From the Division of Molecular Neurobiology,
Department of Basic Medical Science, Institute of Medical Science,
University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan, the § Department of Pediatrics, Keio University
School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan, the ¶ Department of Neurosurgery, Japanese Red Cross
Medical Center, 4-1-22 Hiro-o, Shibuya-ku, Tokyo 150-8935, Japan, the
Department of Neurosurgery, University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-8655, Japan, the Laboratories for
** Molecular Neurogenesis and
§§ Developmental Neurobiology, Brain Science
Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan, and the
Calcium Oscillation Project,
International Cooperative Research Project, Japan Science and
Technology Corporation, 3-4-4, Shirokanedai, Minato-ku,
Tokyo 108-0071, Japan
Received for publication, January 21, 2003, and in revised form, March 4, 2003
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ABSTRACT |
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To understand the molecular mechanism of
ligand-induced gating of the inositol 1,4,5-trisphosphate
(IP3) receptor (IP3R)/Ca2+
release channel, we analyzed the channel properties of deletion mutants
retaining both the IP3-binding and channel-forming domains of IP3R1. Using intrinsically IP3R-deficient
cells as the host cells for receptor expression, we determined that six
of the mutants, those lacking residues 1-223, 651-1130, 1267-2110,
1845-2042, 1845-2216, and 2610-2748, did not exhibit any measurable
Ca2+ release activity, whereas the mutants lacking residues
1131-1379 and 2736-2749 retained the activity. Limited trypsin
digestion showed that not only the IP3-gated
Ca2+-permeable mutants lacking residues 1131-1379 and
2736-2749, but also two nonfunctional mutants lacking residues 1-223
and 651-1130, retained the normal folding structure of at least the
C-terminal channel-forming domain. These results indicate that two
regions of IP3R1, viz. residues 1-223 and
651-1130, are critical for IP3-induced gating. We also
identified a highly conserved cysteine residue at position 2613, which
is located within the C-terminal tail, as being essential for channel
opening. Based on these results, we propose a novel five-domain
structure model in which both N-terminal and internal coupling domains
transduce ligand-binding signals to the C-terminal tail, which acts as
a gatekeeper that triggers opening of the activation gate of
IP3R1 following IP3 binding.
Inositol 1,4,5-trisphosphate
(IP3)1 is a
second messenger that is produced by hydrolysis of phosphatidylinositol
4,5-bisphosphate in response to activation by extracellular stimuli of
the G protein- or tyrosine kinase-coupled receptors on the plasma
membrane in various cell types (1). IP3 mediates the
release of Ca2+ from intracellular storage sites such as
the endoplasmic reticulum by binding to the IP3 receptor
(IP3R)/Ca2+ release channel.
IP3-induced Ca2+ release (IICR) regulates
numerous physiological processes, including fertilization, cell
proliferation, development, muscle contraction, secretion, learning,
and memory. In this signal transduction pathway, the IP3R
works as a switch that converts the information carried by
extracellular stimuli into intracellular Ca2+ signals.
IP3-gated intracellular Ca2+ release channels
are composed of four IP3R subunits (2). There are at least
three types of IP3Rs (IP3R1, IP3R2,
and IP3R3) (3), and they exist as both homo- and
heterotetramers (4). The structure of IP3Rs has
traditionally been divided into three functional domains (3, 5): the
N-terminal ligand-binding domain; the modulatory/coupling domain; and
the C-terminal transmembrane/channel-forming domain, which contains six
putative membrane-spanning regions. The transmembrane region is
required for the intermolecular interaction in the formation of a
tetrameric complex (6-9), and it is likely that the C-terminal cytoplasmic region just following the putative membrane-spanning regions has a supportive role in the association among the subunits (6,
9). An ion conduction pore has been proposed to be located in the
hydrophobic segment between the fifth and sixth transmembrane regions
(10, 11). The primary sequence of the transmembrane domain adjacent to
the pore-forming segment is highly homologous to that of the ryanodine
receptor (RyR), another type of intracellular Ca2+ release
channel, suggesting that these two channels might share a common
structure for the conduction of Ca2+ ions.
Each IP3R subunit has a single high affinity
IP3-binding site (2). The IP3-binding core, a
minimum essential region for specific IP3 binding (12),
resides among residues 226-578 of mouse IP3R1 (2749 amino
acids) (13), and it contains 11 essential basic amino acids for
IP3 binding (14). The N-terminal 225 residues, which are
close to the IP3-binding core, have been thought to function as a suppressor for IP3 binding because their
deletion from the N-terminal 734-amino acid region results in
significant enhancement of IP3-binding activity (12, 15).
IICR is a positively cooperative process (16-18), i.e. the
binding of at least two IP3 molecules to a single
tetrameric IP3R channel is required for channel opening.
IP3 binding elicits a large conformational change in the
N-terminal cytoplasmic portion of the IP3R (19).
Furthermore, the C-terminal cytoplasmic region following the
transmembrane domain is thought to be involved in the
IP3-induced gating of the receptor because monoclonal
antibody (mAb) 18A10, whose epitope is located in the C-terminal
portion of mouse IP3R1 (13, 20, 21), has an inhibitory
effect on IICR, without causing any decrease in the affinity of the
receptor for IP3 (21). Controlled trypsinization induces
fragmentation of mouse IP3R1 into five major fragments, and
all four N-terminal cytoplasmic fragments, which contain the IP3-binding core, are associated directly or indirectly
with the remaining C-terminal fragment, which contains the channel
domain (22). The trypsinized IP3R retains significant IICR
activity, indicating that intramolecular interaction within a subunit
and/or intermolecular interaction between neighboring subunits could effect functional coupling between IP3 binding and
channel opening (22). However, the sites of the interfaces between the
cytoplasmic fragments and the channel domain and the molecular
mechanism of their coupling remain to be elucidated.
IICR has been shown to occur in a quantal manner in permeabilized cells
and isolated endoplasmic reticulum membranes (23, 24). The addition of
submaximal concentrations of IP3 in the presence of
Ca2+ pump inhibitors leads to the partial release of
sequestered Ca2+, and the amount of released
Ca2+ varies with the concentration of IP3 (24).
Although the Ca2+ release terminates abruptly, because it
can be reinitiated by an additional increment in IP3
concentration (24), the rapid termination of Ca2+ release
is not due to ordinary inactivation or desensitization of the receptor.
Purified IP3Rs reconstituted into lipid vesicles reveal a
quantal Ca2+ flux (17, 25), indicating that the quantal
release of Ca2+ is an intrinsic property of the
IP3R. Similar behavior was observed for the RyR, which
mediates Ca2+-induced Ca2+ release from
intracellular Ca2+ stores (26), but has not been observed
for other ligand-gated ion channels on the plasma membrane, suggesting
that the quantal release is a fundamental and unique property of the
intracellular Ca2+ release channels.
To understand the molecular basis of the ligand-induced gating of the
IP3R, we analyzed a series of internal deletion mutants and
site-directed mutants of mouse IP3R1 expressed in
intrinsically IP3R-deficient R23-11 cells (27). We found
that at least two regions and a cysteine residue are essential for
IP3-dependent gating of IP3R1.
These findings provide us with new insight into the gating mechanism of
the IP3R.
Plasmid Constructions--
For transfection of mouse wild-type
IP3R1 cDNA, pBact-STneoB-C1 (28) was used. Seven
deletion mutant cDNAs of mouse IP3R1, D651-1130,
D1131-1379, D1267-2110, D1692-1731, D1845-2042, D1845-2216, and
D2610-2748 (6), were subcloned into pAneo (27) at the SalI
sites. To construct D1-223, an XhoI site was introduced at nucleotide 998 of mouse IP3R1 by PCR using green
fluorescent protein-fused IP3R-D223 2 as
the template DNA. An XhoI-KpnI fragment isolated
from green fluorescent protein-fused IP3R-D223 was ligated
to a SalI-KpnI fragment of pBact-STneoB-C1. The
resultant plasmid, pBact-STneoB-D1-223, uses nucleotides 998-1000,
which correspond to an intrinsic methionine residue at position 224, as
a start codon (ATG) for transcription. To construct D2736-2749, a
KpnI-XhoI fragment isolated from enhanced green
fluorescent protein-fused IP3R/ Generation of Wild-type and Deletion Mutant
IP3R1-expressing Cell Lines--
R23-11 cells (27) were
cultured in RPMI 1640 medium supplemented with 10% fetal calf serum,
1% chicken serum, 50 µM 2-mercaptoethanol, 4 mM glutamine, 100 units/ml penicillin, and 100 µg/ml
streptomycin at 39.5 °C in 5% CO2. Expression plasmids
were linearized and transfected into R23-11 cells by electroporation as
previously described (27) or by lipofection (Effectene, QIAGEN
Inc.).3 Several stable clones
were selected in medium containing 2 mg/ml G418 (Sigma) ~7-10 days
after transfection. Expression of the IP3R and its mutants
was confirmed by immunoblotting with mAbs 4C11 and/or 18A10 using cell
lysates boiled in SDS-PAGE sample buffer (5 mM EDTA, 50 mM Tris-HCl, pH 6.8, 100 mM dithiothreitol, 2%
SDS, and 10% glycerol). Immunoblot analysis was performed as described
previously (22).
Preparation of Membrane Fractions from Stable Cells Expressing
Mouse IP3R1 and Its Mutants--
Membrane fractions were
prepared in accordance with the protocol for mouse cerebella described
by Michikawa et al. (18), with minor modifications. Cells
were collected by centrifugation, washed twice with cold
phosphate-buffered saline, and homogenized in ice-cold homogenization
buffer (5 mM NaN3, 0.1 mM EGTA, 1 mM 2-mercaptoethanol, and 20 mM HEPES-NaOH, pH
7.4) containing protease inhibitors (0.1 mM
phenylmethylsulfonyl fluoride, 10 µM leupeptin, 10 µM pepstatin A, and 10 µM E-64) by 40 strokes in a chilled glass-Teflon Potter homogenizer at 1000 rpm. The
homogenate was centrifuged at 1100 × g for 10 min at
2 °C. The supernatant was centrifuged at 100,000 × g in a Beckman TLA100.3 rotor for 30 min at 2 °C. The
pellet was resuspended in an appropriate volume of wash buffer (600 mM KCl, 5 mM NaN3, 20 mM Na4P2O5, 1 mM 2-mercaptoethanol, and 10 mM HEPES-HCl, pH
7.2) containing protease inhibitors. The suspension was centrifuged at
1100 × g for 10 min, and the supernatant was
centrifuged at 63,000 × g for 30 min at 2 °C. The
pellet was finally suspended in an appropriate volume of
Ca2+ release buffer (110 mM KCl, 10 mM NaCl, 5 mM KH2PO4, 1 mM 2-mercaptoethanol, and 50 mM HEPES-KOH, pH
7.2) containing protease inhibitors to a final concentration of ~15
mg/ml protein. Ca2+ release buffer was passed over Chelex
100 (Bio-Rad) to eliminate any extra free Ca2+ before use.
The membrane fractions were either used immediately or frozen in liquid
nitrogen and stored at [3H]IP3 Binding Assay Using Membrane
Fractions--
The IP3 binding assay was performed as
described previously (6). The membrane fractions (50-200 µg/tube)
were incubated with 9.6 nM
[3H]IP3 (PerkinElmer Life Sciences) in 100 µl of binding buffer (50 mM Tris-HCl, pH 8.0, 1 mM EGTA, and 1 mM 2-mercaptoethanol) for 10 min
at 4 °C. After centrifugation, the pellets were dissolved in
Solvable (PerkinElmer Life Sciences), and the radioactivities were
measured with a scintillation counter (Beckman LS6500). Nonspecific binding was measured in the presence of 10 µM unlabeled
IP3 (Dojindo Laboratories).
IICR Assay for Membrane Fractions--
The membrane fractions
were suspended in Ca2+ release buffer supplemented with 1 µg/ml oligomycin (Sigma), 2 mM MgCl2, 25 µg/ml creatine kinase (Roche Applied Science), 10 mM
creatine phosphate (Sigma), and 2 µM Fura-2 (Molecular
Probes, Inc.) and used at a concentration of 200-300 µg/ml protein.
Fluorescence was recorded at 510 nm with alternate excitation of 340 and 380 nm (F340 and F380, respectively). Using a CAF-110
spectrofluorometer (Japan Spectroscopic Co.), signals were recorded
every 0.01 s with MacLab Version 3.6 (ADInstruments) at
30 °C. When the Ca2+ uptake induced by the addition of 1 mM ATP reached a steady level, 2 µM
thapsigargin was added to eliminate active Ca2+ uptake
through intrinsic Ca2+ pumps. The rate of leakage from the
membrane fractions following the addition of thapsigargin was almost
linear. When the ratio of fluorescence intensity
(F340/F380) reached 1.2, corresponding to ~170 nM free Ca2+, various
concentrations of IP3 were added. At the end of each experiment, 2 mM CaCl2 and 10 mM
EGTA were added successively for normalization and calibration
(30).
Limited Trypsin Digestion of Mutant Receptors--
Limited
trypsin digestion was performed as described previously (22).
Microsomal fractions (0.25-5 mg/ml) of wild-type and mutant
IP3R1-expressing cells were incubated with 0.01-10 µg/ml trypsin in trypsinization buffer (120 mM KCl, 1 mM EDTA, 1 mM 2-mercaptoethanol, and 20 mM Tris-HCl, pH 8.0) at 35 °C for 10 min. The reaction
was terminated by the addition of 50 µg/ml soybean trypsin inhibitor
(Sigma) and 0.1 mM phenylmethylsulfonyl fluoride. After the
addition of an equal volume of SDS-PAGE sample buffer, reaction
mixtures were incubated at 55 °C for 30 min. The digested proteins
were separated by 8% SDS-PAGE and then analyzed by Western blotting
with anti-IP3R1 antibodies N1, 4C11, 10A6, 1ML1, and 18A10 and the anti-(1718-31) antibody (see Fig. 1A)
(22).
Expression of Deletion Mutants of IP3R1 in
Intrinsically IP3R-deficient R23-11 Cells--
As
previously reported (6), we constructed 17 internal deletion mutants of
mouse IP3R1. Among these mutants, we selected seven (Fig.
1B) containing both the
IP3-binding region (residues 226-578) and the putative
transmembrane domain (residues 2276-2589) to investigate the critical
regions for the coupling between ligand binding and channel opening. In
addition, we constructed two mutants lacking residues 1-223 and
2736-2749, respectively (Fig. 1B). To express these mutant
receptors, we introduced the mutant cDNAs into R23-11 cells and
established stable cell lines by selection with 2 mg/ml G418. Fig.
2A illustrates the results
from Western blot analysis of the membrane fractions prepared from
these stable cell lines using anti-IP3R1 polyclonal
antibody 1ML1, whose epitope lies within residues 2504-2523 of
IP3R1 (Fig. 1A) (10). All of the mutant
receptors except D2610-2748 were detected with an appropriate
molecular mass (Fig. 2A). Because D2610-2748 was not well
recognized by antibody 1ML1, we confirmed the expression of D2610-2748
by Western blot analysis using anti-IP3R1 mAb 4C11 (20). As
shown in Fig. 2B (open arrowhead), an additional
signal with a low molecular mass was detected, indicating that
degradation (or truncation) of D2610-2748 occurs in R23-11 cells.
[3H]IP3-binding Activities of Deletion
Mutant IP3Rs--
The IP3-binding activities
of the internal deletion mutant receptors expressed in R23-11 cells
were measured by equilibrium [3H]IP3 binding
analysis as described previously (6). There was no significant
IP3-binding activity in the membrane fraction obtained from
the R23-11 cells (data not shown). Therefore, we measured the
ligand-binding activity of the exogenously expressed IP3R using membrane fractions obtained from the stable cell lines. The
IP3-binding properties of wild-type and deletion mutant
IP3Rs are summarized in Table
I. Wild-type IP3R1 expressed
in R23-11 cells showed a single high affinity IP3-binding
site with a dissociation constant of 20 ± 5 nM
(n = 3). This value is close to those of native
IP3R1 expressed in the mouse cerebellum (31) and
cDNA-derived IP3R1 expressed in L cells (28), NG108-15
cells (6), and Sf9 cells (32). Mutant receptors D1131-1379,
D1692-1731, D2610-2748, and D2736-2749 exhibited binding affinity
similar to that of wild-type IP3R1 (Table I). The
IP3-binding affinity of mutant receptors D1267-2110,
D1845-2042, and D1845-2216 was 2-3-fold lower (Table I), and mutant
D651-1130 had 7.5-fold lower affinity for IP3 (Table I).
Mutant D1-223 exhibited, however, significantly higher affinity for
IP3 (Table I), consistent with a previous report showing
that residues 1-223 act as a suppressor for IP3 binding (12). It has been reported that IP3 binding to the
IP3R is not cooperative (31, 33), and the same property
holds true for the wild-type receptor and all of the mutant receptors
except D1-223 expressed in R23-11 cells (Table I). Both the Western blot (Fig. 2) and IP3 binding (Table I) analyses showed
that the amount of IP3R protein expressed in each cell line
was different. The amounts of the mutant IP3Rs expressed
were in the range of 1.5-9.2 pmol/mg of protein (Table I); and
therefore, we used two stable cell lines (KMN13 and
KMN107)2 expressing different amounts of wild-type
IP3R1 as controls in the following experiments. The
Bmax values for KMN13 and KMN107 were 13 ± 9 and 0.77 ± 0.2 pmol/mg of protein, respectively (Table I).
IICR Activity of Wild-type and Deletion Mutant
IP3Rs--
To investigate the Ca2+ release
activity of the mutant IP3Rs, IICR from the membrane
fractions prepared from each stable cell line was measured in the
presence of the Ca2+ pump inhibitor thapsigargin. No
Ca2+ release was observed from membrane fractions prepared
from R23-11 cells even after the addition of 10 µM
IP3 (Fig. 3A),
indicating that using R23-11 cells as host cells for transfection
allows evaluation of definite Ca2+ release activity by
exogenously expressed IP3Rs. Fig. 3B shows the
time course of the Ca2+ release mediated by recombinant
wild-type IP3R1 after the addition of various
concentrations of IP3. Both the rate and amplitude of
Ca2+ release depended on the concentration of
IP3 added, indicating that IP3R1 expressed in
R23-11 cells exhibits the quantal Ca2+ release that is
known to be an intrinsic property of native IP3R1 (17, 25).
As previously reported (11), D1692-1731, which corresponds to the
SII Limited Trypsin Digestion of Mutant IP3Rs--
Mouse
cerebellar IP3R1 is trypsinized into five major fragments
(I-V) (Fig. 1A) (22). Limited proteolysis provides direct evidence of protein folding (36). To probe the tertiary structure of
the mutant IP3Rs, we analyzed the trypsinized fragments of recombinant IP3R1. Because R23-11 cells contain an
intrinsic mAb 4C11-reactive protein whose molecular mass is similar to
that of tryptic fragment II of IP3R1 (data not shown), we
analyzed four tryptic fragments (I and III-V) of the recombinant
receptors. As shown in Fig.
4A, wild-type
IP3R1 expressed in R23-11 cells was digested into the same
four fragments, indicating that recombinant IP3R1 retains
native structure. We found that trypsin digestion of the
Ca2+-releasing mutant D2736-2749 generated the same four
trypsinized fragments (Fig. 4B), indicating that D2736-2749
folds in the same manner as wild-type IP3R1. Trypsinization
of the other functional mutant, D1131-1379, also generated fragments
IV and V (Fig. 4C). However, D1131-1379, which exhibited
markedly decreased Ca2+ release activity (Fig.
3A), was digested with much lower concentrations of trypsin
(Fig. 4C). This difference in trypsin sensitivity suggests that the deletion of amino acids 1131-1379 influences the structure of
the C-terminal channel domain. Tryptic fragments of the three functionless mutants, D1-223, D651-1130, and D2610-2748, are shown in Fig. 4 (D-F, respectively). Fragments III-V were
generated by trypsin digestion of D1-223 (Fig. 4D).
Trypsinization of D651-1130, which lacks a cleavage site between
fragments II and III (22), generated fragments IV and V (Fig.
4E). Both D1-223 and D651-1130 exhibited trypsin
sensitivity similar to that of wild-type IP3R1, suggesting
that at least the C-terminal channel domains of these mutants fold
correctly. By contrast, only fragments I and III were generated by
trypsin digestion of D2610-2748 (Fig. 4F), and fragments IV
(40 kDa) and V (91 kDa) were not detected (open arrows). These results indicate that deletion of residues 2610-2748 induces a
significant distortion of the folding structure of the C-terminal channel domain of IP3R1.
Identification of the Cysteine Residue Essential for
IP3-induced Gating of IP3R1--
The
C-terminal cytoplasmic region following the transmembrane domain has
been found to be highly conserved in both the IP3R and RyR
families (13), indicating that this region is involved in the formation
of a critical structure that is required for some common functions of
the intracellular Ca2+ release channels. One of the
remarkable features of this region is that it contains two cysteine
residues that are conserved in all of the intracellular
Ca2+ release channels. Both the IP3R and RyR
channels are known to be modified by sulfhydryl reagents (37-39);
therefore, we analyzed whether or not the two conserved cysteine
residues, Cys2610 and Cys2613 (Fig. 5), are
essential for the gating function in mouse
IP3R1. We generated three
mutant receptors (C2610S, C2613S, and C2610S/C2613S) in which
Cys2610 and/or Cys2613 was replaced with serine
(Fig. 1B). We also constructed a mutant receptor (C1976S)
in which Cys1976 was replaced with serine (Fig.
1B). All of the mutated cDNAs were introduced into
R23-11 cells, and stable cell lines expressing mutant receptors were
established. Equilibrium [3H]IP3 binding
assay showed that all of the cysteine mutants bound IP3
with affinities similar to that of wild-type IP3R1 (Table II). The expression levels of the mutant
receptors were in the range of 1-10 pmol/mg of protein (Table II), and
the amounts of all of the mutant receptors expressed in each
established cell line were higher than the expression level of
wild-type IP3R1 in KMN107 cells.
We then examined the Ca2+ channel activities of the mutant
receptors by measuring IICR from the membrane fractions prepared from
the stable cell lines. We found that substitution of serine for
Cys2610 and/or Cys2613 completely abolished the
Ca2+ release activity, whereas no such effect was apparent
upon substitution of serine for Cys1976 (Fig.
6). Limited trypsin digestion of C1976S,
C2613S, and C2610S/C2613S generated fragments I and III-V (Fig.
7, A, C, and
D, closed arrows), indicating that these mutants
retain a normal structure. By contrast, trypsin digestion of C2610S
generated fragments I, III, and IV (Fig. 7B, closed
arrows), but not fragment V (open arrow), indicating that the single amino acid substitution at Cys2610 induces
a significant structural alteration of the C-terminal channel domain of
IP3R1.
Most cells, including mammalian cultured cells, express two or all
three types of the IP3R (4, 40). Thus, measurement of the
actual channel activities of recombinant IP3Rs in most cells is difficult because of the background activities of the endogenous IP3Rs. In this study, we used R23-11 cells,
which intrinsically lack all of the three IP3Rs (27), as
the host cells to exclude the background effects of the endogenous
IP3Rs. Under the conditions used, neither
IP3-binding activity nor IP3-elicited
Ca2+ release activity was detected in membrane fractions
prepared from R23-11 cells (Fig. 3A). Wild-type
IP3R1 expressed in KMN13 cells revealed affinity for
IP3 (Kd = 20 ± 5 nM)
(Table I) similar to that of native (31) and recombinant (6, 28, 32)
mouse IP3R1 and mediated Ca2+ release from
microsomal vesicles in an IP3-dependent manner
(Fig. 3B). Wild-type IP3R1 expressed in KMN13
cells revealed quantal Ca2+ release (Fig. 3B),
which is thought to be an intrinsic property of native
IP3Rs (17, 25), suggesting that recombinant
IP3R1 in R23-11 cells functions properly. Using this
system, we determined that six of the mutants investigated in this
study, those lacking residues 1-223, 651-1130, 1267-2110,
1845-2042, 1845-2216, and 2610-2748, did not exhibit any measurable
Ca2+ release activity, whereas the mutants lacking residues
1131-1379 and 2736-2749 retained the activity.
[3H]IP3 binding analysis showed that all of
these nonfunctional mutants except D1-223 possessed lower
IP3-binding affinity (Table I). However, 98.5% of the
receptors of even the lowest affinity mutant, D651-1130, whose
Kd is 150 nM (Table I), would have been
occupied when 10 µM IP3 was applied.
Therefore, the decrease in IP3-binding affinity is unlikely
to be the primary cause of the loss of function of these mutants. The
limited trypsin digestion of the crude membrane fractions prepared from
the mutant-expressing cells showed that not only the
IP3-gated Ca2+-permeable mutants, D1131-1379
and D2736-2749, but two nonfunctional mutants, D1-223 and D651-1130,
generated fragments IV and V (Fig. 4). All of these mutants except
D1131-1379 exhibited trypsin sensitivity similar to that of wild-type
IP3R1, indicating that these mutants retain a normal
folding structure in at least the C-terminal channel-forming domain.
Immunocytochemical staining suggested that all of the mutants were
localized on the Ca2+ stores of the cells (data not shown).
Hence, we concluded that at least two regions, viz. regions
1-223 and 651-1130, are required for the
IP3-dependent gating of IP3R1.
The Critical Region 1-223 Is Known as a Suppressor of
IP3 Binding--
Deletion of the residues on the
N-terminal side of the IP3-binding core has a complicated
effect on the IP3-binding activity (12). A short deletion
of the N-terminal 31 residues from the N-terminal 734-amino acid region
results in a significant reduction in binding activity even though the
mutant includes the entire IP3-binding core sequence. Such
effects are also found in serial N-terminal deletions up to 215 residues. However, the binding activity recovers when deleted up to the
first 220, 223, or 225 amino acids. The mutant lacking the first 223 amino acids shows >10-fold higher affinity for IP3 than
does the parental N-terminal 734-amino acid region. Based on these
results, Yoshikawa et al. (41) proposed that the first
~225 N-terminal amino acids function as a suppressor of
IP3 binding. In this study, we found that D1-223 had
10-fold higher affinity for IP3 than did wild-type
IP3R1 (Table I). In addition, we found that the
IP3 binding of D1-223 was positively cooperative (Table
I), indicating that the intersubunit interaction may be elicited (or
modified) in the tetrameric complex composed of D1-223. Limited
trypsin digestion showed that the mutant is likely to retain the normal
folding structure of the C-terminal channel-forming domain (Fig.
4D). Surprisingly, D1-223 did not exhibit any measurable
Ca2+ release activity (Fig. 3A). These data
therefore clearly indicate that residues 1-223 are required for the
functional coupling between IP3 binding and channel opening.
Homer (42)- and calmodulin (43)-binding sites are localized in region
1-223 of IP3R1. Homer forms an adaptor complex
that couples between group 1 metabotropic glutamate
receptors and IP3Rs, and it has recently been reported to
be capable of associating with RyR1 and up-regulating its
Ca2+ release activity (44). The Homer-binding motif
(PPXXFR) is present in residues 49-54 (PPKKFR) of mouse
IP3R1 (42). Calmodulin interacts with residues 49-81 and
106-128 in a Ca2+-dependent and
Ca2+-independent manner (43). These binding proteins within
the critical region 1-223 may modulate the gating of
IP3R1.
The Critical Region 651-1130 Is Close to the
IP3-binding Core--
In region 651-1130 of
IP3R1, the alternative splicing site (between amino acids
917 and 918) referred to as SIII is present (45), but the
functional significance of the SIII segment (nine residues) has not yet
been elucidated. There are three possible Ca2+-binding
sites within regions 660-745, 741-849, and 994-1059 (46), suggesting
that region 651-1130 is involved in the
Ca2+-dependent regulation of IP3R
function. Recently, Bosanac et al. (14) unveiled the
three-dimensional structure of the IP3-binding domain
(residues 224-604) that covers the IP3-binding core
(residues 226-578) of IP3R1 in the presence of
IP3. The IP3-binding domain forms an asymmetric
boomerang-like structure that consists of an N-terminal
Recently, Hamada et al. (49) showed that the purified
IP3R from mouse cerebellum contains two distinctive
structures: a windmill-like structure and a square-shaped structure.
Ca2+ reversibly promotes transition from the square- to the
windmill-shaped structure, with relocation of the four peripheral
IP3-binding domains. This observation predicts the presence
of a hinge region that changes its conformation drastically following
Ca2+ binding to the receptor. Hamada et al. (49)
examined the Ca2+-dependent structural change
of the purified IP3R by limited protease digestion
analysis. The results show that a 38-kDa fragment detected using
anti-IP3R1 mAb 4C11 is specifically generated by cleavage in a solution containing CaCl2, but not in an
EDTA-containing solution. The epitope of antibody 4C11 was mapped
within residues 679-727 in IP3R1 (13); and therefore,
region 651-1130 found in this study is a strong candidate for the
hinge region. The presence of Ca2+-binding sites within
this region (46) also supports this hypothesis.
Nonfunctional Mutants D1267-2110, D1845-2042, and
D1845-2216--
Because the nonfunctional mutants D1267-2110,
D1845-2042, and D1845-2216 do not have cleavage sites between tryptic
fragments IV and V (Arg1931, Arg1923, or
Lys1924) (22), we could not evaluate the folding structure
of the C-terminal channel-forming domain of these mutants. Therefore,
we did not determine whether the deleted regions include critical
regions for activation gating or whether the deletions simply distort the structure of the receptor. Notably, the deleted regions in these
mutants include (or are close to) the Ca2+ sensor region
found in IP3R1 (50). Cytoplasmic Ca2+ is a
co-agonist for the IP3R (51); and thus, the
IP3- and Ca2+-binding signals must be combined
on the IP3R. More detailed analysis of the regions deleted
in these mutants may help us to better understand the molecular
mechanism of gating, in particular, the Ca2+-dependent processes during channel opening.
Region 2610-2748 May Be Required for the Correct Folding of
IP3R1--
The C-terminal region following the sixth
transmembrane region may play some part in channel gating of the
IP3R because mAbs that recognize this region have been
reported to either inhibit (21) or enhance (52) IICR. In addition, it
has been suggested that the C terminus is involved in subunit assembly
of the IP3R channel complex (6, 9). It has been shown that,
although a truncation mutant of IP3R1 that lacks all of the
transmembrane regions and the succeeding C terminus (amino acids
2218-2749) is present as a monomer, a deletion mutant that
lacks only the transmembrane regions (amino acids 2112-2605) forms
dimers (6), suggesting that the C-terminal 144 amino acids (positions
2606-2749) are involved in the intersubunit interaction of
IP3R1. Facilitation of multimer formation of mutant
IP3Rs with two or more transmembrane regions is observed if
the mutants are fused to the C-terminal 145 residues (9); however,
recombinant IP3R1 lacking the C-terminal 145 residues forms
tetramers (9), suggesting that this C-terminal region is not essential
for the formation of the tetrameric channel complex. In this study, we
found that deletion of amino acids 2610-2748 completely abolished the
activity of the IP3R channel. D2610-2748 was not well
recognized by antibody 1ML1 (Fig. 2A). Limited trypsin
digestion of mutant D2610-2748 did not generate tryptic fragments IV
and V (Fig. 4F), suggesting that deletion of residues
2610-2748 affects the folding structure around the cleavage sites
between tryptic fragments IV and V.
The Essential Cysteine Residue in the C-terminal Tail--
We
found that the site-directed mutants C2610S, C2613S, and C2610S/C2613S
did not exhibit any measurable Ca2+ release activity (Fig.
6). As shown in Fig. 7, limited trypsin digestion of C2613S and
C2610S/C2613S generated all four tryptic fragments (I and III-V),
whereas trypsinization of C2610S generated only three tryptic fragments
(I, III, and IV). These results indicate that Cys2613 is
required for the functional coupling between IP3 binding
and channel opening. The results of trypsinization of C2610S suggest that substitution of Cys2610 disrupts the correct folding
in at least the C-terminal channel-forming domain of the
IP3R. This is puzzling, however, because C2610S/C2613S generated all four tryptic fragments. The effect of a single amino acid
substitution at Cys2610 could be explained if the cysteine
at position 2613 in the C2610S mutant elicits an artificial
modification (such as disulfide formation, S-nitrosylation, or palmitoylation) that induces a
serious distortion in the folding structure of the C-terminal channel
domain. These modifications might be prevented in the presence of
Cys2610. This explanation suggests a direct or indirect
structural interaction between Cys2610 and
Cys2613 in wild-type IP3R1.
These cysteine residues are also conserved in the RyR family (Fig. 5).
Involvement of the cysteine residues in intracellular Ca2+
channel gating has been postulated on the basis of evidence that thiol
reagents such as oxidized glutathione and thimerosal enhance the
Ca2+-mobilizing activity of both IP3R and RyR
channels (1). There are nine cysteine residues conserved in both
families (13). As shown in Fig. 6, substitution of the conserved
cysteine residue at position 1976 did not affect the activity of
IP3R1, whereas substitution of Cys2610 or
Cys2613 caused loss of function of IP3R1. It is
possible that thiol reagents directly attack the cysteine residues at
positions 2610 and/or 2613 and enhance channel activity. Further
studies are required to elucidate the exact target sites of thiol
reagents and the mechanism of the enhancement of channel gating induced
by these reagents.
Cysteine residues located in the C-terminal region following the
transmembrane domain are known to be involved in the gating of some ion
channels on the plasma membrane, such as cyclic nucleotide-gated channels (53-55) and voltage-dependent and inwardly
rectifying K+ channels (56, 57). In the presence of
oxidants, a certain C-terminal cysteine residue in both these plasma
membrane channels reacts with a cysteine residue located in the
N-terminal region in the same or different subunit. This reaction
depends on the states of the channels, and the formation of disulfide
bonds results in channel potentiation. On the N-terminal side of the
transmembrane domain, the IP3R possesses 18 cysteine
residues that are conserved in the IP3R family. Thus,
examining the interaction between the N and C termini via the formation
of disulfide bonds in the presence of some oxidants according to the
gating states would be useful in understanding the conformational
changes that occur during IP3R gating.
A Novel Five-domain Structure Model for the
IP3R--
We found that two regions, viz.
regions 1-223 and 651-1130, and Cys2613 are crucial for
the IP3-induced gating of IP3R1. How do they contribute to the activation gating of IP3R1? Recently, the
open pore conformation of K+ channels (MthK) was resolved
at a resolution of 3.3 Å (58, 59). Structural comparison between KcsA
and MthK (closed and open K+ channels) revealed that
pore-lining inner helices form the channel gate and that bending of the
inner helices causes channel opening. In the bent configuration, the
inner helices form a wide (12 Å) entryway. The gating hinge is a
glycine residue located in the middle of the inner helices. The glycine
residue is highly conserved in voltage- or ligand-gated channels with
two or six membrane-spanning segments per subunit, suggesting that the
bending of the inner helices is a common mechanism for channel opening.
The IP3R has been proposed to have the same structural
arrangement of the pore-forming domain as the voltage-gated
K+ channels (10); therefore, the IP3R pore may
also be equipped with the same gating mechanism. Pore-lining inner
helices that contain the gating hinge in the MthK channel correspond to
the sixth membrane-spanning segment of the IP3R. It is of
interest to note that a glycine residue is also present within the
sixth membrane-spanning segment of all three types of the
IP3R. This structural similarity suggests that the channel
gate is formed by the sixth membrane-spanning helix of the
IP3R. One of the striking differences between the
IP3R and other ligand-gated channels with six
membrane-spanning segments, such as cyclic nucleotide-gated channels,
is in the location of the ligand-binding site. Both the
IP3-binding site and the Ca2+-binding sites are
positioned on the N-terminal side of the transmembrane domain of the
IP3R, whereas in other ligand-gated channels, the ligand-binding sites are located in the C-terminal cytoplasmic region
that is close to the pore-lining inner helices. In these ligand-gated
channels, the ligand-binding signals may be transferred directly to the
pore domain and cause bending of the inner helices to open the
channels. The ligand-binding signals of the IP3R may be
transferred to the pore domain in a different manner. Based on the
results presented here, we propose a novel five-domain structure model
in which the C-terminal tail works as a gatekeeper for
activation-induced gating of the IP3R (Fig.
8). In this model, conformational changes
in the IP3-binding domain caused by IP3 binding
are transmitted through both the N-terminal and internal coupling
domains to the C-terminal tail, which then triggers channel opening.
Cys2613 in the C-terminal tail may be critical for
receiving the IP3-binding signal and/or for triggering
channel opening. Further studies on the structure and function of the
IP3R using the described experimental approach may provide
us with an exact answer for the long-asked question, "How does the
binding of IP3 at the N terminus gate the C-terminal
Ca2+ permeation pore?"
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
18A10 (29) was ligated to
a KpnI-SalI fragment of pBact-STneoB-C1. To
substitute serine for cysteine at position 1976, 2610, or 2613 or at
both positions 2610 and 2613 of mouse IP3R1, site-directed
mutagenesis was performed with a MutanK kit (Takara) using primers
containing the appropriate substitutions
(5'-GTGGTTTTCAGACAGCAGCTG-3' for nucleotides 6245-6265, 5'-CAGATGAAGCTCGTGGTTTTT-3' for nucleotides
8146-8166, 5'-TTCCAAGCCGGAGATGAAGCA-3' for
nucleotides 8156-8176, and
5'-AAGCCGGAGATGAAGCTCGTGGT-3' for nucleotides
8150-8172). The EcoRI fragment from the EcoRI
site (nucleotide 6979) internal to the 3'-end of the mouse
IP3R1 isolated from pBactS-C1 (13) was subcloned into
pBluescript SK(+). The BamHI fragment (2532 bp) of the mouse
IP3R1 isolated from pBactS-C1 (13) was subcloned into
pUC118. These plasmids were used as template DNAs. After the mutated
EcoRI or BamHI fragments were put back into
pBactS-C1, the mutated cDNAs were subcloned into pBact-STneoB (28)
at the SalI sites. All PCR products and mutations were
confirmed by DNA sequencing.
80 °C until used.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Deletion and site-directed mutants of
IP3R1. A, schematic diagram of the primary
structure of mouse IP3R1. SI, SII, and SIII represent the
regions spliced out in alternative splicing variants. The striped
box represents the IP3-binding core (residues
226-578). The black boxes (M1,2,3,4,5,6)
represent putative membrane-spanning regions. The regions containing
epitopes for antibodies N1, 4C11, 10A6, 1ML1, and 18A10 and the
anti-(1718-31) antibody are indicated by thick horizontal
lines. Five tryptic fragments (I-V) (22) are indicated by
thin horizontal lines. B, structures of the
deletion and point mutants. Open boxes represent deleted
regions. Open circles denote the sites of single amino acid
substitutions of serine for cysteine.
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Fig. 2.
Western blot analysis of wild-type and mutant
IP3R1 molecules expressed in R23-11 cells.
A, membrane proteins prepared from stable cell lines were
analyzed by Western blot analysis using antibody 1ML1. Lane
1, R23-11 cells (10 µg); lane 2, high level
wild-type IP3R1-expressing cells (KMN13; 1 µg);
lane 3, low level wild-type IP3R1-expressing
cells (KMN107; 10 µg); lanes 4-11, cells expressing
D1-223, D651-1130, D1131-1379, D1692-1731, D1845-2042,
D1845-2216, D1267-2110, and D2736-2749 (10 µg each), respectively.
Molecular mass markers are shown on the left in kilodaltons.
B, shown are the results from Western blot analysis with mAb
4C11. Lane 1, R23-11 cells (1 µg); lane 2, high
level wild-type IP3R1-expressing cells (KMN13; 0.1 µg);
lane 3, cells expressing D2610-2748 (1 µg). Molecular
mass markers are shown on the left in kilodaltons.
IP3-binding properties of the deletion mutants
alternative splicing variant of IP3R1
observed in peripheral tissues (34, 35), exhibited Ca2+
release after the addition of 10 µM IP3 (Fig.
3A). Among the eight artificial mutants (D1-223,
D651-1130, D1131-1379, D1267-2110, D1845-2042, D1845-2216,
D2610-2748, and D2736-2749), only D1131-1379 and D2736-2749
possessed measurable Ca2+ release activity (Fig.
3A). Under the conditions employed, we could detect IICR
from membrane fractions prepared from the low level
IP3R1-expressing KMN107 cells (Fig. 3A), which
contained 0.77 ± 0.2 pmol of IP3-binding sites/mg of
protein. The expression levels of all the internal deletion mutants in
each stable cell line were higher than the expression level of
wild-type IP3R1 in KMN107 cells (Table I). These findings
suggest that none of the mutants except D1131-1379 and D2736-2749 act
as IP3-gated Ca2+ release channels.
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Fig. 3.
IP3-dependent
Ca2+ release activity of wild-type and mutant
IP3R1. A, representative time courses of
the Ca2+ release from the microsomal fractions containing
wild-type (WT) and mutant IP3R1.
Ca2+ release was monitored in the presence of 2 µM thapsigargin. IP3 (10 µM)
was added at the times indicated by the arrowheads. Constant
leakage (see "Experimental Procedures") was subtracted from each
trace. The Ca2+ release activity of mutant
IP3R1 was measured in at least three independent
experiments. B, time courses of Ca2+ release
through wild-type IP3R1 following the addition of different
IP3 concentrations. IP3 was added at time
0.
View larger version (44K):
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Fig. 4.
Fragmentation of wild-type and mutant
IP3R1 by limited trypsin digestion. Crude microsomal
fractions (0.25-5 mg/ml protein) were treated with various
concentrations of trypsin. Tryptic fragments I and III-V (22) were
detected by Western blotting using antibodies N1 and 10A6, the
anti-(1718-31) antibody, and antibody 1ML1, respectively. Each major
tryptic fragment is indicated by a closed arrow.
A, wild-type IP3R1; B, D2736-2749;
C, D1131-1379; D, D1-223; E,
D651-1130; F, D2610-2748. Trypsin concentrations
(micrograms/ml) are indicated above each lane. Molecular mass markers
are shown on the left in kilodaltons.
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Fig. 5.
Conserved cysteine residues in the C-terminal
tail. The C-terminal 48-amino acid sequences next to the
transmembrane regions in all three types of IP3Rs and RyRs
are compared. Asterisks and colons indicate
identical and similar residues, respectively. Cys2610 and
Cys2613 in mouse IP3R1 are indicated by
arrowheads. These amino acid sequences were aligned using
the ClustalW algorithm. The GenBankTM/EBI Data Bank
accession numbers are as follows: mouse IP3R1
(mIP3R1), X15373; rat IP3R1
(rIP3R1), J05510; human IP3R1
(hIP3R1), D26070; Xenopus
IP3R (XIP3R), D14400; rat
IP3R2 (rIP3R2), X61677; human
IP3R2 (hIP3R2), D26350; rat
IP3R3 (rIP3R3), L06096; human
IP3R3 (hIP3R3), D26351;
Drosophila IP3R
(DIP3R), D90403; Caenorhabditis
elegans IP3R (CIP3R),
AJ243179; rabbit RyR1 (rbRyR1), X15750; rabbit RyR2
(rbRyR2), U50465; and rabbit RyR3 (rbRyR3),
X68650.
IP3-binding properties of the cysteine mutants
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Fig. 6.
Western blot analysis and
IP3-dependent Ca2+ release activity
of the cysteine mutants. A, Western blot analysis of
mutants C1976S, C2610S, C2613S, and C2610S/C2613S with antibody 1ML1.
Lane 1, R23-11 cells (10 µg/ml); lane 2, high
level wild-type IP3R1-expressing cells (KMN13; 1 µg);
lane 3, low level wild-type IP3R1-expressing
cells (KMN107; 10 µg); lanes 4-7, cells expressing
C1976S, C2610S, C2613S, and C2610S/C2613S (10 µg each), respectively.
Molecular mass markers are shown on the left in kilodaltons.
B, time courses of the Ca2+ release from the
microsomal fractions containing cysteine mutants. Ca2+
release was monitored in the presence of 2 µM
thapsigargin. IP3 (2.5 µM) was added at the
times indicated by the arrowheads. Constant leakage was
subtracted from each trace.
View larger version (47K):
[in a new window]
Fig. 7.
Limited trypsin digestion of the cysteine
mutants of IP3R1. Crude microsomal fractions (0.25-5
mg/ml protein) were treated with various concentrations of trypsin.
Tryptic fragments I and III-V were detected by Western blotting using
antibodies N1 and 10A6, the anti-(1718-31) antibody, and antibody
1ML1, respectively. Each major tryptic fragment is indicated by a
closed arrow. A, C1976S; B, C2610S;
C, C2613S; D, C2610S/C2613S. Trypsin
concentrations (micrograms/ml) are indicated above each lane. Molecular
mass markers are shown on the left in kilodaltons.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-trefoil
domain (residues 224-436) and a C-terminal
-helical domain
(residues 437-604) containing three armadillo repeat-like folds.
IP3 fits into a cleft formed by these two arms of the
boomerang. Our data described here suggest that region 651-1130, which
immediately follows the IP3-binding core, is essential for
IP3-induced gating of the channel. What kind of roles does this region have? It is known that other proteins containing armadillo repeats such as
-catenin (47) and importins (48) have >10 repeats.
Based on the analysis of the amino acid sequence of IP3R1, Bosanac et al. (14) suggested that, in IP3R1,
the armadillo repeat-like folds extend to the C-terminal region of the
IP3-binding core. It is predicted that many
-helical
domains are formed over the entire region 651-1130 (14), and deletion
of residues 1131-1379 did not abolish the
IP3-dependent Ca2+ release activity
(Fig. 3A). We therefore speculate that the armadillo repeat-like fold-containing
-helical domain, which is essential for
IP3-induced gating, is formed within residues 440-1130.
This region might constitute part of the bridge that connects the
IP3-dependent conformational change in the
IP3R with channel opening.
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Fig. 8.
Five-domain structure model of
IP3R1. In this model, the structure of
IP3R1 is divided into five functional domains,
viz. an N-terminal coupling domain (residues 1-225), a
ligand-binding domain (residues 226-578), an internal coupling domain
(residues 579-2275), a transmembrane domain (residues 2276-2589), and
a gatekeeper domain (residues 2590-2749). The signal of
IP3 binding is transferred through both the N-terminal and
internal coupling domains to the gatekeeper domain, which triggers a
conformational change in the activation gate formed within the
transmembrane domain.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. T. Kurosaki for the kind gift of the DT40 and R23-11 cell lines. We thank Drs. T. Inoue, M. Hattori, A. Mizutani, and K. Hamada for valuable discussions and Drs. A. Miyawaki, H. Mizuno, and C. Hirashima for support and advice during the course of this work. We thank Dr. M. Ikura for critical reading of the manuscript. We also thank Drs. M. Kurosaki and T. Yasuda for assistance with the cell culture technique and helpful discussions. We thank Y. Ueno and M. Iwai for excellent technical assistance. K. U. thanks Drs. N. Matsuo, T. Takahashi, and Y. Kojima for providing this research opportunity.
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FOOTNOTES |
---|
* This work was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶¶ To whom correspondence should be addressed. Tel.: 81-3-5449-5316; Fax: 81-3-5449-5420; E-mail: mikosiba@ims.u-tokyo.ac.jp.
Published, JBC Papers in Press, March 5, 2003, DOI 10.1074/jbc.M300646200
2 Y. Tateishi, M. Hattori, T. Michikawa, M. Iwai, K. Uchida, T. Nakayama, T. Nakamura, T. Inoue, and K. Mikoshiba, unpublished data.
3 H. Miyauchi, K. Uchida, T. Kirino, T. Michikawa, and K. Mikoshiba, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; IICR, IP3-induced Ca2+ release; RyR, ryanodine receptor; mAb, monoclonal antibody.
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