From the
Feedback regulation of Ca
In many nonexcitable cells, the depletion of intracellular
Ca
The signal that couples store depletion to the
activation of CRAC channels has not yet been identified, and in
principle it could encompass diffusible and/or membrane-associated
molecules. In a recent study, Randriamampita and Tsien (12) isolated a fraction from the cytoplasm of stimulated Jurkat
leukemic human T cells that triggers Ca
Little is known about how CRAC channels are turned off once
activated, although several reports have demonstrated that increased
[Ca
This paper describes two
mechanisms by which elevated [Ca
[Ca
While effects of okadaic acid
are often accepted as evidence for actions of protein phosphatase 1 or
protein phosphatase 2A, the results of further experiments argue
against such a conclusion. First, 100 nM 1-norokadaone, a
compound similar in structure to okadaic acid but with little or no
inhibitory activity against protein phosphatase 1 and protein
phosphatase 2A(26, 29) , produced effects that were
indistinguishable from those of okadaic acid (Fig. 5). Moreover,
calyculin A (0.1-1 µM), an unrelated compound with
subnanomolar efficacy for inhibiting protein phosphatase 1 and protein
phosphatase 2A(27, 29) , lacked a significant effect on
slow inactivation (Fig. 5). These results are inconsistent with a
mechanism involving protein phosphatase 1 or protein phosphatase 2A.
The possible involvement of the
Ca
Because the mechanism by which depletion activates CRAC channels is
not yet clear, we cannot infer how store refilling inhibits them.
Although induction of an inhibitory signal by replete stores cannot be
ruled out, the simplest explanation consistent with the capacitative
entry hypothesis is that refilling terminates the channel activation
signal, whether it be a diffusible activator or a protein-protein
coupling mechanism. The time course of slow inactivation may reflect
the lifetime of the activation signal if stores refill rapidly and the
open channel lifetime is relatively brief. TG increases the peak
[Ca
The pharmacological profile of
store-independent slow inactivation argues against the involvement of
several well known phosphatases. Although okadaic acid largely blocked
inactivation and is known to be a potent inhibitor of phosphatases 1,
2A, and 3(29, 32) , calyculin A, an even more potent
antagonist of these phosphatases(29) , lacked any effect on the
inactivation process. Furthermore, 1-norokadaone, which lacks
significant activity against all three phosphatases(29) ,
blocked slow inactivation with the same efficacy as okadaic acid.
Calcineurin (protein phosphatase 2B) also does not appear to be
required for slow inactivation, because cyclosporin A and FK506 lacked
an effect even at doses 30-100-fold greater than their IC
Store-dependent and -independent mechanisms of slow inactivation
constitute two parallel pathways by which intracellular Ca
Stores were depleted under the conditions listed, with 1
µM TG or 20 µM IP
We thank Ricardo Dolmetsch and Chris Fanger and Drs.
Markus Hoth, Neil Clipstone, and Paul Driedger for helpful discussions;
Supriya Kelkar for invaluable assistance with cell culture; and Dr.
Markus Hoth for critical comments on the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
release-activated
Ca
(CRAC) channels was studied in Jurkat leukemic T
lymphocytes using whole cell recording and
[Ca
]
measurement
techniques. CRAC channels were activated by passively depleting
intracellular Ca
stores in the absence of
extracellular Ca
. Under conditions of moderate
intracellular Ca
buffering, elevating
[Ca
]
to 22 mM initiated an inward current through CRAC channels that declined
slowly with a half-time of
30 s. This slow inactivation was evoked
by a rise in [Ca
]
, as
it was effectively suppressed by an elevated level of EGTA in the
recording pipette that prevented increases in
[Ca
]
. Blockade of
Ca
uptake into stores by thapsigargin with or without
intracellular inositol 1,4,5-trisphosphate reduced the extent of slow
inactivation by
50%, indicating that store refilling normally
contributes significantly to this process. The store-independent
(thapsigargin-insensitive) portion of slow inactivation was largely
prevented by the protein phosphatase inhibitor, okadaic acid, and by a
structurally related compound, 1-norokadaone, but not by calyculin A
nor by cyclosporin A and FK506 at concentrations that fully inhibit
calcineurin (protein phosphatase 2B) in T cells. These results argue
against the involvement of protein phosphatases 1, 2A, 2B, or 3 in
store-independent inactivation. We conclude that calcium acts through
at least two slow negative feedback pathways to inhibit CRAC channels.
Slow feedback inhibition of CRAC current is likely to play important
roles in controlling the duration and dynamic behavior of
receptor-generated Ca
signals.
stores by inositol 1,4,5-trisphosphate
(IP
)
(
)is the primary mechanism by
which cell surface receptors activate Ca
influx
across the plasma membrane(1) . This phenomenon was first
proposed by Putney and termed capacitative Ca
entry(2) . Multiple types of ion channels underlying
capacitative Ca
entry have been identified in
different cells on the basis of their Ca
permeability
and activation by agents that empty Ca
stores (for
review, see Ref. 3). The depletion-activated Ca
channels present in mast cells and T cells, referred to as
calcium release-activated Ca
(CRAC) channels, are
distinguished from other types of depletion-activated Ca
channels by their high selectivity for Ca
over
monovalent and other divalent cations(4, 5, 6, 7, 8, 9) and their
extremely small unitary conductance(7, 8) . CRAC
channels have been shown to underlie the mitogen-stimulated
Ca
influx that is essential for T cell activation
following T cell receptor engagement(7, 9) .
Furthermore, periodic changes in CRAC channel activity have been shown
to generate oscillations in the level of intracellular free
Ca
([Ca
]
)(4, 10) ,
which may serve to enhance signaling through the T cell
receptor(11) .
influx when
applied to the exterior of astrocyte, neuroblastoma, and macrophage
cell lines, apparently without releasing Ca
from
stores. This activity was attributed to calcium influx factor, a small
(<500 M
) nonproteinaceous phosphate-containing
factor that they proposed as a diffusible messenger responsible for
activating capacitative Ca
entry. Additional
activation mechanisms involving GTP-binding
proteins(13, 14, 15) , tyrosine
kinases(16) , cGMP(17, 18) , and direct physical
coupling between the store's membrane and the plasma membrane (19) have been proposed (for reviews, see Refs. 1 and 3).
]
may play a role.
Feedback inhibition by intracellular Ca
appears to
occur through multiple mechanisms. After entering the cell,
Ca
binds to sites probably residing on the CRAC
channel itself(20) , eliciting rapid inactivation over tens of
milliseconds(8, 20) . In addition, slow inactivation of I
on a time scale of seconds has been observed
following abrupt elevation of
[Ca
]
or global rises
in
[Ca
]
(4, 6, 7, 8) ,
but the underlying mechanisms have not been determined. A central tenet
of the capacitative Ca
entry hypothesis is that
refilling of intracellular Ca
stores should terminate
Ca
influx. This prediction has been confirmed in
intact cells using fluorescent Ca
indicators(10, 21, 22) . However, the
ability of store refilling to close CRAC channels, which is an
important test of the role of CRAC channels in capacitative
Ca
entry, has not yet been examined directly in
patch-clamp experiments. Furthermore, the presence of additional
negative feedback pathways through which Ca
may turn
off CRAC channels has not been explored.
]
slowly feeds back to inactivate CRAC channels. One mechanism
is dependent on store refilling, while the other operates even when
refilling is completely prevented with thapsigargin. Thus, with the
inclusion of fast inactivation(8, 20) , it appears that
intracellular Ca
feeds back by at least three
independent pathways to control capacitative Ca
entry. Autoregulatory feedback on CRAC channels by Ca
is likely to be essential in determining the character of
receptor-stimulated Ca
signals in nonexcitable cells.
A portion of this work has been reported previously in abstract form
(23).
Cells and Reagents
Jurkat E6-1 human
leukemic T cells were maintained in complete medium containing RPMI
1640 and 10% heat-inactivated fetal bovine serum, 2 mM
glutamine, and 25 mM HEPES, in a 6% CO humidified
atmosphere at 37 °C. Log phase cells (0.2-1.2
10
/ml) were used in all experiments. Thapsigargin, okadaic
acid, 1-norokadaone, and calyculin A (LC Pharmaceuticals, Woburn, MA)
were prepared as stocks of 1 mM or 100 µM in
Me
SO. Cyclosporin A (2 mg/ml in 2% ethanol) and FK506 (50
µg/ml in ethanol) were the generous gift of Drs. N. Clipstone and
G. Crabtree at Stanford University.
Patch-Clamp Recording
Patch-clamp experiments were
conducted in the standard whole cell recording
configuration(24) . Extracellular Ringer's solution
contained the following: 155 mM NaCl, 4.5 mM KCl, 1
mM MgCl, 2 or 22 mM CaCl
, 10
mMD-glucose, and 5 mM Na-HEPES (pH 7.4).
Ca
-free Ringer's contained 3 mM MgCl
. Internal solutions contained the following: 140
mM cesium aspartate, 10 mM Cs-HEPES (pH 7.2), and
either 0.66 mM CaCl
, 11.68 mM EGTA, and
3.01 mM MgCl
(12 mM EGTA solution) or
0.066 mM CaCl
, 1.2 mM EGTA, and 2.01
mM MgCl
(1.2 mM EGTA solution). Free
[Ca
] in both of these solutions as measured
with indo-1 was 5 nM; free [Mg
]
was calculated to be 2 mM. Recording electrodes were pulled
from 100-µl pipettes (VWR), coated with Sylgard near their tips,
and fire-polished to a resistance of 2-6 megaohms when filled
with cesium aspartate pipette solution. The patch-clamp output
(Axopatch 200, Axon Instruments, Foster City, CA) was filtered at 1.5
kHz with an 8-pole Bessel filter (Frequency Devices, Haverhill, MA) and
digitized at a rate of 5 kHz. Stimulation and recording were performed
with an Apple Macintosh computer driving an ITC-16 interface
(Instrutech, Elmont, NY) and using PulseControl software extensions
(Jack Herrington and Richard Bookman, University of Miami) to Igor Pro
(WaveMetrics, Inc., Lake Oswego, OR). Command potentials were corrected
for the -12 mV junction potential that exists between the
aspartate-based pipette solutions and Ringer's solution. Cells
were allowed to settle onto but not firmly adhere to glass coverslip
chambers shortly prior to each experiment. Adherent and nonadherent
cells behaved identically in experiments with 1.2 mM EGTA
; however, currents obtained with 12
mM EGTA
were not as sustained in adherent
cells as in nonadherent ones. The reason for this difference was not
investigated further. I
was induced through the
store depletion protocol described previously(20) . Following
formation of the gigaseal, each cell was exposed to
Ca
-free Ringer's, and the whole cell recording
configuration was established. This procedure was sufficient to
activate I
maximally, because additional
pretreatment with 1 µM thapsigargin did not further
increase the average initial size of Ca
current (). After 3 min,
[Ca
]
was elevated to
2-22 mM, and steady-state I
was
measured in all experiments as a 5-10-ms average at the end of
200-ms pulses from -12 mV (the holding potential) to -132
mV delivered once every 2 s. In the experiment shown in Fig. 1,
peak I
was measured from a 1-ms average
beginning 3 ms after the start of each hyperpolarizing pulse to
minimize contributions from uncompensated capacitative current (time
constant, <1 ms) and fast inactivation. All data are corrected for
leak and residual capacitative current measured in the absence of
Ca
. Leak conductances were
20-100 picosiemens. Series resistance compensation was not
employed, since the series resistance (4-25 megaohms) produced
voltage errors of <3 mV. Cell capacitance was determined either from
the settings of the whole cell capacitance-compensation circuitry, or
by integrating currents elicited by 10-mV depolarizing steps. External
solutions were changed by positioning the cell
1 mm inside one
barrel of a perfusion tube array through which the desired solutions
flowed (<0.1 ml/min). Experiments were conducted at 22-25
°C. Where normalized data are presented, current amplitudes were
divided by the current's maximal value, and the time at which the
maximum occurred was defined as time zero. Data are presented as mean
± S.E. Statistical significance of results was assessed using
the Mann-Whitney U test, and differences are considered
significant if p < 0.01. [Ca
]
Measurements with
Indo-1-For experiments combining patch-clamp recording with
[Ca
]
measurements,
pipette solutions were supplemented with 100 µM indo-1
pentapotassium salt (Molecular Probes, Eugene, OR). Recording
conditions have been described in detail previously(20) .
Briefly, cells were illuminated using a 75W xenon arc lamp and a 360
± 5-nm interference filter (Omega Optical, Brattleboro, VT)
mounted on a Nikon Diaphot inverted microscope equipped with a Nikon
Fluor 40
objective (numerical aperature, 1.3) and a
transistor-transistor logic-controlled shutter to control the duration
of illumination. The emission signal was collected from an area
adjusted to be slightly larger than the cell. Emitted light was split
with a 440-nm dichroic mirror and passed through 405 ± 15 and
480 ± 12.5 nm interference filters (Chroma Technology Corp.,
Brattleboro, VT) to two photomultiplier tubes (HC124-02,
Hamamatsu Corp., Bridgewater, NJ).
[Ca
]
was estimated
from the relation [Ca
]
= K*(R - R
)/(R
- R). Background fluorescence was measured in the cell-attached
mode and was subtracted from subsequent fluorescence signals before
calculation of the 405/480 ratio, R. K*, R
, and R
were determined
from in vivo calibrations as described previously(20) .
Figure 1:
Fast and slow inactivation of I. A, after depletion of intracellular
Ca
stores, exposure of a Jurkat T cell to 22 mM Ca
induces a slowly
decaying inward current. Data points were measured every 2 s from the
peak current (
) or steady-state current (
) elicited during
brief hyperpolarizing voltage pulses shown in B (see
``Experimental Procedures''). Collection times for the traces
in B and C are indicated by i-iii and a-c, respectively. B, selected current
responses to 200-ms voltage pulses from -12 to -132 mV.
Rapid inactivation of I
is induced by the
sudden increase in Ca
influx upon hyperpolarization. Dashed line indicates zero current level. C,
current/voltage relation for the inward current. Responses to voltage
ramps (200-ms duration) from -120 to +50 mV show that
current is inward in this voltage range as expected for I
. Data in B and C are not
corrected for leak or capacitative currents. Internal solution, cesium
aspartate + 1.2 mM EGTA.
Fast and Slow Inactivation of I
I by
Intracellular Ca
was
induced in Jurkat leukemic T cells by the passive depletion of
intracellular Ca
stores. Depletion was achieved in
these experiments by incubating each cell in Ca
-free
Ringer's solution for 3 min while dialyzing its interior with a
pipette solution in which [Ca
] was buffered
to 5 nM. After such treatment, elevation of
[Ca
]
from 0 to 22
mM rapidly elicited an inward current (Fig. 1) whose
properties identified it as I
(4, 5, 6, 7, 8, 9, 20) .
These properties included a dependence on
Ca
(Fig. 1A),
rapid inactivation during hyperpolarizing voltage pulses (Fig. 1B), an inwardly rectifying current-voltage
relation with no clear reversal potential up to +50 mV (Fig. 1C), voltage-independent gating, and a lack of
significant current noise. As illustrated in Fig. 1A, I
decayed slowly in cells dialyzed with a
relatively low amount of EGTA (1.2 mM). In this paper, we
refer to the slow decay in current as slow inactivation; the use of
this terminology is not intended to imply any specific type of
mechanism. In all experiments, we measured I
during brief hyperpolarizing voltage pulses to -132 mV
delivered every 2 s (as in Fig. 1B). This protocol
optimizes the size of the current and allows independent measurement of
fast and slow inactivation, as described previously(20) . The
decay in the peak currents (Fig. 1A, squares)
reflects the slow inactivation process alone, whereas decay of the
steady-state currents (Fig. 1A, circles) is
determined by both fast and slow inactivation. Thus, the fact that both
measures of I
decay with the same time course
suggests that the fast and slow inactivation processes are independent.
In further support of this conclusion, none of the pharmacological
treatments described below affected fast inactivation.
]
and current
measurements were combined in indo-1-loaded cells to examine the
calcium dependence of slow inactivation. When depleted cells dialyzed
with low [EGTA]
(1.2 mM) were
exposed to 22 mM Ca
,
[Ca
]
remained
relatively constant for
10 s before climbing to micromolar levels,
as would be expected since Ca
influx via I
should eventually overcome the capacity of
EGTA to buffer Ca
(Fig. 2A). The
increase in [Ca
]
was
associated with a progressive inactivation of I
over a period of
100 s. As the current declined,
[Ca
]
reached a peak
and decreased, presumably as the rate of Ca
entry
fell below that of Ca
efflux by
Ca
-ATPases in the plasma membrane. Increasing the
buffering power of the pipette solution with 12 mM EGTA
restricted [Ca
]
to
levels below 100 nM for the duration of the experiment and
reduced the slow decay of the current (Fig. 2B). The
ability of increased intracellular Ca
buffering to
prolong the current is summarized for 10-17 experiments in Fig. 2C. These results demonstrate that slow
inactivation is Ca
-dependent.
Figure 2:
Slow inactivation of I is Ca
-dependent. A, in the presence of
1.2 mM EGTA, the induction and subsequent slow decline of I
is associated with a delayed rise and fall of
[Ca
]. B, 12 mM EGTA in
the pipette largely suppresses the [Ca
]
increase, and I
is more sustained. All currents
shown in this and subsequent figures were measured at the end of
hyperpolarizing voltage pulses to -132 mV as described in Fig.
1B. C, the effect of intracellular buffering on slow
inhibition of I
from experiments like those in A and B. Current amplitude is normalized to the
maximum amplitude reached in each experiment after exposure to 22
mM Ca
, and time is
plotted from this point onward. Plotted values are the mean ±
S.E. of 10-17 cells.
During whole cell recording, soluble components diffuse out of the
cell and may cause rundown of channel activity. To test whether slow
inactivation of I is due to a washout
phenomenon, we examined whether it can be reversed by removal of
extracellular Ca
. Inhibition of I
in the presence of 22 mM
Ca
was allowed to reach steady
state; at this point, Ca
was
removed for a variable period and then reapplied to measure the extent
to which I
had recovered. In the cell depicted
in Fig. 3, a 60-s exposure to Ca
-free
conditions allowed nearly complete recovery of the current's
amplitude. The extent of recovery varied among the cells tested. In six
cells in which slow inactivation reduced I
to a
level of 11 ± 3%, subsequent incubation in 0
Ca
for 100-150 s allowed
recovery to 54 ± 14% of the initial peak amplitude. The source
of the variation is not known. However, evidence presented below
suggests that most of the inactivation in these experiments is due to
reuptake of Ca
by stores; thus, a variable degree of
reemptying following removal of Ca
may contribute to the different amounts of recovery that were
observed. Regardless, these results further support the Ca
dependence of slow inactivation and demonstrate that it is not
simply due to nonspecific rundown or to washout of a factor essential
for maintenance of I
.
Figure 3:
Slow inactivation is reversible. After I declined to a steady-state level in 22 mM Ca
, the cell was bathed in
Ca
-free Ringer's solution for 60 s and then
reexposed to 22 mM Ca
. In this cell, the
current recovered almost completely, indicating that inactivation is
not due to washout of a diffusible factor.
Refilling of Ca
In the experiments described above,
CaStores Contributes
to Slow Inactivation
stores were depleted passively, without increasing
the permeability of the endoplasmic reticulum membrane to
Ca
or inhibiting its Ca
-ATPases.
Under these conditions, stores would be expected to refill efficiently
following a rise in
[Ca
]
, thereby causing
the slow inactivation of I
. To test this
hypothesis, we measured the time course of I
in
the presence of a high dose of TG (1 µM) that fully blocks
Ca
-ATPases in the endoplasmic reticulum (9, 10, 25)
and hence prevents Ca
reuptake. Cells were incubated
in Ca
-free Ringer's + TG for 3 min prior
to exposure to 22 mM
Ca
. Under these conditions with
1.2 mM EGTA
, slow inactivation of I
was greatly reduced despite a large increase
in [Ca
]
(Fig. 4A, ). In 21 cells treated
with TG, I
decayed over 100 s to a steady-state
level of
50% of its initial value (Fig. 4B).
Consistent with the time course of the current,
[Ca
]
declined only
partially over the same time period (Fig. 4A, ). These results demonstrate that store refilling
contributes to slow Ca
-dependent inactivation of I
. The failure of TG to fully hinder this
process is not due to an inability to prevent store refilling.
Experiments with TG were repeated with 20 µM IP
in the internal solution to attempt to increase the overall
extent of store depletion. The release of stored Ca
by 20 µM IP
was confirmed in separate
experiments by a large [Ca
]
spike occurring within seconds of breaking into cells in
Ca
-free Ringer's; such transients were not
observed in the absence of IP
(data not shown).
Furthermore, IP
alone prevented slow inactivation to about
the same extent as TG alone (
50%; n = 3 cells). As
summarized in Fig. 4B, IP
and TG together
had the same effect as TG alone, indicating that store refilling is not
occurring and therefore cannot explain the failure of TG to prevent I
inactivation. Rather, these results reveal a
second process of Ca
-dependent slow inactivation
occurring independently of changes in Ca
store
content.
Figure 4:
Store refilling contributes to slow
inactivation. A, 1 µM TG partially blocks the
slow decline in I and
[Ca
] in one cell exposed to 22 mM Ca
with 1.2 mM EGTA. TG was present during the 3-min preincubation in
Ca
-free Ringer's. B, TG limits slow
inactivation to an average final extent of
50%. Addition of 20
µM IP
to the internal solution produces no
further effect, indicating that incomplete emptying of stores is not
responsible for the partial effect of TG on current inhibition. Data
are the average normalized currents ± S.E. from 14-21
cells in experiments like those shown in A.
Effects of Phosphatase Inhibitors on Slow
Inactivation
Okadaic acid, a potent inhibitor of phosphatases 1
and 2A (26, 27) has been reported to enhance
capacitative Ca influx in Xenopus oocytes(28) . We therefore tested the ability of okadaic
acid and other phosphatase inhibitors to suppress the TG-insensitive
component of slow inactivation. Okadaic acid inhibits protein
phosphatases 1 and 2A in vitro with IC
values of
1-50 nM(27, 29) . A 3-min preincubation
with 100 nM okadaic acid + TG significantly reduced the
extent of slow inactivation over that seen with TG alone (Fig. 5). On average, the current inactivated only
25% over
100 s, comparable with the amount the current declines when the
[Ca
]
rise is mostly
suppressed with 12 mM EGTA
(Fig. 2C).
Figure 5:
Effects of phosphatase inhibitors on
store-independent I inactivation. Bars indicate the average (± S.E.) extent of current decline
after 100 s in 22 mM Ca
with 1.2 mM EGTA and various inhibitors. The dashed
line shows the average extent of inactivation observed with 1.2
mM EGTA + 1 µM TG (defining the maximum
possible level of store-independent inactivation). Conditions include
100 nM okadaic acid (n = 21), 100 nM 1-norokadaone (n = 6), 0.1-1.0 µM calyculin A (n = 11), 0.17 µM CsA (n = 14), 1.7 µM CsA (n =
20), and 62 nM FK506 (n = 10). Of these, only
okadaic acid, 1-norokadaone, and 1.7 µM CsA reduce the
extent of inactivation to a statistically significant degree (p < 0.01).
Okadaic acid pretreatment did
not affect the maximum amplitude of I attained
in the presence of TG (3.0 ± 0.2 pA/pF; n = 14)
relative to control (), suggesting that an okadaic
acid-sensitive process does not contribute to the activation of I
. In addition, 100 nM okadaic acid
applied in the absence of TG lacked any statistically significant
effect on the time course or extent of slow inactivation (decay
half-time, 36 s; final level, 0.26 ± 0.10; n =
14) compared with control (see Fig. 2C). This result
implies that store refilling assumes the major role in turning off CRAC
channels under the conditions of these experiments (i.e. passive store depletion and normal endoplasmic reticulum
Ca
-ATPase activity).
/calmodulin-dependent protein phosphatase 2B
(calcineurin) in the store-independent inactivation of I
was addressed using the immunosuppressants
cyclosporin A and FK506. These compounds are extremely potent
inhibitors of protein phosphatase 2B when complexed with their
respective binding proteins, the cyclophilins and FKBP(30) .
IC
values for the inhibition of calcineurin in Jurkat cell
lysates are
5 nM for CsA and
0.5 nM for
FK506(31) . As summarized in Fig. 5, CsA prevented
TG-insensitive slow inactivation to the same extent as okadaic acid
when applied at 1.7 µM but not at 170 nM.
Treatment of cells with 62 nM FK506 had no significant effect.
These results argue against a necessary role for protein phosphatase
2B/calcineurin in the Ca
-dependent inactivation of I
.
Three Independent Mechanisms for Feedback Regulation of
CRAC Channels
The results of this study and a previous one (20) demonstrate that intracellular Ca regulates CRAC channels by at least three distinct mechanisms.
The three modes of feedback are readily distinguished by their
kinetics, by their site of action relative to CRAC channels, and by
their pharmacological profiles. Fast inactivation (Fig. 1B) occurs within milliseconds of Ca
entry and is controlled by binding to sites located within
several nanometers of the pore, probably residing on the CRAC channel
itself(20) . The two forms of slow inactivation described in
this study are roughly 1000-fold slower and are driven by a global
rather than a local rise in [Ca
]
(see below). None of the pharmacological agents employed in
this study had any effect on fast inactivation, whereas thapsigargin
selectively inhibited the store-dependent component of slow
inactivation, and okadaic acid, 1-norokadaone, and a high concentration
of cyclosporin A inhibited the store-independent component. The
independence of fast and slow inactivation is also consistent with the
invariance of fast inactivation during the induction and decline of I
as stores empty and refill(20) . For
these reasons, we conclude that fast and slow inactivation occur
through distinct mechanisms that operate in parallel to regulate CRAC
channels. These studies provide the first evidence that capacitative
Ca
entry is controlled through mechanisms independent
of Ca
store content. Thus, the results raise the
interesting possibility that additional signaling pathways may modulate I
.
The Calcium Dependence of Slow Inactivation
The
ability of high levels of an intracellular Ca buffer
(12 mM EGTA) to reduce the extent of slow inactivation agrees
with previous observations that intracellular Ca
buffering enhances the amplitude and duration of I
in whole-cell
recordings(4, 6) . Due to its slow Ca
binding rate, 12 mM EGTA cannot reduce
[Ca
]
in microdomains
near CRAC channels(20) , but it can suppress increases in bulk
[Ca
]
(Fig. 2B). Thus, the effect of high
[EGTA] suggests that most of the Ca
binding
sites subserving slow inactivation are farther from the channel than
those underlying fast inactivation(20) .
Slow Inactivation by Store Refilling
A critical
prediction of the capacitative Ca entry hypothesis is
that store refilling should terminate influx. This prediction has been
supported by previous reports that store refilling is temporally
correlated with decreased influx of
Ca
or Mn
(21, 22). However, the effect of
refilling on the CRAC channels themselves, as well as the possible
existence of additional inactivation pathways, has not been explored.
Our findings that TG inhibits
50% of the slow inactivation of I
therefore constitute an important
confirmation that I
underlies capacitative
Ca
influx in Jurkat T cells and additionally reveal a
store-independent inactivation mechanism operating in parallel.
]
without affecting
the peak current amplitudes (), suggesting that stores in
passively depleted cells do in fact rapidly sequester
Ca
. The average current with 1.2 mM EGTA
decays with a half-time of 32 s (Fig. 2C). These kinetics are similar to the time course
with which depletion-induced Mn
influx in neutrophils
decays upon refilling of stores (t = 20 s at 25
°C)(22) . Thus, our results are consistent with the action
of a relatively long-lived CRAC channel activator, a feature that may
have important implications for the generation of
[Ca
]
oscillations (see
below).
Slow Inactivation by a Store-independent
Mechanism
A second mechanism of slow
Ca-dependent inactivation of I
was revealed by the current's decline in the presence of 1
µM TG, a condition that precludes store refilling.
Store-independent inactivation is largely inhibited by okadaic acid, an
observation that may explain the potentiating effect of okadaic acid on
capacitative Ca
entry described previously in Xenopus oocytes(28) . In those studies, okadaic acid
reversed the decay of a Ca
-activated Cl
current in 5-hydroxytryptamine-stimulated oocytes, leading to the
speculation that a phosphatase acts to limit the lifetime of a
diffusible CRAC channel activator(28) . In view of our present
results, the gradual decline in Ca
-activated
Cl
current seen in the oocytes may have resulted from
the Ca
-dependent inactivation of I
by the store-independent (okadaic acid-sensitive) mechanism.
Further experiments in oocytes using 1-norokadaone and calyculin A
could test this hypothesis.
values for inhibition of protein phosphatase 2B in Jurkat cells.
Taken together, our results argue against a role for protein
phosphatases 1, 2A, 2B, and 3 in mediating slow inactivation of CRAC
channels. The inhibitory effects we observed may be nonspecific. The
mechanism underlying store-independent slow inactivation is unknown,
but could in principle result from a decrease in the CRAC channel
activation signal, production of a channel inhibitor, or a change in
the channels themselves.
Physiological Role of Slow I
Slow inactivation is likely to play an
essential role in the generation of
[CaInactivation
]
oscillations in T
cells. Oscillations in Jurkat and human T cells are triggered by
several stimuli that release moderate amounts of Ca
from intracellular stores, such as cross-linking of the T cell
antigen receptor and low doses of ionomycin or thapsigargin and other
smooth endoplasmic reticulum Ca-ATPase
inhibitors(4, 10, 33, 34) . Periodic
Ca
influx via I
appears to be
responsible for [Ca
]
oscillations in these cells(4, 10) , supporting a
simple oscillation model in which fluctuations in store content and
phasic opening and closing of CRAC channels are coupled through changes
in [Ca
]
(10) .
To enable this model system to oscillate, time lags must exist between
store depletion and CRAC channel opening and between
[Ca
]
elevation, store
refilling, and CRAC channel closure. Previous work has shown that CRAC
channels open with a time constant of 20-30 s following store
depletion with IP
(6, 8) , demonstrating a
lag between depletion and channel activation. This study describes two
mechanisms that operate with similarly slow kinetics (half-times,
30 s) to control channel closure following
[Ca
]
elevation.
feeds back to control the amplitude and duration of capacitative
Ca
influx. The relative contribution of these two
mechanisms to Ca
signaling under physiological
conditions is not known. The relatively small effect of okadaic acid
observed in the absence of TG (Fig. 5) suggests that the
store-dependent mechanism is dominant; however, it should be noted that
the passive method of depletion employed in these experiments would be
expected to favor store refilling. The store-independent inactivation
mechanism is likely to play a greater role under physiological
conditions, in which elevated intracellular IP
promotes
Ca
release and hinders store refilling. The existence
of a mechanism that regulates CRAC channels independently of store
content reveals an unsuspected degree of complexity in the regulation
of capacitative Ca
entry.
Table: I and
[Ca
]
following store
depletion
present where
noted. Unless otherwise stated, all experiments were conducted with 1.2
mM EGTA
. I
was
measured during voltage steps to -132 mV as described under
``Experimental Procedures.'' I
is the
maximal current observed after elevation of
[Ca
]
from 0 to 22
mM; this occurred within 10 s of the solution change.
Normalized I
is the maximum current divided by
membrane capacitance and reflects the surface density of open channels.
Peak [Ca
]
is the
highest value observed after elevation of
[Ca
]
from 0 to 22
mM after store depletion. Steady-state
[Ca
]
is the value
measured
120 s after the solution change.
[Ca
]
did not reach a
peak or a steady-state value in the presence of 12 mM EGTA
; for these experiments, peak
[Ca
]
was measured
20 s after elevation of [Ca
]
from 0 to 22 mM. Data are presented as mean
± S.E. (n).
,
inositol 1,4,5-trisphosphate; [Ca
], free
intracellular Ca
concentration; I
, Ca
release-activated
Ca
current; TG, thapsigargin; CsA, cyclosporin A.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.