Calcio Astrocito B2 +++

J Physiol 589.
16 (2011) pp 3909–3927 3909
Regulation of bradykinin-induced activation

of volume-sensitive outwardly rectifying anion channels
by Ca2+ nanodomains in mouse astrocytes
Tenpei Akita and Yasunobu Okada
Department of Cell Physiology, National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki 444-8585, Japan
Non-technical summary Cell volume regulation is an essential function involved not only in
homeostasis, but also in cell migration, fission and programmed cell death. The volume-sensitive
outwardly rectifying (VSOR) anion channel provides the main pathway for anion transport across
the cell membrane during the regulation. We previously demonstrated that an inflammatory
mediator, bradykinin, activates the VSOR channels in the major glial cells, astrocytes, in the
The Journal of Physiology
brain derived from mice and the channels send signals to adjacent neurons through the release
of glutamate. Here we demonstrate that this activation is controlled in the immediate vicinity of
Ca2+ -permeable channel proteins in the astrocytes via high concentrations of intracellular Ca2+ ,
so-called ‘Ca2+ nanodomains’. This mechanism would provide a basis for responding quickly and
certainly to even a minute amount of bradykinin released from surrounding tissues (e.g. slightly
damaged blood vessel walls) with local control of cell shape changes and signal transmission by
astrocytes.
Abstract Volume-sensitive outwardly rectifying (VSOR) anion channels play a key role in a
variety of essential cell functions including cell volume regulation, cell death induction and
intercellular communications. We previously demonstrated that, in cultured mouse cortical
astrocytes, VSOR channels are activated in response to an inflammatory mediator, bradykinin,
even without an increase in cell volume. Here we report that this VSOR channel activation must
be mediated firstly by ‘nanodomains’ of high [Ca2+ ]i generated at the sites of both Ca2+ release
from intracellular Ca2+ stores and Ca2+ entry at the plasma membrane. Bradykinin elicited a
[Ca2+ ]i rise, initially caused by Ca2+ release and then by Ca2+ entry. Suppression of the [Ca2+ ]i
rise by removal of extracellular Ca2+ and by depletion of Ca2+ stores suppressed the VSOR
channel activation in a graded manner. Quantitative RT-PCR and suppression of gene expression
with small interfering RNAs indicated that Orai1, TRPC1 and TRPC3 channels are involved
in the Ca2+ entry and especially the entry through TRPC1 channels is strongly involved in the
bradykinin-induced activation of VSOR channels. Moreover, Ca2+ -dependent protein kinases Cα
and β were found to mediate the activation after the [Ca2+ ]i rise through inducing generation of
reactive oxygen species. Intracellular application of a slow Ca2+ chelator, EGTA, at 10 mM or a fast
chelator, BAPTA, at 1 mM, however, had little effect on the VSOR channel activation. Application
of BAPTA at 10 mM suppressed significantly the activation to one-third. These suggest that the
VSOR channel activation induced by bradykinin is regulated by Ca2+ in the vicinity of individual
Ca2+ release and entry channels, providing a basis for local control of cell volume regulation and
intercellular communications.
(Received 28 February 2011; accepted after revision 17 June 2011; first published online 20 June 2011)
Corresponding author Y. Okada: Department of Cell Physiology, National Institute for Physiological Sciences, National
Institutes of Natural Sciences, Okazaki 444-8585, Japan. Email: [email protected]

C 2011 The Authors. Journal compilation
C 2011 The Physiological Society DOI: 10.1113/jphysiol.2011.208173
3910 T. Akita and Y. Okada J Physiol 589.16
Abbreviations 2-APB, 2-aminoethoxydiphenyl borate; CM-DCF, 5-(and-6)-chloromethyl-2 ,7 -dichlorofluorescein;

DCPIB, 4-(2-butyl-6,7-dichloro-2-cyclopentyl-indan-1-on-5-yl) oxybutyric acid; DPI, diphenyleneiodonium; NMDG,
N -methyl-D-glucamine; NOX, NADPH oxidase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; ROS,
reactive oxygen species; RVD, regulatory volume decrease; siRNA, small interfering RNA; VSOR, volume-sensitive
outwardly rectifying.
Introduction the bradykinin B2 receptor coupled to Gαq proteins

(Liu et al. 2009). Thus the receptor activation should
The volume-sensitive outwardly rectifying (VSOR) anion elicit intracellular Ca2+ release through InsP 3 receptors
channel is the most influential regulator of cell volume and generation of a PKC activator, diacylglycerol. Large
in most types of animal cells. The channel is typically amplitudes of [Ca2+ ]i rises due to Ca2+ release are indeed
activated by an increase in cell volume, and opening generated in astrocytes in response to bradykinin. How
of the channel provides a pathway for outflux of intra- the [Ca2+ ]i rises and PKC activation are linked to VSOR
cellular anions, especially Cl− ions. This outflux of anions, channel activation is, however, yet to be determined.
usually accompanied by an outflux of K+ ions, facilitates Many reports have long suggested the Ca2+ independence
regulatory volume decrease (RVD) of the cells. The of VSOR channel activation induced by cell swelling
molecular identity of VSOR channel is, however, not (reviewed by Okada, 1997 and Hoffmann et al. 2009).
determined yet (reviewed by Okada et al. 2009). Indeed, the swelling-induced VSOR channel activation
The VSOR channel is permeable significantly to is not necessarily accompanied by detectable [Ca2+ ]i
amino acids, like glutamate or aspartate. Thus the rises in some types of cell (reviewed by Hoffmann
conditions for opening VSOR channels may also et al. 2009), suggesting non-essential contribution of
induce intercellular signal transmission mediated by Ca2+ to the swelling-induced activation. In our previous
the excitatory amino acid. We previously demonstrated study, suppression of observable bradykinin-induced
that an inflammatory mediator, bradykinin, activates [Ca2+ ]i rises by treatment with the membrane-permeable
VSOR channels in cultured mouse cortical astrocytes acetoxymethyl (AM) ester form of BAPTA had in fact
and the glutamate released via VSOR channels evokes no effects on VSOR channel activation (Liu et al. 2009).
[Ca2+ ]i rises in adjacent co-cultured neurons through However, a more thorough examination is required
NMDA receptor activation (Liu et al. 2009). In injured because Ca2+ action may be limited within very
brains, the amount of excitatory amino acids released narrow regions, narrower than the resolution limit of
via astrocytic VSOR channels may become very conventional optical microscopy, around the orifices of
massive and it may cause severe excitotoxic neuronal open Ca2+ channels, so called ‘Ca2+ nanodomains’. In
damage. The damage can indeed be alleviated by the nanodomains, more than 10 μM of high [Ca2+ ]i rises
application of a specific blocker of VSOR channels, 4-(2- proportional to the amplitudes of single-channel Ca2+
butyl-6, 7-dichloro-2-cyclopentyl - indan - 1 - on - 5 - yl) currents are generated and only a fast Ca2+ chelator,
oxybutyric acid (DCPIB; Zhang et al. 2008). BAPTA, at very high concentrations can have access to
Activation of VSOR channels is not necessarily them (Neher, 1998b; Augustine et al. 2003). In the case of
accompanied by an increase in cell volume. The the swelling-induced activation of VSOR channels, it has
bradykinin-induced activation of VSOR channels in our been reported that large amplitudes of the current through
previous study was, in fact, not associated with volume VSOR channels are still maintained even in the presence
increases. Such a volume-independent activation of VSOR of intracellular BAPTA at 5–10 mM (Kubo & Okada, 1992;
channels may be mediated by generation of reactive Pedersen et al. 1998). However, a different mechanism
oxygen species (ROS) through NADPH oxidases (NOX), may regulate the activation mediated by Gαq -coupled
as reported in human cervix HeLa cells (Shimizu et al. receptors without cell swelling. Moreover, since the [Ca2+ ]i
2004), rat hepatoma cells (Varela et al. 2004), rat micro- rises within 20 nm of Ca2+ channel pores must be less
glia (Harrigan et al. 2008) and rabbit cardiomyocytes affected even by BAPTA (Neher, 1998b), it is necessary
(Browe & Baumgarten, 2004; Deng et al. 2010), although to examine the effect of thorough removal of Ca2+ both
it is not a universal mechanism as demonstrated in in the extracellular solution and in the intracellular Ca2+
mouse Ehrlich ascites tumour cells (Lambert et al. 2009). stores and also to examine the dose dependence of the
The bradykinin-induced VSOR channel activation in activation on the intracellular Ca2+ chelator, in order to
astrocytes was found to be mediated by ROS (Liu elucidate exactly whether the Ca2+ in the nanodomains
et al. 2009). The prototypical NOX, NOX2/gp91phox, is may participate partly or not and to determine the distance
reported to be expressed and driven by activation of range of Ca2+ action in the process if it is involved.
protein kinase C (PKC) or [Ca2+ ]i rises in astrocytes Ca2+ nanodomain-mediated regulation is well known
(Pawate et al. 2004; Abramov et al. 2005). The bradykinin in the exocytosis of neurotransmitters (Neher, 1998b;
action on VSOR channel activation is mediated by

C 2011 The Physiological Society
J Physiol 589.16 VSOR channel regulation via Ca2+ nanodomains 3911
Augustine et al. 2003) and at the excitation–contraction from 44 mother mice in this study) were killed by
coupling in cardiac muscle cells (Cheng & Lederer, 2008). cervical dislocation, and cerebral cortices were obtained
The nanodomain mediation confers fast, accurate and by removing olfactory bulbs, cerebellums, brainstems,
localized regulation of Ca2+ -dependent processes on a basal ganglia, hippocampi and meninges from the brains.
single cell. Furthermore, Ca2+ -sensing molecules involved The cortices were minced and digested with papain
in the same cellular function but with different Ca2+ (Sigma-Aldrich), and the cells were dissociated by gentle
sensitivities in a single cell may be distributed at different pipetting with Pasteur pipettes. The cells were plated
distances from the centre of a nanodomain, modulating and cultured in flasks at 37◦ C in a humidified 5%
the function over different time scales, as seen in the CO2 –95% air environment with minimum essential
modulation of neuronal membrane excitability (Akita & medium supplemented with 10% fetal bovine serum,
Kuba, 2000) and in the regulation of intracellular Ca2+ 40 U ml−1 penicillin and 100 μg ml−1 streptomycin. The
release itself (Akita & Kuba, 2008). Thus, estimating the cells were used up within 1 month after the beginning
distance range of Ca2+ action is very important to realize of cultivation. For experiments, the cells in confluent
the significance of a Ca2+ -dependent process. It is quite cultures were first trypsinized for 1–2 min with gentle
conceivable that Ca2+ nanodomain-mediated regulation rocking. After discarding the trypsin solution, the
would play a role also in local cell volume regulation cells were detached from the flask bottoms by gentle
during cell shape changes, migration or fission, although pipetting with the culture medium, then transferred to
the evidence is not elucidated yet. polyethyleneimine-coated glass coverslips at a density
In this study we provide the first evidence for Ca2+ of 5 × 105 cells cm−2 and cultured overnight before use.
nanodomain-mediated regulation of the cell volume Immunocytochemistry revealed that >90% of the cells
regulator, VSOR channels. First we demonstrate that the were glial fibrillary acidic protein-positive, an astrocyte
bradykinin-induced VSOR channel activation in mouse marker, under this condition. The cells with relatively
astrocytes is indeed Ca2+ dependent; the activation was flat and rectangular cell bodies, suggesting protoplasmic
suppressed in parallel with the suppression of intra- type-1 astrocytes, were selected for experiments.
cellular Ca2+ release and store- or receptor-operated Ca2+
entry, and Ca2+ -dependent types of PKC were indeed
involved in the process. By introducing different types Solutions
of Ca2+ chelators at given concentrations directly into
The extracellular bathing solution used for whole-cell
the cells via whole-cell patch pipettes, we found that only
patch-clamp recordings contained (in mM): 135
BAPTA at tens of millimolar concentrations could inter-
N -methyl-D-glucamine (NMDG)-Cl, 2 CaCl2 , 1 MgCl2 ,
fere significantly with VSOR channel activation; that is, the
10 Hepes and 5 glucose (pH 7.4, 280 mosmol (kg H2 O)−1 ).
Ca2+ action should take place within the nanodomains.
The standard whole-cell patch pipette solution contained
A preliminary report of this work has been made in a
(in mM): 135 NMDGCl, 1 MgCl2 , 3 Na2 ATP, 5 Hepes and 1
meeting (Akita & Okada, 2010).
EGTA with 0.2 CaCl2 (estimated free [Ca2+ ] ≈ 15 nM; pH
7.4, 275 mosmol (kg H2 O)−1 ). When high concentrations
of EGTA or BAPTA were added to the pipette solution,
Methods the Cl− concentration was reduced to keep the same
Ethical approval osmolality as that of the standard solution and CaCl2 was
added to maintain the same free [Ca2+ ]. For [Ca2+ ]i and
All procedures in this study were performed according to ROS measurements, we used Ringer solution containing
the Guidelines for Care and Use of Laboratory Animals (in mM): 135 NaCl, 5 KCl, 2 CaCl2 , 1 MgCl2 , 5 NaHepes,
of the Physiological Society of Japan. Experimental 6 Hepes and 5 glucose (pH 7.4, 285 mosmol (kg H2 O)−1 ).
protocols were reviewed and approved in advance by When Ca2+ was removed from the bathing solutions,
the Institutional Animal Care and Use Committee of equimolar Mg2+ (with or without 0.1 mM EGTA, as
the National Institutes of Natural Sciences. The authors mentioned in text) was added to the solutions.
have read and the experiments comply with the policies
and regulations of The Journal of Physiology given by
Drummond (2009). Patch-clamp studies
Whole-cell currents were measured with the whole-cell
patch-clamp technique, as described previously (Liu et al.
Cell preparation
2009). Patch pipettes were fabricated from borosilicate
Astrocytes in primary culture were prepared from the glass capillaries and had a resistance of 2–3 M when
cerebral cortices of 2-day-old ddY mouse pups, as filled with the pipette solution. The currents were recorded
described previously (Liu et al. 2009). The pups (obtained with an EPC10 amplifier controlled via Patchmaster

software (HEKA Elektronik, Lambrecht/Pfalz, Germany). (Mm00444690 m1), TRPC4 (Mm00444284 m1), TRPC5
The current signals were filtered at 1 kHz and sampled (Mm00437183 m1), TRPC6 (Mm01176083 m1), TRPC7
at 5 kHz. Liquid junction potentials were calculated (Mm00442606 m1), TRPM7 (Mm00457998 m1), PKCα
with JPCalc in pCLAMP10 software (Molecular Devices, (Mm00440858 m1), PKCβ (Mm00435749 m1), PKCγ
Sunnyvale, CA, USA) and corrected on-line. The series (Mm00440861 m1) and β-actin (Mm00607939 s1) as an
resistance during the recording was kept at <10 M endogenous reference. PCR was made in a 50 μl reaction
and compensated for by 70%. All the experiments were volume containing 50 ng of cDNA in each well of 96-well
performed at 25–30◦ C. plates, and four replicate wells were taken for each target
gene. Cycling conditions consisted of the initial enzyme
activation at 95◦ C for 10 min followed by 40 cycles of
Calcium imaging denaturation at 95◦ C for 15 s and annealing at 60◦ C for
1 min. No amplification signals when reverse transcription
[Ca2+ ]i changes were measured at 25–30◦ C as the changes
was omitted were detected for all probes (data not
in the ratio of fura-2 fluorescence in the cytoplasm. Cells
shown). mRNA expression levels were compared by the
were loaded with the indicator by incubation in the
comparative C T method, using the level of TRPC1
culture medium containing 0.5 μM fura-2 AM (Biotium)
mRNA as a calibrator. All measurements were repeated at
for 30 min at 37◦ C, then washed with Ringer solution for
least three times using the mRNA preparations isolated
5 min in the recording chamber mounted on the stage of
from different cultures.
an Olympus IX71 inverted microscope (Olympus Japan).
The cytoplasmic fura-2 was excited at 340 nm and 380 nm
(14 nm bandwidth) with a monochromator (Polychrome
Immunocytochemistry
IV, TILL Photonics, Gräfelfing, Germany), and detected
at 510 nm (80 nm bandwidth). The fluorescence images Cells were fixed with ice-cold methanol for
were obtained through a 40× water immersion objective 10 min at −20◦ C, and permeabilized with 0.5%
(UApo40XW340, NA1.15, Olympus Japan) with a Roper saponin (Sigma-Aldrich) and 0.025% Triton X-100
NTE (Roper Scientific, Trenton, NJ, USA) or a Hamamatsu (Sigma-Aldrich) dissolved in phosphate-buffered saline
ORCA-R2 (Hamamatsu Photonics) cooled CCD camera (PBS) for 30 min at room temperature. Antibody
controlled via MetaFluor software (Molecular Devices). reactions were performed at room temperature for 2 h for
The image pairs obtained with 340 nm light and 380 nm primary antibodies and for 1 h for secondary antibodies.
light were recorded at 2 s intervals. Image analysis Non-specific binding of antibodies was blocked by adding
was performed off-line with ImageJ software (National 4% bovine serum albumin (Sigma-Aldrich) throughout
Institutes of Health). The region of interest was defined the permeabilization and the antibody reactions. The
to include most of the cytoplasm for each cell. The ratios staining was observed by epifluorescence illumination
of the fluorescence intensities obtained with 340 nm light with the Polychrome IV monochromator through the
(F 340 ) to those with 380 nm light (F 380 ) in the same regions Olympus 40× water immersion objective (NA 1.15)
were calculated after background subtraction. The cells after mounting the coverslips with SlowFade Gold
showing oscillatory [Ca2+ ]i changes before bradykinin (Molecular Probes). The primary antibodies used were
application were discarded from analysis. rabbit anti-Orai1 (1:100; Abcam, Cambridge, UK), rabbit
anti-TRPC1 (1:200; Osenses, Keswick, SA, Australia),
rabbit anti-TRPC3 (1:200; Alomone Labs, Jerusalem,
Quantitative RT-PCR Israel) and rabbit anti-TRPM7 (1:100; Alomone Labs).
The secondary antibody was Alexa Fluor 546 goat
The total RNA in the cells was extracted with an
anti-rabbit IgG (1:1000; Molecular Probes). Consistency
RNeasy Mini kit (Qiagen, Hilden, Germany), and its
of staining patterns was confirmed by comparing the
content and purity were assessed with a NanoDrop 1000
staining results with the antibodies at three different
spectrophotometer (Thermo Fischer Scientific). The total
concentrations (1:100, 1:200, 1:500) for all the primary
RNA of >10 μg with a purity of >2.0 in the absorbance
antibodies. Negative controls were obtained by omission
ratio (260 nm/280 nm) was constantly obtained from a
of primary antibodies.
confluent culture in a 25 cm2 flask. Reverse transcription
with elimination of genomic DNA was made by using a
QuantiTect kit (Qiagen) with a GeneAmp 9700 thermal
Small interfering RNA transfection
cycler (Applied Biosystems). Quantitative real-time PCR
was performed in an Applied Biosystems 7300 system with For gene knockdown studies with small interfering RNA
TaqMan Gene Expression Assay kits (Applied Biosystems) (siRNA), we used Dharmacon ON-TARGETplus
for mouse Orai1 (assay ID: Mm00774349 m1), TRPC1 SMARTpool siRNA reagents (Thermo Fischer
(Mm00441975 m1), TRPC2 (Mm00441984 m1), TRPC3 Scientific) designed against mouse Orai1 (catalogue

no. L-056431-02), TRPC1 (L-043863-00), TRPC3 Drugs

(L-049680-00), TRPM7 (L-040716-00), PKCα
All drugs were made up as 1000–10,000× stock solutions,
(L-040348-00) and PKCβ (L-048412-00). This reagent
stored frozen and then thawed and diluted with the
contains a mixture of four different siRNAs targeting
bathing solution on the day of use. Bradykinin, DCPIB,
the same gene. We also used an ON-TARGETplus
2-aminoethoxydiphenyl borate (2-APB), thapsigargin,
Non-Targeting Pool reagent (D-001810-10), containing
BAPTA, phorbol 12-myristate 13-acetate (PMA) and
four different siRNAs but not targeting any known
Gö6976 were purchased from Sigma-Aldrich. EGTA was
genes, for negative control. Cells at 30–50% confluency
from Dojindo (Kumamoto, Japan).
were transfected with the siRNA mixture at 1 nM using
Lipofectamine RNAiMAX (Invitrogen) in Opti-MEM
I Reduced Serum Medium (Invitrogen) without serum Statistics
and antibiotics at 37◦ C in a humidified 5% CO2 –95%
air environment. The transfection medium was replaced Data are expressed as means ± SEM, and error bars in
with the culture medium after 8–10 h. The effect of graphs indicate SEM. Statistical multiple comparisons
knockdown was first assessed by quantitative RT-PCR were made by Peritz’s closed testing procedure with the
3 days after the transfection. Selective suppression of F statistic after ANOVA tests. Differences were judged as
the mRNA level to 10–20% was confirmed for all target significant when the P of making at least one incorrect
genes, as shown in Supplemental Fig. S1. Maximal assertion in one set of multiple comparisons, called the
functional suppression was determined by measuring familywise error rate, was <0.05. The effect of a treatment
bradykinin-induced [Ca2+ ]i rises for channel knockdown on the same sample set was assessed by Student’s paired
and VSOR currents for PKC knockdown 3–5 days after t test.
the transfection and comparing them with those in
non-targeting siRNA-transfected cells. The maximum
was seen at 3 days for knockdown of Orai1, PKCα and
PKCβ, and at 4–5 days for TRPC1 and TRPC3. Results
Bradykinin-induced [Ca2+ ]i rises are produced by
intracellular Ca2+ release and store- or
receptor-operated Ca2+ entry
ROS imaging
We previously demonstrated that application of 1 μM
Intracellular ROS production was tracked at 25–30◦ C bradykinin under isotonic conditions activates VSOR
from the increase in fluorescence of 5-(and-6)- channels through bradykinin B2 receptor activation and
chloromethyl-2 ,7 -dichlorofluorescein (CM-DCF) in the this is accompanied by prominent [Ca2+ ]i rises in
cytoplasm. Cells were loaded with the indicator cultured mouse cortical astrocytes (Liu et al. 2009). To
by incubation in Ringer solution containing the elucidate their Ca2+ sources, we examined the effects
membrane-permeant (acetylated) reduced form of of removing extracelluar Ca2+ and the Ca2+ in intra-
CM-DCF (CM-H2 DCFDA; Molecular Probes) at 10 μM cellular Ca2+ stores on the time course of the [Ca2+ ]i
for 30 min at 37◦ C, then washed with Ringer solution rises. The bradykinin-induced [Ca2+ ]i rise consisted
for 5 min in the recording chamber. The indicator was of two rising phases: the initial large transient phase
excited at 490 nm (8 nm bandwidth) with the Polychrome followed by a small sustained phase (Fig. 1Aa). The initial
IV monochromator, and detected at 540 nm (50 nm band- transient subsided within 1 min after the beginning of
width). Great care was taken to reduce photo-oxidation bradykinin application, whereas the second sustained
of the indicator by using weak excitation light with a phase continued over >15 min in the presence of
short exposure time. The images were obtained through a bradykinin. Removal of extracellular Ca2+ completely
20× dry objective (UPlanSApo, NA0.75, Olympus Japan) abolished the sustained phase and reduced weakly the
with the ORCA-R2 camera controlled via MetaFluor amplitude of the initial transient (from 0.37 ± 0.02 in
software at 10 s intervals. Image analysis was performed the fura-2 ratio (F 340 /F 380 ) to 0.26 ± 0.02, Fig. 1Ab).
off-line with ImageJ. The region of interest was defined Pretreatment of the cells with a blocker of Ca2+ pumps
to include most of the cytoplasm for each cell. The on the Ca2+ stores, thapsigargin (1 μM), in addition to
fluorescence increase was detected as the increase in the removal of extracellular Ca2+ eradicated the initial
normalized fluorescence intensity relative to the initial transient (Fig. 1Ac). Thus most of the initial transient
level (F/F 0 ). The initial level was determined as the average component must be caused by intracellular Ca2+ release
over 1 min after the beginning of measurement in the from Ca2+ stores, and the second sustained component
same region. The intensity was measured after background must be caused exclusively by Ca2+ entry at the plasma
subtraction. membrane. The Ca2+ entry would be generated in

response to depletion of Ca2+ stores or to bradykinin Bradykinin-induced activation of VSOR anion

receptor activation. Indeed, application of the blocker channels depends both on Ca2+ release
of store- or receptor-operated Ca2+ entry, Gd3+ (1 μM) and on Ca2+ entry
or 2-APB (30 μM), in the presence of extracellular Ca2+
selectively suppressed the second component without Then we examined the effects of removal of extracellular
affecting the initial component (Fig. 1Ad and e). When Ca2+ and depletion of Ca2+ stores on VSOR
the extent of suppression was evaluated from the time channel activation. The bradykinin application induced
integral of the second component from 1 to 15 min development of an anion current, showing mild outward
after stimulation, the effects of Gd3+ and 2-APB were rectification and weak inactivation at high voltages, over
found to be statistically similar to that of extracellular 15–20 min under whole-cell voltage clamp conditions
Ca2+ removal (Fig. 1B). Thus Ca2+ -permeable channels (Fig. 2A). We previously checked that this current was
sensitive to both Gd3+ and 2-APB must be involved blocked by anion channel blockers DIDS, phloretin
in the Ca2+ entry responsible for the second and DCPIB (the latter two should preferentially and
component. specifically block VSOR channels, respectively), and that
the current generation required intracellular ATP and had
Figure 1. Effects of Ca2+ depletion and blockers of Ca2+ entry on bradykinin-induced [Ca2+ ]i rises in
cultured mouse astrocytes
Aa, bradykinin-induced rise in fura-2 ratio (F 340 /F 380 ) in the cytoplasm averaged over 78 cells. Ab,
bradykinin-induced F 340 /F 380 rise in the absence of extracellular Ca2+ (n = 75). EGTA was not added in the
bathing solution. Ac, bradykinin response in the absence of extracellular Ca2+ observed after pretreatment with
1 μM thapsigargin (Thap.) for 30 min (n = 69). Thapsigargin was included in the loading solution but not in the
bathing solution. Ad, response in the presence of 1 μM Gd3+ (n = 90). Ae, response in the presence of 30 μM
2-APB (n = 85). Cells were preincubated in the Gd3+ or 2-APB containing solution for 10 min at room temperature
before the measurements. Insets in Aa–e show typical F 340 /F 380 traces observed in single cells for 15 min. All traces
in this figure were recorded with a Roper NTE camera. B, comparison of the time integrals of net F 340 /F 380 rises
(areas in (F 340 /F 380 ) × seconds) from basal levels (indicated by dashed lines in Aa–e) over 1–15 min after the
beginning of bradykinin application. The areas for comparison are indicated as shaded regions in Aa–e. All the
areas except that in the control conditions (Aa) had negative values. This was presumably due to a temporary
facilitating effect of bradykinin on Ca2+ extrusion (Ac). There are no statistical differences between these negative
values (judged by multiple comparisons).

Figure 2. Effects of Ca2+ depletion on bradykinin-induced whole-cell currents through VSOR anion
channels
A, time course of an increase in the whole-cell current through VSOR channels during application of 1 μM
bradykinin and its suppression by 10 μM DCPIB. Bradykinin and DCPIB were applied through bath perfusion
under isotonic conditions. The time course was monitored by repetitively applying a pair of alternating step
voltage pulses to ±40 mV (0.5 s duration) from the holding potential of 0 mV every 5 s. The arrowhead indicates
the 0 current level. To examine the changes in voltage dependency of the current, test voltage pulses were
applied before application of bradykinin (open diamond), at the maximum plateau of the bradykinin-induced
current enhancement (open square) and after suppression to the minimum caused by DCPIB (filled diamond). The
corresponding current responses are shown in the right panel on an expanded time scale. Test voltage pulses
consisted of the pulses (0.5 s duration) ranging from −100 mV to +100 mV in 20 mV increments with fixed
pre-pulses (0.1 s duration) to −100 mV and post-pulses (0.1 s duration) to −80 mV. B, bradykinin-induced VSOR
current in the absence of extracellular Ca2+ and its enhancement by restoration of extracellular Ca2+ to 2 mM.
C, bradykinin-induced VSOR current in the absence of extracellular Ca2+ observed after pretreatment with 1 μM
thapsigargin and its suppression by 10 μM DCPIB. D, the bradykinin-induced current observed after thapsigargin
treatment and adding 10 mM BAPTA in the pipette solution and 0.1 mM EGTA in the Ca2+ -free bathing solution,
and its very weak suppression by 10 μM DCPIB. The current responses indicated by the symbols in B–D are those
elicited by test voltage pulses as in A. E, current–voltage (I–V) relationships of the whole-cell currents under the
conditions shown in A–D. Symbols represent the means ± SEM of the peak current amplitudes elicited by test
voltage pulses in current density (pA pF−1 ). The peak amplitude was determined at 20 ms after the beginning of
the pulse. The numbers of cells analysed were 17 for control in the presence of extracellular Ca2+ as in A (open
squares), 15 for removal of extracellular Ca2+ (open circles) and Ca2+ restoration (open triangles) as in B, 14 for
thapsigargin pretreatment (filled circles) as in C, and 16 for thorough removal of Ca2+ (filled triangles) and DCPIB
treatment (crosses) as in D. The mean membrane capacitance of the cells used here was 51.3 ± 3.3 pF.

sensitivity to extracellular hypertonicity but not to Gd3+ , VSOR channel activation in response to hypotonic stimuli
consistent with the properties of VSOR channels (Liu in HeLa cells (Numata et al. 2007b).
et al. 2009). Although the inactivation of the current in Our quantitative RT-PCR study indicated that the
this preparation is much weaker than that of the typical mRNAs of Orai1, TRPC1, TRPC3 and TRPM7 were
swelling-induced VSOR current as seen in epithelial cells expressed abundantly, whereas others were scanty or
(see review by Okada, 1997), the current is quite similar absent in our astrocytes (Fig. 3A). Immunostaining with
to the swelling-induced currents in this preparation (see the antibodies against Orai1, TRPC1, TRPC3 and TRPM7
the supplemental figure of Liu et al. 2009) and in neurons proteins indeed suggested actual expression of these
(Leaney et al. 1997; Inoue et al. 2005), microglia (Harrigan channels (Fig. 3B). To examine the contribution of each
et al. 2008; Schlichter et al. 2011) and cardiomyocytes type of channel to the bradykinin-induced [Ca2+ ]i rise, we
(Baumgarten & Clemo, 2003). transfected the cells with the siRNA targeting the channel
The maximum amplitude of the VSOR current was and examined its effects on the [Ca2+ ]i rise.
suppressed by half in the absence of extracellular Ca2+ The transfection with the siRNA for Orai1, TRPC1 or
(nominally Ca2+ free). The peak amplitude of the TRPC3 channels significantly suppressed selectively the
current (in current density) elicited by depolarization second sustained component of the [Ca2+ ]i rise (Fig. 3Cb,
to +100 mV was 30.0 ± 2.6 pA pF−1 (open circles in d and e vs. Ca; P < 0.05 judged by multiple comparisons,
Fig. 2B and E), whereas it was 63.4 ± 10.0 pA pF−1 in marked with asterisks in Fig. 3D) without affecting the
the presence of Ca2+ (P < 0.05 by multiple comparisons; initial transient component, whereas the transfection with
open squares in Fig. 2A and E). Indeed, restoration of TRPM7 siRNA had effects on neither the initial nor the
extracellular Ca2+ restored the amplitude to a similar second components (Fig. 3Cc vs. Ca; Fig. 3D). Thus Orai1,
level (49.6 ± 3.8 pA pF−1 , open triangles in Fig. 2B and TRPC1 and TRPC3 channels must be involved in the Ca2+
E). Pretreatment with thapsigargin in addition to the entry after intracellular Ca2+ release. No involvement of
removal of extracellular Ca2+ further suppressed the TRPM7 channels would be explained by the absence of
amplitude to one-quarter (16.3 ± 1.7 pA pF−1 , P < 0.05 large changes in cell volume during bradykinin application
by multiple comparisons; filled circles in Fig. 2C and (Liu et al. 2009), because the channels must be activated
E). When the residual Ca2+ was thoroughly removed by through direct mechanical stress (Numata et al. 2007a).
adding Ca2+ chelators both on the extracellular (0.1 mM In our conditions of siRNA transfection (see Methods and
EGTA) and intracellular (10 mM BAPTA included in the Supplemental Fig. S1A for details), the effects of siRNAs
pipette solution) sides, the amplitude was further reduced for Orai1, TRPC1 and TRPC3 channels were similar when
(9.9 ± 1.0 pA pF−1 , P < 0.05 by multiple comparisons; they were assessed from the decreases in time integral of
filled triangles in Fig. 2D and E) and the DCPIB-sensitive the [Ca2+ ]i rise over 1–20 min after the beginning of the
current component became very small although it was still rise (Fig. 3D).
statistically significant (suppression to 6.9 ± 0.6 pA pF−1 ,
P < 0.01 by paired t test, crosses in Fig. 2D and E).
Thus the results strongly suggest that bradykinin-induced Ca2+ entry through TRPC1 channels is strongly
activation of VSOR anion channels depends on intra- involved in bradykinin-induced activation of VSOR
cellular [Ca2+ ]i rises originating both from intracellular
anion channels
Ca2+ release and from Ca2+ entry.
Then we examined the effects of suppression of Orai1,
TRPC1 and TRPC3 channels on bradykinin-induced
activation of VSOR channels. Transfection with TRPC1
Orai1, TRPC1 and TRPC3 channels are involved siRNA significantly suppressed the VSOR current
activation when it was compared at 20 min after the
in store- or receptor-operated Ca2+ entry
beginning of bradykinin application (11.1 ± 1.4 pA pF−1
As Gd3+ - and 2-APB-sensitive channels responsible for at +100 mV; open circles in Fig. 4B and C) with that
store- or receptor-operated Ca2+ entry, Orai1 and the in the control non-targeting siRNA-transfected cells
canonical type of TRP (TRPC) channels are known in (18.9 ± 2.1 pA pF−1 ; open squares in Fig. 4A and C;
many types of cells (Birnbaumer, 2009). So we checked P < 0.05 judged by multiple comparisons, marked with
their expressions in our astrocytes and examined their the asterisk in Fig. 4D). A notable thing was that in 70%
roles in bradykinin-induced [Ca2+ ]i rises and VSOR of the TRPC1 knockdown cells (22 of 31 cells) the current
channel activation. In addition, we also checked the development ceased within 9 min (9.2 ± 0.8 min, average
expression of TRPM7 channels, which are also Ca2+ over 22 cells) after the beginning of bradykinin application
permeable and Gd3+ and 2-APB sensitive, because our and then the current decreased gradually over >10 min.
group previously demonstrated that the Ca2+ entry An example is shown in Fig. 4B. Such a waxing and
through these channels assists the RVD process driven by waning pattern of development was seen only in 2 of

29 non-targeting siRNA-transfected cells; even in these could be induced when the extracelluar Ca2+ was removed
two cells the waning phase was seen >15 min after the during the course of the current development (Fig. 4E).
beginning of bradykinin application. This suggests that In Orai1 siRNA-transfected cells, such a pattern of the
the Ca2+ entry through TRPC1 channels participates in current decrease was seen in 40% (13 of 32 cells); thus
VSOR channel activation especially in the later phase of the majority showed a monotonic increase in current
the current development. This seemed consistent with the amplitude during bradykinin application, as shown in
suppression of especially the later phase of the [Ca2+ ]i rise Supplemental Fig. S2A. In TRPC3 siRNA-transfected cells,
in TRPC1 siRNA-transfected cells (Fig. 3Cd). In further just half of the cells showed the waxing and waning
support of this, a similar pattern of the current decrease pattern (Supplemental Fig. S2Ba and b). On average,
Figure 3. Effects of siRNA-mediated knockdown of Orai1, TRPC1, TRPC3 and TRPM7 channels on
bradykinin-induced [Ca2+ ]i rises
A, comparison of the mRNA expression levels of Gd3+ - and 2-APB-sensitive Ca2+ entry channels by quantitative
RT-PCR. The expression levels are normalized to that of TRPC1. The levels of TRPC2 (0.013), TRPC4 (0.008) and
TRPC6 (0.005) were substantially low and those of TRPC5 and TRPC7 were not detected. The results were obtained
from a total of 30 pups born from 3 mother mice. B, immunostaining of the cells with the antibodies against
the channels showing high mRNA expression levels. The staining only with secondary antibodies is shown as a
negative control. C, bradykinin-induced F 340 /F 380 rise in the cells transfected with non-targeting siRNA as controls
(a, averaged over 240 cells) and the rises in the cells transfected with Orai1 siRNA (b, n = 253), TRPM7 siRNA
(c, n = 211), TRPC1 siRNA (d, n = 262) and TRPC3 siRNA (e, n = 242). The traces in this figure were recorded
with a Hamamatsu ORCA-R2 camera. D, comparison of the areas of net F 340 /F 380 rises over 1–20 min (indicated
by shaded regions in Ca–e). The areas in the cells transfected with the siRNAs for Orai1, TRPC1 and TRPC3 were
significantly suppressed (marked with asterisks) compared to those in the others (P < 0.05, judged by multiple
comparisons). There are no significant differences between the areas in these 3 types of transfected cell. The area
in TRPM7 siRNA-transfected cells is not statistically different from that in non-targeting siRNA-transfected cells.

however, when the amplitudes were compared at 20 min the current developed gradually and reached a plateau
after the beginning of bradykinin application, significant 15–20 min after the beginning of bradykinin action,
suppression was seen only in TRPC1 siRNA-transfected whereas the [Ca2+ ]i rise reached a peak immediately after
cells (Fig. 4D). Thus, considering the similar suppression the bradykinin action and decayed rapidly to a small
of the [Ca2+ ]i rises in these three types of transfected cell sustained level. This difference implies that Ca2+ would
(Fig. 3D), the results suggest that the Ca2+ entry through not act directly on VSOR channels. A plausible mediator
TRPC1 channels would be preferentially involved in the is evidently the Ca2+ -dependent types of PKC. So we
VSOR channel activation in the majority of the cells. explored this possibility.
Application of a PKC activator, PMA (1 μM), instead
of bradykinin, indeed induced development of a current
showing mild outward rectification and weak inactivation
Bradykinin-induced activation of VSOR anion
at high voltages (open circles in Fig. 5A and C), which
channels is mediated by Ca2+ -dependent PKCα and β was blocked by DCPIB (crosses in Fig. 5A and C). Thus
The foregoing results strongly support the dependence of PMA mimicked the bradykinin action on VSOR channel
bradykinin-induced VSOR channel activation on [Ca2+ ]i activation. Furthermore, the bradykinin-induced VSOR
rises. However, the time course of the current development current activation was strongly suppressed in the presence
was evidently different from that of the [Ca2+ ]i rise; of a specific inhibitor of Ca2+ -dependent types of PKC,
Figure 4. Effects of TRPC1 knockdown on bradykinin-induced VSOR currents

Bradykinin-induced VSOR currents were monitored during application of voltage pulses as in Fig. 2A. A,
bradykinin-induced VSOR current in non-targeting siRNA-transfected cells. B, an example showing a waxing
and waning pattern of current development induced by bradykinin in TRPC1 siRNA-transfected cells. This pattern
was seen in the majority of the cells (22 of 31 cells). C, I–V relationships measured 20 min after the beginning
of bradykinin application in these transfected cells. Filled symbols indicate the mean values before application of
bradykinin, and open symbols indicate those after 20 min. The numbers of cells analysed were 29 for non-targeting
siRNA (squares) and 31 for TRPC1 siRNA (circles). D, comparison of the peak amplitudes of the currents evoked
by voltage pulses to +100 mV between the cells transfected with the siRNAs for Orai1 (n = 32), TRPC1 and
TRPC3 (n = 27) and the cells with non-targeting (NT) siRNA. Only the amplitude in TRPC1 knockdown cells
was significantly suppressed (asterisk, P < 0.05 by multiple comparisons). The examples of current traces and
the I–V relationships in Orai1-knockdown and TRPC3-knockdown cells are shown in Supplemental Fig. S2. E,
effect of extracellular Ca2+ removal during the course of bradykinin-induced current development. The trace is a
representative of three separate experiments.

Gö6976 (1 μM; open triangles in Fig. 5B and C), whereas waning pattern of current development in response
prominent [Ca2+ ]i rises were still preserved (Fig. 5D). to bradykinin; the current reached a plateau within
To consolidate this pharmacological result, we also 8–9 min and it was sustained or declined very slowly
examined the effects of transfection with the siRNAs over 10 min (17 of 29 PKCα knockdown cells as in
for Ca2+ -dependent PKCs on VSOR current activation. Fig. 6C, and 20 of 31 PKCβ knockdown cells as in
Quantitative RT-PCR indicated that, among three types Fig. 6Da). The rest of the cells showed monotonic
(α, β and γ) of Ca2+ -dependent PKC, PKCα was found current development. On average, when the currents at
to be the major type in our astrocytes (Fig. 6A). The +100 mV were compared at 20 min after the beginning
expression of PKCβ mRNA was much lower than that of bradykinin application, both the amplitude in PKCα
of PKCα, but its level was comparable to that of TRPC1 knockdown cells (10.8 ± 1.3 pA pF−1 ; open circles in
mRNA. The expression of PKCγ mRNA was at an Fig. 6C and E) and that in PKCβ knockdown cells
almost negligible level. Thus we examined the effects (12.6 ± 1.4 pA pF−1 ; open triangles in Fig. 6Da and E)
of knockdown of PKCα and PKCβ one by one. In were significantly smaller than that in non-targeting
our transfection conditions, both the siRNAs for PKCα siRNA-transfected cells (18.9 ± 2.1 pA pF−1 ; open squares
and PKCβ suppressed selectively the mRNA levels of in Fig. 6B and E; P < 0.05 by multiple comparisons).
targeted PKCs to 10% (Supplemental Fig. S1B). Compared However, the typical mild outward rectification of the
with the bradykinin-induced current development in VSOR current characteristic became obscured especially
non-targeting siRNA-transfected cells (Fig. 6B), 60% in PKCβ knockdown cells (open triangles in Fig. 6Db
of both the PKCα knockdown cells and the PKCβ and E), suggesting that contamination of other types
knockdown cells exhibited a very mild waxing and of current became noticeable. By extracting the VSOR
Figure 5. Involvement of Ca2+ -dependent PKC in bradykinin-induced activation of VSOR currents

Development and suppression of VSOR currents were monitored during application of voltage pulses as in Fig. 2A.
A, development of DCPIB-sensitive whole-cell current induced by 1 μM PMA under isotonic conditions in the
absence of bradykinin. B, inhibition of bradykinin-induced VSOR current in the presence of a specific blocker
of Ca2+ -dependent PKCs, Gö6976 (1 μM). Cells were preincubated with Gö6976 for 30 min at 37◦ C, and the
responses were measured in the presence of Gö6976 at 25–30◦ C in the bathing solution. C, I–V relationships of
the currents shown in A and B. PMA and Gö6976 were dissolved in the bathing solution with 0.01% DMSO. Thus
the control bradykinin response was taken in the presence of 0.01% DMSO (open squares; n = 17). The current
response induced by PMA (open circles; n = 14) was similar to the control and blocked by 10 μM DCPIB (crosses;
n = 14). The response induced by bradykinin was greatly suppressed in the presence of Gö6976 (P < 0.001 by
ANOVA, open triangles; n = 21). D, bradykinin-induced [Ca2+ ]i rise in the presence of Gö6976 averaged over 140
cells. The amplitude and the area of the [Ca2+ ]i rise over 1–15 min (shaded region) were significantly larger than
those of the control (P < 0.05 by ANOVA). The traces were recorded with a Roper NTE camera.

Figure 6. Effects of siRNAs for PKCα and PKCβ on bradykinin-induced VSOR currents
A, mRNA expression levels of Ca2+ -dependent PKCα, β and γ , determined by quantitative RT-PCR. The levels are
normalized to that of TRPC1 mRNA. The results were obtained from total 30 pups born from 3 mother mice. B and
C, bradykinin-induced VSOR currents in non-targeting siRNA-transfected cells and in PKCα siRNA-transfected cells,
respectively. The currents were monitored during application of voltage pulses as in Fig. 2A. Da, bradykinin-induced
current and its suppression by 10 μM DCPIB in PKCβ siRNA-transfected cells. Db, comparison of the I–V
relationships before (filled triangles) and 20 min after the beginning of bradykinin application (open triangles),
and after suppression by DCPIB (filled diamonds). Data were from 19 cells. Asterisks indicate the significant
differences between the currents before bradykinin application (filled triangles) and after bradykinin application
with DCPIB treatment (filled diamonds) at P < 0.01 by paired t test. Dc, I–V relationship of the DCPIB-sensitive
current component. Crosses indicate the mean ± SEM of the differences between the currents just before and
after the DCPIB treatment in individual cells shown in Db. E, I–V relationships of bradykinin-induced VSOR currents
in the siRNA-transfected cells shown in B–D. Filled symbols indicate the mean ± SEM of the amplitudes before
application of bradykinin, and open symbols indicate those after 20 min. The numbers of cells analysed were 29
for non-targeting siRNA-transfected cells (squares), 29 for PKCα siRNA-transfected cells (circles) and 31 for PKCβ
siRNA-transfected cells (triangles).

current component with DCPIB (crosses in Fig. 6Dc), we around the sensing sites is very high and because the
found that the contaminating current was contained only chelators pass through the narrow space between the
in the negative potential range, suggesting its inwardly sensing sites and Ca2+ sources so quickly that Ca2+ is
rectifying property. (The asterisks in Fig. 6Db indicate the less likely to be captured (Neher, 1998b). Based on the
significant differences in amplitude between the currents failure of BAPTA-AM treatment to suppress the VSOR
before bradykinin application (filled triangles) and after channel activation in our previous study (Liu et al. 2009),
DCPIB treatment (filled diamonds); P < 0.01 by paired we examined the effects of applying Ca2+ chelators at
t test.) Since no significant shift in reversal potential given concentrations directly into the cells through patch
was seen after the DCPIB treatment, a current through pipettes on the VSOR channel activation.
Cl− -permeable ClC-2-like channels bearing a similar By default, we included 1 mM EGTA into the pipette
inwardly rectifying property (Makara et al. 2003) would solution for VSOR current recordings (see Methods for
probably be involved to some extent. In any case, since details), whereas we had included 10 mM EGTA in our
the bradykinin-induced current at positive potentials was previous study (Liu et al. 2009). However, this difference
found to be purely the VSOR current, the reductions in did not yield a significant difference in the maximum
amplitude at +100 mV (Fig. 6E) indicate that both PKCα amplitude of VSOR current induced by bradykinin.
and β must be involved in the VSOR channel activation. The amplitude at +100 mV in the presence of intra-
Thus, these data confirm that the VSOR channel activation cellular 10 mM EGTA was 49.3 ± 6.4 pA pF−1 in this
induced by bradykinin is mediated by Ca2+ -dependent study (open circles in Fig. 8A and D), whereas it was
PKCα and PKCβ, not by the direct interaction of Ca2+ 63.4 ± 10.0 pA pF−1 in the presence of 1 mM EGTA (n.s.
with the channel. by multiple comparisons; open squares in Fig. 8D).
The amplitude was still not significantly affected, when
BAPTA, whose Ca2+ -binding speed is 200 times faster
Bradykinin-induced ROS generation is mediated by than EGTA, at 1 mM was included in the pipette solution
Ca2+ -dependent PKC (44.3 ± 8.5 pA pF−1 ; filled circles in Fig. 8B and D).
When the concentration of BAPTA was increased to
Moreover, we also examined whether the PKC activation
10 mM, the amplitude was suppressed significantly to
is linked to ROS generation. Bradykinin-induced ROS
about one-third (23.1 ± 2.7 pA pF−1 , P < 0.05 by multiple
generation detected with CM-DCF fluorescence (Fig. 7A)
comparisons; filled squares in Fig. 8C and D). Thus the
was strongly suppressed in the presence of Gö6976,
results should indicate that Ca2+ -sensing sites of the
whereas the responsiveness of CM-DCF to exogenous
molecules responsible for VSOR channel activation are
H2 O2 was not affected (Fig. 7B). We also checked that the
located or moved so close to Ca2+ sources upon bradykinin
ROS generation was abolished in the presence of a NOX
receptor activation that only the fast Ca2+ chelator BAPTA
inhibitor, diphenyleneiodonium (DPI; 10 μM, Fig. 7C)
at >10 mM can interfere with Ca2+ binding to the sensing
as we showed previously (Liu et al. 2009), and also in
sites. The extracellularly applied BAPTA-AM at 50 μM in
the absence of extracellular Ca2+ after pretreatment with
our previous study (Liu et al. 2009) would have not been
thapsigargin (Fig. 7D). Similar ROS generation was also
concentrated in the cytoplasm at >1 mM.
produced by PMA instead of bradykinin (Fig. 7E). Since
we previously demonstrated that the bradykinin-induced
VSOR current and associated glutamate release were pre-
vented in the presence of ROS scavengers and several NOX Discussion
inhibitors (Liu et al. 2009), the result confirms that PKC
This study elucidated the dependence of
activation precedes and is linked serially to ROS generation
bradykinin-induced VSOR channel activation on
through NOX for activation of VSOR channels.
Ca2+ nanodomains under isotonic conditions. The Ca2+
involved in the VSOR channel activation was provided
High concentrations of intracellular BAPTA initially by Ca2+ release from intracellular Ca2+ stores
are required to inhibit bradykinin-induced activation and then by store- or receptor-operated Ca2+ entry at
the plasma membrane. In the Ca2+ entry, Orai1, TRPC1
of VSOR anion channels
and TRPC3 channels were found to be involved in our
Ca2+ -dependent processes must be inhibited if the Ca2+ preparations, and the entry through TRPC1 channels
binding to its sensing sites is interrupted sufficiently seemed to be preferentially used for the VSOR channel
by exogenously applied Ca2+ chelators. However, if the activation. The Ca2+ must be utilized at least in part for
distance between the sensing sites and Ca2+ sources activation of Ca2+ -dependent PKCα and β, and these
is shorter than 100 nm, higher concentrations of the PKCs induced ROS generation through NOX. The ROS
chelators with faster Ca2+ -binding kinetics are required for must be involved in VSOR channel activation, although
interruption, because the amount of Ca2+ to be captured the mechanism is not yet known.

Our study further indicated that the sites of Ca2+ to a [Ca2+ ]i in the range of >1 μM as those of PKCs
action must be very close to the Ca2+ source channels. (Keranen & Newton, 1997), and considering that low
How close the distance is can be roughly estimated by concentrations of BAPTA should not affect drastically
using a theoretical model of Ca2+ nanodomains (Neher, the VSOR channel activation, the Ca2+ sensors would
1998a). The model predicts that no significant [Ca2+ ]i probably sense the [Ca2+ ]i rises within ∼20 nm of the
rises would be generated at the sites >40 nm away from channel pores. The sensors involved would include those
individual open channel pores in the presence of 10 mM of phospholipase C and PKC. Activation of these enzymes
BAPTA (Fig. 8E). Assuming that the Ca2+ sensors respond necessitates their binding to the plasma membrane,
Figure 7. Bradykinin-induced ROS generation through NOX and its sensitivity to a blocker of
Ca2+ -dependent PKC and to Ca2+ depletion
A, comparison of the time courses of increases in normalized fluorescence (F/F 0 ) of CM-DCF with and without
application of bradykinin. Bradykinin at 1 μM (+ Bradykinin) or a blank bathing solution (– Bradykinin) was applied
5 min after the beginning of observation (at time 0, indicated by the arrow). The fluorescence was measured at 10 s
intervals, and each point in the plot represents the averaged F/F 0 value over 1 min. To check the responsiveness
of CM-DCF to exogenous ROS, 1 mM H2 O2 was applied at 30 min in the ‘− Bradykinin’ condition. The traces are
the averages over 148 cells in the ‘+ Bradykinin’ condition (squares) and 164 cells in the ‘− Bradykinin’ condition
(circles), respectively. The larger F/F 0 rise at 30 min in the ‘+ Bradykinin’ condition is significant at P < 0.01 by
ANOVA. The F/F 0 rise in the ‘− Bradykinin’ condition would be mostly caused by the photo-oxidation of CM-DCF,
since similar amplitudes of the rises were generated even in the presence of a NOX blocker, DPI, as shown below
in C. B, suppression of bradykinin-induced F/F 0 rise in the presence of 1 μM Gö6976. Gö6976 was included also
during dye loading. The traces are from 154 cells in the ‘+ Bradykinin’ condition (squares) and 139 cells in the
‘− Bradykinin’ condition (circles). C, suppression of the F/F 0 rise in the presence of 10 μM DPI. The traces are the
averages over 227 cells in the ‘+ Bradykinin’ condition (squares) and 230 cells in the ‘− Bradykinin’ condition
(circles). D, suppression of the F/F 0 rise in the absence of extracellular Ca2+ observed after pretreatment with
thapsigargin. Thapsigargin at 1 μM was included in the loading solution but not in the bathing solution. EGTA at
0.1 mM was added to the 0 Ca2+ bathing solution. The traces are from 202 cells in the ‘+ Bradykinin’ condition
(squares) and 195 cells in the ‘− Bradykinin’ condition (circles). E, F/F 0 rise induced by 1 μM PMA. PMA or a blank
solution (– PMA) was applied at time 0 (indicated by the arrow). The traces are from 205 cells in the ‘+ PMA’
condition (squares) and 181 cells in the ‘− PMA’ condition (circles). The larger F/F 0 rise at 30 min in the ‘+ PMA’
condition is significant at P < 0.01 by ANOVA.

because not only Ca2+ but also the activated G proteins, synergistically the NOX activity with the phosphorylation
the substrate phosphatidylinositol 4,5-bisphosphate in the (Berthier et al. 2003). Furthermore, although how ROS
membrane and the resultant diacylglycerol are required activate VSOR channels is totally unknown, the ROS
for their activation (Drin & Scarlata, 2007; Gallegos signalling involved in normal cellular responses tends
& Newton, 2008). Thus it is evidently reasonable that to be specifically coupled to its targets, and unwanted
these enzymes rely on the Ca2+ in the nanodomains spread of ROS is immediately quenched by a wealth of
generated just adjacent to the activated G protein-coupled scavengers, like superoxide dismutase and glutathione,
receptors. in the cytoplasm (Terada, 2006). Therefore, our results
It is well known that the ROS generator NOX can be strongly suggest that the bradykinin-induced VSOR
activated by PKC through phosphorylation of its subunit channel activation is regulated in the immediate vicinity
p47phox (Bedard & Krause, 2007), and Ca2+ may enhance of individual Ca2+ release and entry channels.
Figure 8. Effects of intracellular EGTA and BAPTA on bradykinin-induced VSOR currents

A–C, bradykinin-induced VSOR currents in the presence of intracellular EGTA at 10 mM (A), BAPTA at 1 mM (B) and
BAPTA at 10 mM (C). Voltage pulse protocols were the same as those in Fig. 2A. The Ca2+ chelators were applied
via patch pipettes. D, I–V relationships at different concentrations of intracellular Ca2+ chelators. The numbers of
the cells analysed were 17 for 1 mM EGTA (open squares), 15 for 10 mM EGTA (open circles), 13 for 1 mM BAPTA
(filled circles), 14 for 10 mM BAPTA (filled squares) and 16 for 10 mM BAPTA observed after pretreatment with
1 μM thapsigargin and removal of extracellular Ca2+ (filled triangles). Significant suppression in the amplitude
at +100 mV was seen only in the presence of 10 mM BAPTA (filled squares and triangles; P < 0.05 by multiple
comparisons). E, model of steady-state spatial [Ca2+ ]i profiles of a single ‘Ca2+ nanodomain’ in the presence of
Ca2+ chelators at different concentrations. The profiles were drawn based on the equation proposed by Neher
(1998a):
iCa r
[Ca2+ ]i (r ) = exp − + [Ca2+ ]i (∞) ,
4π F D Ca r λ

where λ = k D Ca[B] , [B] = KD
2+ [BT ], r is the distance from a Ca2+ channel pore, i Ca is the single-channel
Bon K D +[Ca ]i (∞)
Ca2+ current (0.1 pA, assuming that i Ca through a TRPC channel is slightly smaller than that through a
voltage-gated Ca2+ channel), F is the Faraday constant, DCa is the diffusion coefficient of free Ca2+ (223 μm2 s−1 ;
Allbritton et al. 1992), λ is the length constant of the profile, kBon is the Ca2+ binding rate constant of the
chelator (2.5 × 106 M−1 s−1 for EGTA, 5 × 108 M−1 s−1 for BAPTA; Naraghi, 1997), [B] is the concentration of
the Ca2+ -free chelator, [BT ] is the total concentration of the chelator, and K D is the dissociation constant of the
chelator (0.438 μM, based on the in vivo calibration of a BAPTA-type Ca2+ indicator by Akita & Kuba (2000); the
K D values of EGTA and BAPTA are assumed to be the same). [Ca2+ ]i (∞) must be the basal [Ca2+ ]i in the bulk
cytoplasm (0.1 μM). Note that [Ca2+ ]i is plotted on a logarithmic scale. The calculated λ values were 331 nm,
105 nm, 23.4 nm and 7.4 nm in the presence of 1 mM EGTA, 10 mM EGTA, 1 mM BAPTA and 10 mM BAPTA,
respectively. The profile in the absence of chelators, indicated by the dotted line, was calculated by putting λ at
infinity, i.e. making the exponential term unity.

It must be noted, however, that this mechanism is et al. 2009). In our recording conditions, however, we have
different from the principal mechanism of VSOR channel never seen such a rapid current enhancement just after the
activation induced by osmotic cell swelling in response to beginning of bradykinin application when Ca2+ release
hypotonic stimuli. The swelling-induced VSOR current began. This would be due to the difference in the mode of
is known to be relatively resistant to intracellular BAPTA current recording or in the amount of Ca2+ chelators in the
at 5–10 mM (Kubo & Okada, 1992; Pedersen et al. 1998), pipette solution. Park et al. (2009) recorded the currents
as mentioned in Introduction. This is in contrast to in a perforated patch clamp mode, whereas we recorded
the ∼70% suppression of bradykinin-induced current the currents in the conventional whole-cell patch clamp
by BAPTA at 10 mM (Fig. 8C and D). Furthermore, the mode with the pipette solution containing 1 mM EGTA
Ca2+ -dependent enzyme cascade should not work unless by default to fix the basal Ca2+ concentration to ∼15 nM
the [Ca2+ ]i rise is accompanied by generation of the (see Methods for details). Therefore, it is possible that
membrane-associated components, as described above. the rapidly activated current component would have been
In this sense, the cascade depends not only on Ca2+ but eliminated by intracellular dialysis with 1 mM EGTA in
also on the receptor activation per se. By contrast, the our conditions. In other words, the activation of rapidly
VSOR channel activation induced by hypotonic stimuli responsive Ca2+ -activated Cl− channels, if any, would be
was shown to be never abolished by interruption of any much more loosely coupled to Ca2+ sources than the
known types of signalling cascade (Okada et al. 2009), VSOR channel activation is in astrocytes.
and the activation correlates directly with the increase in The characteristics of the current through VSOR
surface area of the cell as a result of cell swelling (Okada, channels, especially the outward rectification and
1997), although the correlation may be modulated more inactivation at high voltages, are known to have some
or less by some signalling cascades. Thus the mechanism variations between cell types, but whether these are due
of hypotonicity-induced VSOR channel activation seems to some modifications on the same channel molecule or
to depend much more on the event associated with others is totally unknown (see reviews by Okada, 1997;
intrinsic structural changes of the cell. However, the Nilius et al. 1997a; Hoffmann et al. 2009). As mentioned
Ca2+ -dependent cascade may contribute to a part of in Results, the VSOR current in astrocytes is similar to
the VSOR channel activity during osmotic swelling. For that in cardiomyocytes, where ClC-3 has been proposed
example, the chemical transmitters released during hypo- as a candidate for VSOR channels (Duan et al. 1997)
tonic stimuli, like ATP, may contribute to the activity although this has been challenged by many reports
through their binding to Gq-coupled receptors (Wang including those from our groups (Gong et al. 2004;
et al. 1996; Darby et al. 2003; Mongin & Kimelberg, 2005). Wang et al. 2005). Since the ClC-3 current may exhibit
As another example, the δ-type of phospholipase C is similar characteristics to the VSOR current, one may
reported to be activated directly by Ca2+ without the help argue that the bradykinin-induced current in astrocytes
of G-proteins (Kim et al. 1999; Mogami et al. 2003), so might be caused by the activation of ClC-3. However,
this might generate diacylglycerol sufficiently to induce the same group that proposed ClC-3 as the candidate
the PKC-NOX signalling in response to the [Ca2+ ]i rises (Duan et al. 1999) clearly demonstrated that a site in the
caused by hypotonic stimuli. These may explain the partial amino terminus of ClC-3 can be directly phosphorylated
dependence of swelling-induced VSOR channel activation by PKC and this causes suppression of ClC-3 activity.
on PKC (Verdon et al. 1995; Du & Sorota, 1999; Gong et al. Evidently, this does not match with our case, where
2004) or ROS (Varela et al. 2004; Ren et al. 2008) reported PKC activation causes ROS generation for VSOR
in some types of cell, although the dependency on PKC channel activation. Therefore, the bradykinin-induced
or ROS is known to be quite variable between cell types current would be unlikely to be caused by
(reviewed by Okada, 1997; Nilius et al. 1997a; Hoffmann ClC-3.
et al. 2009). With its properties of slow and sustained activation, the
The Ca2+ nanodomain-mediated control of the enzyme VSOR channel is specialized for cell volume regulation in
cascade produced slow but prolonged activation of large response to hypotonic exposure and to apoptotic stimuli,
VSOR currents, even when the [Ca2+ ]i rise in the bulk both of which follow relatively long time courses of
cytoplasm had almost disappeared. This activation pattern >10 min and >1 h, respectively (reviewed by Okada et al.
is entirely different from that of the rapidly responsive 2001). Thus the Ca2+ nanodomain-mediated regulation of
so-called Ca2+ -activated Cl− channels. The prototypical VSOR channels would provide a firm basis for local control
Ca2+ -activated Cl− current is activated immediately in of cell volume regulation, e.g. retraction of a part of a cell
response to the beginning of a [Ca2+ ]i rise, and reaches causing cell shape change or migration (Schneider et al.
a peak and decays rapidly in parallel with the [Ca2+ ]i 2008), and of intercellular communications on a long time
peak and decay (Nilius et al. 1997b; Machaca & Hartzell, scale, even when Ca2+ signals are transient and confined
1999). A similar activation pattern of a Ca2+ -activated to a subcellular fine structure level (Koizumi et al. 1999;
Cl− current was also reported in mouse astrocytes (Park Conklin et al. 2005).

Bradykinin is widely distributed throughout the brain Akita T & Kuba K (2000). Functional triads consisting of
(Kariya et al. 1985) and known to be released during ryanodine receptors, Ca2+ channels, and Ca2+ -activated K+
trauma, stroke and inflammation in the brain (Ellis et al. channels in bullfrog sympathetic neurons: Plastic
1989; Kamiya et al. 1993; Gröger et al. 2005). We speculate modulation of action potential. J Gen Physiol
that one possible in situ role for local regulation of the 116, 697–720.
Akita T & Okada Y (2010). Regulation of bradykinin-induced
VSOR channel in the brain in response to bradykinin
activation of volume-sensitive outwardly rectifying (VSOR)
might be as follows. A group of astrocytes in the brain anion channels via ‘Ca2+ nanodomains’ in mouse cortical
is known to form part of the blood–brain barrier, astrocytes. J Physiol Sci 60, S66.
surrounding capillary endothelial cells with their fine end- Allbritton NL, Meyer T & Stryer L (1992). Range of messenger
feet processes (Abbott et al. 2006). Bradykinin is known action of calcium ion and inositol 1,4,5-trisphosphate.
to be produced easily when the endothelial cell walls Science 258, 1812–1815.
are damaged by for example hypertension. Thus, when Augustine GJ, Santamaria F & Tanaka K (2003). Local calcium
damage occurs, a small amount of bradykinin may leak signaling in neurons. Neuron 40, 331–346.
out and act on a part of an astrocyte endfoot. If this Baumgarten CM & Clemo HF (2003). Swelling-activated
small amount of bradykinin induces a minute amount of chloride channels in cardiac physiology and
InsP 3 production in the cell, the [Ca2+ ]i rise, if it occurs, pathophysiology. Prog Biophys Mol Biol 82, 25–42.
Bedard K & Krause KH (2007). The NOX family of
may be limited as a local transient event (Callamaras et al.
ROS-generating NADPH oxidases: physiology and
1998; Koizumi et al. 1999), and may not propagate as Ca2+ pathophysiology. Physiol Rev 87, 245–313.
waves throughout the astrocyte. Even in this case, however, Berthier S, Paclet MH, Lerouge S, Roux F, Vergnaud S,
a number of VSOR channels around the local [Ca2+ ]i Coleman AW & Morel F (2003). Changing the conformation
rise would likely be activated based on the nanodomain state of cytochrome b558 initiates NADPH oxidase
regulation of VSOR channels. And not only Cl− ions but activation: MRP8/MRP14 regulation. J Biol Chem 278,
also excitatory amino acids are released focally through the 25499–25508.
VSOR channels. Furthermore, because of its slow kinetics, Birnbaumer L (2009). The TRPC class of ion channels: a
VSOR channel activation would still be maintained for critical review of their roles in slow, sustained increases in
a while even after the [Ca2+ ]i rise diminishes, and this intracellular Ca2+ concentrations. Annu Rev Pharmacol
would facilitate a local retraction of the endfoot process, Toxicol 49, 395–426.
Browe DM & Baumgarten CM (2004). Angiotensin II (AT1)
providing a space for the repair of the injured site by
receptors and NADPH oxidase regulate Cl− current elicited
microglia. To elucidate such a role for VSOR channel, fine by β1 integrin stretch in rabbit ventricular myocytes. J Gen
changes in cell structure and in cell volume induced by Physiol 124, 273–287.
bradykinin have to be examined carefully, which remains Callamaras N, Marchant JS, Sun XP & Parker I (1998).
for future studies. Activation and co-ordination of InsP3 -mediated elementary
In conclusion, this study demonstrated for the first Ca2+ events during global Ca2+ signals in Xenopus oocytes. J
time that VSOR channel activation may be mediated by Physiol 509, 81–91.
Ca2+ nanodomains, especially when their generation is Cheng H & Lederer WJ (2008). Calcium sparks. Physiol Rev 88,
associated with the activation of Gq-coupled bradykinin 1491–1545.
receptors. Therefore, it would also be intriguing to see Conklin MW, Lin MS & Spitzer NC (2005). Local calcium
whether activation of other types of Gq-coupled receptors transients contribute to disappearance of pFAK, focal
complex removal and deadhesion of neuronal growth cones
involved in normal cellular functions, like those for ATP or
and fibroblasts. Dev Biol 287, 201–212.
glutamate, may induce similar VSOR channel activation Darby M, Kuzmiski JB, Panenka W, Feighan D & MacVicar BA
and local cell volume regulation. (2003). ATP released from astrocytes during swelling
activates chloride channels. J Neurophysiol 89, 1870–1877.
Deng W, Baki L & Baumgarten CM (2010). Endothelin
signalling regulates volume-sensitive Cl− current via
NADPH oxidase and mitochondrial reactive oxygen species.
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duct cells. J Membr Biol 147, 173–183. Sports, Science and Technology (MEXT).


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J Physiol 589.

16 (2011) pp 3909–3927 3909

Regulation of bradykinin-induced activation

Abbreviations 2-APB, 2-aminoethoxydiphenyl borate; CM-DCF, 5-(and-6)-chloromethyl-2 ,7 -dichlorofluorescein;

Introduction the bradykinin B2 receptor coupled to Gαq proteins

no. L-056431-02), TRPC1 (L-043863-00), TRPC3 Drugs

response to depletion of Ca2+ stores or to bradykinin Bradykinin-induced activation of VSOR anion

Figure 4. Effects of TRPC1 knockdown on bradykinin-induced VSOR currents

Figure 5. Involvement of Ca2+ -dependent PKC in bradykinin-induced activation of VSOR currents

Figure 8. Effects of intracellular EGTA and BAPTA on bradykinin-induced VSOR currents

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