Am J Physiol Cell Physiol 306: C856–C863, 2014.
First published February 19, 2014; doi:10.1152/ajpcell.00368.2013.
Adenosine triphosphate regulates the activity of guinea pig Cav1.2 channel
by direct binding to the channel in a dose-dependent manner
Rui Feng,1,2 Jianjun Xu,2 Etsuko Minobe,2 Asako Kameyama,2 Lei Yang,2 Lifeng Yu,1 Liying Hao,1*
and Masaki Kameyama2*
1
Department of Pharmaceutical Toxicology, School of Pharmacy, China Medical University, Shenyang, China;
and 2Department of Physiology, Graduate School of Medical and Dental Sciences, Kagoshima University, Sakuragaoka,
Kagoshima, Japan
Submitted 6 December 2013; accepted in final form 3 February 2014
adenosine-triphosphate; calmodulin; L-type Ca2⫹ channel; phosphorylation
L-TYPE CA2⫹ CHANNEL
(LTCC) is a type of voltage-dependent
calcium channel. LTCC mediates the influx of Ca2⫹ from the
extracellular side into the cytoplasm and, hence, plays a fundamental role in many physiological processes, such as muscle
contraction, secretion of neurotransmitters, and gene expression (4, 15). LTCC is composed of ␣1-, ␣2␦-, ␤1– 4-, and
␥-subunits, and has several isoforms, including Cav1.1,
Cav1.2, Cav1.3, and Cav1.4. The Cav1.2 channel is the predominant type of LTCC in the heart and consists of ␣1C
subunit and auxiliary ␤- and ␣2␦-subunits (56). Multiple factors have been identified to regulate the Cav1.2 channel
through various mechanisms, including protein kinase phosphorylation and direct interaction with the channel. It is reported that the activity of the Cav1.2 channel is modulated by
cAMP-dependent protein kinase (6, 16) and calmodulin (CaM)
* L. Hao and M. Kameyama contributed equally to this work.
Address for reprint requests and other correspondence: L. Hao, Dept. of
Pharmaceutical Toxicology, School of Pharmacy, China Medical Univ., 92
Beier Road, Shenyang 110001, China.
C856
kinase II-mediated phosphorylation (14, 15). Some studies
indicate that CaM is directly tethered to the Cav1.2 channel
and mediates the channel function (38, 50, 53). Free calcium
ions (Ca2⫹), free magnesium ions (Mg2⫹), and calpastatin are
reported to bind to the Cav1.2 channel to regulate its activity
(5, 8, 22, 31).
In addition to the regulating factors, intracellular adenosine
triphosphate (ATP) is also known to affect the activity of Ca2⫹
channels in heart muscle (21, 33, 46). One of the unique
characteristics of cardiac LTCC is known as run-down. It is
reported that CaM plus ATP induces the activity of Ca2⫹
channels after run-down (51). Many studies demonstrate the
importance of CaM in the regulation of LTCC (38, 50, 53).
However, the function of CaM requires ATP to induce the
activity of LTCC (51). Although the functions of ATP have
been examined in cardiac Ca2⫹ channel, the mechanism is
poorly understood.
It is well known that the effect of ATP on Ca2⫹ channel is
attributed mainly to the promotion of protein phosphorylation.
Previous studies show that the role of ATP in the regulation of
Cav1.2 channel activities involves the cAMP-dependent protein kinase-mediated phosphorylation pathway (23, 30, 35, 37,
43). However, several studies indicate that ATP modulates the
Cav1.2 channel by phosphorylation-independent mechanisms
(23, 30, 35, 36, 37, 43, 51, 52). The phosphorylation-independent pathway includes the allosteric effects of ATP binding,
changes of actin cytoskeleton, and ATP-dependent phospholipases, etc. (19). It has been found that ATP binding to a
low-affinity site shifts the Na⫹/Ca2⫹ exchanger from an inactive state to an active state and the activating effect of ATP to
RyR1 occurs through direct binding. However, the phosphorylation-independent mechanism for the activation of the cardiac Ca2⫹ channel by ATP remains unknown. The present
study is to investigate the mechanism by which ATP regulates
Cav1.2 channel activity.
MATERIALS AND METHODS
Sample preparation. Ventricular tissue was obtained from adult
guinea pig hearts using collagenase. A female guinea pig (weight
400 – 600 g) was anesthetized with pentobarbital sodium (30 mg/kg
ip), and the aorta was cannulated in situ while the animals were under
artificial respiration. The dissected heart was mounted on a Langendorff apparatus and perfused first with Tyrode solution at 37°C for 3
min and then with nominally Ca2⫹-free Tyrode solution for 6 min,
and finally with Ca2⫹-free Tyrode solution containing collagenase
(0.08 mg/ml; Yakult) for 8 –15 min. The enzyme solution was then
washed out with a high-K⫹ and low-Ca2⫹ solution (storage solution).
Finally, the ventricular tissue was cut into pieces and stored at ⫺80°C
for further experiments. To obtain single cardiac myocytes, the left
0363-6143/14 Copyright © 2014 the American Physiological Society
http://www.ajpcell.org
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Feng R, Xu J, Minobe E, Kameyama A, Yang L, Yu L, Hao L,
Kameyama M. Adenosine triphosphate regulates the activity of guinea pig
Cav1.2 channel by direct binding to the channel in a dose-dependent manner.
Am J Physiol Cell Physiol 306: C856–C863, 2014. First published February
19, 2014; doi:10.1152/ajpcell.00368.2013.—The present study is to investigate the mechanism by which ATP regulates Cav1.2 channel
activity. Ventricular tissue was obtained from adult guinea pig hearts
using collagenase. Ca2⫹ channel activity was monitored using the
patch-clamp technique. Proteins were purified using wheat germ
agglutinin-Sepharose, and the concentration was determined using the
Coomassie brilliant blue technique. ATP binding to the Cav1.2
channel was examined using the photoaffinity method. EDA-ATPbiotin maintains Ca2⫹ channel activity in inside-out membrane
patches. ATP directly bound to the Cav1.2 channel in a dosedependent manner, and at least two molecules of ATP bound to one
molecule of the Cav1.2 channel. Low levels of calmodulin (CaM)
increased ATP binding to the Cav1.2 channel, but higher levels of
CaM decreased ATP binding to the Cav1.2 channel. In addition, Ca2⫹
was another regulator for ATP binding to the Cav1.2 channel. Furthermore, ATP bound to GST-fusion peptides of NH2-terminal region
(amino acids 6 –140) and proximal COOH-terminal region (amino
acids 1,509 –1,789) of the main subunit (␣1C) of the Cav1.2 channel.
Our data suggest that ATP might regulate Cav1.2 channel activity by
directly binding to the Cav1.2 channel in a dose-dependent manner. In
addition, the ATP-binding effect to the Cav1.2 channel was both
CaM- and Ca2⫹ dependent.
ATP REGULATES CAV1.2 CHANNEL ACTIVITY
the Coomassie brilliant blue technique of Bradford and bovine serum
albumin as the standard.
Preparation of CaM and fragments of Cav1.2. The coding sequences of human CaM [amino acids (aa) 3–149] and the cytoplasmic
regions of Cav1.2 (␣1C), including the NH2-terminal region (NT, aa
6 –140) and three COOH-terminal regions [proximal (CT1, aa 1509 –
1789), middle (CT2, aa 1778 –2003), and distal (CT3, aa 1942–
2169)], were cloned from HEK 293 cDNA by PCR amplication. Then
the sequences of CaM, NT, CT1, CT2, and CT3 were inserted into
pGEX6p-3 vector (GE Healthcare, Piscataway, NJ) to construct the
plasmids of pGEX6p-3-CaM, pGEX6p-3-NT, pGEX6p-3-CT1,
GEX6p-3-CT2, and GEX6p-3-CT3. The correct constructs were confirmed by DNA sequencing. For expression of GST fusion protein, the
correctly constructed plasmids were transformed into the Escherichia
coli BL21 (DE3) strain (Stratagene, La Jolla, CA), and the bacteria
were cultured to an A600 of 0.8. The expression of GST fusion protein
was induced by 1 mM isopropyl ␤-thiogalactopyranoside (IPTG) at
37°C for 4 h. After IPTG induction, bacteria were resuspended in Tris
buffer (50 mM Tris and 150 mM NaCl pH 8.0). The suspension was
treated with lysis buffer (Tris buffer containing 0.1 mg/ml lysozyme
and 5 mM dithiothreitol) for 30 min. In the case of the ␣1C-fragments,
in particular CT1, the bacterial precipitates were treated with 1.5%
n-lauroylsarcosine for 30 min, due to its limited solubility. After
treatment with 1% Triton X-100 for 30 min, the lysate was separated
by centrifugation at 10,000 g for 20 min, and the supernatant was
collected and incubated with glutathione-Sepharose 4B beads (GE
Healthcare, Piscataway, NJ) for 2 h. GST-CaM fusion proteins bound
to the beads were extensively washed with Tris buffer, and GST was
removed by PreScission Protease (GE Healthcare). All steps were
performed on ice or at 4°C. The concentrations purified proteins of
CaM, GST-NT, GST-CT1, GST-CT2, and GST-CT3 were calculated
by the Bradford method using a protein assay kit (Pierce, Rockford,
IL). Bovine serum albumin (Pierce) was used as a standard sample.
The purity of purified proteins was confirmed by densitometry on
sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE).
Immunoblotting. The WGA purified protein (Cav1.2) was treated
with sample buffer for 5 min and then separated by 5.8% SDS-PAGE.
Proteins were electrophoretically transferred from the SDS-PAGE gel
to a PVDF membrane using a wet transfer apparatus (Bio-Rad,
Hercules, CA) at 100 V in transfer buffer on ice for 1 h. After transfer,
the membrane was incubated in blocking buffer (5% blocking reagent;
GE Healthcare) at room temperature for 1 h. The membrane was then
rinsed and incubated with primary antibodies of anti-NH2-terminal
tail (aa 1– 46) of Cav1.2 and anti-COOH-terminal tail (aa 1507–1733)
of Cav1.2 (1:1,000 dilution) at 4°C for 18 h followed by HRPconjugated secondary antibody (1:2,500 dilution) incubation at room
temperature for 1 h. Signal was detected using enhanced chemiluminescence and exposed to Amersham Hyperfilm.
Assay for ATP binding to Cav1.2 and fragments of Cav1.2. The
WGA purified Cav1.2 protein (⬃2 ␮g) was incubated with 8N3ATP2=3=-biotin-LC-hydrazide (azido-ATP-biotin) or 8-azidoadenosine 5=triphosphate (azido-ATP) in the presence of Ca2⫹, CaM, and Mg2⫹ in
Tris buffer (20 mM Tris·HCl and 1 mM EDTA) on ice in the dark for
2 h, and 254-nm UV light was used to induce a covalent linkage
within the active site. Fragments of Cav1.2 (⬃2 ␮g each, GST-NT,
GST-CT1, GST-CT2, and GST-CT3) were mixed with azido-ATPbiotin (0.38 mM) in 250 nM Ca2⫹ buffer. The molar ratio of CaM to
Cav1.2 was 2:1. After binding, the compound (ATP bound to Cav1.2/
Cav1.2 fragments) was treated with SDS sample loading buffer for 5
min and separated on a 5.8% SDS-PAGE and then transferred to a
PVDF membrane using a wet transfer apparatus (Bio-Rad) at 100 V
in transfer buffer on ice for 1 h. Then, after blocking, the membranes
were incubated with streptavidin-HRP (1:10,000). Signal was detected using enhanced chemiluminescence and exposed to Amersham
Hyperfilm. The amount of bound ATP was quantified by scanning
with ATTO E-graph (ATTO, Tokyo, Japan) and the optical density
was analyzed by CS analyzer software (ATTO).
AJP-Cell Physiol • doi:10.1152/ajpcell.00368.2013 • www.ajpcell.org
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ventricular myocytes were dispersed and filtered through a stainless
steel mesh (105 ␮m). The isolated myocytes were treated with storage
solution containing alkaline protease [Nagase NK-103, Osaka, Japan
(0.05 mg/ml); and DNase I type IV, (0.02 mg/ml), Sigma-Aldrich, St.
Louis, MO] at 37°C for 5 min and washed twice with storage solution
followed by centrifugation at 800 rpm for 3 min. The isolated cells
were stored at 4°C in storage solution until use in the experiments.
Experiments were carried out after being approved by the Committee
of Animal Experimentation (Kagoshima University, Kagoshima, Japan).
Reagents. Tyrode solution contained the following (in mM): 135
NaCl, 5.4 KCl, 0.33 NaH2PO4, 1.0 MgCl2, 5.5 glucose, 1.8 CaCl2,
and 10 HEPES-NaOH buffer (pH 7.4). The storage solution was
composed of the following (in mM): 70 KOH, 50 glutamic acid, 40
KCl, 20 KH2PO4, 20 taurine, 3 MgCl2, 10 glucose, 10 HEPES, and
0.5 EGTA (pH was adjusted to 7.4 with KOH). The basic internal
solution (I.O. solution) contained the following (in mM): 120 potassium aspartate, 30 KCl, 1 EGTA, 0.5 MgCl2, 0.5 CaCl2 (free [Ca2⫹] ⫽
80 nM), and 10 HEPES-KOH buffer (pH 7.4). Protease inhibitor
cocktail was purchased from Roche (Mannheim, Germany). Leupeptin was obtained from Peptide Institute (Osaka, Japan). 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and
n-lauroylsarcosine were purchased from Sigma-Aldrich. Wheat germ
agglutinin (WGA)-Sepharose was obtained from GE Healthcare
(Uppsala, Sweden). Antibody against NH2-terminal tail of the Cav1.2
channel was from Alomone Labs (No. ACC-013; Jerusalem, Israel).
Antibody against COOH-terminal tail of the Cav1.2 channel was from
StressMarq Biosciences (SMC-300D; Victoria, Canada). Goat antimouse horseradish peroxidase (HRP)-conjugated IgG was from
Chemicon (Temecula, CA). (6-Aminohexyl)-ATP-biotin [(6AHATP)-biotin] and 2=/3=-O-(2-aminoethyl-carmamoyl)-ATP-biotin
[(EDA-ATP)-biotin] were from Jena Bioscience (Jena, Germany).
8-Azido-(EDA-ATP)-biotin and 8-azido-ATP were from Alt Bioscience (Lexington, KY). Streptavidin HRP conjugate was from Thermo
Scientific (Basingstoke, UK). Polyvinylidene difluoride (PVDF)
membrane was from Millipore (Billerica, MA).
Patch clamp. Ca2⫹ channel activity was monitored using the
patch-clamp technique. The myocytes were superfused with I.O.
solution. Channel activity was elicited by a depolarizing pulse from a
holding potential of ⫺70 to 0 mV for 200 ms at a rate of 0.5 Hz with
patch-clamp amplifier 200B (Axon, Molecular Devices) and fed to a
computer at a sampling rate of 3.3 kHz, where the capacity and
leakage currents were subtracted digitally. The NPo value was used to
represent the channel activity, where N is the number of active
channels in the patch and Po is the time-averaged open state probability of the channels at each depolarizing pulse. NPo was calculated
based on the equation NPo ⫽ I/i, where I was the mean current during
the 5- to 105-ms period after the onset of the test pulses and i was the
unitary current amplitude. In each experiment, basal activity was
recorded in the cell-attached mode for 2 min and then in the inside-out
patch mode. Test solutions were applied by moving the patch into a
small inlet of the perfusion chamber, which was connected to a
microinjection system.
Purification of Cav1.2 protein. The guinea pig ventricular tissue (8
g) was cut into pieces and homogenized at 3,600 rpm on ice in buffer
A solution containing the following (in mM): 100 sucrose, 20
Tris·HCl, 10 EGTA, 5 EDTA, and protease inhibitor cocktail (pH
7.5). Homogenates were centrifuged for 20 min at 10,000 g and 4°C,
and the supernatant was ultracentrifuged for 1 h at 10,000 g and 4°C.
The pellet was resuspended on ice in 3 ml buffer A. The resuspended
pellet was incubated on ice with CHAPS (final concentration 1%) for
1 h and then ultracentrifuged for 30 min at 16,000 g and 4°C. The
supernatant was saved as soluble protein. The soluble protein (1 ml)
and WGA-Sepharose (0.2 ml) were mixed for 2 h. The WGASepharose was then packed in a small column and washed with 2 ml
buffer B solution containing the following (in mM): 20 Tris·HCl and
1 EDTA (pH 7.5). The protein concentration was determined using
C857
C858
ATP REGULATES CAV1.2 CHANNEL ACTIVITY
Curve fitting of the bound (ATP) was performed with SigmaPlot
10.0 software, assuming that free (ATP) in our experimental conditions was nearly equal to total (ATP). According to the law of mass
action, bound (ATP) (Y) in the two-site model was expressed by the
two Hill equations as follows.
Y ⫽ Bmax 1 · X ⁄ (Kd1 ⫹ X) ⫹ Bmax 2 · X ⁄ (Kd2 ⫹ X)
In this equation, Bmax1, Kd1, Bmax2, and Kd2 denote Bmax and Kd for the
high- and low-affinity sites, respectively. Bmax is the number
of binding sites, and Kd is binding affinity. Data were presented as
means ⫾ SE.
Statistical analyses. All data were analyzed using the SPSS 13.0
software package (SPSS, Chicago, IL). The data are expressed as
means ⫾ SE. Student’s t-test and Dunnett’s test were used to analyze
the statistical significance. P ⬍ 0.05 was considered statistically
significant.
EDA-ATP-biotin maintains Ca2⫹ channel activity in insideout membrane patches. To test whether ATP and CaM affect
Ca2⫹ channel activity in inside-out membrane patches by
reversing the run-down of Ca2⫹ channel, we examined the
effects of MgATP, 6AH-ATP-biotin (Fig. 1A), and EDA-ATPbiotin (Fig. 1B), together with CaM, on Ca2⫹ channel activity
in inside-out membrane patches using the patch-clamp technique. Our data showed that MgATP plus CaM had effects on
Ca2⫹ channel activity (Fig. 1, C and F), and EDA-ATP-biotin
plus CaM also prevented the run-down of Ca2⫹ channel
(Fig. 1, D and F), but 6AH-ATP-biotin plus CaM had no
effect on Ca2⫹ channel activity (Fig. 1, E and F). Therefore,
our results demonstrate that EDA-ATP-biotin maintained
Ca2⫹ channel activity in inside-out membrane patches. Thus
A
B
6AH-ATP-biotin
EDA-ATP-biotin
H
(CH2)6NH
biotin
biotin
D
i.o.
i.o.
CaM (1 µM) + ATP (1 mM)
4
3
2
CaM + EDA-ATP-biotin (1 mM)
3
NPo
NPo
NH(C2H4)NHCO
1
2
bi
o
C
1
0
0
0
1
2
3
4
5
6
7
8
E
0
9 10 11 12
Time (min)
1
2
3
4
5
F
3
2
bio
1
0
0
1
2
3
4
5
6
7
8
9 10 11 12
Relative NPo (%)
CaM + 6AH-ATP-biotin (1 mM)
6
7
8
9 10 11 12
Time (min)
NS
i.o.
NPo
Fig. 1. Effect of biotin labeled ATP on Ca2⫹
channel activity in inside-out membrane
patches. A: molecular formula of 6AH-ATPbiotin. B: molecular formula of EDA-ATPbiotin. C: effect of 1 mM MgATP on NPo
(where N is the number of active channels in
the patch and Po is the time-averaged open
state probability of the channels at each depolarizing pulse) in the presence of 1 ␮M
calmodulin (CaM) and 250 nM Ca2⫹ (n ⫽ 5;
i.o., basic internal solution). D: effect of 1
mM EDA-ATP- biotin on NPo in the presence
of 1 ␮M CaM, 250 nM Ca2⫹, and 1 mM
MgCl2 (n ⫽ 4). E: effect of 1 mM 6AH-ATPbiotin on NPo in the presence of 1 ␮M CaM,
250 nM Ca2⫹, and 1 mM MgCl2 (n ⫽ 6).
F: mean channel activity induced by CaM and
ATP. Data were normalized to the preceding
channel activity in cell-attached mode and are
shown as means ⫾ SE. **P ⬍ 0.01, compared with ATP group.
**
100
Time (min)
AJP-Cell Physiol • doi:10.1152/ajpcell.00368.2013 • www.ajpcell.org
75
NS
50
25
(n=5)
(n=6)
(n=4)
0
ATP
6AH-ATPbiotin
EDA-ATPbiotin
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.5 on June 17, 2017
RESULTS
we selected azido-ATP-biotin for subsequent binding experiments as EDA-ATP-biotin and azido-ATP-biotin had similar structures.
ATP directly binds to the Cav1.2 channel in a dosedependent manner. To investigate how different concentrations of ATP bind to the Cav1.2 channel, we employed ATP
binding assay. First, we purified the Cav1.2 channel protein
from guinea pig heart by using WGA-Sepharose (Fig. 2A).
Second, we identified the proteins by anti-Cav1.2 antibody
against peptides corresponding to residues 1– 46 of NH2
terminus and 1,507–1,733 of intracellular carboxyl terminus, and both antibodies detected the full-length protein
(Fig. 2B). Third, we used different concentrations of ATP to
incubate with the Cav1.2 channel, and the results indicate
that ATP directly bound to the Cav1.2 channel (Fig. 3A) in
a dose-dependent manner (Fig. 3, B and C). The Bmax and Kd
were as follows: Bmax1 ⫽ 1.09, Kd1 ⫽ 0.07 mM; Bmax2 ⫽ 1.1,
Kd2 ⫽ 0.54 mM (Fig. 3C). These results suggest that ATP
directly bound to the Cav1.2 channel in a dose-dependent
manner and at least two ATP molecules directly bound to
one Cav1.2 channel with distinct binding affinity.
ATP binding to the Cav1.2 channel is dependent on CaM. To
examine how CaM affects ATP binding to the Cav1.2 channel,
we investigated the ATP-binding effect to the Cav1.2 channel
in the presence of different amount of CaM. With the increase
of the amount of CaM, the effect of ATP binding to the Cav1.2
channel was first increased and then decreased. In addition, the
maximal amount of ATP bound to the Cav1.2 channel was
observed when the amount of CaM was equal to that of Ca2⫹
channel. When the CaM:Cav1.2 ratio was lower than 1 (mol/
mol), increasing the amount of CaM promoted ATP binding to
C859
ATP REGULATES CAV1.2 CHANNEL ACTIVITY
A
B
Cav1.2 from g.p. heart
Immunoblot (anti-α1C)
Mr’
(kDa)
Mr’
(kDa)
250
Density (A.U.)
200
Fig. 2. Purification of the Cav1.2 channel from guinea
pig (g.p.) heart. A: Coomassie brilliant blue staining of
the Cav1.2 channel in the 5.8% SDS-PAGE gel (left)
and densitometric analysis showing a relatively high
purity of the channel protein. The molecular mass was
220 kDa. B: immunoblot analysis with anti-Cav1.2
antibodies raised against NH2-terminus (NT) and
COOH terminus (CT). A.U., arbitrary units.
250
200
150
15
Epitope
0
possible binding of ATP to the NH2- and COOH-terminal
tails of the channel, since these regions were known to
interact with CaM (3, 32, 40, 41, 56). First, the Cav1.2
␣1C-subunit was divided into the NT region and three CT
regions (CT1, CT2, and CT3) (Fig. 5A). Secondly, these
four regions were constructed into a GST-tagged vector for
protein expression. The purified GST-fusion proteins of
GST-NT, GST-CT1, GST-CT2, and GST-CT3 were confirmed by Coomassie brilliant blue (Fig. 5B). Thirdly, these
purified GST-fusion proteins were incubated with azidoATP-biotin in the presence of Ca2⫹ (250 nM) and CaM (2
mol/mol of peptide) and photolabeled by UV. The representative (Fig. 5C) and quantitative results (Fig. 5D) of the
ATP-binding assay were shown. As shown in Fig. 5C, CT1
and NT, but not CT2 and CT3, were photolabeled with
azido-ATP-biotin. The relative amounts of bound ATPbiotin were calculated based on the amount of bound ATPbiotin of CT1 (Fig. 5D). The bound amounts were 100% for
CT1, 5.0 ⫾ 0.7% for CT2, 5.2 ⫾ 1.1% for CT3, and 62.2 ⫾
4.6% for NT (n ⫽ 6). These results showed that ATP bound
to NT and CT1 regions of Cav1.2 ␣1C-subunit.
Azido-ATP-Bio (0.38 mM)
+
+
Azido-ATP (3.8 mM)
−
+
B
0
0.1
0.38 0.75
1.5
[Azido-ATP-Bio] (mM)
Bound ATP-Bio (A.U.)
C
A
CT
2.5
2
1.5
1
0.5
kDa1 = 0.07 mM
kDa2 = 0.54 mM
0
0.01
0.1
1
10
[Azido-ATP-Bio] (mM)
Fig. 3. Analysis of ATP binding to the Cav1.2 channel. A: ATP (azido-ATP-biotin: 0.38 mM) was used for ATP binding assay. In the presence of ATP unlabeled
with biotin (azido-ATP: 3.8 mM), the bound intensity was significantly decreased showing the specific binding. B: ATP band intensity was increased with
increasing concentrations of ATP (azido-ATP-biotin: 0, 0.1, 0.38, 0.75, and 1.5 mM). C: curve of ATP (azido-ATP-biotin: 0, 0.1, 0.38, 0.75, 1.5, and 3 mM)
binding to the Cav1.2 channel in the presence of CaM and Ca2⫹. The CaM:Cav1.2 molar ratio was 2:1, and Ca2⫹ concentration was 250 nM. Bound ATP was
plotted against Cav1.2 protein on a mol/mol basis. Data are shown in molar ratio (ATP/Cav1.2 protein) expressed as means ⫾ SE (n ⫽ 4 –10). The Bmax and
Kd were as follows: Bmax1 ⫽ 1.09, Kd1 ⫽ 0.07 mM; Bmax2 ⫽ 1.1, and Kd2 ⫽ 0.54 mM, where Bmax1, Kd1, Bmax2, and Kd2 denote Bmax and Kd for the high- and
low-affinity sites, respectively.
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the Cav1.2 channel (Fig. 4A). By contrast, when CaM:Cav1.2
ratio was larger than 1, increasing amount of CaM inhibited
ATP binding to Ca2⫹ channel (Fig. 4A). This observation
indicates that ATP binding to Cav1.2 was dependent on CaM
and the activity of the Cav1.2 channel was retained only in the
presence of both ATP and CaM.
ATP binding to the Cav1.2 channel is dependent on Ca2⫹.
To examine how Ca2⫹ affects ATP binding to the Cav1.2
channel, we examined the ATP-binding effect to the Cav1.2
channel in the presence of CaM (CaM:Cav1.2 ⫽ 2:1) and
different concentrations of Ca2⫹ (Fig. 4B). The ATP-binding
effect was the highest when the concentration of Ca2⫹ was 80
nM but was lower under other concentrations (Fig. 4B). This
result suggests that ATP binding to Cav1.2 was dependent on
the concentration of Ca2⫹.
ATP binds to NH2- and COOH-terminal tails of the Cav1.2
channel. The specific ATP-binding regions of the Cav1.2
channel were further analyzed. The finding that azido-ATPbiotin binds to the Cav1.2 Ca2⫹ channel in CaM- and
Ca2⫹-dependent manners implies that the ATP-binding regions of the channel might be close to the CaM/Ca2⫹interacting regions. To test this hypothesis, we examined
NT
C860
ATP REGULATES CAV1.2 CHANNEL ACTIVITY
B
[Azido-ATP-Bio]=0.38 mM
[Ca2+]=250 nM
3
***
2.5
***
**
2
*
1.5
1
0.5
0
0
1
2
3
4
2.5
**
2
***
1.5
***
0.5
0
0
200
400
2:1
1:1
1:1
DISCUSSION
Cav1.2 controls depolarization-induced Ca2⫹ entry in cardiomyocytes, and this process is regulated by numerous cytoplasmic factors. Many studies suggest that ATP maintains the
“basal” activity of LTCC (36, 51, 52). One of the unique
characteristics of LTCC is known as run-down. ATP plus CaM
prevent or reverse the run-down of Ca2⫹ channels, but CaM
alone (without ATP) cannot maintain the “basal” activity of the
Ca2⫹ channel, suggesting that ATP is essential for the regulation of LTCC. However, the mechanism by which ATP regulates LTCC is complex. Protein kinase A-mediated phosphorylation of LTCC is suggested to mediate the effect of ATP on
LTCC. However, accumulated evidence shows that ATP has
an activating effect on Ca2⫹ channels independent of protein
0:1
[Ca2+]
(nM)
B
Cav1.2 (Guinea pig)
I
II
III
800 1000 1200
80
250
500
1000
phosphorylation. A study showed that the whole cell Ca2⫹
current in guinea pig myocytes was enhanced by ATP independent of protein phosphorylation (36). Our previous studies
reveal that a phosphorylation-independent action of ATP might
be involved in the regulation of the Ca2⫹ channel (51, 52).
Therefore, a direct interaction between ATP and the Ca2⫹
channel may exist in addition to the indirect mechanism of
channel phosphorylation. In this study, we demonstrated that
ATP directly bound to the Cav1.2 channel, providing a novel
mechanism by which ATP stabilizes Cav1.2 channel activity.
Recently, a number of biotin-labeled ATPs have been used
for ATP binding experiments (9, 45). In this work, we investigated two kinds of biotin-labeled ATP for reversing the
activity of the Cav1.2 channel in cardiac myocytes using the
A
NH2
600
[Ca2+] (nM)
CaM/Cav1.2 (mol/mol)
CaM/Cav1.2
***
1
5
(mol/mol)
IV
1509-1789
NT
CT1
CT1
CT2
CT3
NT
250
150
100
75
COOH
6-140
kDa
50
1778-2003
37
CT2
1942-2169
CT3
25
(CBB staining)
D
C
Azido-ATP-Bio binding
CT1
CT2
CT3
NT
[Azido-ATP-Bio] = 0.38 mM
[Ca2+] = 250 nM
CaM/Cav1.2 = 2
150
Bound ATP-Bio
(% of CT1)
Fig. 5. Analysis of ATP binding to the NH2terminal and COOH-terminal regions of the
␣1C-subunit of the Cav1.2 channel. A: schematic drawing of NH2-terminal region (NT)
and 3 COOH-terminal regions (CT1, CT2,
and CT3) of the ␣1C-subunit of the Cav1.2
channel. B: Coomassie brilliant blue staining
results of purified GST-NT, GST-CT1, GSTCT2, and GST-CT3. NT, CT1, CT2, and CT3
regions were constructed into pGEX6p-3 vector and expressed in vitro. C: representative
results of ATP binding assay. Purified GSTfusion proteins (2 ␮g) were incubated with
ATP (azido-ATP-biotin: 0.38 mM) in 250 nM
Ca2⫹ buffer. The molar ratio of CaM:Cav1.2
was 2:1. D: quantitative results of ATP binding assay. The relative amounts of bound
ATP-biotin were calculated based on the
amount of bound ATP-biotin of CT1. Data
are shown as means ⫾ SE (n ⫽ 6). ***P ⬍
0.001, all compared with CT1.
[Azido-ATP-Bio]=0.38 mM
CaM/Cav1.2 ratio=2
3
Bound ATP-Bio (A.U.)
Bound ATP-Bio (A.U.)
A
100
***
50
0
CT1
Azido-ATP-Bio (0.38 mM)
AJP-Cell Physiol • doi:10.1152/ajpcell.00368.2013 • www.ajpcell.org
***
***
CT2
CT3
NT
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.5 on June 17, 2017
Fig. 4. Analysis of ATP binding to the Cav1.2
channel in the presence of CaM or Ca2⫹. A: ATP
(azido-ATP-biotin: 0.38 mM) binding to the
Cav1.2 channel in the presence of CaM with
CaM:Cav1.2 molar ratios of 0:1; 0.5:1; 1:1;
2:1; 4:1 and a Ca2⫹ concentration of 250 nM.
Bound ATP was plotted against Cav1.2 protein on a mol/mol basis. Data are shown in
molar ratio (ATP/Cav1.2 protein) expressed
as means ⫾ SE (n ⫽ 4 –7). ***P ⬍ 0.001;
**P ⬍ 0.01; *P ⬍ 0.05, all compared with
CaM:Cav1.2 molar ratios of 0:1 group. B: ATP
(azido-ATP-biotin: 0.38 mM) binding to the
Cav1.2 channel in the presence of Ca2⫹ (0, 80,
250, and 500 nM and 1 ␮M) and CaM (CaM
to Cav1.2 molar ratio was 2:1). Data are
shown as means ⫾ SE (n ⫽ 4 – 6). ***P ⬍
0.001; **P ⬍ 0.01; *P ⬍ 0.05, all compared
with the Ca2⫹ (80 nM) group.
ATP REGULATES CAV1.2 CHANNEL ACTIVITY
0.5:1, 1:1], whereas the ATP-binding effect to the Cav1.2
channel was decreased with higher amounts of CaM [CaM:
Cav1.2 (mol/mol) ⫽ 1:1, 2:1, or 4:1]. These results indicate
that CaM plays a dual role on ATP binding to the Cav1.2
channel and that ATP and CaM might have both synergistic
and antagonistic effect when bound to the Cav1.2 channel.
Other study also reports that ATP competes with CaM binding
to TRPV1 (26, 28). The effect of CaM [CaM:Cav1.2
(mol/mol) ⫽ 1:1, 2:1, or 4:1] on ATP binding to the Cav1.2
channel was similar to that on ATP binding to TRPV1. Furthermore, it is reported that the direct binding of CaM to
channels and the formation of distinct structures by the resulting CaM-channel complexes are essential for both Ca2⫹dependent facilitation and inactivation. Therefore, we speculate that ATP, together with CaM, regulates Ca2⫹-dependent
facilitation and inactivation of the Cav1.2 channel.
Intracellular Ca2⫹ regulates the Cav1.2 channel by the
mechanisms known as Ca2⫹-dependent facilitation and inactivation (10, 24, 38, 40, 55). In our work, we determined the
ATP-binding effect under different concentrations of Ca2⫹ and
demonstrated that the ATP-binding effect to the Cav1.2 channel was the strongest in the presence of 80 nM Ca2⫹, which is
a physiological concentration. According to our previous
study, one Cav1.2 molecule could bind to two CaM molecules
(3). Therefore, we performed the binding experiments in the
presence of CaM:Cav1.2 (mol/mol) 2:1. Our other results (data
not shown) indicate that CaM could bind to the Cav1.2 channel
in the presence of 80 nM Ca2⫹, although the binding effect was
relatively weak. However, ATP could strongly bind to the
Cav1.2 channel in the presence of 80 nM Ca2⫹. Thus we
speculate that ATP and CaM might compete for binding to the
Cav1.2 channel in the presence of 80 nM Ca2⫹. When [Ca2⫹]
was zero, the CaM was Ca2⫹-free apoCaM, which was reported to bind to the Cav1.2 channel (27). In our study, ATP
also bound to the Cav1.2 channel without Ca2⫹, but the
ATP-binding effect was weak compared with that in the
presence of 80 nM Ca2⫹. Moreover, when [Ca2⫹] is higher
(250 nM, 500 nM, and 1 ␮M), the ATP-binding effect to the
Cav1.2 channel is not strong, because CaM in this condition is
Ca2⫹ CaM that can bind to the Cav1.2 channel (10, 48). We
speculate that Ca2⫹ CaM inhibits ATP binding to the Cav1.2
channel with increasing [Ca2⫹]. This suggests that Ca2⫹ is
another pivotal factor for regulating the ATP-binding effect.
In summary, we demonstrate for the first time that ATP can
bind to the Cav1.2 channel in a dose-dependent manner, and
we propose that at least two molecules of ATP can bind to one
molecule of the Cav1.2 channel. CaM had dual effects on ATP
binding to the Cav1.2 channel. Low levels of CaM increased
ATP binding to the Cav1.2 channel, but higher levels of CaM
decreased ATP binding to the Cav1.2 channel. Furthermore,
Ca2⫹ was another regulator for ATP binding to the Cav1.2
channel. Our findings were consistent with findings of previous
studies (33, 36, 51), indicating that ATP may be an important
regulator of the Cav1.2 channel activity through direct binding
to the channel. This direct binding provides a novel mechanism
by which ATP stabilizes Cav1.2 channel activity.
ACKNOWLEDGMENTS
We thank E. Iwasaki for secretarial assistance.
AJP-Cell Physiol • doi:10.1152/ajpcell.00368.2013 • www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.5 on June 17, 2017
patch-clamp method. We found that EDA-ATP-biotin had the
same biologic activity as MgATP, indicating that EDA-ATPbiotin can be used for examining the mechanism of the cardiac
Cav1.2 channel. So we used the azido-ATP-biotin, an EDAATP-biotin analog, for the binding experiments. Our results
showed that ATP could bind to the Cav1.2 channel directly.
This observation was consistent with other studies (18, 47).
ATP-binding cassette (ABC) family is a group of proteins to
which ATP can bind (7, 25, 29, 42, 49). ATP can exert the
effects of purinergic receptor (P2XR) through binding to the
ABC family proteins (2, 34, 44). Moreover, it is well known
that ATP can bind to ryanodine receptor 1 (RyR1) (20) and
transient receptor potential cation channel subfamily V member 1 (TRPV1) (28) and TRPV6 (1). Our results strongly
indicate that the Cav1.2 channel may be another ATP binding
protein.
Our previous study showed that millimolar concentrations of
ATP were required to prevent run-down of the Cav1.2 channel
(51). In this study, we used different concentrations (0, 0.1,
0.38, 0.75, 1.5, and 3 mM) of ATP for the binding to the
Cav1.2 channel. The results showed that the binding effect of
ATP to the Cav1.2 channel was concentration-dependent (Fig.
2). The binding result suggested that at least two ATP molecules were likely to bind to one molecule of the Cav1.2
channel. The Kd value of the high-affinity (Kd1) site for ATP
was much higher than that of the low-affinity (Kd2) site. The Kd2
value was similar to our previous patch-clamp experiments
(data not shown), suggesting that this binding site has lower
affinity. We speculate that one ATP molecule binds to the
high-affinity site and another ATP molecule binds to the
low-affinity site. It is reported that three regions in RyR1 are
involved in ATP binding (20) and that a single P2X receptor
has three intersubunit-ATP binding sites (17). Therefore, further experiments are needed to identify the accurate sites of the
Cav1.2 channel that participate in ATP binding.
In addition, a number of studies indicated that CaM can bind
to both NH2- and COOH-terminal tails of the Cav1.2 channel
(3, 32, 40, 41, 56). Our previous study revealed that the
fragments of the COOH-terminal tail of the Cav1.2 channel
(preIQ-IQ peptide in CT1 region) bind with two CaM molecules at the same time (3). It is proposed that the first CaM
binding to one site plays a role in Ca2⫹-dependent facilitation
and the second CaM binding to the other site triggers Ca2⫹dependent inactivation (13). In this study, we found that ATP
bound to both NH2- and COOH-terminal tails of the channel.
Furthermore, the ATP binding was both CaM and Ca2⫹ dependent. Thus we speculate that ATP might play a role in
CaM- and Ca2⫹-dependent regulations of channel activity (see
below).
In the patch-clamp experiments of our previous study, we
found that ATP or CaM alone could not prevent or reverse the
run-down of the Cav1.2 channel. It is well established that
CaM can bind to the Cav1.2 channel (3, 12, 13, 32, 39, 41, 54,
56), and our results suggest that ATP can also bind to the
Cav1.2 channel. It is reported that one molecule of the Cav1.2
channel can bind to two molecules of CaM. In the present
study, we investigated the ATP-binding effect to the Cav1.2
channel in the presence of different amounts of CaM [CaM:
Cav1.2 (mol/mol) ⫽ 0:1, 0.5:1, 1:1, 2:1, 4:1]. We found that
the ATP-binding effect to the Cav1.2 channel was increased
with lower amounts of CaM [CaM:Cav1.2 (mol/mol) ⫽ 0:1,
C861
C862
ATP REGULATES CAV1.2 CHANNEL ACTIVITY
GRANTS
This work was supported by grants from the Natural Science Foundation of
China (31071004), Grants-in-Aid from MEXT Japan (21390059, 23790251,
and 25460294), and a grant from the Kodama Memorial Foundation for
Medical Research.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: R.F. and E.M. performed experiments; R.F. and J.X.
analyzed data; R.F., J.X., L. Yang, and L. Yu interpreted results of experiments; R.F. and A.K. prepared figures; R.F. drafted manuscript; L.H. and M.K.
conception and design of research; L.H. and M.K. approved final version of
manuscript.
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