From the Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709 and the § Department of Molecular Genetics, Institute for Liver Research, Kansai Medical University, Moriguchi 570-8506, Japan
Received for publication, December 21, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We investigated the putative roles of
phospholipase C, polyphosphoinositides, and inositol
1,4,5-trisphosphate (IP3) in capacitative calcium
entry and calcium release-activated calcium current
(Icrac) in lacrimal acinar cells, rat
basophilic leukemia cells, and DT40 B-lymphocytes. Inhibition of
phospholipase C with U73122 blocked calcium entry and
Icrac activation whether in response to a
phospholipase C-coupled agonist or to calcium store depletion with
thapsigargin. Run-down of cellular polyphosphoinositides by
concentrations of wortmannin that block phosphatidylinositol 4-kinase
completely blocked calcium entry and Icrac. The
membrane-permeant IP3 receptor inhibitor,
2-aminoethoxydiphenyl borane, blocked both capacitative calcium
entry and Icrac. However, it is likely that
2-aminoethoxydiphenyl borane does not inhibit through an action on the
IP3 receptor because the drug was equally effective in
wild-type DT40 B-cells and in DT40 B-cells whose genes for all three
IP3 receptors had been disrupted. Intracellular application
of another potent IP3 receptor antagonist, heparin, failed
to inhibit activation of Icrac. Finally, the
inhibition of Icrac activation by U73122 or
wortmannin was not reversed or prevented by direct intracellular application of IP3. These findings indicate a requirement
for phospholipase C and for polyphosphoinositides for activation of capacitative calcium entry. However, the results call into question the
previously suggested roles of IP3 and IP3
receptor in this mechanism, at least in these particular cell types.
Activation of cell surface receptors coupled to phospholipase C
(PLC)1 leads to generation of
the second messenger inositol 1,4,5-trisphosphate (IP3).
IP3 is known to bind to and activate receptors present on
intracellular calcium stores, the endoplasmic reticulum, allowing calcium to be released into the cytosol (1). In most cells, the
emptying of these calcium stores subsequently activates calcium influx
across the plasma membrane through the "capacitative calcium entry"
pathway (2, 3). It is unclear how empty intracellular stores signal
activation of plasma membrane capacitative calcium entry. However, a
rise in cytosolic calcium is not required, nor is the activation of
plasma membrane receptors, because agents such as the calcium-ATPase
inhibitor thapsigargin and the calcium ionophore ionomycin, which
deplete calcium stores independently of receptor-coupled events, can
fully activate capacitative calcium entry (3). Two general models are
proposed as underlying mechanisms for capacitative calcium entry
activation. One is based on the requirement for a diffusible messenger
generated upon store depletion (4, 5). The other hypothesizes a
conformational coupling between proteins on the intracellular stores
(e.g. the IP3 receptor) and capacitative calcium
entry channels or associated proteins in the plasma membrane (6,
7).
The diffusible messenger hypothesis proposes that a decrease in the
concentration of stored calcium leads to the release of a factor that
diffuses to the plasma membrane and activates capacitative calcium
entry channels. Evidence in support of this model comes from reports of
an unidentified calcium influx factor (4, 5, 8, 9) and patch
clamping experiments in Xenopus oocytes (10).
Evidence from Xenopus oocytes indicates that if the signal is diffusible then its diffusion is somewhat limited, because the
signal remains confined to the area of calcium release
(11-13).
The conformational coupling model proposes the direct relay of a signal
through protein-protein interactions. In the model's simplest form,
IP3 receptors in the endoplasmic reticulum interact with
capacitative calcium entry channels in the plasma membrane (6, 7). A
change in the conformation of the IP3 receptor, which
occurs after a drop in endoplasmic reticulum luminal calcium (14), may
then be transmitted directly to the capacitative calcium entry channels
causing them to open (6, 7). This theory was originally proposed by
analogy with ryanodine receptors on the sarcoplasmic reticulum stores,
which bind directly to dihydropyridine calcium channels in the plasma
membrane of skeletal muscle (15). The theory has subsequently gained
some experimental support. A number of studies suggest that the
regulation of capacitative calcium entry is dependent upon an intimate
interaction between the endoplasmic reticulum and plasma membrane.
Pharmacological or physical dislocation of the plasma membrane away
from the endoplasmic reticulum prevents activation of capacitative
calcium entry by store depletion (16-19).
However, there is more direct evidence that an IP3
receptor-capacitative calcium entry channel complex bridges the
gap between endoplasmic reticulum and plasma membrane. Calcium flux
through endogenous capacitative calcium entry channels (20) or
overexpressed TRP3 channels (a candidate capacitative calcium entry
channel (21)) can be recorded in the cell-attached configuration but ceases when the patch is excised. The addition of IP3 and
the IP3 receptor to the patch (but not the IP3
receptor alone) reconstitutes capacitative calcium entry activity. A
similar requirement for the IP3 receptor is revealed by the
use of the IP3 receptor inhibitors 2-aminoethoxydiphenyl
borane (2-APB) and xestospongin C, which uncouple store depletion from
the activation of capacitative calcium entry (22). Thus, there is
substantial evidence that IP3 receptors are required for
activation of capacitative calcium entry channels and also evidence
that these IP3 receptors need to be liganded with
IP3.
The requirement for IP3 for capacitative calcium entry
presents something of a paradox, because, as discussed above, many laboratories have confirmed that store depletion alone, in the absence
of phospholipase C activation, is capable of full activation of
capacitative calcium entry. Thus, it has been suggested that the
requirement for IP3 must normally be fulfilled by its basal production, presumably by a phospholipase C located in close proximity to the channel-IP3 receptor complex (23). The experiments
in the present study were therefore designed to address the question of
whether basal PLC activity and basal levels of IP3 in
resting cells are required and are sufficient to support a role for the IP3-liganded IP3 receptor in the context of the
conformational coupling capacitative calcium entry model.
To this end, we monitored capacitative calcium entry channel activity
directly by measuring the calcium release-activated calcium current
(Icrac) in RBL cells and indirectly by measuring the cytosolic calcium concentration in RBL, mouse lacrimal, and DT40
cells. In the last case, we used both wild-type DT40 cells and cells
whose IP3 receptor genes were disrupted by targeted homologous recombination (24). IP3 receptor function was
inhibited pharmacologically with the membrane-permeant IP3
receptor inhibitor 2-APB and the membrane-impermeant inhibitor, low
molecular weight heparin. Basal IP3 formation was prevented
with a phospholipase C inhibitor (U73122), and the levels of the
precursor polyphosphoinositides were decreased by use of a
phosphatidylinositol 4-kinase inhibitor (wortmannin). Intracellular
calcium stores were subsequently depleted, independent of PLC and the
IP3 receptor, using thapsigargin and ionomycin, to test
whether capacitative calcium entry could still be activated by store
depletion. Our results show that maneuvers that are expected to disrupt
basal PLC activity prevent activation of capacitative calcium entry
upon store depletion. However, the specific function of PLC is
uncertain, since we are unable to demonstrate a role for
IP3 or IP3 receptors in this pathway.
Cell Culture and Measurement of Intracellular Calcium--
Rat
basophilic leukemia cells stably expressing the muscarinic m1 receptor
(RBL-2H3 m1) were a gift from Dr. M. Beaven (National Institutes of
Health, Bethesda, MD) (25). Cells were cultured in Earle's minimal
essential medium with Earle's salts, 10% fetal bovine serum, 2 mM L-glutamine, 50 units/ml penicillin, and 50 mg/ml streptomycin, (37 °C, 5% CO2). For experiments,
cells were passaged onto glass coverslips (number 1 1/2) and
used 12-36 h after plating.
Mouse lacrimal cells were isolated as described previously (26).
Briefly, the excised glands from three mice (male CD-1; 30-40 g) were
finely minced and treated for 1 min with 0.2 mg/10 ml trypsin (Sigma).
The cells were then removed from the trypsin by centrifugation,
followed by a 5-min incubation with 2.5 mg/10 ml soybean trypsin
inhibitor (Sigma) in the presence of 2.5 mM EGTA. Finally,
the acinar cells were isolated after treating the tissue with 5 mg/10
ml collagenase (Roche Molecular Biochemicals) for 10 min. Throughout,
all enzyme solutions were prepared in Dulbecco's modified Eagle's
medium. Following isolation, the cells were washed and suspended in
sterile Dulbecco's modified Eagle's medium containing 10% fetal
bovine serum, 2 mM glutamine, 50 units/ml penicillin, and
50 units/ml streptomycin. The cells were allowed to attach to glass
coverslips (number 1) coated with Matrigel. Lacrimal cells were
incubated on glass coverslips at least 3 h before use.
Lacrimal cells attached to glass coverslips were mounted in a Teflon
chamber and incubated with 0.5 µM Fura-2/AM (Molecular Probes, Inc., Eugene, OR) for 30 min at room temperature. The cells
were then washed and bathed in a HEPES-buffered physiological saline
solution (HBSS; 120 mM NaCl, 5.3 mM KCl, 0.8 mM MgSO4, 1.8 mM CaCl2,
11.1 mM glucose, 20 mM HEPES, pH 7.4) at room
temperature at least 20 min before Ca2+ measurements were
made. In some experiments, a nominally Ca2+-free medium was
used, which was identical in composition except for the omission of
added CaCl2.
The fluorescence of Fura-2-loaded lacrimal cells was monitored with a
photomultiplier-based system, mounted on a Nikon Diaphot 300 inverted
microscope equipped with a Nikon × 40 (1.3 NA) Neofluor objective. The fluorescence light source was a Deltascan D101 (Photon Technology International Ltd.), equipped with a light path
chopper and dual excitation monochromators. The light path chopper
enabled rapid interchange between two excitation wavelengths (340 and
380 nm), and a photomultiplier tube monitored the emission fluorescence
at 510 nm, selected by a barrier filter (Omega). All experiments were
performed at room temperature. The data are expressed as a ratio of
Fura-2 fluorescence due to excitation at 340 nm to that due to
excitation at 380 nm
(F340/F380).
The immortalized chicken B-lymphocyte cell line, DT-40 (RIKEN Cell Bank
number RCB1464), and a mutant version with genes for all three
IP3 receptor types disrupted (RIKEN Cell Bank number RCB1467) were maintained in suspension with RPMI 1640 supplemented with
10% fetal bovine serum, 1% chicken serum, 4 mM glutamine, 50 units/ml penicillin, 50 units/ml streptomycin, and 50 µg/ml 2-mercaptoethanol. The cells were maintained in culture at 40 °C in
a humidified 95% air, 5% CO2 incubator, and at a cell
density that ranged between 25 × 104 and 1 × 106 cells/ml. Both cell types were allowed to attach to
glass coverslips (number 1) coated with Matrigel, and maintained in the
RPMI 1640 medium described above. Both DT-40 cell types, attached to
glass coverslips, were mounted in a Teflon chamber and incubated with either 1 µM Fura-2/AM (Molecular Probes) for 30 min at
40 °C or 1 µM fluo-4/AM (Molecular Probes) for 15 min
at 40 °C. The cells were then washed and bathed in a
modified HBSS (136.9 mM NaCl, 5.4 mM KCl, 0.81 mM MgSO4, 0.44 mM
KH2PO4, 0.34 mM
Na2HPO4, 0.34 mM
NaHCO3, 1.8 mM CaCl2, 5.5 mM glucose, 10 mM HEPES, pH 7.4) at room
temperature at least 20 min before Ca2+ measurements were
made. In some experiments, a nominally Ca2+-free medium was
used, which was identical in composition except for the omission of
added CaCl2. In addition, calcium entry in DT-40 cells
loaded with Fura-2 was determined in the presence of 0.5 mM
CaCl2, whereas calcium entry with fluo-4 was determined in
the presence of 0.25 mM CaCl2, in both
instances to avoid saturation of the fluorescence signal due to the
very large increases in [Ca2+]i in this cell type.
The fluorescence intensities of Fura-2- and fluo-4-loaded DT-40 cells
were monitored with a camera-based imaging system (Universal Imaging)
mounted on a Zeiss Axiovert 35 inverted microscope equipped with a
Zeiss × 40 (1.3 NA) fluor objective. Both fluorescence excitation
and emission wavelengths were selected by filters (Chroma).
For Fura-2 measurements, a Sutter Instruments filter changer enabled
alternative excitation at 340 and 380 nm, and the emission fluorescence
was monitored at 510 nm with a Paultek Imaging camera (model PC-20)
equipped with a GenIISys intensifier (Dage-MTI, Inc.). The images of
multiple cells collected at each excitation wavelength were
subsequently processed using the MetaFluor software (Universal Imaging
Corp., West Chester, PA) to provide ratio images. After background
fluorescence correction, these images were further processed to convert
the fluorescence ratios into [Ca2+] values
(Kd(Fura-2) = 135 nM). Individual
cells in the field of view were selected with suitable regions of
interest, and their calcium changes with time were extracted.
All experiments were performed at room temperature.
Fluo-4 measurements were performed with the same imaging system, except
single wavelength excitation was performed at 485 nm, and the emission
fluorescence was monitored at 510 nm. Regions of interest were used to
select Individual cells in the field of view so that the time course of
changes in fluorescence intensity could be monitored and extracted.
Electrophysiology--
Patch clamp experiments were conducted in
the standard whole cell recording configuration, using RBL-2H3 m1 cells
(27). Patch pipette (2-4 megaohms; Garner glass, type 7052)
solutions contained 140 mM Cs-Asp, 2 mM
MgCl2, 10 mM HEPES, 1 mM MgATP, and
10 mM
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-Cs4 (with free calcium set to 100 nM,
calculated using MaxChelator software, version 6.60), pH 7.2. Bath
solution (HBSS) was as described above, except CaCl2 was
increased to 10 mM for calcium-HBSS, or omitted for
nominally calcium-free HBSS (10 mM MgCl2 was
included in nominally calcium-free HBSS).
In all experiments, upon forming the whole cell configuration,
the cell membrane potential was held at 0 or +20 mV. Once every 5 s, the membrane potential was stepped to Measurement of [3H]Inositol-labeled
Phosphatidylinositol 4-Phosphate (PIP) and Phosphatidylinositol
4,5-Bisphosphate (PIP2)--
RBL-2H3 cells were labeled
with [3H]inositol and incubated in the presence or
absence of methacholine, wortmannin, or both according to the protocol
for examining effects on Icrac (see "Results"). 3H-Labeled lipids were extracted,
deacylated, and separated by HPLC as previously described (28, 29). The
[3H]inositol-labeled polyphosphoinositide levels were
determined by liquid scintillation spectroscopy of the HPLC fractions
corresponding to the retention times of authentic PIP and
PIP2 standards.
Materials--
IP3, caged IP3,
ionomycin, U73122, and U73343 were from Calbiochem. Thapsigargin was
from LC laboratories. Heparin and wortmannin were from Sigma.
Cs4-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid and Fura-2 were from Molecular Probes.
Inhibition of IP3 Receptor Function with 2-APB, but Not
Heparin, Blocks Icrac and Capacitative Calcium
Entry--
RBL-2H3 cells have a well defined calcium release-activated
calcium current (Icrac), activated upon
intracellular calcium store depletion (30). We used the whole cell
patch clamp technique to measure Icrac in
RBL-2H3 cells, stably transfected with the muscarinic m1 receptor (25).
Extracellular application of ionomycin (500 nM) led to
rapid and full activation of Icrac (Fig.
1A). Subsequently,
extracellular application of the IP3 receptor inhibitor 2-APB (100 µM) rapidly blocked
Icrac. Cells incubated with 2-APB for 3 min
before exposure to ionomycin failed to show any detectable activation of Icrac (Fig. 1B). The
inhibition was not readily reversible upon removal of 2-APB (Fig. 1,
A and B).
In contrast to the results with 2-APB, blockade of IP3
receptor function with the competitive IP3 receptor
antagonist heparin (10 mg/ml) failed to block ionomycin-activated
Icrac (Fig. 1C). Heparin is
cell-impermeant and was delivered to the cell interior by inclusion in
the patch pipette. An effective concentration clearly entered the cell,
because 200 s after forming the whole cell mode, flash photolysis
of caged IP3 (30 µM) failed to release stored
calcium or activate Icrac in cells exposed to
heparin (Fig. 1D, open circles). In
the absence of heparin, Icrac was readily activated by UV flash photolysis of caged IP3 (Fig.
1D, closed circles).
Heparin has previously been reported to have no effect on the rise in
cytosolic calcium induced by thapsigargin-activated capacitative
calcium entry in mouse lacrimal cells (31). However, the
IP3 receptor antagonist 2-APB (30 µM) does
prevent capacitative calcium entry in response to store depletion with
thapsigargin in mouse lacrimal cells (Fig. 1E).
Capacitative Calcium Entry Is Sensitive to Inhibition of PLC by
U73122--
Methacholine, a muscarinic receptor agonist, evokes a
sustained rise in intracellular calcium in mouse lacrimal cells, when added at both a low (1 µM) and high (100 µM) concentration (Fig. 2A). The calcium signal is
composed of the IP3-mediated release of stored calcium and
plasma membrane capacitative calcium entry (32). We aimed to prevent
this agonist response by blocking activation of PLC and IP3
generation with a membrane-permeable PLC inhibitor, U73122 (33). A
3-5-min pretreatment of cells with 10 µM U73122 has
previously been documented to fully and irreversibly prevent PLC
activation upon agonist stimulation (33, 34). Likewise, a 5-min
preincubation of mouse lacrimal cells with 10 µM U73122
was sufficient to prevent the intracellular calcium response to low or
high doses of methacholine, consistent with a blockade of
agonist-activated PLC (Fig. 2A). A 5-min pretreatment of
cells with 1-5 µM U73122 resulted in variable degrees of inhibition of the calcium signal (data not shown). A 5-min
preincubation of cells with 10 µM U73343, a less potent
analogue of U73122, had no effect on the agonist-evoked responses (not
shown).
The addition of the Ca2+-ATPase inhibitor thapsigargin to
lacrimal cells in the absence of extracellular calcium caused a
transient increase in intracellular calcium, due to release of calcium
from stores. Upon the addition of extracellular calcium, a second more sustained rise in intracellular calcium concentration occurs, due to
calcium influx via capacitative calcium entry (Fig. 2B). A
5-min pretreatment of cells with 10 µM U73122 had no
effect on the ability of thapsigargin to release intracellular calcium stores but fully prevented the rise in intracellular calcium
concentration upon the readdition of extracellular calcium (Fig.
2B). U73343 had no effect on thapsigargin-induced
capacitative calcium entry (Fig. 2B). Thus, inhibition of
PLC activity with U73122 appears to result in a specific block of
capacitative calcium entry after store depletion.
Icrac Is Sensitive to Inhibition of PLC Activity by
U73122--
To ensure the effects of U73122 were not indirect (for
example, due to changes in cell membrane potential or stimulation of
the calcium removal processes), we examined the effects of this reagent
on Icrac in RBL-2H3 m1 cells, measured under
voltage-clamped conditions. The extracellular addition of either
thapsigargin (1 µM) or ionomycin (500 nM) was
sufficient to deplete intracellular calcium stores and fully activate
Icrac in control cells (Fig. 3, black circles).
A 5-min pretreatment of cells with U73122 (10 µM),
prevented activation of Icrac upon store
depletion with either thapsigargin (Fig. 3A, open
circles) or ionomycin (Fig. 3B, open
circles). A 5-min pretreatment of cells with 5 µM U73122 (a dose that failed to consistently block
either agonist or thapsigargin-induced calcium signals in mouse
lacrimal cells) failed to block activation of
Icrac in RBL-2H3 m1 cells, although
Icrac was inhibited to 38 ± 4%
(n = 9) of controls (see also Fig.
4B). A 5-min pretreatment of
cells with the inactive analogue U73343 (10-15 µM) did
not affect activation of Icrac upon store
depletion with either thapsigargin or ionomycin (Fig. 3B,
gray circles).
The addition of 10 µM U73122 to RBL-2H3 cells once
Icrac had been activated resulted in a much
smaller inhibition of Icrac and never caused a
complete block. Icrac in U73122-treated cells
averaged 73 ± 12% of controls. This suggests that
Icrac is much less sensitive to inhibition of
PLC once it is initiated (Fig. 3, C and D).
The Addition of Exogenous IP3 and Diacylglycerol Fails
to Restore U73122-inhibited Capacitative Calcium Entry--
In the
context of the conformational coupling model, the apparent dependence
of capacitative calcium entry on PLC might reflect a requirement for
IP3 on the channel-associated IP3 receptor (6, 7, 23). Thus, we next addressed whether the addition of exogenous IP3 to the patch pipette would overcome the block of
Icrac activation in cells treated with U73122.
Intracellular delivery of F-IP3 (50 or 500 µM), a slowly metabolizable analogue of IP3,
rapidly activated Icrac in control cells after
forming the whole cell mode (Fig. 4A). However, a 5-min
pretreatment of cells with 10 µM U73122 fully
prevented activation of Icrac by delivery of 50 µM F-IP3 (Fig. 4A). A combination
of 100 µM IP3 together with 100 µM 1-oleyl-2-acetyl-sn-glycerol, a membrane
permeant diacylglycerol, also failed to restore
Icrac in response to a combination of
thapsigargin and ionomycin (not shown, n = 4).
Because the addition of exogenous IP3 was not sufficient to
restore Icrac after inhibition of PLC, we tried
to increase endogenous IP3 levels. Hence, we aimed to
inhibit PLC activity partially using submaximal U73122 (5 µM, 5 min pretreatment); subsequently, we added a high
dose of methacholine (100 µM) in an attempt to drive
residual PLC activity. Pretreatment of RBL-2H3 m1 cells with 5 µM U73122 inhibited ionomycin-activated
Icrac to 38 ± 4% of control levels. The
subsequent addition of 100 µM methacholine failed to
increase Icrac significantly (Fig.
4B). In RBL-2H3 m1 cells not pretreated with U73122, a
time-matched addition of 100 µM methacholine caused
Icrac activation, showing that the failure of
response in U73122-treated cells is not due to a general lack of
responsiveness after 600 s in the whole cell mode (Fig.
4B, dotted trace).
Inhibition of Phosphatidylinositol 4-Kinase Inhibits
Icrac--
Another method to disrupt PLC activity is to
prevent production of the substrate for the enzyme. Concentrations of
wortmannin 100-fold greater than those required to inhibit
phosphatidylinositol 3-kinase (10-100 nM) are reported to
inhibit phosphatidylinositol 4-kinase (10-100 µM) (35).
PIP is the immediate precursor of PIP2, which is the
substrate for PLC and the precursor of IP3 (36). Previous
studies have demonstrated that inhibition of phosphatidylinositol
4-kinase, which phosphorylates phosphatidylinositol to PIP, results in
inhibition of capacitative calcium entry in platelets (37). In SH-SY-5Y
cells at least, although PIP is depleted by a 10-min preincubation with
10 µM wortmannin, depletion of PIP2 is facilitated by
agonist stimulation (38). Hence, we attempted to reduce cellular PIP
and PIP2 levels by incubating cells with wortmannin for an extended
period (10-20 µM; 40 min) and to further deplete PIP2 to
low levels with agonist stimulation (methacholine, 10-15 min). A final
15-20 min in the presence of 20 µM wortmannin but in the
absence of methacholine ensured that Icrac would
not be preactivated by the agonist. We then assessed the effect of this
protocol on Icrac (Fig.
5). This pretreatment of RBL 2H3 m1 cells
almost completely inhibited Icrac activated by
thapsigargin and ionomycin (13 ± 3% of control cells, Fig. 5A, open circles). Control cells
pretreated with 100 µM methacholine, for 10 min, in the
absence of wortmannin, displayed normal Icrac activation upon treatment with thapsigargin and ionomycin (107 ± 15% of control cells, Fig. 5A, closed
circles). This indicates that methacholine pretreatment
per se is not responsible for the inhibition.
In cells pretreated with 20 µM wortmannin for only 5 min,
in the absence of agonist (a treatment shown not to deplete PIP2 levels
in a previous study (38)), Icrac was not
inhibited (95.8 ± 3% of controls, n = 4, not
shown). Moreover, a 40-60-min pretreatment of cells with 10 nM wortmannin to inhibit phosphatidylinositol 3-kinase, but
not phosphatidylinositol 4-kinase, also failed to inhibit
Icrac (106 ± 11% of controls,
n = 5, not shown).
The Addition of Exogenous IP3 Fails to Restore
Wortmannin-inhibited Icrac--
In cells pretreated with
20 µM wortmannin (40 min) and 100 µM
methacholine (10 min) to inhibit phosphatidylinositol 4-kinase and
deplete polyphosphoinositides, we attempted to overcome the inhibition
of Icrac activation by the addition of exogenous
IP3. Intracellular delivery of 500 µM
F-IP3 failed to restore the Icrac activation in wortmannin-pretreated cells (Fig. 5B).
Effect of Wortmannin on Levels of PIP and PIP2 in
RBL-2H3 Cells--
The above results suggest that the inhibitory
action of wortmannin is not likely to be due to a diminished ability to
produce IP3 from PIP2. Thus, we utilized a
[3H]inositol labeling protocol to examine the effects of
wortmannin on levels of PIP and PIP2 in RBL-2H3
cells. The results are summarized in Table
I. Using a protocol similar to that for
the Icrac measurements, wortmannin efficiently
depleted cellular PIP, whether or not combined with agonist treatment.
Despite this striking effect on PIP levels, PIP2 levels
were little affected. PIP2 was reduced somewhat only with
the combination of wortmannin and agonist, but it seems unlikely that
this would be sufficient to completely prevent PLC-mediated formation
of IP3.
2-APB and Wortmannin Block Capacitative Calcium Entry in DT-40
IP3 Receptor Knockout Cells--
Sugawara et
al. (24) generated a line of DT-40 cells (an immortalized B-cell
line) with the genes for all three IP3 receptor types
disrupted by targeted homologous recombination. These cells were
negative for all three types of IP3 receptors by Northern analysis. The knockout cells were shown to produce normal phospholipase C responses following activation of appropriate surface membrane receptors, but produced no detectable [Ca2+]i
signal (24). However, normal capacitative calcium entry responses to
thapsigargin were observed. We have utilized the cell line with all
three IP3 receptors knocked out to examine the apparent
roles of IP3 receptors and inositol lipids in capacitative calcium entry in this cell type. We have used fluorimetric analysis of
[Ca2+]i changes; for technical reasons, we have
not been able to successfully measure store-operated ion currents in
this cell type.
It has been argued that because of the site of gene disruption, these
cells could, in theory, produce a truncated form of IP3
receptor that might still serve to couple to plasma membrane channels
(21). Thus, we measured specific binding of
[3H]IP3 to membranes prepared from wild-type
DT-40 cells and the IP3 receptor knockout cells. The
wild-type cells contained 16.8 ± 2.1 fmol/mg of protein of
specific IP3 binding sites. The knockout cells were found
to contain 1.3 ± 1.7 fmol/mg of protein of specific IP3 binding sites, a value not significantly different from zero.
The data shown in Fig. 6 confirm the
earlier observations of Sugawara et al. (24). The wild type
cells exhibited irregular [Ca2+]i oscillations in
response to activation of the B-cell receptor in ~70% of the cells.
In the IP3 receptor knockout cells, no response to B-cell
receptor activation was seen in any of the cells (Fig. 6A).
Fig. 6B confirms that DT-40 cells exhibit a capacitative calcium entry response to thapsigargin and that this response is
quantitatively and qualitatively indistinguishable in the wild type and
mutant cell lines.
In the protocol depicted in Fig. 7, DT-40
cells were sequentially treated with an activator of the B-cell
receptor and with thapsigargin. In this experiment, examining multiple
cells simultaneously, the asynchrony of the oscillatory responses to
B-cell receptor activation resulted in a somewhat blunted average
response. In Fig. 7A, the absence of the agonist response in
the knockout cells, together with a normal response to thapsigargin, is
again evident. A similar experiment, carried out in the presence of 75 µM 2-APB, is shown in Fig. 7B. 2-APB
completely blocked the B-cell receptor response in the wild-type cells
and reduced the thapsigargin response to a small, transient response in
both the wild-type and knockout cells. This response is similar to that
seen in the absence of external Ca2+ (not shown) and
presumably reflects intracellular release of Ca2+ from
internal stores.
Unfortunately, we were not able to examine the effects of U73122 on
Ca2+ signaling in DT-40 cells, because concentrations of
the drug required for phospholipase C inhibition were toxic to the
cells and on their own caused large irregular rises in
[Ca2+]i. However, we were able to utilize
wortmannin to examine the possible role of phosphorylated inositol
lipids. As shown in Fig. 8, wortmannin
treatment caused a marked attenuation of the Ca2+ entry
response to thapsigargin, and this effect was similar in the wild type
and mutant cell lines.
The current study employed a variety of pharmacological reagents
that would be expected to disrupt the phospholipase C-IP3 pathway at distinct points. The rationale for this approach was to gain
insight into the role played by this pathway in coupling intracellular
Ca2+ store depletion to the activation of capacitative
calcium entry channels in the plasma membrane. The model under
consideration is one in which basal activity of phospholipase C and
basal levels of IP3 in the vicinity of coupling complexes,
are required for signaling to take place. Surprisingly, our
findings call into question a role for IP3 in signaling
capacitative calcium entry, yet the data support a critical role of a
phospholipase C and phosphorylated inositol lipids.
In a previous study, Berven and Barritt (39) observed inhibition by
U73122 of thapsigargin-induced calcium entry in hepatocytes, but since
they had no reason to suspect a role for phospholipase C in this
process, they concluded that this represented a nonspecific effect of
the drug. However, our results suggest that the inhibitory effect of
this drug is indeed likely due to its action against PLC. First,
U73122, but not the less potent isomer U73343, completely inhibited
both capacitative calcium entry and Icrac. Second, the minimum concentration of the drug that inhibited calcium entry and Icrac was also the minimum
concentration reported to inhibit PLC-dependent signaling.
Third, U73122 was much less effective when applied after activation of
Icrac had already taken place, indicating that
it is not acting as a direct channel blocker.
At the concentrations employed in the current study, wortmannin
inhibits PI 4-kinase, and under these conditions the drug blocked
capacitative calcium entry and Icrac. As for
U73122, inhibition did not occur if the drug was added after activation had taken place. However, while this drug effectively depleted cellular
PIP levels, PIP2 levels were minimally affected.
The original rationale for examining the roles of PLC and PIP2 was
based on a model according to which PLC-mediated breakdown of PIP2
would provide IP3, a requirement for coupling
IP3 receptors to plasma membrane capacitative calcium entry
or CRAC channels (23). In support of this idea, we found that the
membrane-permeant IP3 receptor antagonist, 2-APB,
completely inhibited capacitative calcium entry and
Icrac. This result confirms the previous
findings of Ma et al. (22). These investigators concluded
that 2-APB was unlikely to be acting as a channel blocker, because it
was able to block the activation of TRP3 channels when the channels were activated by a PLC-linked stimulus but not when the channels were
activated by diacylglycerol. We have confirmed this observation in a
stable, TRP3-expressing cell line in our own
laboratory.2 However, in the
case of capacitative calcium entry channels it seems unlikely that the
inhibitory action of 2-APB involves IP3 receptors. In the
current study, in DT-40 cells lacking all three types of
IP3 receptors (24), 2-APB was as effective in inhibiting capacitative calcium entry as in wild type cells. While 2-APB was able
to block Icrac in RBL cells, unlike U73122 and
wortmannin it was equally effective when applied after the current was
activated. Furthermore, high concentrations of another potent
IP3 receptor antagonist, heparin, failed to inhibit the
development of Icrac. Finally, we have recently
observed that 2-APB is more effective in inhibiting
Icrac when applied to the outside of the cell
rather than intracellularly (40). Thus, we believe that at least in the
RBL and DT40 cell types, the main inhibitory action of 2-APB involves
an action on the store-operated channels, not involving interactions
with IP3 receptors.
While our findings implicate a role for PLC and polyphosphoinositides,
it appears that this is not due to a requirement for the production of
IP3. Inhibition of Icrac by U73122
or wortmannin was not reversed by direct intracellular application of
IP3 or by the other product of PIP2 breakdown,
diacylglycerol. We found that wortmannin was as effective as an
inhibitor in the IP3 receptor knockout cells as in wild
type cells. In addition, wortmannin caused depletion of PIP but had
little effect on PIP2. This raises the issue of what the
functions of polyphosphoinositides and PLC are in capacitative calcium
entry. For the case of PLC, there may be an analogy with the
Drosophila photoreceptor, where PLC is absolutely essential
for signaling to TRP (41, 42), yet IP3 receptors are not
required (43). However, in the case of Drosophila TRP, the
function of calcium store depletion in signaling is controversial
(44).
While we cannot at present assign a specific role for
polyphosphoinositides with certainty, it seems unlikely that their
function is simply to act as a precursor for formation of
IP3 and diacylglycerol. Rosado and Sage (19) utilized a
similar strategy to reveal a requirement for polyphosphoinositides in
platelet signaling and suggested a role for these lipids in
cytoskeletal rearrangements that accompanied store depletion.
The precise role played by both PLC and the polyphosphoinositides must
await further study. Based on our current findings, the role of
IP3 and the IP3 receptor seems at present
unclear. The best evidence for the conformational coupling model comes from studies of exogenously expressed TRP channels (21) or from studies
of store-operated channels in endothelial cells (45) and A431 epidermal
cells (20). In each of these cases, the properties of the currents and
single channels are clearly different from those expected of
Icrac. Thus, it is possible that capacitative calcium entry is signaled by a conformational coupling mechanism in
some cell types but not in others. There is an obvious analogy with the
modes of coupling between ryanodine receptors and L-type calcium
channels. This direct mode of interaction apparently occurs only in
skeletal muscle, while coupling in other systems (heart for example)
involves a diffusible messenger, in this case calcium itself.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
100 mV (for 20 ms to assess
Icrac), and then a voltage ramp to +60 mV over a
period of 160 ms was applied. Currents are normalized to cell
capacitance. All voltages are corrected for a 10-mV liquid junction
potential. Membrane currents were amplified with an Axopatch-1C
amplifier (Axon Instruments, Burlingham, CA). Voltage clamp protocols
were implemented, and data acquisition was performed with PCLAMP 6.1 software (Axon Instruments). Currents were filtered at 1 kHz and digitized at 200-µs intervals.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (21K):
[in a new window]
Fig. 1.
The IP3 receptor inhibitor 2-ABP,
but not heparin, blocks Icrac and
capacitative calcium entry. A and B,
extracellular application of 500 nM ionomycin
(IONO) was used to deplete intracellular calcium stores and
activate Icrac in RBL-2H3 m1 cells. The
IP3 receptor inhibitor 2-APB (100 µM) was
applied extracellularly either after (A, n = 4) or before (B, n = 3) application of
ionomycin for the time indicated by the bars. C,
12 mg/ml heparin was included in the patch pipette (open
circles, n = 6; controls are
filled circles, n = 6). 200 or
400 s (not shown) after forming the whole cell mode (time 0),
ionomycin (500 nM) was added to deplete intracellular
calcium stores and activate Icrac. D,
30 µM caged IP3 was included in the patch
pipette in the absence (filled circles,
n = 7) or presence (open circles,
n = 10) of 12 mg/ml heparin. 200 s after forming
the whole cell mode (time 0), UV light was applied to release the caged
IP3, allowing intracellular calcium store depletion and
Icrac activation. E, Fura-2-loaded
mouse lacrimal cells were treated with 1 µM thapsigargin
to deplete intracellular calcium stores and activate capacitative
calcium entry, either in the presence (i, n = 5) or absence (ii, n = 5) of 30 µM 2-APB. For patch clamp experiments, the whole cell
configuration was established at time 0, at which time the voltage
protocol described under "Experimental Procedures" was initiated.
Icrac was measured once every 5 s at
100 mV. Current is normalized against cell capacitance, and
the current density (pA/picofarads; pA/pF) is plotted.
Values are mean ± S.E.
View larger version (13K):
[in a new window]
Fig. 2.
The phospholipase C inhibitor U73122 blocks
both agonist-activated PLC and thapsigargin- activated capacitative
calcium entry. Mouse lacrimal cells, loaded with Fura-2, were
treated with low (1 µM) and high (100 µM)
doses of methacholine (MeCh) (A) or with
thapsigargin (B) to deplete intracellular stores and
activate capacitative calcium entry. As indicated, cells were either
untreated or pretreated, for 5 min, with a PLC inhibitor U73122 (10 µM, n = 3) or its inactive analogue
U73343 (10 µM, n = 3). Nominally
calcium-free HBSS was replaced with HBSS containing 1.8 mM
calcium as indicated.
View larger version (35K):
[in a new window]
Fig. 3.
The phospholipase C inhibitor U73122 blocks
Icrac. A-D, RBL-2H3 m1
cells were first exposed to 10 µM U73122 or 10 µM U73343 for 5 min or were untreated. Subsequently,
thapsigargin (TG, 1 µM) or ionomycin
(IONO, 500 nM) was applied extracellularly to
deplete intracellular calcium stores and activate
Icrac. HBSS containing 10 mM calcium
was replaced with nominally calcium-free HBSS at the times indicated.
Filled circles represent control experiments,
without U73122. In B, the gray circles
represent cells treated with 10 µM U73343. Values are
mean ± S.E. (n 8).
View larger version (28K):
[in a new window]
Fig. 4.
The addition of exogenous IP3
fails to relieve the inhibition of Icrac
produced by inhibition of PLC. A, RBL-2H3 m1 cells were
pretreated for 5 min with either 10 µM U73122
(n = 18, open symbols) or 10 µM U73343 (n = 5, gray
symbols) or were untreated (n = 21, black symbols). After pretreatment, the whole
cell configuration was established (time 0), and 50 or 500 µM (not shown) F-IP3 was delivered to the
cell interior through the patch pipette. B, RBL-2H3 m1 cells
were exposed to 5 µM U73122 for 5 min (black
symbols, n = 10), after which time 500 nM ionomycin (IONO) was applied extracellularly
(as indicated). After a further 200 s, 100 µM
methacholine (MeCh) was added. In control cells, not treated
with U73122 (broken trace, no symbols), the
addition of 100 µM methacholine 600 s after forming
the whole cell mode rapidly activated Icrac.
HBSS containing 10 mM calcium was replaced with nominally
calcium-free HBSS at the times indicated.
View larger version (29K):
[in a new window]
Fig. 5.
Wortmannin-induced inhibition of
Icrac. A, RBL-2H3 m1 cells
were pretreated with 20 µM wortmannin for 20-30 min, and
100 µM methacholine (MeCh) was added for a
further 10-15 min, followed by a 10-15-min recovery period in the
absence of agonist. The whole cell mode was then established, and 1 µM thapsigargin/ionomycin was applied extracellularly as
indicated to activate Icrac. Extracellular
calcium was removed as indicated. Traces are mean ± S.E.
(n 4). B, RBL-2H3 m1 cells were
pretreated with 20 µM wortmannin and 100 µM
methacholine as described above. After this pretreatment, the whole
cell mode was established, and 500 µM F-IP3,
included in the patch pipette, was delivered to the cell interior.
pA/pF, pA/picofarads
Effect of pretreatment with methacholine, wortmannin, or both on levels
of [3H]inositol-labeled polyphosphoinositides in RBL-2H3
cells
View larger version (32K):
[in a new window]
Fig. 6.
Calcium signaling in wild type
(WT) and IP3 receptor knockout
(IP3R-KO) DT-40 B
lymphocytes. A, wild-type or IP3 receptor
knockout cells were treated with 500 ng/ml of anti-IgM to activate the
B-cell receptor. In the wild-type cells (solid
line), 69.5% of the cells responded with a pattern of
irregular [Ca2+]i oscillations, as shown in three
example traces. In the knockout cells (dotted
line), no [Ca2+]i signal was observed
in any cells. The results are representative of three experiments.
B, wild-type (solid line) or
IP3 receptor knockout (dotted line)
cells were treated with 1 µM thapsigargin in the absence
of extracellular Ca2+, and then Ca2+ was
restored as indicated. The results are representative of three
experiments.
View larger version (18K):
[in a new window]
Fig. 7.
Capacitative calcium entry in wild-type and
IP3 receptor knockout DT-40 B lymphocytes is blocked by
2-APB. A, wild-type (WT; solid
line) and IP3 receptor knockout
(IP3R-KO; dotted
line) cells were treated sequentially with 500 ng/ml of
anti-IgM and then with 1 µM thapsigargin (TG)
in the presence of extracellular Ca2+. B, the
protocol was the same as in A, except the cells were
pretreated with 75 µM 2-APB. The results are
representative of three experiments.
View larger version (24K):
[in a new window]
Fig. 8.
Capacitative calcium entry in wild-type and
IP3 receptor knockout DT-40 B lymphocytes is blocked by
wortmannin. Capacitative calcium entry in response to 1 µM thapsigargin was determined in wild-type
(A) and IP3 receptor knockout (B)
cells (solid lines). In both cell lines, the
response was blocked by pretreatment with 20 µM
wortmannin for 1 h (dotted lines). Data are
representative of three different experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful for helpful critiques provided by Dr. Melisa Ho and Dr. Nina Storey.
![]() |
FOOTNOTES |
---|
* 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.
Present address: Eli Lilly and Company, Ltd., Lilly Research
Centre, Erl Wood Manor, Sunninghill Road, Windlesham, Surrey GU20 6PH,
United Kingdom.
¶ To whom correspondence should be addressed. E-mail: putney@niehs.nih.gov; Fax: 919-541-7879.
Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M011571200
2 G. St. J. Bird and J. W. Putney, Jr., unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PLC, phospholipase C; Icrac, calcium release-activated calcium current; IP3, inositol 1,4,5-trisphosphate; 2-APB, 2-aminoethoxydiphenyl borane; HBSS, HEPES-buffered physiological saline solution; PIP, phosphatidylinositol 4-phosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; HPLC, high pressure liquid chromatography; F-IP3, 3-deoxy-3-fluoro-inositol 1,4,5-trisphosphate.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Berridge, M. J. (1993) Nature 361, 315-325[CrossRef][Medline] [Order article via Infotrieve] |
2. | Putney, J. W., Jr. (1986) Cell Calcium 7, 1-12[Medline] [Order article via Infotrieve] |
3. | Putney, J. W., Jr. (1997) Capacitative Calcium Entry , Landes Biomedical Publishing, Austin, TX |
4. | Randriamampita, C., and Tsien, R. Y. (1993) Nature 364, 809-814[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Trepakova, E. S.,
Csutora, P.,
Hunton, D. L.,
Marchase, R. B.,
Cohen, R. A.,
and Bolotina, V. M.
(2000)
J. Biol. Chem.
275,
26158-63261 |
6. | Irvine, R. F. (1990) FEBS Lett. 263, 5-9[CrossRef][Medline] [Order article via Infotrieve] |
7. | Berridge, M. J. (1995) Biochem. J. 312, 1-11[Medline] [Order article via Infotrieve] |
8. |
Thomas, D.,
and Hanley, M. R.
(1995)
J. Biol. Chem.
270,
6429-6432 |
9. |
Csutora, P.,
Su, Z.,
Kim, H. Y.,
Bugrim, A.,
Cunningham, K. W.,
Nuccitelli, R.,
Keizer, J. E.,
Hanley, M. R.,
Blalock, J. E.,
and Marchase, R. B.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
121-126 |
10. | Parekh, A. B., Terlau, H., and Stühmer, W. (1993) Nature 364, 814-818[CrossRef][Medline] [Order article via Infotrieve] |
11. | Petersen, C. C. H., and Berridge, M. J. (1996) Pflüg. Arch. 432, 286-292[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Bird, G.,
St, J.,
Takahashi, M.,
Tanzawa, K.,
and Putney, J. W., Jr.
(1999)
J. Biol. Chem.
274,
20643-20649 |
13. | Jaconi, M., Pyle, J., Bortolon, R., Ou, J., and Clapham, D. (1997) Curr. Biol. 7, 599-602[Medline] [Order article via Infotrieve] |
14. | Oldershaw, K. A., and Taylor, C. W. (1993) Biochem. J. 292, 631-633[Medline] [Order article via Infotrieve] |
15. | Meissner, G. (1994) Annu. Rev. Physiol. 56, 485-508[CrossRef][Medline] [Order article via Infotrieve] |
16. | Patterson, R. L., van Rossum, D. B., and Gill, D. L. (1999) Cell 98, 487-499[Medline] [Order article via Infotrieve] |
17. |
Rosado, J. A.,
and Sage, S. O.
(2000)
J. Physiol. (Lond.)
526,
221-229 |
18. | Yao, Y., Ferrer-Montiel, A. V., Montal, M., and Tsien, R. Y. (1999) Cell 98, 475-485[Medline] [Order article via Infotrieve] |
19. |
Rosado, J. A.,
and Sage, S. O.
(2000)
J. Biol. Chem.
275,
9110-9113 |
20. |
Zubov, A. I.,
Kaznacheeva, E. V.,
Alexeeno, V. A.,
Kiselyov, K.,
Muallem, S.,
and Mozhayeva, G.
(1999)
J. Biol. Chem.
274,
25983-25985 |
21. | Kiselyov, K., Xu, X., Mozhayeva, G., Kuo, T., Pessah, I., Mignery, G., Zhu, X., Birnbaumer, L., and Muallem, S. (1998) Nature 396, 478-482[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Ma, H.-T.,
Patterson, R. L.,
van Rossum, D. B.,
Birnbaumer, L.,
Mikoshiba, K.,
and Gill, D. L.
(2000)
Science
287,
1647-1651 |
23. | Putney, J. W., Jr. (1999) Cell 99, 5-8[Medline] [Order article via Infotrieve] |
24. |
Sugawara, H.,
Kurosaki, M.,
Takata, M.,
and Kurosaki, T.
(1997)
EMBO J.
16,
3078-3088 |
25. |
Choi, O. H.,
Lee, J. H.,
Kassessinoff, T.,
Cunha-Melo, J. R.,
Jones, S. V.,
and Beaven, M. A.
(1993)
J. Immunol.
151,
5586-5595 |
26. |
Parod, R. J.,
Leslie, B. A.,
and Putney, J. W., Jr.
(1980)
Am. J. Physiol.
239,
G99-G105 |
27. | Hamill, O. P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F. J. (1981) Pflüg. Arch. 391, 85-100[Medline] [Order article via Infotrieve] |
28. | Anderson, K. E., Stephens, L. R., and Hawkins, P. T. (1999) in Signal Transduction: A Practical Approach (Milligan, G., ed) , pp. 283-300, Oxford University Press, London |
29. | Auger, K. R., Serunian, L. A., and Cantley, L. C. (1990) in Methods in Inositide Research (Irvine, R. F., ed) , pp. 159-166, Raven Press, New York |
30. | Hoth, M., and Penner, R. (1992) Nature 355, 353-355[CrossRef][Medline] [Order article via Infotrieve] |
31. | Bird, G., St, J., Rossier, M. F., Hughes, A. R., Shears, S. B., Armstrong, D. L., and Putney, J. W., Jr. (1991) Nature 352, 162-165[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Kwan, C. Y.,
Takemura, H.,
Obie, J. F.,
Thastrup, O.,
and Putney, J. W., Jr.
(1990)
Am. J. Physiol.
258,
C1006-C1015 |
33. | Smith, R. J., Sam, L. M., Justen, J. M., Bundy, G. L., Bala, G. A., and Bleasdale, J. E. (1990) J. Pharmacol. Exp. Ther. 253, 688-697[Abstract] |
34. |
Broad, L. M.,
Cannon, T. R.,
and Taylor, C. W.
(1999)
J. Physiol. (Lond.)
517,
121-134 |
35. | Downing, G. J., Kim, S., Nakanishi, S., Catt, K. J., and Balla, T. (1996) Biochemistry 35, 3587-3594[CrossRef][Medline] [Order article via Infotrieve] |
36. | Berridge, M. J., and Irvine, R. F. (1984) Nature 312, 315-321[Medline] [Order article via Infotrieve] |
37. | Jenner, S., Farndale, R. W., and Sage, S. O. (1996) FEBS Lett. 381, 249-251[CrossRef][Medline] [Order article via Infotrieve] |
38. | Linseman, D. A., McEwen, E. L., Sorensen, S. D., and Fisher, S. K. (1998) J. Neurochem. 70, 940-950[Medline] [Order article via Infotrieve] |
39. | Berven, L. A., and Barritt, G. J. (1995) Biochem. Pharmacol. 49, 1373-1379[CrossRef][Medline] [Order article via Infotrieve] |
40. |
Braun, F.-J.,
Broad, L. M.,
Armstrong, D. L.,
and Putney, J. W., Jr.
(2001)
J. Biol. Chem.
276,
1063-1070 |
41. | Bloomquist, B. T., Shortridge, R. D., Schneuwly, S., Pedrew, M., Montell, C., Steller, H., Rubin, G., and Pak, W. L. (1988) Cell 54, 723-733[Medline] [Order article via Infotrieve] |
42. |
McKay, R. R.,
Chen, D. M.,
Miller, K.,
Kim, S.,
Stark, W. S.,
and Shortridge, R. D.
(1995)
J. Biol. Chem.
270,
13271-13276 |
43. | Acharya, J. K., Jalink, K., Hardy, R. W., Hartenstein, V., and Zuker, C. S. (1997) Neuron 18, 881-887[Medline] [Order article via Infotrieve] |
44. | Montell, C. (1999) Annu. Rev. Cell Dev. Biol. 15, 231-268[CrossRef][Medline] [Order article via Infotrieve] |
45. |
Vaca, L.,
and Kunze, D. L.
(1994)
Am. J. Physiol.
267,
C920-C925 |