(Received for publication, July 11, 1995; and in revised form, September 12, 1995)
From the
The least understood aspect of the agonist-induced
Ca signal is the activation and regulation of the
Ca
release-activated Ca
influx
(CRAC) across the plasma membrane. To explore the possible role of
heterotrimeric G proteins in the various regulatory mechanisms of CRAC,
continuous renal epithelial cell lines stably expressing
and the constitutively active
were
isolated and used to measure CRAC activity by the Mn
quench technique. Release of intracellular Ca
by
agonist stimulation or thapsigargin was required for activation of CRAC
in all cells. Although the size of the internal stores was similar in
all cells, CRAC was 2-3-fold higher in
- and
-expressing cells. However, the channel was
differentially regulated in the two cell types. Incubation at low
[Ca
]
, inhibition of
the NOS pathway, or inhibition of tyrosine kinases inhibited CRAC
activity in
but not
cells.
Treatment with okadaic acid prevented inhibition of the channel by low
[Ca
]
and the protein
kinase inhibitors in
cells. These results suggest
that expression of
dominantly activates CRAC by
stabilizing a phosphorylated state, whereas expression of
makes CRAC activation completely dependent on phosphorylation by
several kinases. G proteins may also modulate CRAC activity
independently of the phosphorylation/dephosphorylation state of the
pathway to increase maximal CRAC activity. Furthermore, our results
suggest a general mechanism for regulation of CRAC that depends on
coupling of receptors to specific G proteins.
The [Ca]
signal
evoked by G protein-dependent Ca
mobilizing agonists
such as bradykinin (BK) (
)and carbachol involves activation
of inositol 1,4,5-trisphosphate-mediated Ca
release
from internal stores (IS), which is followed by activation of a plasma
membrane located Ca
influx pathway to increase
[Ca
]
. Subsequently IS
and plasma membrane Ca
pumps remove Ca
from the cytosol restoring
[Ca
]
to basal
levels(1, 2) . Periodic repetition of this sequence
results in [Ca
]
oscillations(1, 2) .
A poorly understood
aspect of the Ca signal is the nature and regulation
of the Ca
release-activated Ca
influx (CRAC) pathway, although significant advances have been
made in recent years(1, 3) . Ca
release from IS is required and sufficient for activation of
Ca
entry(3, 4) . Ca
release initiated by agonist stimulation or by inhibition of IS
Ca
pumps activates the same CRAC pathway(3) .
Patch clamp experiments in several cell
types(5, 6, 7, 8) have identified a
Ca
release-activated Ca
current
(I
) which can also be followed by measurement of
Mn
influx through I
and quenching of
Fura 2 fluorescence(8, 9, 10) .
Multiple
regulatory mechanisms of I have been reported. CRAC and
I
appear to be sensitive to
[Ca
]
(11, 12, 13) ,
where high [Ca
]
rapidly and partially inactivates the
influx(11, 13) . In multiple cell types, CRAC is
inhibited by tyrosine kinase inhibitors, suggesting that tyrosine
kinase-dependent phosphorylation regulates
I
(14, 15, 16, 17) .
In basophils (18) and lacrimal acinar cells (19) a
GTP-dependent and GTP
S-inhibitable mediator is required for
channel activation. In pancreatic acini (20, 21, 22, 23) intestinal cells (24) and macrophages, (25) cGMP modulates CRAC
activity.
The mechanism linking Ca release from IS
and activation of I
is not well defined. When the
channel is regulated by cGMP, Ca
release from IS
activates a nitric-oxide synthase (NOS) to generate NO, increase cGMP
and stimulate Ca
influx(22, 23) .
However, the channel is not activated by cGMP without depletion of IS
Ca
(22) . Recently, much attention has been
given to activation of I
by a diffusible messenger(s).
In Xenopus oocytes (26) and basophils(18) ,
sustained activation of I
requires a cytosolic factor
that can be stabilized by inhibition of protein phosphatases with
okadaic acid (OA)(26) . A small, P
-containing
molecule, named Ca
influx factor, was extracted from
Jurkat T cells and was suggested to be the diffusible messenger
responsible for activation of CRAC(27) . Subsequent studies in
oocytes further support (28, 29) and challenge (30, 31) the suggestion that Ca
influx factor is the universal activator of Ca
influx required for reloading of IS upon termination of cell
stimulation.
The family of heterotrimeric G proteins couples
receptor-initiated events in the plasma membrane to effector molecules
inside the cell. The subunits contain the primary information
that determines the specificity of receptor and effector interactions.
G
is widely expressed in mammalian tissues and
regulates phospholipase C
isoforms, thereby regulating inositol
1,4,5-trisphosphate production and Ca
release from
IS. The functions of the
chains of the
/
family are only beginning to be
understood.
is widely expressed in mammalian
tissues(32, 33) . Expression of
increases Na
/H
exchange
activity(34, 35, 36) , transforms cells in
tissue culture, and increases expression of immediate early
genes(37, 38, 39) . More recently we showed
that stable overexpression of G
in the continuous MCT
renal epithelial cell line increases expression and activity of the
inducible form of NOS through a transcriptional mechanism. (
)
Because of the central role of G proteins in
Ca signaling we used MCT cells stably expressing
and the activated mutant
to
study the role of G proteins in regulating CRAC activity. We report
here that expression of
and
markedly increase CRAC influx (2-3-fold) initiated by agonist
stimulation or depletion of IS with Tg. In cells expressing
CRAC activation required elevated
[Ca
]
, the activity of
protein kinases and could be stably activated by OA, an inhibitor of
protein phosphatases. In
-expressing cells,
depletion of IS from Ca
maximally activated CRAC
independent of [Ca
]
and cellular kinases/phosphatase activities. These findings
suggest that (a) G proteins modulate CRAC influx by direct and
indirect interactions and (b) CRAC influx is regulated by
multiple kinase/phosphatase-dependent events.
Fura 2 was purchased from Molecular Probes; tissue culture
medium, serum, and G-418 were from Life Technologies, Inc.
[H]Arginine was from Amersham Corp. Tissue
culture plasticware was from Falcon. Other chemicals were from Sigma
or, if molecular biology grade, from Fisher.
Figure 1:
Rate of
CRAC influx in Neo control cells and cells expressing and
. Cells loaded with Fura 2 and
incubated in solution A were stimulated with 50 nM BK or
exposed to 0.1 µM Tg. When
[Ca
]
was stabilized,
Mn
influx was initiated by addition of 0.25 mM Mn
to the medium. After about 2 min the cells
were lysed by addition of 50 µM digitonin (Dig.)
to obtain the 100% quench at each condition. The effect of digitonin on
fluorescence of BK-stimulated cell was deleted for clarity. Each
experiment shown represents at least 8 similar observations with cells
from different subpassages.
Stable expression of the G protein
subunits
and the GTPase-deficient
(
) dramatically increased CRAC influx in BK
or Tg-stimulated cells. The basal rate of Mn
influx
was similar in all cells, although it tended to be slightly higher in
cells (17 ± 8%, n = 8
relative to paired Neo control or
cells). After
stimulation of the pooled
-expressing cells with BK,
CRAC activity was 6.78-fold higher than that measured in
controls. Treatment of
cells with Tg was more
effective than BK stimulation in increasing CRAC activity. Tg increased
CRAC activity by 16.05-fold above control, which was about 2.49-fold
higher than BK in the same cells and 3.01 ± 0.08-fold higher (n = 9) than in Tg-treated Neo control cells. In cells
from the clone expressing
, BK and Tg stimulation
caused similar increases in CRAC activity, which was 10.14- and
11.25-fold above control
, respectively. These
rates were between 3.18 ± 0.27-fold (n = 4) (BK)
and 1.9 ± 0.17-fold (n = 9) (Tg) above the rates
measured in similarly treated Neo controls. Similar results were
observed in three additional
clones and in three
clones expressing wild type
, although in all cases
expression of
increased CRAC activity more than
expression of
. In the case of
,
recent experiments showed that expression of the GTPase-deficient
increased Ca
and
Mn
influx rates similar to
(data
not shown).
Since CRAC activity increases with increased
Ca depletion of
IS(3, 44, 45, 46) , a possible
explanation for the increased activity of CRAC in
-
and
-expressing cells is an increased IS pool
size and depletion during Tg treatment. This was not the case since
measurement of pool size by exposing cells in Ca
-free
medium to high concentrations of ionomycin (47) produced
similar increases in [Ca
]
in
all cell types. Thus, 5 µM ionomycin increased
[Ca
]
of cells incubated in
albumin- and Ca
-free medium to 848 ± 66 (Neo),
861 ± 58 (
), and 797 ± 55
(
) in 3-5 experiments from different
subpassages. In addition, Tg caused similar
[Ca
]
increase in all cells (see
below).
Figure 2:
Dependence of CRAC activity on
[Ca]
. Neo,
, or
cells were incubated in
Ca
-free solution A (for experiments a-c in each case) or solution A containing 1.5 mM CaCl
(trace d in each case) and stimulated with 0.1
µM Tg. At different times after exposure to Tg (a and d, 4 min; b, 30 s) cells were exposed to a
mixture of Ca
and Mn
(a and b) or Mn
(d) to measure
the rate of Mn
quench. Cells incubated in
Ca
-free medium and Tg for about 4.5 min were exposed
to 1.5 mM CaCl
for about 30 s to increase
[Ca
]
(trace
c) before exposure to Mn
for measurement of
fluorescence quench rate.
The time courses of CRAC activation during
IS Ca depletion and during restoration of
[Ca
]
for the three cell types
were measured using the protocol of Fig. 2and are illustrated
in Fig. 3. In
cells, activation of CRAC
required Ca
release from IS, but the sustained CRAC
activity did not require a sustained elevation of
[Ca
]
. CRAC activity was near
maximal after 3 min of incubation with Tg, and remained active when
[Ca
]
was reduced to resting
levels (Fig. 3, panel A). CRAC activity did not change
with a subsequent increase in [Ca
]
to about 350 nM (Fig. 3, panelB). In contrast, in cells expressing
,
activation of CRAC required Ca
depletion and
persistently high [Ca
]
. During
the Ca
depletion protocol, CRAC activity mirrored
[Ca
]
, increasing during the
first min of incubation with Tg and returning to the basal rate as
[Ca
]
was reduced. Increasing
[Ca
]
then reactivated CRAC (Fig. 3, panel B, open circles). CRAC
activation was time-dependent, requiring a 2-min incubation at 37
°C, although [Ca
]
was
maximal 20-25 s after addition of
[Ca
]
(Fig. 2). The
requirement for [Ca
]
to
activate CRAC in the Neo control cells was intermediate between that of
the cells expressing
and the cells expressing
. However, CRAC activity was 2-3-fold
higher in the cells expressing the G proteins than in the Neo cells (Fig. 3, panel B). The requirement of high
[Ca
]
for activation of CRAC in
cells is likely to be independent of NOS, since
these cells show high NOS activity in the unstimulated state (see
below). Hence expression of
increased CRAC activity
and stabilized it in a
[Ca
]
-dependent form and
expression of
increased CRAC activity and
stabilized it in a
[Ca
]
-independent form.
Figure 3:
Time course of CRAC activation during
Ca depletion (A) and
[Ca
]
restoration (B). The protocol of figure 2 was used to measure rates of
Ca
influx during Ca
depletion and
[Ca
]
restoration. 100%
control was taken as the rate of quench in Fig. 2(trace
d) of
cells. At different times during Tg
treatment in Ca
-free medium (25 s, 1, 2, 3, or 5 min)
of the different cell types a mixture of Ca
and
Mn
was added to measure the rate of fluorescence
quench (panel A). Then 1.5 mM Ca
was added to increase [Ca
]
as in Fig. 2(trace c) and Mn
quench rates were measured 0.5, 1, 2 or 5 min after addition of
Ca
(panel B). The results in the figure are
the mean of 3 (Neo), 5 (
), and 4
(
) experiments. S.E. were between 5 and 12% of
the signals and are not shown for clarity.
Figure 4:
NOS activity in intact MCT cells. Cells
were preincubated for 15 min in arginine-deficient medium and
[H]arginine. They were then stimulated with 50
nM BK in the presence or absence of 5 mML-NAME for an additional 10 min. The reactions were
stopped with trichloroacetic acid, extracted with ether, and
[
H]citrulline separated from
[
H]arginine by column chromatography. Each value
is the mean of triplicate samples ± S.E. The experiment shown is
representative of three others with similar
results.
Fig. 5depicts representative tracings, whereas Fig. 6summarizes the results of multiple experiments. Treatment
of control, , or
cells with
the arginine analog L-NAME, which inhibits NOS
activity(48) , or NO
, an NO
donor(22, 49) , had no effect on CRAC activity when IS
were loaded with [Ca
]
(Fig. 6). However, L-NAME almost abolished CRAC
activity in
cells, whether it was activated by BK (Fig. 6) or Tg ( Fig. 5and Fig. 6). Inhibition of
CRAC by L-NAME was completely reversed by bypassing NOS
inhibition with NO
to supply the cells
with NO and activate the soluble guanylyl cyclase. cGMP was as
effective as NO
in reversing the
inhibitory effect of L-NAME (data not shown).
Figure 5:
Effect of NOS inhibition and a NO donor on
CRAC activity in cells expressing and
. Cells in solution A were treated for 10 min at
37 °C with or without 5 mML-NAME. Samples of the
cells were also incubated for 2 min with 15 mM
NO
before stimulation with 0.1 µM Tg. After 4 min of exposure to Tg, fluorescence quench
measurements were initiated by addition of Mn
to the
incubation medium.
Figure 6:
Inhibition of Ca influx
by L-NAME and reversal by NO
in
cells expressing
but not
. The
procedure used to treat the cells and measure fluorescence quench was
the same as that described in the legend to Fig. 5. The various
cell types were stimulated with 50 nM BK or 0.1 µM Tg as indicated. The rate of fluorescence quench in Tg-treated
cells was taken as 100% control. The figure shows
the mean ± S.E. of 3-4
experiments.
In contrast
to the finding in cells, L-NAME and
NO
had no effect on BK or Tg-activated
CRAC activity in
cells. Thus, L-NAME
did not inhibit CRAC activity in BK- or Tg-treated cells and increasing
cGMP with NO
did not increase CRAC
activity beyond that measured in the absence of
NO
. Again, in Neo controls both
NO-sensitive and NO-insensitive components of CRAC activity (a mixture
of the behavior observed in
- and
-overexpressing cells) was found, with lower
maximal CRAC activity.
Figure 7:
Effect of genistein on CRAC activity.
Cells were incubated with the indicated concentration of genistein or
daidzein for 5 min at 37 °C before stimulation with 50 nM BK (data not shown) or 0.1 µM Tg. After 4 min of
incubation in the presence of Tg, Mn was added to
measure the rate of Fura 2 fluorescence quench. The upper panel shows the traces recorded from Neo cells treated with 50
µM genistein or 50 µM daidzein and
cells treated with 10 µM genistein or
10 µM daidzein. Daidzein up to 100 µM had no
effect in either cell type. The lower panel shows the mean
± S.E. of 4 (
and Neo) or 5
(
) experiments of cells treated with Tg. The 100%
control in each case was taken as the rate of quench measured in
Tg-treated cells in the absence of drugs.
Figure 8:
Effect of okadaic acid on CRAC activity in
cells expressing .
-overexpressing
cells were treated with 0.1 µM OA or 0.1 µM of the nonactive analog 1-nor-okadaone for 5 min at 37 °C
before addition of 10 µM genistein. After an additional
5-min incubation at 37 °C, treated and untreated cells were
stimulated with 0.1 µM Tg for 4 min before addition of
Mn
to measure the rate of fluorescence
quench.
Figure 9:
Okadaic acid reverses CRAC inhibition by
low [Ca]
, NOS, and PTK
inhibitors. Cells were treated with 0.1 µM OA, 5 mML-NAME, or 10 µM genistein as indicated in
the figure and then stimulated with 0.1 µM Tg. After
stabilization of [Ca
]
,
fluorescence quench was measured by addition of Mn
to
the incubation medium. Samples of cells were also maintained in
Ca
-free medium during treatment with OA and
stimulation with Tg to measure the effect of OA on CRAC activity of
cells at low
[Ca
]
as detailed in Fig. 2, time point a. The figure shows the averages of
3 (Neo), 2 (
), or 4 (
)
experiments. Please compare the effect of low
[Ca
]
, L-NAME
and genistein in this figure to those in Fig. 2(middle
panel), 6, and 7.
The most important finding of this set of experiments is that
treatment with OA completely prevented the inhibition of CRAC activity
by low [Ca]
(Fig. 9), L-NAME (Fig. 9), and genistein ( Fig. 8and Fig. 9) in
-overexpressing cells. OA prevented
the inhibition of CRAC by low [Ca
]
and the kinase inhibitors whether CRAC was activated by Tg
treatment (Fig. 9, middle columns) or BK (data not
shown). The nonactive analogue of OA, 1-nor-okadaone, had no effect ( Fig. 8for Tg and genistein).
The activation and regulation of the CRAC pathway have not
been characterized in molecular terms. Recent studies suggest that
Ca influx factor, a small P
-containing
molecule (27) that is sensitive to cellular
phosphatases(50) , may be the ubiquitous activator of CRAC.
This suggestion has been both challenged (30, 31) and
supported (28, 29) by studies using oocytes and
lacrimal acinar cells. In a number of experimental systems CRAC is
regulated by the NO metabolic pathway (20, 21, 22, 23, 24, 25) ,
GTP-dependent and GTP
S-inhibited processes (18, 19) , protein-tyrosine
kinases(14, 15, 16, 17) , and
cytochrome P-450 metabolites(51, 52) . No individual
regulatory mechanism can account for all aspects of CRAC activity in
all cases, and it is possible that several mechanisms may be operative
in one cell type(3, 17, 24) . A common
feature of the CRAC regulatory mechanisms described so far appears to
be involvement of a phosphorylation/dephosphorylation mechanism.
Indeed, inhibition of protein phosphatases with OA prolongs and
augments CRAC activity(26, 50) .
In the present
study we used a new approach, that of overexpressing specific G protein
subunits to study the regulation of CRAC. Our results suggest
that G proteins can regulate CRAC activity, both by influencing the
phosphorylation/dephosphorylation state of the system and by a
phosphorylation-independent mechanism. These two modes are illustrated
in Fig. 10. Below we discuss the evidence in favor of such
regulatory effects.
Figure 10: A model summarizing the effect of heterotrimeric G proteins on CRAC activity.
Release of Ca from the IS of
Neo control cells by BK stimulation or treatment with Tg increased CRAC
activity. Tg was more effective than BK in activating CRAC because it
mobilized all the Ca
present in IS, whereas BK
mobilized about 60% of the stored Ca
. This was the
case whether Ca
was measured in cell populations or
at the single cell level. A similar situation existed in
-overexpressing cells. In
-expressing cells BK and Tg were equally
effective in mobilizing IS Ca
and activating CRAC. A
close relationship between Ca
mobilization and
Ca
influx has been reported previously in several
cell types(3, 44, 45, 46) . Our
studies extend these findings to show that even when CRAC activity is
up-regulated its regulation by Ca
content of the IS
is maintained. An additional point of these experiments is that
overexpression of the
subunits did not alter the fundamental
features of Ca
signaling. The cells must have
adjusted to overexpression of
, and in particular the
constitutively activated
, to prevent persistent
activation of the Ca
signaling system. This
adjustment probably represents activation of the normal ``turn
off'' mechanism for signals initiated by
or
.
Neo control cells express a number of G protein
chains including
and
(34) . In these cells CRAC activity has two
components. The first requires persistently high
[Ca
]
and is sensitive to
inhibition by inhibitors of NOS and protein-tyrosine kinases. The
second component is relatively independent of
[Ca
]
and is not sensitive to
inhibition by these agents. Expression of specific heterotrimeric G
protein
subunits stabilizes CRAC activity in one of these forms.
Overexpression of
increased total CRAC activity and
stabilized the form that requires persistently high
[Ca
]
and is sensitive to
inhibition by L-NAME and genistein. A 2-min incubation at high
[Ca
]
was required for maximal
activation of CRAC in
cells (Fig. 3B). This [Ca
]
dependence of CRAC activation is not likely to reflect NOS
activation since during agonist stimulation NOS is maximally activated
within 30 s (22, 23) and
cells show
high NOS activity also in the unstimulated state (Fig. 4). High
[Ca
]
may influence CRAC
activity through activation of a
[Ca
]
-dependent protein
kinase(s), since the requirement for
[Ca
]
can be bypassed by OA.
Such will be a new mechanism of CRAC regulation. Inhibition of a
phosphatase by
was required to unmask this
mechanism. Expression of
also made CRAC activity
completely dependent on an active NOS pathway. Finally CRAC activity in
-overexpressing cells was completely blocked by
genistein at a concentration at least 5-fold lower than that reported
for other systems(15, 16, 17) . When taken
together these observations suggest that
stabilizes
the CRAC pathway in a general inactive state.
Overexpression of
also increased total CRAC activity, but
stabilized the second,
[Ca
]
-independent, L-NAME- and genistein-insensitive form of the pathway.
Activation of CRAC still required Ca
release from IS,
but [Ca
]
could be reduced to
resting levels (Fig. 2) without inactivating CRAC (Fig. 3). Inhibition of the NO pathway ( Fig. 5and Fig. 6) or protein-tyrosine kinases (Fig. 7) did not
inhibit CRAC activity. Thus, once CRAC was activated by Ca
release from IS of
-expressing cells, it
could not be inactivated by reducing
[Ca
]
or inhibiting protein
kinase activities.
The simplest interpretation of these observations
is that expression of stabilizes a dephosphorylated
state of CRAC to make CRAC activity completely dependent on protein
kinases, whereas expression of
constitutively
activates CRAC by stabilizing a phosphorylated state of the channel
itself or an accessory regulatory protein (Fig. 10). Such an
interpretation is supported by the effect of the protein phosphatase
inhibitor OA, which caused CRAC activity in
cells to
behave much like that in
cells. These findings
can help explain previous observations concerning regulation of CRAC
activity. In pancreatic
acini(20, 21, 22, 23) , intestinal
cells(24) , and macrophages(25) , but not other
cells(3) , CRAC activity is regulated by cGMP through the NO
pathway. Similarly, in some (14, 15, 16, 17) but not necessarily
all (3) cells, CRAC activity is inhibited by protein kinase
inhibitors. It is possible that the relative expression and/or
influence of
and
on CRAC activity
determines the dominant regulatory mode of CRAC in a given cell type.
Expression of or
increases
CRAC activity by a mechanism beyond affecting the phosphorylation state
of the CRAC pathway (Fig. 10). The maximal activity of CRAC in
and
cells was approximately
3-fold higher than that in Neo controls or cells overexpressing other
wild type or constitutively active
subunits(40) . We
could not find a condition, including long incubation at high
[Ca
]
of Tg-treated cells,
increased cGMP by a NO donor, or treatment with OA of control cells or
cells exposed to Tg and cGMP, which augmented CRAC activity in Neo
cells to the level seen in the cells that overexpress
and
. Furthermore, the effect of
and
was specific to these
subunits
in that similar overexpression of
reduced CRAC
activity in the same cell type(40) . Augmentation of CRAC
activity did not require activated
subunits. The properties of
the [Ca
]
signal in
- and
-expressing cells was
similar. Recently we isolated MCT cells overexpressing
and found that their
[Ca
]
signal resembled that of
-overexpressing cells. Thus,
and
appear to up-regulate CRAC activity beyond
affecting the phosphorylation state of the pathway. It is not clear at
present whether expression of
and
increased the number of CRAC channels or the activity of existing
channels.
In summary, by expressing different G protein
subunits such as
,
(40) , and
and
(present study) we were
able to show that CRAC activity can exist in a dephosphorylated state
(
overexpression) or phosphorylated state
(
overexpression) that modulates the activity of
the pathway. This may explain the diversity of CRAC activity and its
regulation observed in different cell types.