 12-36 h there after.
Electrophysiology--
Patch clamp experiments were performed at
20-22 °C in the tight-seal whole cell, cell-attached and inside-out
configurations (18). Patch pipettes were pulled from borosilicate glass
(Corning glass, 7052) and fire polished. Membrane currents, filtered at 1-2 kHz, were recorded using an Axopatch-200B amplifier (Axon Instruments, Burlingame, CA). Voltage clamp protocols were implemented and data acquisition performed with pCLAMP 7.0 Software (Axon Instruments). Solution changes were accomplished by bath perfusion. The
time required for a complete change was around 2 s. All voltages were corrected for a liquid junction potential.
For whole cell experiments with Ca2+ as charge carrier,
unless stated otherwise, the patch pipette (2-5 megaohm) solutions had the following composition (in mM): 140 Cs+-aspartate, 2 MgCl2, 1 MgATP, 10 Cs+-BAPTA (with free calcium set to 100 nM,
calculated using MaxChelator software, version 6.60), 10 HEPES (pH 7.2 with CsOH). The bath solutions contained (in mM) 140 NaCl,
4.7 KCl, 10 CsCl, 1,13 MgCl2, 10 glucose, 10 CaCl2, 10 HEPES (pH 7.2 with NaOH). Divalent free whole
cell measurements, with Na+ as charge carrier, were done
with pipettes containing (in mM) 128 Cs+-aspartate, 12 Cs+-BAPTA, 0.9 CaCl2 (free Ca2+ ~5 nM), 10 HEPES
(pH 7.2 with CsOH), and bath solutions containing (in mM)
150 Na+ methane sulfonate, 2 EDTA, 10 HEPES (pH 7.2 with
NaOH). CRAC channels were opened by passive store-depletion with high
BAPTA (12 mM) in the pipette solution, or by addition of
ionomycin or thapsigargin to the bath. Cells were held at a potential
of 0 mV. Every 1, 2, or 5 s either voltage ramps from
During cell-attached and inside-out recordings data were collected from
10-s records at the given membrane potential, digitized at 5 or 10 kHz
and filtered digitally for analysis and presentation. The pipette
(5-10 megaohm) solutions contained (in mM) 150 NaCl, 2 EDTA, 10 HEPES (pH 7.2 with NaOH). In cell-attached experiments the
bath solution contained (in mM) 145 KCl, 5 NaCl, 10 MgCl2, 10 HEPES (pH 7.2 with KOH) to nullify the cell's
resting potential. 90 s before excision of the patch, the bath was
perfused with intracellular solution, which contained (in
mM) 145 K+-glutamate, 5 NaCl, 2 EDTA, 10 HEPES
(pH 7.2 with KOH). Single-channel analysis was performed with the
pCLAMP 6 software. po values were calculated for
consecutive 1-s periods.
CRAC Currents and Monovalent Currents in RBL-2H3 m1
Cells--
Ca2+ release-activated Ca2+
currents can be activated by a variety of procedures that share the
common property of emptying intracellular IP3-sensitive
stores. Stores can be depleted actively by exposure to receptor
agonists that elevate IP3 levels, external application of
Ca2+-ionophores like ionomycin, or the presence of
IP3 in the patch pipette solution (19). Passive methods for
store depletion rely on a constitutive and ill-defined leak of
Ca2+ from stores. If store refilling is prevented by
inhibition of SERCA pumps or by high concentrations of cytoplasmic
Ca2+ chelators, the stores gradually lose their
Ca2+.
In the whole cell measurements shown in Fig.
1, we used the latter method and
activated CRAC channels by breaking into the cell, while including high
concentrations of the Ca2+-chelator, BAPTA (12 mM), in the pipette solution. With external Ca2+ present (Fig. 1a), Ca2+ ions
permeate through the open CRAC channels and the Ca2+-influx
during progressive channel activation could be monitored as a
developing small inward current, of 30 to 60 pA (2-3 pA/pF) (n = 5). Single-channel currents underlying the
macroscopic Ca2+ currents were too small to be resolved,
even at
With divalent free solutions and Na+ as the permeant ion,
however, single-channel events have been observed during whole cell measurements in Jurkat T-lymphocytes (11). We first sought to determine
whether similar currents could be observed with rat basophilic leukemia
(RBL-2H3 m1) cells. The current development in Fig. 1b
illustrates activation of a large inward current under divalent
cation-free conditions (n = 6). The latency for current activation after break in, and the development time to reach the peak
macroscopic current, were characteristically 1.5 to 2 times longer for
the current in divalent-free solutions, than for
Icrac in Ca2+-containing solutions.
In the absence of divalent cations, dialysis of the cell with the
pipette solution typically activated macroscopic currents up to 2000 pA
(~130 pA/pF), or ~40-fold greater than Icrac. The current-voltage relationship of the
fully developed current showed a modest inward rectification. External
addition of 10 µM Mg2+ inhibited the
monovalent currents in a voltage-dependent manner (data not shown).
During the initial activation of the large cation current (see Fig.
1b), we were able to detect single-channel openings in RBL-2H3 m1 cells (Fig. 1c). We recorded single-channel
inward currents of 3.3 to 4 pA, during voltage steps to Stable Activation of Single Cation Channels in Excised Plasma
Membrane Patches--
To gain access to the cytoplasmic surface of the
channels, and to limit the number of investigated channels, we
attempted to record the activity of individual channels in excised
plasma membrane patches. Sodium ions (150 mM) were used as
the primary charge carriers. Starting from cell-attached patches with
no channel activity, we excised the patches into divalent cation-free
solutions, while clamping the cell's membrane potential to
When patches were excised into divalent-free media, the channels
apparently needed no additional factors to be activated, such as
IP3 or exogenously added IP3 receptors (7, 20).
However, if fragments of endoplasmic reticulum stores were still
attached to the patches, the absence of ATP, Ca2+, and
Mg2+ from the intracellular solutions could have induced
store depletion, and thus caused or contributed to channel activation.
Continuous channel activity began an average of 70 to 80 s after
excision (Fig. 2c) and was stable during recording time for at least 20 min, even after extensive washing. In 32% of the
experiments the progressive opening of 6 and more channels in a patch
could be observed, which may suggest clustering of CRAC channels in specific membrane domains.
Single-channel Properties of Excised Channels, Activated by
Divalent Cation-free Solutions--
We examined the single-channel
properties of channels in inside-out patches, in divalent cation-free
solutions. Fig. 3a shows recordings of 2 active channels in an excised inside-out patch, at
different membrane potentials. The channels had a high open probability
(0.94 ± 0.03 at Effect of Ca2+-store Depletion on the Activation of
Monovalent Currents and CRAC Currents--
The results in Fig. 1 show
that the activation of the large cation current by divalent cation-free
solutions was considerably slower than the activation of
Icrac, despite the expectation that passive
store emptying by BAPTA should occur at a similar rate in the two
conditions. This would indicate that the activation of cation channels
by divalent cation-free solutions involves a mechanism other than, or
in addition to store depletion. Alternatively, it is possible that the
absence of divalent cations slows the mechanism for coupling store
depletion to channel activation. To address these possibilities, we
carried out experiments in which Ca2+-stores were emptied
and CRAC channels thus activated prior to going whole cell or excision
into divalent cation-free media. Before sealing on the cells, we
applied either 1 µM of the SERCA inhibitor, thapsigargin
plus 500 nM of the ionophore ionomycin, or 500 nM ionomycin alone. The incubation time with these drugs was minimally 5 min, by which time stores should be completely depleted
and the currents should have fully developed (21). Subsequently, we
patched the cells, and established either the whole cell configuration
with either Ca2+ or Na+ as charge carriers, or
excised the patch (inside-out mode) at
Prior depletion of Ca2+-stores resulted in preactivation of
Icrac with about 2 pA/pF (7/7) when measured
with Ca2+ as the charge carrier (Fig.
4, a and b, inset).
However, directly after switching to Na+ in divalent free
solutions, the current only increased 4-fold (Fig. 4b,
inset), corresponding to about 10% of activation compared with
the peak macroscopic divalent free currents (Fig. 4b). This 4-fold activation may correspond to the current seen in Jurkat cells by
Kerschbaum and Cahalan (11) in the absence of external divalent
cations, but with Mg2+ present in the cytoplasm. However,
in the RBL-2H3 cells no larger, transient inward current was observed.
Similar kinetics for RBL-1 cells have been reported by Fiero et
al. (22). Following this 4-fold jump, which was only seen in the
preactivated cells, a slower activation to the full, maximally
conducting state was observed, and the kinetics of this process were
similar in control and preactivated cells. Likewise, the appearance of
single-channel openings following excision into divalent cation-free
solutions occurred with a similar delay when stores were previously
emptied (Fig. 4c).
Taken together, these results demonstrate that the delay time for whole
cell current activation by divalent cation-free solutions, as well as
the delay time for channel activation following excision into divalent
cation-free solutions does not appear to depend on depletion of
Ca2+-stores. However, they do not rule out a requirement
for initial store depletion, followed by a subsequent amplification
step, which is rate-limiting. Nonetheless, the mechanism by which
observable single-channel activation occurs appears to be at least
somewhat distinct from the mechanism of activation of
Icrac by store depletion. Note, however, that
activation of an intermediate activity state of the channels, similar
to that seen with low divalent cations outside and with
Mg2+ inside (11, 22), did appear to be hastened by prior
depletion of Ca2+-stores. The significance of this
observation will be considered subsequently under "Discussion."
Modulation of Channel Activity in Excised Patches by Internal
Ca2+, Mg2+, and pH--
To further address the
issue of whether the channels activated by divalent cation-free
solutions represent CRAC channels, we next examined the modulation of
these channels by internal divalent cations and by pH to compare their
behavior which was shown previously for CRAC channels.
Krause et al. (23) reported from whole cell studies in RBL-1
cells, that Icrac activation changed from
spontaneous to storeoperated mode, if the cytosolic
[Ca2+] is elevated to more than 50 nM. The
spontaneous Icrac was inactivated at a resting
cytosolic [Ca2+] of 105 nM. On the basis of
their finding, after reaching full channel activity in the inside-out
mode, we perfused the bath with intracellular solution containing 100 nM free Ca2+ (Fig.
5a). This concentration
completely inhibited channel activity in 50.5 ± 9.5 s in 4 patches, while in 3 other patches channel activity was unaffected. In
the 4 patches in which channel activity was inhibited, restoration of
low Ca2+ conditions reversed the inhibition in only 1 of
the 4 experiments. The inhibitory effect of Ca2+ was rather
specific and not due to inhibition of monovalent currents by divalent
ions per se, because similarly low concentrations of free
Mg2+ failed to inhibit. However, if 100 µM
Mg2+ or more was added to the inner surface of the active
channels, inhibition developed in 13.4 ± 3.1 s (12 out of 12 patches) (Fig. 5b). The activity was restored by divalent
free buffers (not shown). In both cases, as the inhibition developed,
single channel conductance was unaffected indicating that the divalent
cations suppress open probability.
In the whole cell mode in Jurkat T-lymphocytes, the inactivation of
Na+ current by intracellular Mg2+ could be
reduced by increasing the intracellular pH (14). The experiment in Fig.
5c demonstrates a similar effect of increasing pH for the
channels in excised patches. After inhibition by 100 µM
Mg2+ (pH 7.2), channel activity could be rescued by
increasing the pH in the perfusion solution to pH 8.2 (4 out of 7 cells). The time needed for complete reversal in the cells that
responded to the pH change was 42 ± 10 s. We also carried
out experiments similar to those described by Kerschbaum and Cahalan
(14) in which the channels were inhibited by addition of a much higher concentration of Mg2+ (2.4 mM). Channel
activity was blocked in 7 of 7 experiments, but in three attempts there
was no recovery on shifting to pH 8.0 (not shown).
From the experiments depicted in Fig. 5, some interesting observations
can be made. First, the results provide evidence that the
single-channels are the channels underlying the large whole cell
current in divalent cation-free solutions. Second, the inhibition of
the channels with Ca2+ and Mg2+ concentrations
in the physiological range explains the inability to detect the same
channels in the cell-attached mode. In the cell-attached mode, neither
addition of 1 µM thapsigargin (n = 14)
nor addition of 500 nM ionomycin (n = 13)
evoked detectable single-channel activity. Note that under these
conditions, whole cell currents (Icrac, pA
magnitude) can be seen indicating that single channels must be opening;
but these channels are not detectable because in the presence of
Ca2+ or Mg2+ the single channel conductance
and/or frequency of opening is below the level of detection. Third, the
ability of low, physiological concentrations of Ca2+ to
inhibit the channels is consistent with their identity as CRAC channels.
Effects of the IP3-receptor Antagonist 2-APB
(2-Aminoethoxydiphenyl Borane) on Icrac
Activity--
Recent reports have supported a conformational coupling
model for SOC activation, whereby IP3 receptors in the
endoplasmic reticulum sense the Ca2+ filling status of the
stores and transfer this information via direct coupling to SOCs in the
plasma membrane (5-7). Ma et al. (16) concluded from their
studies, using the membrane permeant IP3 receptor inhibitor
2-APB that IP3 receptors are essential not only for opening
of SOCs, but also for maintaining their activation. On this basis, we
examined the effects of 2-APB on macroscopic currents, and on channel
activity in excised patches.
The results shown before in Fig. 2 demonstrate that no added
IP3 or added IP3 receptors are necessary to
maintain activity of CRAC channels in excised patches under divalent
cation-free conditions. This is in contrast to previous reports
demonstrating that Trp-3 channel activity (24) and store-operated
channel activity in A431 cells (7) were rapidly lost in the absence of
added IP3 and IP3 receptors. This may indicate
that once activated under divalent-free conditions, CRAC channel
activity is independent of Ca2+-store depletion and
coupling to IP3 receptors (although we cannot unequivocally
rule out the stable retention of fragments of endoplasmic reticulum and
IP3 receptors). Thus, we were initially not surprised to
find that addition of 100 µM 2-APB to the medium
surrounding activated channels in excised patches in most instances
failed to inhibit channel activity (for example, Fig.
6d). However, with this
protocol 2-APB is added to the cytoplasmic side of the plasma membrane,
while in previous studies of Ma et al. (16), the drug was
always added to the outside. Experiments illustrated in Fig. 6,
a and b, show that in the whole cell
configuration, with either Ca2+ or Na+ as
charge carrier, 2-APB blocked the current completely when applied
externally, but had significantly less effect when applied in the
pipette. Finally, although 2-APB blocked channel activity when applied
to the cytoplasmic side of excised patches in only 2 of 11 experiments
(Fig. 6d), if included in the patch pipette, channel
activation did not occur in any of 10 separate experiments (Fig.
6c).
These results suggest that 2-APB may be acting as a direct blocker of
SOCs, rather than as an IP3 receptor antagonist as supposed in earlier studies. However, because of the protocols used, we considered the possibility that once the channels were activated by
divalent cation-free solutions, they would lose their sensitivity to
the effects of 2-APB as an IP3 receptor inhibitor. Thus, we also added 2-APB to the external medium before establishment of a
surface membrane seal with the patch pipette and therefore prior to
excision (Fig. 6e). However, channel activation was seen in 3 of 5 patches in this series of experiments, and this is the expected
frequency of successes from control experiments (about 70%). Thus, we
conclude that at least in RBL-2H3 m1 cells, the site of inhibition of
CRAC channels by 2-APB is external and may be on the channels
themselves. It seems unlikely that the drug's action involves
IP3 receptors under the plasma membrane.
Although not as yet thoroughly investigated in this regard, 2-APB may
be relatively selective for CRAC channels among plasma membrane
Ca2+ channels. The current study shows that CRAC channels
are directly blocked by this drug, but L-type calcium channels (15),
Trp-3 channels (16), and arachidonic acid-activated channels are
not.2 Thus, the sensitivity
of the channels to 2-APB seen under divalent cation-free conditions can
be taken as an additional piece of evidence that they do in fact
represent a high conductance state of CRAC channels. Also, based on the
stability of channel activity in divalent cation-free solutions, we
conclude that when activated in this way, interactions between channels
and IP3-liganded IP3 receptors are not required
for activation, or at least for maintenance of the activated state.
Our studies on RBL-2H3 m1 cells have shown that in the complete
absence of intra- and extracellular divalent cations large macroscopic
inward currents develop, when measured in whole cell mode. This
behavior is similar to that described for Jurkat T-lymphocytes by
Kerschbaum and Cahalan (11). During the initial phase of activation,
single-channels with conductances of 33 to 40 pS could be detected,
resulting in an estimate of 260 to 500 channels per cell, calculated
from the ratio of macroscopic to unitary currents. By excision into
divalent cation-free media we found similar channels in inside-out
patches, with conductances of 25 to 39 pS (averaging 31 pS). Since the
conductances for single-channels in excised patches and in whole cell
measurements are similar to each other, and the channels have similar
open probabilities and lack of selectivity, we conclude that they are
the same channels.
Whole cell currents as well as currents in excised patches were blocked
by the drug 2-APB, a relatively specific inhibitor of SOC activation
(16). Channel activity in excised patches was also inhibited by low
Ca2+ concentrations, as low as 100 nM.
Similarly low concentrations of Mg2+ did not inhibit,
showing that the inhibiting effect of Ca2+ is specific, and
not due to a general inhibition of monovalent currents by divalent
cations. Furthermore, the inhibition of channel activity in excised
patches caused by 100 µM Mg2+ could be
rescued by increasing the pH in the intracellular solution. A similar
pH effect was shown for whole cell monovalent currents in Jurkat cells
(14); however, in contrast to the findings in Jurkat cells, in RBL-2H3
cells inhibition by higher (2.4 mM) concentrations of
Mg2+ could not be reversed.
We have also carried out experiments to investigate the role of
intracellular Ca2+ store depletion on the kinetics of
activation of Icrac and the large currents and
single-channels activated under divalent cation-free conditions. Our
results indicate that two currents were preactivated by store
depletion, Icrac and an intermediate-sized
current which is similar to one seen with no divalent cations outside
but with Mg2+ inside. However, the large current in whole
cell mode, and the single-channel activity in excised patches did not
develop more rapidly when stores were first depleted. So, given this
independence of store depletion for the current and single-channel
kinetics, what is the evidence that the channels underlying the large
monovalent currents are in fact CRAC-channels? We believe that they are
indeed CRAC channels due to the following observations. (i) The ability of low, physiological concentrations of Ca2+ to inhibit the
channels in excised patches is consistent with their identity as CRAC
channels. We know of no other channel which is so exquisitely sensitive
to inhibition by Ca2+. (ii) We found that the channels
observed under divalent cation-free conditions are sensitive to 2-APB;
this drug appears to be relatively selective for CRAC channels among
membrane Ca2+ channels in inhibiting SOC activation; for
example, the drug does not block voltage-dependent calcium
channels or arachidonic acid-activated channels (15).2
Therefore, although the investigated channels seem likely to be CRAC
channels, activation by store depletion may be unnecessary in the
complete absence of divalent cations. Alternatively, activation by
store depletion may precede subsequent further activation by divalent
cation-free solutions such that the latter is
rate-limiting.3
Although the kinetics of currents activated by divalent cation-free
solutions appear independent of store depletion, there is another line
of evidence that may link these currents to the process of calcium
store depletion. While the large, macroscopic current was not
pre-activated by store depletion, a current much smaller than the
maximal macroscopic, yet larger than Icrac was observed in the pre-activated cells when extracellular divalent cations
were removed. This current may correspond to the intermediate sized
current seen by Kerschbaum and Cahalan (11) in the absence of external
divalent cations, but with intracellular Mg2+ present. In
the latter case, it was shown that the intermediate current resulted
from single channel openings identical to those underlying the larger
macroscopic current. In our experiments, an intermediate current was
activated by calcium store depletion (Fig. 4B, inset), and
if the single channels underlying this current are the same as those
for the macroscopic current, then this is further evidence that the
large openings are CRAC channels. However, we have not been successful
in observing single channel openings in RBL-2H3 cells with
Mg2+ present.
According to the conformational coupling model for SOC activation,
IP3 receptors are essential for activation and maintenance of channel activity (5, 6, 16). Recent findings have also implicated a
requirement for IP3 in this activation mechanism (7).
However, there is data in conflict with this idea, at least in some
cell types. For example, heparin is known not to inhibit SOC activation
(25) or Icrac (9), and SOC activation occurs in
cells apparently devoid of IP3 receptors (26). In the
current study, once excised single-channels were activated by divalent
cation-free solutions, they did not run down for at least 20 min. This
stable activation, elicited solely by excision into divalent
cation-free media, was independent of added IP3 or
IP3 receptors. However, the previous studies implicating
coupling of SOCs to IP3 receptors were not performed in the
complete absence of divalent cations. Thus, two distinct modes of
activation may exist, one requiring coupling to IP3
receptors, and one dependent on removal of divalent cations and
independent of IP3 receptors. This conclusion is similar to
one drawn by Krause et al. (23) who concluded that low
cytoplasmic Ca2+ per se was sufficient to
activate an Icrac-like current in RBL-1 cells.
Another important finding from the current work, with significant
impact on the interpretation of results in previous investigations, is
the effect of 2-APB on the divalent cation-free activated CRAC channels. The relatively specific blockade of CRAC channels by 2-APB
has until now been attributed to its antagonistic effect on
IP3 receptors (15), thereby blocking their conformational coupling to the channels (16). Our results from whole cell and excised
patch measurements, however, demonstrate that 2-APB inhibits CRAC
channels also under conditions where their activation and maintenance
of activity appear not to be IP3 or IP3
receptor dependent. Additionally, we find that the site of inhibition
of CRAC channels by 2-APB is likely on the external side of the
channels themselves rather than on IP3 receptors under the
plasma membrane. However, Ma et al. (16) clearly
demonstrated that 2-APB could only block TRP3 channels when activated
through an IP3-dependent mechanism, and not
when activated more directly by diacylglycerol. We have confirmed this
finding with our own independently generated TRP3-expressing cell
line.2 Thus, while we doubt that 2-APB blocks CRAC channels
by inhibiting IP3 receptors, we consider it still an open
question as to whether the drug acts by blocking CRAC channels
directly, or by interfering with their mechanism of activation.
Taken together, the findings in the current study demonstrate that the
current through single CRAC channels can be amplified and observed
under divalent cation-free conditions in RBL-2H3 m1 cells. The
mechanism of this activation is not known. It may result simply from
the dissociation of divalent cations from regulatory sites on or near
the channels, or it could be due to the dissociation of another factor
(or factors) whose regulation of the channels depends on divalent
cations. Soluble factors do not seem to be absolutely required for
inactivation by Ca2+ and Mg2+ since these
cations blocked channel activity when added several minutes after
excision. However, we were generally unsuccessful in reversing the
Ca2+ block by returning the patches to a
low-Ca2+ medium. Furthermore, we found that following
excision of patches in media containing 100 nM
[Ca2+], lowering of [Ca2+] did not lead to
the appearance of channel activity (data not shown). This may indicate
that Ca2+ binding to inhibitory sites is irreversible in
the absence of a factor or structure that is lost following patch excision.
Regardless of the underlying mechanism, the use of divalent cation-free
solutions has provided conditions for the first measurement and
characterization of single CRAC channel activity in excised plasma
membrane patches. Understanding the behavior of CRAC channels in the
absence of divalent cations may provide an important first step for the
design of future experiments addressing the mechanisms of regulation of
CRAC channels under more physiological conditions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
100 to +60
mV or voltage steps from 0 to
100 mV were delivered for 200 ms. Currents were sampled at 5 kHz during voltage ramps and 25 kHz during
voltage steps. All whole cell data were corrected for leak currents.
Leak current was collected in response to voltage ramps or steps
immediately after establishing the whole cell configuration.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
120 mV membrane potential.
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Fig. 1.
CRAC currents and monovalent currents in
RBL-2H3 m1 cells. a and b, during whole cell
recordings (representative plots are shown), intracellular
Ca2+ stores were passively depleted with 12 mM
BAPTA after going whole cell at 0 s. Currents, measured during
voltage-steps to 100 mV were plotted versus time, either
with 10 mM Ca2+ (n = 5)
(a) or 150 mM Na+ (n = 6) (b) as charge carriers. The solutions used in
a and b were the same, except, for the
Ca2+ recordings, EDTA in the bath was substituted with 10 mM Ca2+. b, only in divalent free
solutions, single-channel events could be resolved during the voltage
steps. c shows a sample trace, taken from a voltage step to
100 mV, applied at 287 s (star) in the measurement in
b. The unitary current amplitude is ~4 pA.
100 mV,
giving conductances of 33 to 40 pS (n = 6, assuming a
reversible potential of 0 mV, see below). Based on the whole cell
current amplitudes and the unitary current of the single-channels, we
calculated the number of channels per cell to be 260 to 500 channels
(n = 6). This suggests an average surface density of
~0.22 channels per µm2.
73 mV. In
65 out of 95 inside-out patches this procedure induced a channel activity, with an average conductance of 31 pS (25 pS to 39 pS) (Fig.
2, a and b). We
conclude that the channel activity seen after excision corresponds to
the channels underlying the macroscopic current activated by divalent
cation-free solutions; the single-channel conductance found in excised
patches was similar to the conductance recorded in the whole cell mode
(Fig. 1c), the channels appear to have similar lack of
selectivity, and the channels have similar, high open probability under
total divalent cation-free conditions.
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Fig. 2.
Single-channel activity in excised plasma
membrane patches. a, single-channel activity was
elicited in 65 out of 95 attempts by excision of patches into low
buffered divalent cation-free solutions, at 73 mV Vm in the
inside-out mode. b, a sample trace of an excised patch at
73 mV Vm is shown at a higher resolution, for comparison with
the whole cell single-channel recording in Fig. 1c.
c, Continuous channel activity in the inside-out mode
started, under the given conditions, after an average time of 70-80 s
(n = 34). Following convention, unitary inward currents
are displayed as downward deflections.
113 mV, n = 5). The duration of
the closures from the open state decreased with hyperpolarization, from
= 1.19 ± 0.13 (± S.E.) ms at
73 mV to
= 0.50 ± 0.06 (± S.E.) ms at
113 mV (n = 7 cells). From traces such as those shown in Fig. 3a, current
amplitudes were measured manually or determined from all points
amplitude histograms (Fig. 3b), over a voltage range from
113 to +67 mV. The resulting current-voltage relationships (Fig.
3c) gave an average single-channel conductance of 31 pS (25 to 39 pS). Moreover, the IV-relationship was linear with a reversal
potential close to 0 mV. As the major cation in the intracellular
solution was K+, while that in the pipette was
Na+, this is indicative of a channel that passes monovalent
cations indiscriminately (12). The IV-relationship is similar to that for the whole cell current, except the latter is slightly inwardly rectifying, due to a small effect of voltage on open probability (not
shown). Another specific channel feature, the mean open time, was
calculated to be 14.9 ± 1.2 (± S.E.) ms (n = 7)
(Fig. 3d).
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Fig. 3.
Kinetic properties of channels, activated by
divalent cation-free solutions in excised patches. a,
examples of Na+-inward currents at different membrane
potentials show high open probability of the single-channels. The
duration of closures from the open state decreased with
hyperpolarization. b, from all-points amplitude histograms,
calculated from 10 s recordings at different voltages, as depicted
for 93 mV (unitary current amplitude 2.7 pA), the mean
current-voltage relationship in c was constructed.
c, the averaged data indicate a single-channel conductance
of 31 pS. Each point represents the average of five to eight
experiments. d, in the experiment depicted, the open dwell
time histogram gave a time constant of 16.1 ms. The line
represents a single exponential fit to the data recorded at
73 mV.
The total number of events was 479. In addition to these short openings
(average time constant of 14.9 ± 1.2 ms), occasionally also
intermediate (38.9 ± 4.4 ms), and long openings (83.1 ± 8.9 ms) occurred.
73 mV Vm into
divalent-free solutions. In the whole cell experiments, the pipette
solutions included high concentrations of BAPTA to passively deplete
stores in the control cells.
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Fig. 4.
Effects of Ca2+ store
depletion on the activation of monovalent currents and CRAC
currents. a and b, the time courses are
taken from whole cell measurements of control cells and cells
preincubated for a minimum of 5 min with 1 µM
thapsigargin, 500 nM ionomycin, added to the bath
solution. Breaking in at 0 s was performed with pipette solutions
containing 12 mM BAPTA ([Ca2+] = 5 nM). a, with Ca2+ as charge carrier
Icrac could be preactivated, with a current
density of ~2pA/pF (tracing in the figure is representative of
three), compared with control cells (representative of six). To
establish baseline current in control and preactivated cells, at
370 s Ca2+ was replaced with equimolar
Mg2+ which does not permeate CRAC channels. b,
preactivation of the same extent, with extracellular Ca2+
present, is shown in the inset (0-25 s). Directly after
switching to Na+ in divalent free solutions the current
density, compared with controls, increased 4-fold (inset).
The ensuing current kinetics were similar in preactivated (average of
6 ± S.E.) and control cells (average of 6 ± S.E.).
c, also, the appearance of single-channel openings following
excision into divalent cation-free buffers ( 73 mV Vm)
occurred with a similar delay when stores were previously depleted with
500 nM ionomycin (72 ± 22 s, n = 3), compared with controls (70-80 s, see Fig. 2).
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Fig. 5.
Modulation of channel activity by
Ca2+, Mg2+, and pH.
a-c, representative open probability plots picture the time
course of channel activity in inside-out patches, following application
of the indicated ions to the inner surface of the cell membrane. The
plots were created from recordings after patch excision into divalent
free solutions, while the membrane was held at a potential of 73 mV.
The additions were made several minutes after excision (time 0 does not
correspond to the time of excision). a, addition of 100 nM Ca2+ to the patch was enough to inhibit the
channels in 50.9 ± 9.5 s (± S.E.), in 4 out of 7 trials.
b, the inhibition time with 100 µM
Mg2+ (pH 7.2) was faster, with 13.4 ± 3.1 s (12 out of 12). c, the full channel activity could be rescued by
increasing the pH to pH 8.2. The time needed was 42 ± 10 s
(4 out of 7).
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Fig. 6.
Effects of the IP3
receptor antagonist 2-APB on
Icrac and single-channel activity.
The action of the membrane permeable 2-APB (100 µM) was
tested either in whole cell or inside-out patches ( 73 mV Vm),
adding it to intra- or extracellular solutions. a and
b, the external addition of 2-APB before going whole cell
fully inhibited current development with either Ca2+
(average of three) or Na+ (average of three) as charge
carrier. Included in the pipette solutions, 2-APB inhibited the current
only by ~50%, seen in: a, (average of two, or ~40%,
seen in b, average of four. Control traces in a
and b are averages of four and five experiments,
respectively. For a, means ± S.E. are shown. For
b, S.E. values are not shown because of the density of data
points. The maximal currents were (pA/pF): control,
129 ± 29;
2-APB in pipette,
81 ± 24; 2-APB in bath,
10 ± 2. c, if 2-APB was included in the pipette solutions, in all
experiments (10 out of 10) the channels remained silent after patch
excision. d, 2-APB infrequently had an inhibitory effect (2 out of 11), when added to active inside-out patches. The patches
remained active in 9 out 11 trials. e, cells were also
pretreated with 2-APB for 5 min before sealing. After patch excision
into divalent free solutions with 2-APB present, the channel activity
remained in 3 out of 5 patches. Therefore, the success rate for
activation was the same as in untreated cells (60-70%). The closed
state of the channels in c-e is indicated as
c.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Christian Erxleben and Dr. Jerrel Yakel for reading the manuscript and helpful comments and Dr. Michael Beaven for providing the RBL-2H3 m1 cells.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 919-541-3298;
Fax: 919-541-1898; E-mail: braun2@niehs.nih.gov.
Published, JBC Papers in Press, October 19, 2000, DOI 10.1074/jbc.M008348200
2 D. Luo, L. M. Broad, G. St. J. Bird, and J. W. Putney, Jr., unpublished observation.
3 While this paper was under review, a manuscript appeared describing studies of single channel behavior in Jurkat and T-cells by utilizing divalent cation-free solutions in the whole cell patch clamp mode (Fomina, A. F., Fanger, C. M., Kozak, J. A., and Cahalan, M. D. (2000) J. Cell Biol. 150, 1435-1444). In contrast to the current study, Fomina et al. found that the appearance of the large macroscopic current was hastened by pre-activation of SOCs, and they concluded, as have we, that the observable channels underlying the large macroscopic currents are the channels responsible for Icrac.
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ABBREVIATIONS |
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The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; SOC, store-operated channel; RBL, rat basophilic leukemia cells; 2-APB, 2-aminoethoxydiphenyl borane; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; CRAC, Ca2+ release-activated Ca2+ channel.
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