(Received for publication, November 7, 1995; and in revised form, January 25, 1996)
From the
The rat basophilic cell line RBL-1 is known to express high
levels of the Ca current activated by store
depletion, known as Ca
release-activated
Ca
current (I
), the main
Ca
influx pathway so far identified in nonexcitable
cells. We show here that, as reported in other cell types, metabolic
drugs strongly inhibit the Ca
influx operated by
store depletion in RBL-1 cells also. We have tested the hypothesis that
intracellular adenine and/or guanine nucleotide levels act as coupling
factors between I
and cell metabolism. Using the whole
cell configuration of the patch-clamp technique, we demonstrate that
addition of ADP to the intracellular solution significantly reduces
I
induced by inositol 1,4,5-trisphosphate. This
phenomenon differs from other regulatory pathways of I
,
since it is highly temperature-dependent, is observable only in the
presence of low intracellular Ca
buffering capacity,
and requires a cytosolic factor(s) which is rapidly lost during cell
dialysis. Moreover, the inhibition is specific for ADP and is partially
mimicked by ADP
S and AMP, but not by GDP or GTP.
Many receptor agonists convey their message to intracellular
target processes by increasing the cytosolic concentration of ionized
calcium,
[Ca]
(
)(1, 2) .
This phenomenon is usually dependent on both Ca
release from intracellular stores and Ca
influx
across the plasma membrane. Since the early 1980s, it was postulated
that the increased permeability of the plasma membrane to
Ca
was somehow linked to the depletion of internal
Ca
pools, but this hypothesis became widely accepted
only in the last few years (3, 4, 5, 6, 7) . This
influx pathway depends on a new class of Ca
channels,
named ``store-operated Ca
channels'' or
``SOCs''(8) . Although much evidence demonstrates
that activation of SOCs depends on the Ca
content of
InsP
-sensitive stores(5) , the coupling between
stored Ca
and channel activity is still largely
obscure(6) . The prototype of SOCs is the CRAC channel,
initially described in rat mast cells(9) . Channels with
similar characteristics appear to be expressed in many different cell
types (10, 11, 12, 13, 14, 15, 16, 17) .
The main characteristics of CRAC are: (i) voltage independence of
gating, (ii) low unitary conductance (<100 femtosiemens), and (iii)
high Ca
selectivity (>1000:1 over monovalent
cations)(9, 10, 11, 17, 18, 19) .
Among the various possible mechanisms suggested for CRAC activation,
both a diffusible messenger released from empty stores, often referred
to as CIF, for Ca
influx
factor(20, 21) , and direct coupling between the store
and the plasma membrane channels have been
considered(6, 7, 8) . The modulation of CRAC
activity is also a matter of debate. Small G proteins have been
suggested to be involved either in the activation of CRAC itself or in
the modulation of channel
activity(22, 23, 24, 25) .
Similarly, in some cell types, cGMP and CIF have been shown to
up-regulate Ca
influx or currents activated in
response to Ca
store
depletion(8, 16, 26, 27, 28) .
Drugs acting on cytochrome P-450 or on intracellular kinases and/or
phosphatases have also been shown to modulate CRAC-type currents (13, 29, 30, 31, 32) . The
picture emerging from all these observations is rather complex,
suggesting that although CRAC channels are subjected to multiple
control mechanisms, either they are differently modulated in the
various cell types or they are a heterogeneous class of channels with
variable sensitivity and mechanism of activation-modulation.
We have
recently shown that the energy level of the cell controls the influx of
Ca activated by store depletion in a variety of cell
types(33) . The interest of this observation is 2-fold: on the
one hand, the effect of energy level appears as quite a general
phenomenon, in as much as it was observed in cells as diverse as
lymphocytes, hepatocytes, ascites tumor cells and epithelial-derived
tumor cell lines; on the other, it may represent not only a general
mechanism for modulating Ca
influx in physiological
conditions, but also a safety device operative under pathological
situations such as hypoxia, metabolic activation, etc. The mechanism
through which the cell energy level inhibits Ca
influx and whether or not this inhibition concerns the CRAC
channels is still unknown. In this contribution, by using the
patch-clamp technique in the whole-cell configuration, we address this
problem directly and test the effects of adenine and guanine
nucleotides on the current sustained by CRAC channels. We demonstrate
that ADP, but not ATP, GTP, or GDP, is a physiological modulator of
I
. This modulation is rather complex, being highly
sensitive to temperature and intracytosolic Ca
buffering and requiring a soluble factor(s) which is rapidly
washed out during cell dialysis.
Given that I is relatively large and well
characterized in rat mast cells and in the mast cell line
RBL-1(9, 18, 19, 22) , we employed
the latter model system to characterize the effect of metabolic
inhibitors on this Ca
current. We first tested
whether also in the rat cell line RBL-1 the Ca
entry
induced by store depletion was sensitive to the treatment with
mitochondrial poisons. Fig. 1shows that oligomycin, a known
inhibitor of the mitochondrial ATP synthase, caused a reduction of the
[Ca
]
increases induced by
treatment of the cells with the sarcoendoplasmic reticulum
Ca
ATPase (SERCA) inhibitor
thapsigargin(36) . In particular, Fig. 1A shows
that oligomycin hardly affected [Ca
]
in high glucose medium, whereas it clearly reduced the steady
state increase of [Ca
]
caused
by thapsigargin both in glucose-free medium (Fig. 1B)
and in glucose medium but containing 10 mM 2-deoxyglucose (Fig. 1C). Thus, as observed in other cells, the effect
of mitochondrial inhibitors is observed only if glycolysis is
inhibited, indicating that the energy level rather than mitochondrial
inhibition is the key factor regulating, directly or indirectly,
Ca
influx induced by store
depletion(33, 35) . Other mitochondrial inhibitors,
such as sodium azide, KCN, antimycin A, rotenone, and carbonyl cyanide p-fluoromethoxyphenylhydrazone exerted similar effects (not
shown, and see (33) and (35) ).
Figure 1:
Effect of metabolic inhibitors in RBL-1
cells. RBL-1 cells cultured on glass coverslips were loaded with
fura-2/AM as described under ''Experimental Procedures.``
Cells were bathed in standard solution containing 1 mM CaCl and 11 mM (A), 0 mM (B), or 1 mM (C) glucose. A,
and B, Ca
influx was activated by applying
the same solutions containing 1 µM thapsigargin (Tg) in the presence (continuous traces) or absence (dashed traces) of 1.4 µM oligomycin (Ol). Traces are representative of 5 similar
experiments. C, Ca
influx was activated by 1
µM thapsigargin (Tg) and during the
Ca
plateau oligomycin was applied in the presence (continuous trace) or absence (dashed trace) of 10
mM 2-deoxyglucose (2-dGlc). Averaged responses of
10-12 cells representative of 3 similar
experiments.
The experiments
described in Fig. 1strongly suggest that the target of
metabolic drugs is I since the sustained increase of
[Ca
]
caused by thapsigargin is
due mainly to the activation of this current(37) .
We
hypothesized that the level of intracellular ADP or GDP, rather than
that of ATP or GTP, is critical in modulating thapsigargin-induced
Ca influx(33) . Considering that the
patch-clamp technique in the whole-cell configuration gives access to
the cell interior, we could test directly whether indeed the
intracellular nucleotide level affects I
. In order to
activate the current, InsP
was also included in the
intracellular solution. Fig. 2A (left panel)
shows that, shortly after establishing the whole-cell configuration,
there is a rapid activation of an inward current which reached its
maximum within about 1 min. The current-voltage relationship, as
revealed by the voltage ramp from -100 to +100 mV, shown on
the right panel of Fig. 2A, confirms that the
current has the typical characteristics of I
, e.g. inward rectification and reversal potential above +50 mV. In
control conditions (dashed traces), the pipette solution
contained 2 mM ATP. Fig. 2A shows that, if the
pipette contained 0.5 mM ADP in addition to ATP (continuous
traces), neither the amplitude nor the kinetics of the current was
affected. In addition, no significant effect on I
was
detected if the same amount of GDP or GTP was added to the
intracellular solution. Finally, none of the above-mentioned
mitochondrial inhibitors had any direct effect on I
, as
measured under these conditions (not shown).
Figure 2:
Effect of ADP and temperature on
I and capacitative Ca
influx. A,
left, kinetics of I
activation at 25 °C during
perfusion with a standard pipette solution containing 10 mM cesium BAPTA, 50 µM InsP
, 2 mM ATP with (continuous traces) or without (dashed
traces) 0.5 mM ADP. Inward currents, recorded at 0 mV
holding potential and sampled at 2 Hz, were normalized to the cell
capacitance for comparison. Right, normalized current-voltage
relationships derived from subtracting fast ramps, delivered after
break-in (before activation of the current), from current responses at
the time indicated by the arrow. The voltage-pulse protocol,
delivered every 2 s, is schematically shown on the bottom of
the same panel. B, cells loaded with fura-2/AM were suspended
(10
cells/ml) in Ca
- and glucose-free
external solution containing 0.2 mM EGTA. Cell suspensions
were then challenged with 1 µM thapsigargin in the
presence (continuous traces) or in the absence (dashed
traces) of 1.4 µM oligomycin. After 3 min, 0.4 mM MnCl
was added to follow the activation of
capacitative influx. In each panel, the slower traces represent the
basal Mn
influx, recorded in the absence or in the
presence of oligomycin. The vertical calibration on the left side represents the total Mn
-quenchable fura-2
fluorescence at 360 nm. The experiments shown on the left panel were carried out at 25 °C, those on the right panel at 37 °C. C, pooled data of normalized peak current,
measured at 0 mV from current-voltage relationships, as shown in A (right). Cells, voltage-clamped at 25 or at 37 °C,
were dialyzed with the above mentioned internal solution. For
statistics, see Table 1. D, the Mn
influx, induced by thapsigargin in the absence (control) or in
the presence of oligomycin, was estimated from the initial rate of
fluorescence quenching, after subtraction of the basal influx, and
plotted as percentage of control. Pooled data are from 3 similar
experiments (**, p < 0.001).
The
experiments described in Fig. 3were designed to test this
possibility. The pipette solution contained 1.2 mM EGTA
instead of 10 mM BAPTA. Thus, not only the absolute
Ca buffering capacity was reduced, but a
``slow'' buffer such as EGTA was substituted for a
``fast'' buffer such as BAPTA(18) . As shown in Fig. 3A, at 37 °C and in the presence of 1.2 mM EGTA, addition of 0.5 mM ADP to the ATP-containing
solution (continuous traces) significantly reduced the peak
current, measured at a holding potential of 0 mV (upper
panel), by an average factor of 42.0 ± 2.7% (mean ±
S.E. of 3 similar experiments, 29 and 26 cells for each condition). The
inhibition was observed also at -40 mV, when I
was
measured during fast (50 ms) voltage ramps from -100 to +100
mV, delivered every 2 s from a holding potential of 0 mV (see Fig. 3A, lower panel, and Table 1) or
during long pulses (100 ms) to negative voltages (-100 mV, 55
± 15% inhibition, n = 7). Fig. 3B shows the mean peak current at 0 mV, measured in the same batch of
cells, at 25 and 37 °C, with 1.2 mM EGTA as the internal
buffer.
Figure 3:
ADP inhibits I at low
internal Ca
buffering capacity. A, upper,
temporal pattern of I
activation at 37 °C during
perfusion with a standard pipette solution containing 1.2 mM cesium EGTA, 50 µM InsP
, 2 mM ATP with (continuous traces) or without (dashed
traces) 0.5 mM ADP. Inward currents, recorded at 0 mV
holding potential and sampled at 2 Hz, were normalized to the cell
capacitance for comparison. Lower, normalized current-voltage
relationships, obtained as described in Fig. 2A (right panel). B, pooled data of the normalized
peak current at 0 mV, measured as described in Fig. 2C,
with the above-mentioned internal solution (**, P <
0.002).
The observed phenomenon was largely independent of the ATP
concentration since it occurred with solutions containing either 0,
0.5, or 2 mM ATP. Under these conditions, the normalized peak
current in controls was -0.95 ± 0.05 (n =
5), -0.97 ± 0.08 (n = 10), and -1.09
± 0.09 (n = 9) pA/pF respectively, whereas, the
average I inhibition induced by ADP was practically
unchanged. These results support the hypothesis that the effect of
metabolic poisons is not due to a reduction in the ATP level, but
rather to an increase in the ADP concentration(33) . In cells
of similar size and access through the patch pipette, as monitored by
membrane capacitance and series conductance, respectively, other
current properties, such as the delay before current activation and the
time constant to reach the peak current, did not change significantly
in the presence of ADP (see Table 1). However, as expected, a
reduction of the Ca
buffering capacity altered the
slow kinetics of I
. Thus, while with 10 mM BAPTA in the pipette the current hardly decreased from the peak
level onwards, with 1.2 mM EGTA the current at steady-state
was about 50% of the peak current, with or without ADP.
Finally, the
data of Table 1demonstrate that the effect of a reduced
buffering capacity on I inhibition by intracellular ADP
can be appreciated only at the physiological temperature, since at 25
°C the ADP modulation was not statistically significant, even with
1.2 mM EGTA. The lack of inhibition by intracellular perfusion
with ADP, in the presence of 10 mM BAPTA, could reflect a
toxic effect of high BAPTA concentrations, rather than its ability to
trap Ca
. This appears unlikely, since: (i) no effect
of ADP was observed when the EGTA concentration was increased from 1.2
to 10 mM, while (ii) the effect of ADP became statistically
insignificant when 1.2 mM EGTA was substituted with 1.2 mM BAPTA (see Table 1).
Figure 4:
Metabolic inhibitors are effective in
perforated-patch conditions. A, a single cell, loaded with
fura-2/AM, perfused at 37 °C with a standard external solution
containing 1 mM CaCl and 11 mM glucose,
was voltage-clamped at -40 mV in the perforated cell
configuration as described under ''Experimental Procedures.``
Ca
influx was induced by local application of 1
µM thapsigargin (Tg). After establishing of the
Ca
plateau phase, the external solution was switched
to the same one but containing 1 mM KCN (cyanide) in the
absence of glucose. B, conditions as in A. Once the
Ca
plateau was established, the cell was
alternatively clamped at -40, 0, and +40 mV to induce
typical [Ca
]
oscillations driven by the membrane potential. Traces are
representative of 3 similar experiments.
Figure 5:
Effect of nucleotides on I.
Cells were voltage-clamped at 37 °C in the whole-cell configuration
as described in Fig. 3. The standard internal solution contained
1.2 mM cesium EGTA, 50 µM InsP
, 2
mM ATP in the absence (control, n = 16) or
presence of 0.5 mM ADP (n = 13), ADP
S (n = 11), AMP (n = 8), GDP (n = 10), or GTP (n = 4). The normalized peak
current at 0 mV, measured as described in Fig. 2C, is
shown as percentage of control (**, P <
0.001).
The
sensitivity to adenine nucleotide is known to be a characteristic of
metabolism-regulated K channels which have been widely
characterized in either pharmacological or molecular
terms(41) . We thus investigated whether drugs such as
tolbutamide (100 µM) or diazoxide (100 µM),
which are known to affect K
channels, could also
affect Ca
influx through CRAC channels. None of these
drugs affected either the Ca
influx induced by store
depletion (measured in intact cells with Ca
indicators) or the current induced by InsP
(measured
by the patch-clamp technique, not shown).
The selective Ca current, named
I
, activated by store depletion and originally described
in rat mast cells and human T lymphoma cells, has been shown more
recently to be expressed in many eukaryotic cell
types(9, 10, 11, 12, 13, 14, 15, 16, 17) .
Whether or not CRAC or CRAC-like channels are a homogeneous group of
proteins and whether they represent the mammalian homologues of the Drosophila cation channels, the trp/trpl proteins, is
under intense investigation(42, 43, 44) . It
is clear, however, that, as originally shown in Jurkat T cells ((10) ), many physiological stimuli, in addition to
store-depleting drugs, also activate I
or
I
-like
currents(14, 17, 37, 45) . The
physiological relevance of I
has been strengthened by
the discovery of its absence in lymphocytes from a patient with a
primary immunodeficiency associated with a defective T cell
proliferation(46) . As for other key Ca
channels, it is predictable that CRAC channels also are under
strict and multiple control mechanisms. Surprisingly, up to now, little
is known, not only about the molecular mechanism of activation of this
current, but also on its modulation by physiological stimuli. Several
drugs, in fact, have been shown to affect I
or, more
generally, Ca
influx activated by store depletion,
but very few examples of physiological modulation of this current have
been reported so
far(30, 31, 32, 33, 40) .
From this point of view, the demonstration that Ca
influx induced by depleting InsP
-sensitive
Ca
stores is modulated by the energy level of the
cells appears of major interest since it is found in many different
cell types and because of its pathophysiological
implications(33) .
The present findings not only demonstrate
directly that I is the target through which metabolic
poisons inhibit Ca
influx activated by store
depletion, but they also allow the mechanism of this inhibition to be
defined more precisely. In particular, among the different hypotheses
suggested in order to explain the effect of metabolic stress, we can
now indicate ADP, instead of GDP, as the main factor coupling energy
depletion to I
inhibition. At the same time, we can
exclude that changes in ATP concentration, within a physiological
range, can have a direct modulatory role on the current. In fact,
within the first 30 s of intracellular perfusion, varying the ATP
concentrations, from 0 to 2 mM, did not modify significantly
the current activated by InsP
. Although under these
conditions the equilibrium between the pipette solution and the cytosol
is not complete, we can reasonably assume that the cytosolic ATP
concentration qualitatively reflects that of the intracellular
perfusion buffer. These data obviously do not exclude that
intracellular ATP may affect I
indirectly, by acting on
either store-refilling or through
kinases/phosphates(30, 31, 32) . However, the
affinity of the SERCAs and of the kinases for the nucleotide are in the
µM range and it is unlikely that the ATP concentration can
decrease to such low levels under physiological conditions.
Since inhibition induced by ADP is detectable only at the beginning of cell perfusion when equilibration is not yet complete, the effective concentration of ADP remains unknown. Nonetheless, the physiological intracellular concentration of free ADP is very low, between 30 and 50 µM, and the concentration routinely employed in this study (0.5 mM) is large enough to reflect the 5-10 times changes that occur under metabolic stress(33) .
Modulation
by adenine nucleotides is not a peculiarity of CRAC channels. Several
plasma membrane and intracellular channels are known to be sensitive to
ATP and/or ADP. Among them, the ATP-dependent K channels (K
) expressed in pancreatic
cells, cardiac myocytes, and neurons, and the CFTR Cl
channels expressed in many epithelial cells are the best
characterized(41, 47) . However, the effect of ADP on
I
is quite different from that of ATP or ADP on the
other channels. For example, the control of CFTR or K
channels by adenine nucleotides is observed also in the excised
inside-out configuration of the patch-clamp technique, while that of
ADP on I
is lost after a few tens of seconds of
intracellular dialysis. Indeed, a direct binding of the nucleotides to
the channels themselves, or to closely associated membrane proteins,
has been demonstrated in the case of CFTR and K
channels, respectively. In the case of CRAC channels, our data
suggest a more indirect mechanism of regulation by adenine nucleotides.
Another unique feature of I inhibition by ADP is its
temperature dependence, a characteristic which distinguishes it from
all other mechanisms of I
modulation. A Q
factor, ranging from 3 to 5, suggests the
involvement of a high energy barrier in this inhibitory pathway. The
concomitant requirement for a low Ca
buffering
capacity indicates that Ca
is also involved in the
process. In this respect, both the amount of Ca
buffering and its kinetic characteristics appear relevant. The
simplest interpretation of these findings is that the ADP effect
results from an increase of the known inhibitory effects of
Ca
on
I
(18, 31, 40) . However,
unlike the fast inactivation, discussed in detail by Zweifach and
Lewis(40) , that induced by ADP is quantitatively similar at
different voltages and it is abolished not only by buffering with high
and low BAPTA, but also with high EGTA, suggesting that the
Ca
binding sites underlying ADP modulation are
farther from the channel mouth than those linked to fast
inactivation(40) .
Finally, the need to study I at physiological temperature has unraveled other important
characteristics of this current. In particular, at 37 °C, the
amplitude of the current is only slightly increased with respect to 25
°C, while the time constant to reach the peak current is
significantly shorter. A weak temperature dependence of current
amplitude is not unusual among ion channels. This was, however, not
easily predictable in the case of I
, a current which is
thought to be activated by an intracellular second messenger,
synthesized or released upon depletion of intracellular Ca
stores(13, 20) . In turn, this finding suggests
that either the messenger(s) activating the current is produced (or
released) via a process weakly sensitive to temperature and/or that its
steady-state concentration, even at 25 °C, is sufficient to
activate the current maximally. The decrease in time constant at higher
temperature could be in part attributed to a faster rate of production
of the activatory messenger, but a role for Ca
on
this parameter may be suggested by the slowdown effect exerted by BAPTA
at higher concentration. This problem is presently under investigation.
Last, but not least, increasing the temperature from 25 °C to 37
°C also reduced the delay before current activation, about twice as
short. No striking difference was observed when comparing equal
concentrations of EGTA and BAPTA; however, the higher the buffer
concentration, the shortest the delay, suggesting that Ca
ions exert an inhibitory action at some step during current
recruitment. In the presence of ADP, the delay and the time constant
were not significantly modified.
In summary, we have shown here that
the energy status of the cell directly modulates I through a Ca
- and temperature-dependent
mechanism. This modulation depends primarily, or exclusively, on the
intracellular ADP level. Our findings represent a further example of
the key role played by this adenine nucleotide in coupling cell
metabolism to the signal transduction pathways at the plasma membrane
level.