From the Neurobiotechnology Center and Department of
Neuroscience and § Department of Molecular and Cellular
Biochemistry, Ohio State University, Columbus, Ohio 43210 and the
¶ Departments of Anesthesiology, Biological Chemistry, and
Molecular, Cellular, and Developmental Biology, UCLA,
Los Angeles, California 90095
Received for publication, March 15, 2001, and in revised form, March 30, 2001
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ABSTRACT |
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Homologues of Drosophila Trp
(transient receptor potential) form
plasma membrane channels that mediate Ca2+ entry following
the activation of phospholipase C by cell surface receptors. Among the
seven Trp homologous found in mammals, Trp3 has been shown to interact
with and respond to IP3 receptors (IP3Rs) for
activation. Here we show that Trp4 and other Trp proteins also interact
with IP3Rs. The IP3R-binding domain also
interacts with calmodulin (CaM) in a
Ca2+-dependent manner with affinities ranging
from 10 nM for Trp2 to 290 nM for Trp6. In
addition, other binding sites for CaM and IP3Rs are present
in the Binding of many cell surface receptors by hormones,
neurotransmitters, and growth factors leads to the activation of
phospholipase C, which in turn produces diacylglycerol and
inositol 1,4,5-trisphosphate (IP3).1
Diacylglycerol activates protein kinase C, while IP3
triggers Ca2+ release from internal Ca2+ stores
by activating a set of intracellular Ca2+ release channels,
referred to as IP3 receptors (IP3Rs). The
release of Ca2+ from internal stores in turn activates
store-operated channels (SOCs) located on the plasma membrane, allowing
Ca2+ influx from the extracellular space. The
store-operated Ca2+ influx, also known as capacitative
Ca2+ entry (1, 2), plays critical roles in controlling the
duration and the frequency of cytosolic Ca2+ changes
(2-4).
In contrast to the well defined roles of IP3 and
IP3Rs in Ca2+ release, the molecular makeup of
channels that mediate Ca2+ influx and their gating
mechanism(s) remain to be elucidated. Drosophila transient
receptor potential (Trp) protein and its mammalian homologues have been
shown to form either Ca2+ selective or nonselective cation
channels that mediate Ca2+ influx in response to
phospholipase C activation (5-7). To date, seven trp genes
have been cloned from mammalian species (6, 8), probably reflecting the
heterogeneity of Ca2+ influx channels or pathways found in
different cells (4, 9). Expression of individual Trp proteins in
heterologous systems revealed that Trp channels may be activated by a
number of intermediaries involved in the phospholipase C-stimulated
signaling cascade, including Ca2+ (10), diacylglycerol
(11), and activated IP3Rs (12-14). Although store
depletion induced by an intracellular Ca2+-ATPase
inhibitor, thapsigargin, appears to be sufficient to open some Trp
channels (e.g. Trp1 (15), Trp2 (16), and Trp4 (17)), it
remains controversial whether all Trp proteins participate in forming
SOCs (18, 19). Perhaps the answer lies within the structural
organization of the channel, which could be composed of four different
Trp subunits (5). A recent example showed that coexpression of two
Drosophila store-insensitive Trp proteins, Trp Recent studies showed that IP3Rs are involved in the
activation of Trp3. Following the initial demonstration that human Trp3 (hTrp3) in inside-out membrane patches was activated by
IP3Rs in the presence of IP3 (12), Boulay
et al. (14) identified the binding domains involved in the
Trp-IP3R interaction, which were found to be located in the
N terminus of type 3 IP3R (IP3R3) and the C
terminus of Trp3. Overexpression of short peptide fragments containing
these binding sites altered the activity of endogenous store-operated
Ca2+ influx in HEK293 cells (14). While the association
with IP3Rs has also been shown for Trp1 and Trp6 by
coimmunoprecipitation (14, 23, 24), it remains to be determined whether
direct interaction with IP3Rs is common for all Trp
proteins. In this study, we examined murine Trp4 (mTrp4) for
interaction with the first and the stronger Trp3-binding domain of
IP3R3 (F2q; Glu669-Asp698)
(14). In addition, we examined the interaction between Trp4 and
calmodulin (CaM), which has been shown to bind to the C termini of
Drosophila Trp (25) and TrpL (26, 27) and has been
implicated to cause inactivation of TrpL (28). We report here the
presence of two CaM-binding and at least two IP3R3-binding
sites at the C terminus of Trp4. The first CaM-binding site overlaps
closely with one of the IP3R3-binding site. Common binding
sites for CaM and IP3Rs also exist in other Trp proteins.
In functional studies, we show that currents are activated in
inside-out membrane patches excised from Trp4-expressing HEK293 cells
by calmidazolium (CMZ), an antagonist of CaM, and by a peptide
representing one of the Trp-binding domains of IP3R3.
DNA Constructs--
Fragments of IP3Rs and Trps were
generated by polymerase chain reaction (PCR). All sense primers contain
an NcoI recognition site at the 5' with ATG in frame with
the codon for the first amino acid. The antisense primers start with an
A nucleotide followed by the antisense codon for the last amino acid.
PCR products were subcloned into pCRII (Invitrogen), and nucleotide
compositions were confirmed by DNA sequencing. Glutathione
S-transferase (GST) fusion constructs were made by
subcloning NcoI/EcoRI fragments into a modified
pGEX4T-1 vector (Amersham Pharmacia Biotech), in which an
NcoI site was added after the BamHI site. By
design, the insert in each fusion protein starts with a Met and ends
with a "TAA" stop codon (T comes from the A in the antisense
primer, while AA comes from the pCRII vector). Complementary DNA for
enhanced blue fluorescence protein (EBFP) was excised from pEBFP-C1
(CLONTECH) and subcloned into pAGA (29) at
NcoI/SmaI sites, while that for maltose-binding
protein (MBP) was amplified from pMAL-C2 (New England Biolabs) by PCR,
excised with MfeI/NcoI, and subcloned into pAGA
at EcoRI/NcoI sites. Constructs for EBFP fusion
proteins contained inserts downstream from the EBFP coding region and
were made using existing restriction sites on mouse trp4 at
the 5'-end and XbaI at the 3'-end. MBP fusion
proteins contain the first 322 residues of MBP followed by inserts, of
which the cDNAs were subcloned into
NcoI/EcoRI sites.
In Vitro Binding Experiments--
Preparation of GST fusion
proteins; 35S-labeled CaM, EBFP, and MBP fusion proteins;
and procedures for pull-down experiments are as described (30). For
interaction with CaM, CaM-Sepharose (Amersham Pharmacia Biotech) was
used. The binding buffer used for IP3R3-Trp interaction
contains 100 mM KCl, 2 mM MgCl2,
0.5% Lubrol, and 20 mM Tris-HCl, pH 7.5. The buffer used
for CaM-Trp interaction generally contains 120 mM KCl,
0.5% Lubrol, 20 mM Tris-HCl, 10 mM EGTA, 9.96 mM CaCl2. pH was adjusted to 7.5. The estimated
free Ca2+ concentration for the buffer is 10 µM. However, because EGTA is a poor buffer for
Ca2+ concentration at the micromolar range, the actual free
Ca2+ concentration of this buffer is about 70 µM as determined by spectrofluorometric measurements
using Fura2FF (TEF Laboratories, Austin, TX) as a low affinity
Ca2+ indicator and HEDTA-buffered solutions as standards.
For the determination of Ca2+-dependence of Trp binding to
CaM, HEDTA or nitrilotriacetic acid instead of EGTA was used, and total
CaCl2 was added according to the MaxChelator program (C. Patton, Stanford University) to give rise to desired free
Ca2+ concentrations. 35S-Labeled MBP fusion
proteins containing the CaM-binding sites were incubated with
CaM-Sepharose at room temperature for 30 min in varying free
Ca2+ concentrations. Each sample was washed twice with the
same binding buffer that was used for the incubation; thus, the free
Ca2+ concentration was kept unchanged. Bound proteins were
subjected to SDS-polyacrylamide gel electrophoresis. The radioactivity
of [35S]MBP fusion proteins retained was quantified by
phosphorimaging analysis. The percentage of maximal increase over the
value obtained in 10 mM EGTA with no added Ca2+
was fitted with the Hill equation
I/Imax = Cn/(Cn + (K1/2)n), where
I/Imax represents the relative
binding, C is the Ca2+ concentration,
n is the Hill coefficient, and K1/2 is
the Ca2+ concentration that gives rise to half-maximal binding.
For competition studies, recombinant human CaM was prepared
from bacterial lysate using phenyl-Sepharose (Sigma) as described (31).
Peptide F2v (EYLSIEYSEEEVWLTWTD) was synthesized by Research Genetics.
CaM or peptide F2v, in the desired final concentrations, was included
in the binding and washing buffers containing 50 or 70 µM
free Ca2+.
Affinity Measurement for Trp-CaM
Interaction--
All fluorescence measurements were performed on a
PerkinElmer Life Sciences LS5 Spectrofluorometer at 22 °C. Peptides
for CaM-binding sites of Trp1-7 were synthesized by Waterloo Peptide Synthesis (University of Waterloo, Ontario, Canada). Peptide for the
second CaM-binding site of mTrp4 Cell Lines and Electrophysiology--
HEK293 cells stably
expressing mTrp4 (T4-1 and T4-60 cells) and culture conditions were as
described (33). Cells were seeded in 35-mm dishes 2 days prior to patch
clamp recordings. Conditions for recording from inside-out patches were
essentially as described (34). The pipette solution contained 140 mM Na-Hepes, 5 mM NaCl, and 2 mM
CaCl2, pH 7.5. Inside-out patches were excised from the Trp4 cells into a Ca2+-free intracellular solution
containing 140 mM potassium gluconate, 5 mM
NaCl, 1 mM MgCl2, 5 mM EGTA, 10 mM Hepes, pH 7.5. CMZ, CaM, and peptide F2v were diluted to
the final concentration in the intracellular solution containing no
Ca2+ (for F2v) or 18 µM free Ca2+
(for CMZ) and were applied to the cytoplasmic side of excised patches
through perfusion. A step protocol containing continuous alternating
steps to 40 and The C Terminus of Trp4 Binds to IP3R3 and CaM--
A
previous study showed that a GST fusion protein containing the F2q
fragment of human IP3R3
(Glu669-Asp698) interacted with the C terminus
of hTrp3 (14). The Trp3 peptides shown to interact with
IP3R3 were T3C7 (Met742-Glu795)
and T3C8 (Gln777-Asp797). T3C8 bound to
IP3R3 more weakly than T3C7 and thus represents a partial
IP3R-binding domain of Trp3. Because the sequence homology at the regions that align to T3C7 is relatively low (13, 13, and 17%
identical between Trp3 and Trp1, Trp2, and Trp4, respectively), it was
difficult to predict whether the IP3R-binding domain is conserved among all Trp homologues. Therefore, we tested the binding of
GST-IP3R3F2q to mTrp4. 35S-Labeled MBP and EBFP
fusion proteins containing the N and C terminus of mTrp4, respectively,
were synthesized in vitro and incubated with GST or
GST-IP3R3F2q bound to glutathione-Sepharose. Fig.
1A shows the Trp4 regions
present in the fusion proteins, and Fig. 1B shows that while
MBP (M) or EBFP (E) alone and MBP-Trp4 N terminus
(NT) have no specific interaction with IP3R3F2q,
the EBFP-Trp4 C-terminal fusion protein (CT) binds to
GST-IP3R3F2q. To examine the interaction between Trp4 and
CaM, we incubated the fusion proteins with CaM-Sepharose. Only
EBFP-T4CT (CT) interacted with CaM. The binding of T4CT to
CaM was very weak in the absence of Ca2+, but in the
presence of 70 µM Ca2+, it was greatly
increased (Fig. 1B).
We divided T4CT into two portions. CT1 (residues 659-750) includes the
sequence homologous to T3C7 of Trp3, while CT2 (residues 733-974)
contains the more C-terminal sequence unique to Trp4. Interestingly,
both CT1 and CT2 bind strongly to GST-IP3R3F2q and to
Ca2+/CaM. Another N-terminal construct (NT1) and
the transmembrane region (TM) of Trp4 did not interact with
IP3R3F2q or Ca2+/CaM (Fig. 1C). The
overlapping sequence between CT1 and CT2 is relatively conserved among
the Trp homologues (11 out 20 amino acids are identical between Trp3
and Trp4). However, an EBFP fusion protein containing this sequence
(Cf) did not interact with either IP3R3F2q or
Ca2+/CaM (Fig. 1D). On the other hand, a
construct that contains mostly the sequence equivalent to T3C7 but not
T4Cf (Cc) interacted with both
IP3R3F2q and Ca2+/CaM. These results indicate
that just as T3C7, a similar region at the C terminus of Trp4 also
binds to IP3R3. In addition, there is at least one
additional IP3R-binding site more C-terminal to the
previously defined site. Moreover, both IP3R-binding
regions bind to Ca2+/CaM.
The First CaM-binding Site of Trp4 Closely Overlaps with an
IP3R-binding Site--
To determine how close the first
CaM-binding site of Trp4 is to the site that binds to
IP3R3F2q, we produced MBP- and EBFP-fusion proteins
containing smaller fragments of T4Cc and tested their binding to GST-IP3R3F2q and Ca2+/CaM. Fig.
2 shows the composition of the fusion
proteins and the binding results. In general, Trp4 fragments that bound
to IP3R3F2q also bound to Ca2+/CaM. The
smallest fragment that binds to IP3R3F2q and
Ca2+/CaM with nearly the same strength as T4Cc
is T4Cw (Arg695-Glu724). Removing
two residues from either the N or the C terminus of T4Cw
reduced the interaction with both IP3R3F2q and
Ca2+/CaM (Fig. 2C, T4Cv and
T4Cx). Smaller fragments, such as Ca, Caa, and Cab, had much weaker binding to
IP3R3F2q, and this was also accompanied by greatly reduced
binding to Ca2+/CaM. Therefore, the binding sites for CaM
and for the IP3R cannot be separated by deletion studies,
indicating that a common sequence, referred to as the
CaM/IP3R binding (CIRB) domain, is involved in the
interaction with CaM and with the IP3R.
The Second CaM-binding Site Is Present in Trp4
In contrast to IP3R3F2q, CaM binds to a short, confined
sequence in CT2, CT2p (Arg787-Asn812, Fig. 3,
C and D). Removing two residues from its N
terminus (CT2r) nearly abolished the binding, whereas removing two
residues from its C terminus (CT2q) reduced the binding by 45%,
as determined by phosphorimaging analysis from two independent
experiments. Although CT2p is contained within the CT2
IP3R3F2q-binding region, it does not bind to the
IP3R fragment. Therefore, the binding sites for CaM and
IP3R3F2q in the CT2 region do not overlap as closely as the
CIRB sequence. Because the CaM binding site is not present in TRP4 CIRB Domain Is Conserved among Trp Proteins--
As shown in Fig.
4, regions equivalent to T4Cw
from hTrp1, mTrp2, hTrp3, mTrp5, mTrp6, mTrp7, and
Drosophila Trp and TrpL also interacted with both
GST-IP3R3F2q and Ca2+/CaM. For TrpL, the CIRB
domain is in addition to the two CaM-binding sites (710, 854)
identified previously (26, 27). For Drosophila Trp, the CIRB
domain is within a previously reported CaM-binding region (683)
(25). For Trp3, the CIRB domain is the only CaM-binding domain, because
deletion of this site eliminated the binding and the regulation of Trp3
activity by Ca2+/CaM (34). This may also be the case for
Trp6 and Trp7, since they are very similar to Trp3. Trp1 terminates
soon after the CIRB sequence and therefore is unlikely to have a second
binding site at the C terminus. The C-terminal sequence of mTrp2
(residues 944-1072) and of mTrp5 (residues 762-975) interacted with
both Ca2+/CaM and GST-IP3R3F2q (not shown),
suggesting that the second binding sites for the two modulators are
conserved in Trp2, Trp4
Using synthetic peptides representing the CIRB domains of Trp1-7 and
the second CaM-binding site of Trp4, we determined the affinities to
range from 10 nM for mTrp2 to 290 nM for mTrp6
(Table I). The second CaM-binding site of
Trp4 has lower affinity (600 nM) than the CIRB domains of
all Trp proteins. The apparent affinities for Ca2+, as
measured by in vitro binding assays using
[35S]MBP-Trp fusion proteins and CaM-Sepharose, differed
greatly from 1.0 µM for the second CaM-binding site of
Trp4 to 44.2 µM for the CIRB domain of Trp5 (Table I).
The Hill coefficients of Ca2+ dependence for the CaM-Trp
interactions were about 1.5-3.5.
All IP3Rs Interact with Trp3--
Just as the CIRB
domain is conserved in all Trp homologues, the F2q Trp-binding site is
also conserved in the three known mammalian IP3Rs. Fig.
5A shows that regions
homologous to IP3R3F2q from IP3R1 and
IP3R2 also interacted with the C terminus of Trp3. The
alignment of the three sequences indicates that only the C-terminal halves are conserved. Further experimentation using smaller segments of
IP3R3F2q fused to GST showed that the C-terminal half of
F2q (represented by F2v) is the minimal domain of IP3R3
that interacts with Trp3 (Fig. 5B).
Competition between CaM and IP3R3 for Binding to
Trps--
In in vitro binding assays, we have shown that
for T3C7, the interaction with IP3R3F2q was inhibited by
purified recombinant human CaM and the binding to CaM was blocked by a
synthetic peptide, composed of the sequence of F2v (34). Peptide F2v
appears to be a potent inhibitor for CaM binding to Trp3, because the
competition was observed in a binding buffer that contained
Ca2+, a condition under which CaM has a high affinity for
Trp3. In order to learn the relative abilities of CaM to compete with
the IP3R for binding to different Trps, we tested the
competition between CaM and IP3R3F2q for binding to the
CIRB domains of other mammalian Trp homologues. The addition of CaM
reduced the binding of 35S-labeled MBP-T4Cw to
GST-IP3R3F2q in a dose-dependent manner, with
50% inhibition (IC50) occurring at 2.1 µM
(Fig. 6A), similar to the
value obtained for Trp3 (1.2 µM) (34). The inhibition of
the Trp4-IP3R3 interaction by 20 µM CaM was
about 73%, slightly less than that of Trp3-IP3R3
interaction (88%) (34). In addition, 20 µM CaM also
inhibited the interaction between IP3R3F2q and the CIRB
domains of Trp1, -2, -5, -6, and -7 to various degrees (Fig.
6B). The effect of CaM was
Ca2+-dependent, since the inhibition by CaM was
not observed in a Ca2+-free binding buffer (Fig.
6B). These results indicate that at high Ca2+
concentrations, CaM competes with IP3Rs for binding to the
CIRB domains of all Trp proteins. However, the extent of inhibition varies from Trp to Trp, which is probably related to the differences in
their affinities to CaM. Although the CT2 IP3R-binding site does not overlap as closely to the CaM-binding site as the CIRB sequence, the binding between CT2c and IP3R3F2q was also
blocked by CaM (Fig. 6C).
Peptide F2v blocked the interaction between CaM and the CIRB domain of
Trp1-7 with IC50 values ranging from 1.7 to 90 µM (Fig. 6D). The peptide is more effective in
inhibiting the interaction between CaM and Trp3, -6, and -7 than that
between CaM and Trp1, -2, -4, and -5. The effect of the peptide is
specific because the binding of CaM to Trp4CT2k (residues 781-814),
which does not interact with IP3R3F2q (not shown), was not
inhibited (Fig. 6D).
Peptide F2v and a CaM Antagonist Activate Trp4 in Excised
Inside-out Patches--
CaM bound to the CIRB domain has a general
inhibitory function on Trp3, because in inside-out patches excised from
HEK293 cells expressing Trp3, removing or inactivating CaM led to a
large increase in Trp3 activity (34). In the absence of
Ca2+ and at 5 µM, peptide F2v activated Trp3
by competing with CaM for binding to the CIRB domain. The activated
channel was blocked by CaM (34). Consistent with the finding that F2v
was 10 times less effective in competing with CaM for binding to Trp4
than to Trp3 (Fig. 6D), we found that in inside-out patches,
F2v was also less potent in activating Trp4 than Trp3 (Fig.
7A). At 5 µM,
F2v only caused a small increase in Trp4 activity, which is significantly different (p < 0.05) from the basal
activity in one Trp4 cell line (T4-1) but not in the other one (T4-60).
When the concentration of F2v was increased to 50 µM,
both cell lines showed significant increase in activity
(p < 0.01), which is more than 6 times higher than
that stimulated by 5 µM F2v. Additionally, patches
excised from Trp4 cells were activated by 1 and 10 µM CMZ
(Fig. 7B). Under the same conditions, untransfected HEK293 cells did not show any significant increase in activity after treatment
with peptide F2v or CMZ (Fig. 7, A and B).
The mechanism of activation for SOCs remains mysterious. Three
major hypotheses have been proposed. The first assumes that a small
diffusible soluble factor capable of stimulating SOCs appears in the
cytosol upon store depletion. Although it has been shown that acid
extracts from activated Jurkat cells and platelets or a
Ca2+ store-depleted mutant yeast strain contain such a
factor, which when applied to naive cells could stimulate
Ca2+ influx (35, 36) or cation conductance (37), the
identity of the Ca2+ influx factor has not been determined.
The second hypothesis claims that a secretion-like process involving
the insertion of channel-containing vesicles into the plasma membrane
is required for activating SOCs (38). In agreement with this is the
finding that actin redistribution affected capacitative
Ca2+ entry (39). The third hypothesis is called
conformational coupling and is modeled after the well known mechanism
of excitation-contraction coupling between the L-type Ca2+
channel and the ryanodine receptor in skeletal muscle (40). In this
case, IP3Rs are thought to serve not only as channels for
Ca2+ release but also as sensors for store depletion. The
signal of store depletion is sent to the plasma membrane
Ca2+ entry channels via a direct protein-protein
interaction (2, 41). Consistent with this hypothesis, it has been
demonstrated that IP3 activates cation channels on the
plasma membranes of endothelial cells, macrophages and A431 epithelial
cells (42-44) and that the activity of IP3R1 purified from
rat cerebellum is modulated by luminal Ca2+ (45).
Despite the controversy about whether or not Trp proteins form SOCs,
accumulating evidence has indicated that IP3 and
IP3Rs are involved in the activation of SOCs as well as
Trp3 (12-14, 46). Also, just like the native SOCs, Trp3 activation was
prevented by actin filament condensation induced by calyculin A, a
phosphatase inhibitor (46). Therefore, the conformational coupling
mechanism, and perhaps the secretion-like coupling mechanism as well,
are applicable to Trp-based channels. Using molecular and biochemical approaches, Boulay et al. (14) identified the interacting
domains of Trp3 and IP3R3 and showed that they are involved
in the regulation of native SOCs. In the current study, we further
demonstrate that the interactions between Trp and IP3R are
common for all Trp proteins and for all IP3Rs. Thus,
conformational coupling involving direct physical interaction with
activated IP3Rs may be a common mechanism for the
activation of Trp-based channels. Moreover, we show that the
IP3R-binding domains of the Trp proteins also bind to
Ca2+/CaM and that the binding by CaM inhibits the
association between Trp and IP3Rs. Therefore, the
competition between CaM and IP3Rs for binding to a common
site may play a key role in controlling the gating of Trp-based
channels. Based on the functional study of Trp3 using inside-out
membrane patches and a non-Ca2+-binding CaM mutant, we
concluded that at rest, CaM is tethered to the channel and prevents it
from being spontaneously active (34). Maneuvers that displaced CaM from
Trp3 strongly activated the channel (34). Here, we show that Trp4 is
also strongly activated by inactivating CaM with CMZ, indicating that
CaM probably plays the same inhibitory role in all Trp-based channels.
Consistent with this, CMZ also activates current in patches excised
from cells expressing Trp1 or Trp6 (not shown). Thus, binding to CaM is
essential to prevent the spontaneous activity of Trp channels.
Analysis of genomic sequences revealed that for hTrp1, hTrp4, hTrp5,
Drosophila Trp, and TrpL, the coding sequence for the N-terminal end of the CIRB domain coincides with the beginning of an
exon,2 indicating that
conformational coupling involving binding by IP3Rs and its
regulation by CaM were acquired early in evolution and that there may
be a constraint to preserve these modes of regulation for Trps.
However, the IP3R-deficient Drosophila
photoreceptors showed normal response to light and unaltered function
of Trp and TrpL (47, 48), suggesting that the Drosophila
IP3R is not involved in activating these channels. The fact
that Drosophila Trp and TrpL interacted with human
IP3R3F2q fragment in vitro does not implicate a
similar interaction in vivo. Interestingly, a comparison
between the Drosophila IP3R and the human
IP3R3 showed a very low homology between the two at the F2q
region. An extra 38-amino acid sequence in the Drosophila
IP3R (725) essentially disrupts an otherwise intact
F2q region. In addition, the Drosophila sequence contains
only one of the two tryptophans critical for binding to Trp3 (34).
Therefore, it is unlikely that the Drosophila IP3R would be involved in activating the Trp channels in
the same manner as its mammalian counterparts. Since only a single gene for IP3R is present in the Drosophila genome
(48), it is possible that in the insect's photoreceptors, a different
protein would compete with CaM for binding to the CIRB domains of Trp
and TrpL and hence activate the channels. Furthermore, the mechanism
present here is only one of the several ways by which the Trp channels are activated. The involvement of IP3Rs in the activation
of mammalian Trps does not rule out the participation of other
molecules in Trp gating.
In addition to the CIRB domain, binding sites for CaM and
IP3Rs are present downstream in Trp2, Trp4 Diversity also arises from the differences in the affinities of CIRB
domains of different Trp homologues for CaM and for IP3Rs. The affinities for CaM differ by as much as 29-fold between the CIRB
domains of Trp2 and Trp6 (Table I). Because peptide F2v is more
effective in displacing CaM from Trp3 and the closely related Trp6 and
Trp7 than from the CIRB domain of other Trps, the affinities of the
CIRB domains for the Trp-binding site of IP3R3 may also be
very different. In the context of conformational coupling, one
important step accomplished by IP3Rs is to displace the
inhibitory CaM from the CIRB domain. This may be accomplished mainly by
the binding of the F2v domain of IP3R3 and homologous regions of IP3R1 and IP3R2. The effectiveness
of the F2v may be dependent on local Ca2+ concentrations,
since Trp binding to CaM is greatly enhanced by Ca2+. In
the binding assay shown in Fig. 6D, 70 µM free
Ca2+ was used to facilitate the detection of CaM binding.
Under this condition, peptide F2v blocked CaM binding to the CIRB
domain of Trp3, -6, and -7 more effectively than to that of Trp1, -2, -4, and -5, suggesting that the closely related Trp3, -6, and -7 have
higher affinity for the F2v domain and thus may be more sensitive to
the activation by IP3Rs than the rest of the Trp homologues. However, it is important to note that under resting Ca2+ concentrations of ~0.1 µM, all CIRB
domains would prefer binding to F2v in the in vitro binding
assay. Because it is not clear how CaM is tethered to the channel under
resting conditions when the cytosolic Ca2+ concentration is
low, it is difficult to predict whether the F2v domain alone is
sufficient to displace CaM from all Trp channels in excised patches.
The fact that a higher concentration of peptide F2v is needed to
activate Trp4 than to activate Trp3 agrees well with the binding data,
suggesting that when acting alone, F2v is a weak competitor for the
CaM-Trp4 interaction. This supports our conclusion that peptide F2v
activates Trp3 by displacing CaM from the common binding site.
CaM regulation through competition with another protein appears to be a
common mechanism shared with other Ca2+-permeable channels.
Similar to Trp-based channels, a C-terminal site of the
N-methyl-D-aspartate receptor binds to
Ca2+/CaM and actin-associated Native SOCs may be regulated by IP3Rs and CaM in a similar
manner. However, because the single channel conductance of the endogenous channel is very low (12) and the channel density is much
lower than the expressed Trp channels, it has been difficult for us to
determine confidently whether or not similar mechanisms that stimulate
the Trp channels also stimulate the native SOCs in control HEK cells.
In A431 epithelial cells, IP3 has been shown to activate
SOCs via IP3Rs (53). In whole-cell studies, intracellular infusion of CaM inhibited the SOC in bovine endothelial cells by
slowing down the activation and speeding up the inactivation (54).
Ca2+-dependent inactivation has been documented
for SOCs found in a number of different cells (55, 56). Thus,
conformational coupling involving IP3Rs and its negative
control via CaM may be a primary mechanism of gating for SOCs.
but not the
isoform of Trp4. In the presence of
Ca2+, the Trp-IP3R interaction is inhibited by
CaM. However, a synthetic peptide representing a Trp-binding domain of
IP3Rs inhibited the binding of CaM to Trp3, -6, and -7 more
effectively than that to Trp1, -2, -4, and -5. In inside-out membrane
patches, Trp4 is activated strongly by calmidazolium, an antagonist of
CaM, and a high (50 µM) but not a low (5 µM) concentration of the Trp-binding peptide of the
IP3R. Our data support the view that both CaM and IP3Rs play important roles in controlling the gating of
Trp-based channels. However, the sensitivity and responses to CaM and
IP3Rs differ for each Trp.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and
Trp-like (TrpL), led to the formation of a SOC (20). Thus, the store
sensitivity may be reconstituted with the proper combination of
different Trp subunits. Consistent with this idea, Trp1, Trp3, and Trp4
have been shown to be part of SOCs in human submandibular gland cells,
neurons, and adrenal cortex cells, respectively (15, 21, 22).
MATERIALS AND METHODS
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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was synthesized by the Tufts University Core Facility (Tuft University, Boston, MA).
Phosphodiesterase activity was assayed by monitoring the hydrolysis of
fluorescent 2'-methylanthraniloyl cGMP (8 µM) in 1 ml of solution containing 200 mM MOPS, pH 7.0, 90 mM KCl, 3 mM MgCl2, 2 mM EGTA, 100 µM free Ca2+, 25 nM CaM, and the desired amount of peptide. Samples were
excited at 330 nm, and emission at 450 nm was measured. The
dissociation constant (Kd) with Ca2+/CaM
was calculated from the activation curves of phosphodiesterase by CaM
in the absence and the presence of the peptide as described (32).
40 mV, each for 1 s, from holding at 0 mV was
applied throughout the experiments. Currents were recorded at sampling
frequency of 5 kHz and filtered at 1 kHz. Currents recorded at
40 mV
from 100 episodes under each condition were averaged using pCLAMP 8 (Axon Instruments) after digital filtering at 500 Hz and base-line adjustment.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (60K):
[in a new window]
Fig. 1.
Localization of IP3R-binding
domain and CaM-binding sites at the C terminus of mTrp4.
A, diagram of mTrp4 and its fragments included in the
EBFP or MBP fusion proteins. Numbers in
parentheses indicate the positions of the fragments.
Thick and thin lines denote that the binding to
CaM is positive and negative, respectively. Shaded
boxes in the full-length mTrp4
indicate the locations of
transmembrane (TM) segments. B, representative
binding results showing the autoradiograms of input
35S-labeled EBFP-Trp4 C-terminal (CT), MBP-Trp4
N-terminal (NT) fusion proteins, EBFP (E), and
MBP (M) and those retained by GST, GST-IP3R3F2q,
and CaM. A picture of a Coomassie Blue-stained gel displayed
below the second panel shows the
amount of GST and GST fusion protein used. C and
D, additional binding results showing that two C-terminal
regions of mTrp4
(CT1 and CT2) (C) and Trp4Cc
(D) bind to both IP3R3F2q and
Ca2+/CaM.
View larger version (52K):
[in a new window]
Fig. 2.
Colocalization of binding sites for
Ca2+/CaM and IP3R3F2q on Trp4CT1.
A, compositions of EBFP or MBP fusion proteins containing
subfragments of Trp4Cc. Positions in the full-length
mTrp4 are shown in parentheses. Relative intensities of
the fusion proteins retained by Ca2+/CaM and
GST-IP3R3F2q are indicated by the plus and
minus signs and are summarized from 2-4 binding
experiments. B and C show binding results from
representative experiments.
but Not
Trp4
--
The minimal domains that bind to CaM and
IP3R3F2q at the CT2 region were sought in a similar manner
(Fig. 3). However, it appears that the
CT2 IP3R-binding domain is not confined to a short sequence
like the CIRB domain. When CT2 was divided into CT2a and CT2b, both
retained weak interaction with IP3R3F2q (Fig. 3B). Further analyses suggested that the region from
Gly781 to Ser864 retains significant binding to
IP3R3F2q (Fig. 3C). Interestingly, molecular
cloning and immunoblot analysis have revealed the presence of two major
isoforms of Trp4, Trp4
and Trp4
(33). The
form lacks the 84 amino acids corresponding to Gly781-Ser864 of
the
form. Thus, CT2
binds very weakly to IP3R3F2q
(Fig. 3B).
View larger version (47K):
[in a new window]
Fig. 3.
Determination of binding sites for
Ca2+/CaM and IP3R3F2q on
Trp4CT2 . A, diagrams of
mTrp4
and mTrp4
and compositions of MBP fusion proteins
containing subfragments of Trp4CT2. Positions in respect to the
full-length mTrp4
are shown in parentheses. CT2
contains the CT2 fragment of mTrp4
and lacks the 84-amino acid
region (shown as a dashed line in MBP-CT2
and
black box in mTrp4
). Thick and
thin lines denote that the binding to CaM is
positive and negative, respectively. B-D show binding
results from representative experiments. Like Trp4CT2p, Trp4CT2h-o did
not bind to GST-IP3R3F2q (not shown).
,
CT2
does not bind to Ca2+/CaM (Fig. 3B).
, and Trp5, despite the fact that the
homology among the three Trps is very low at these regions.
View larger version (60K):
[in a new window]
Fig. 4.
The common CaM/IP3R binding site
is present in all Trp proteins. A, amino acid
compositions of mammalian Trp1-7, Drosophila Trp
(DmTrp), and TrpL (DmTrpL) present in the MBP
fusion proteins. Positions for these sequences in the full-length
proteins are shown in parentheses. 35S-Labeled
MBP fusion proteins were tested for binding to GST-IP3R3F2q
and to Ca2+/CaM as described under "Materials and
Methods." Representative binding results are shown in
B.
Affinities of Trp binding to CaM
View larger version (45K):
[in a new window]
Fig. 5.
All IP3Rs interact with the C
terminus of Trp3. A, amino acid compositions of
IP3R1, IP3R2, and IP3R3 included in
the GST fusion proteins. B, amino acid compositions of
subregions of IP3R3F2q included in the GST fusion proteins.
Positions in the full-length proteins are indicated in parentheses.
35S-Labeled hTrp3 C terminus (T3CT,
Asn725-Glu848) was tested for binding to the
GST-IP3R fusion proteins as described under "Materials
and Methods." Representative binding results are shown in
C. Upper panels show
[35S]T3CT retained by the GST fusion proteins as revealed
by autoradiography, while lower panels show the
amount of GST fusion proteins used as revealed by staining with
Coomassie Blue.
View larger version (39K):
[in a new window]
Fig. 6.
Competition between CaM and IP3R3
for binding to the Trp CIRB domains. A, CaM inhibits
Trp4 binding to IP3R3. Varying concentrations of CaM were
included in the binding reactions for 35S-labeled
MBP-T4Cw and GST-IP3R3F2q. The binding buffer
contained 70 µM free Ca2+. After washing,
bound [35S]MBP-T4Cw was separated by
SDS-polyacrylamide gel electrophoresis. The left
panel shows an autoradiogram from a representative
experiment, while the right panel shows averages
of results of phosphorimaging analysis of the relative amount of
[35S]T4Cw retained from three experiments. The
curve is the least-square fit of the equation,
y = 1/(1 + [P]/IC50), where y
is relative binding, [P] is CaM concentration, and IC50 = 2.1 µM is the concentration that causes 50% inhibition.
B, representative experiments show that 20 µM
CaM inhibits the binding of IP3R3F2q to the CIRB domain of
Trp1, -2, -5, -6, and -7 in a Ca2+-dependent
manner. 10 mM HEDTA and EGTA were used to buffer
[Ca2+] to 50 µM and 0 (<10
nM), respectively. C, binding of Trp4CT2c to
IP3R3F2q was inhibited by 20 µM CaM.
[Ca2+] = 50 µM. D, peptide F2v
inhibits binding of CaM to the CIRB domain of Trp1-7. Varying
concentrations of peptide F2v were included in the binding reaction for
35S-labeled CaM and GST fusion proteins containing the CIRB
sequence of Trp1-7 or the second CaM-binding site of Trp4 (CT2k,
781-814). Positions of Trp sequences included in the GST fusion
proteins are indicated in parentheses. The binding buffer
contained 70 µM free Ca2+. After washing,
bound [35S]CaM was separated by SDS-polyacrylamide gel
electrophoresis and revealed by autoradiography. IC50
values were determined from results of phosphorimaging analysis of the
relative amount of [35S]CaM retained by each Trp fragment
from two or three experiments. The percentages of inhibitions for
different concentrations of F2v were fitted with the equation as in
A, except that [P] is the concentration of the
peptide.
View larger version (36K):
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Fig. 7.
Peptide F2v and CMZ stimulate channel
activity in inside-out patches excised from Trp4. Inside-out
patches were excised from untransfected HEK293 cells (control) or two
stable cell lines expressing mTrp4 (T4-60 and T4-1) to a bath
solution that contained either no Ca2+ (A) or 18 µM Ca2+ (B) as described under
"Materials and Methods." Peptide F2v at 5 or 50 µM
(A) and CMZ at 1 or 10 µM (B) were
applied to the cytoplasmic side of the membrane by perfusion.
Bar graphs show averages ± S.E. of the mean
current (sampled from periods of 400 s) at
40 mV from the
numbers of patches indicated in parentheses. Representative
traces for control and T4-60 cells at basal level and 50 µM F2v (A) or 10 µM CMZ
(B) are shown on the right. Dashed
lines indicate closed level. *, p < 0.01;
**, p < 0.05 different from basal level by Student's
t test.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, and Trp5,
suggesting that subtype-specific functions carried by CaM and
IP3Rs also exist and contribute to the diversity in the
regulation of Trp-based channels. Interestingly, the last 240 amino
acids of human Trp4
including the 84-amino acid region that contains
the binding sites for CaM and IP3Rs are encoded by a single
exon.2 In Trp4
, the 84-amino acid region is deleted
through the use of alternative splicing sites, and thus the second
binding sites for CaM and IP3Rs are not present.
Surprisingly, the same region was recently shown to bind to the C
terminus of IP3Rs (49). It remains to be determined how the
bindings of the N and C terminus of IP3Rs and CaM to this
region affect the function of Trp4
and how they differ from
bindings to the more upstream CIRB domain.
-actinin. CaM inhibits
whereas
-actinin promotes channel opening (50, 51). A common binding
site for Ca2+/CaM and the C terminus of the olfactory
cyclic nucleotide-gated channel has been found at the N terminus of the
same channel. CaM binding disrupts the interdomain coupling between the
N and C terminus and inactivates the channel (52). For these channels, it is not known whether CaM is tethered to the channels at rest and
whether it plays any functional role without the presence of any
physiological stimulus. To date, only Trp channels have been shown to
be activated by removing or inhibiting CaM. The relatively harsh
treatment may be effective in releasing the channel from inhibition by
CaM, but this does not result in a complete activation of Trp channels
(34). A full stimulation of Trp channels should therefore include
multiple events, such as the activation of IP3Rs,
Ca2+ store-depletion, and perhaps the increase in the level
of diacylglycerol.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Z. Chen for the initial characterization of Trp4 binding to IP3R3 and CaM, D. Chuang for technical assistance, and Dr. K. Mikoshiba for cDNA of IP3R1 and IP3R3.
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FOOTNOTES |
---|
* The work was supported in part by National Institutes of Health Grants GM54235 (to M. X. Z.) and HL45198 (to L. B.).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 all correspondence should be addressed: The Ohio State
University Neurobiotechnology Center, 168 Rightmire Hall, 1060 Carmack
Rd., Columbus, OH 43210. Tel.: 614-292-8173; Fax: 614-292-5379; E-mail:
zhu.55@osu.edu.
Published, JBC Papers in Press, April 4, 2001, DOI 10.1074/jbc.M102316200
2 M. X. Zhu, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; CaM, calmodulin; CIRB, CaM/IP3 receptor binding; CMZ, calmidazolium; EBFP, enhanced blue fluorescence protein; GST, glutathione S-transferase; hTrp1, -3, -4, and -5, human Trp1, -3, -4, and -5, respectively; IP3R, IP3 receptor; IP3R3, type 3 IP3R; MBP, maltose-binding protein; mTrp2, -4, -5, -6, and -7, murine Trp2, -4, -5, -6, and -7, respectively; PCR, polymerase chain reaction; SOC, store-operated channel; MOPS, 4-morpholinepropanesulfonic acid; HEDTA, N'-(2-hydroxyethyl)ethylenediamine-N,N,N'-triacetic acid.
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REFERENCES |
---|
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---|
1. | Putney, J. W., Jr. (1986) Cell Calcium 7, 1-12[Medline] [Order article via Infotrieve] |
2. | Putney, J. W., Jr. (1990) Cell Calcium 11, 611-624[Medline] [Order article via Infotrieve] |
3. | Berridge, M. J. (1995) Biochem. J. 312, 1-11[Medline] [Order article via Infotrieve] |
4. | Clapham, D. E. (1995) Cell 80, 259-268[Medline] [Order article via Infotrieve] |
5. |
Birnbaumer, L.,
Zhu, X.,
Jiang, M.,
Boulay, G.,
Peyton, M.,
Vannier, B.,
Brown, D.,
Platano, D.,
Sadeghi, H.,
Stefani, E.,
and Birnbaumer, M.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
15195-15202 |
6. | Zhu, X., Jiang, M., Peyton, M., Boulay, G., Hurst, R., Stefani, E., and Birnbaumer, L. (1996) Cell 85, 661-671[Medline] [Order article via Infotrieve] |
7. | Hofmann, T., Schaefer, M., Schultz, G., and Gudermann, T. (2000) J. Mol. Med. 78, 14-25[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Okada, T,
Inoue, R.,
Yamazaki, K.,
Maeda, A.,
Kurosaki, T.,
Yamakuni, T.,
Tanaka, I.,
Shimizu, S.,
Ikenaka, K.,
Imoto, K.,
and Mori, Y.
(1999)
J. Biol. Chem.
274,
27359-27370 |
9. | Fasolato, C., Innocenti, B., and Pozzan, T. (1994) Trends Pharmacol. Sci. 15, 77-83[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Zitt, C.,
Obukhov, A. G.,
Strubing, C.,
Zobel, A.,
Kalkbrenner, F.,
Luckhoff, A.,
and Schultz, G.
(1997)
J. Cell Biol.
138,
1333-1341 |
11. | Hofmann, T., Obukhov, A. G., Schaefer, M., Harteneck, C., Gudermann, T., and Schultz, G. (1999) Nature 397, 259-263[CrossRef][Medline] [Order article via Infotrieve] |
12. | Kiselyov, K., Xu, X., Kuo, T. H., Mozhayeva, G., Pessah, I., Mignery, G., Zhu, X., Birnbaumer, L., and Muallem, S. (1998) Nature 396, 478-482[CrossRef][Medline] [Order article via Infotrieve] |
13. | Kiselyov, K., Mignery, G. A., Zhu, M. X., and Muallem, S. (1999) Mol. Cell 4, 423-429[Medline] [Order article via Infotrieve] |
14. |
Boulay, G.,
Brown, D.,
Qin, N.,
Jiang, M.,
Dietrich, A.,
Zhu, M. X.,
Chen, Z.,
Birnbaumer, M.,
Mikoshiba, K.,
and Birnbaumer, L.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
14955-14960 |
15. |
Liu, X.,
Wang, W.,
Singh, B. B.,
Lockwich, T.,
Jadlowiec, J.,
O'Connell, B.,
Wellner, R.,
Zhu, M. X.,
and Ambudkar, I. S.
(2000)
J. Biol. Chem.
275,
3403-3411 |
16. |
Vannier, B.,
Peyton, M.,
Boulay, G.,
Brown, D.,
Qin, N.,
Jiang, M.,
Zhu, X.,
and Birnbaumer, L.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
2060-2064 |
17. | Philipp, S., Cavalie, A., Freichel, M., Wissenbach, U., Zimmer, S., Trost, C., Marquart, A., Murakami, M., and Flockerzi, V. (1996) EMBO J. 15, 6166-6171[Abstract] |
18. |
Boulay, G.,
Zhu, X.,
Peyton, M.,
Jiang, M.,
Hurst, R.,
Stefani, E.,
and Birnbaumer, L.
(1997)
J. Biol. Chem.
272,
29672-29680 |
19. |
Zhu, X.,
Jiang, M.,
and Birnbaumer, L.
(1998)
J. Biol. Chem.
273,
133-142 |
20. | Xu, X. Z., Chien, F., Butler, A., Salkoff, L., and Montell, C. (2000) Neuron 26, 647-657[Medline] [Order article via Infotrieve] |
21. | Li, H. S., Xu, X. Z., and Montell, C. (1999) Neuron 24, 261-273[Medline] [Order article via Infotrieve] |
22. |
Philipp, S.,
Trost, C.,
Warnat, J.,
Rautmann, J.,
Himmerkus, N.,
Schroth, G.,
Kretz, O.,
Nastainczyk, W.,
Cavalie, A.,
Hoth, M.,
and Flockerzi, V.
(2000)
J. Biol. Chem.
275,
23965-23972 |
23. |
Lockwich, T. P.,
Liu, X.,
Singh, B. B.,
Jadlowiec, J.,
Weiland, S.,
and Ambudkar, I. S.
(2000)
J. Biol. Chem.
275,
11934-11942 |
24. | Rosado, J. A., and Sage, S. O. (2000) Biochem. J. 350, 631-635[CrossRef][Medline] [Order article via Infotrieve] |
25. | Chevesich, J., Kreuz, A. J., and Montell, C. (1997) Neuron 18, 95-105[Medline] [Order article via Infotrieve] |
26. | Warr, C. G., and Kelly, L. E. (1996) Biochem. J. 314, 497-503[Medline] [Order article via Infotrieve] |
27. | Trost, C., Marquart, A., Zimmer, S., Philipp, S., Cavalie, A., and Flockerzi, V. (1999) FEBS Lett. 451, 257-263[CrossRef][Medline] [Order article via Infotrieve] |
28. | Scott, K., Sun, Y., Beckingham, K., and Zuker, C. S. (1997) Cell 91, 375-383[Medline] [Order article via Infotrieve] |
29. |
Sanford, J.,
Codina, J.,
and Birnbaumer, L.
(1991)
J. Biol. Chem.
266,
9570-9579 |
30. |
Qin, N.,
Olcese, R.,
Bransby, M.,
Lin, T.,
and Birnbaumer, L.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
2435-2438 |
31. |
Lee, S. H.,
Kim, J. C.,
Lee, M. S.,
Heo, W. D.,
Seo, H. Y.,
Yoon, H. W.,
Hong, J. C.,
Lee, S. Y.,
Bahk, J. D.,
Hwang, I.,
and Cho, M. J.
(1995)
J. Biol. Chem.
270,
21806-21812 |
32. | Erickson-Viitanen, S., and DeGrado, W. F. (1987) Methods Enzymol. 139, 455-478[Medline] [Order article via Infotrieve] |
33. |
Tang, Y.,
Tang, J.,
Chen, Z.,
Trost, C.,
Flockerzi, V.,
Li, M.,
Ramesh, V.,
and Zhu, M. X.
(2000)
J. Biol. Chem.
275,
37559-37564 |
34. |
Zhang, Z.,
Tang, J.,
Tikunova, S.,
Johnson, J. D.,
Chen, Z.,
Qin, N.,
Dietrich, A.,
Stefani, E.,
Birnbaumer, L.,
and Zhu, M. X.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
3168-3173 |
35. | Randriamampita, C., and Tsien, R. Y. (1993) Nature 364, 809-814[CrossRef][Medline] [Order article via Infotrieve] |
36. |
Csutora, P.,
Su, Z.,
Kim, H. Y.,
Bugrim, A.,
Cunningham, K. W.,
Nuccitelli, R.,
Keizer, J. E.,
Hanley, M. R.,
Blalock, J. E.,
and Marchase, R. B.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
121-126 |
37. |
Trepakova, E. S.,
Csutora, P.,
Hunton, D. L.,
Marchase, R. B.,
Cohen, R. A,
and Bolotina, V. M.
(2000)
J. Biol. Chem.
275,
26158-26163 |
38. | Yao, Y., Ferrer-Montiel, A. V., Montal, M., and Tsien, R. Y. (1999) Cell 98, 475-485[Medline] [Order article via Infotrieve] |
39. | Patterson, R. L., van Rossum, D. B., and Gill, D. L. (1999) Cell 98, 487-499[Medline] [Order article via Infotrieve] |
40. | Meissner, G., and Lu, X. (1995) Biosci. Rep. 15, 399-408[Medline] [Order article via Infotrieve] |
41. | Irvine, R. F. (1990) FEBS Lett. 263, 5-9[CrossRef][Medline] [Order article via Infotrieve] |
42. | Vaca, L., and Kunze, D. L. (1995) Am. J. Physiol. 269, C733-C738[Abstract] |
43. | Kiselyov, K. I., Mamin, A. G., Semyonova, S. B., and Mozhayeva, G. N. (1997) FEBS Lett. 407, 309-312[CrossRef][Medline] [Order article via Infotrieve] |
44. | Kiselyov, K. I., Semyonova, S. B., Mamin, A. G., and Mozhayeva, G. N. (1999) Pflugers Arch. 437, 305-314[CrossRef][Medline] [Order article via Infotrieve] |
45. |
Thrower, E. C.,
Mobasheri, H.,
Dargan, S.,
Marius, P.,
Lea, E. J.,
and Dawson, A. P.
(2000)
J. Biol. Chem.
275,
36049-36055 |
46. |
Ma, H. T.,
Patterson, R. L.,
van Rossum, D. B.,
Birnbaumer, L.,
Mikoshiba, K.,
and Gill, D. L.
(2000)
Science
287,
1647-1651 |
47. | Acharya, J. K., Jalink, K., Hardy, R. W., Hartenstein, V., and Zuker, C. S. (1997) Neuron 18, 881-887[Medline] [Order article via Infotrieve] |
48. | Raghu, P., Colley, N. J., Webel, R., James, T., Hasan, G., Danin, M., Selinger, Z., and Hardie, R. C. (2000) Mol. Cell Neurosci. 15, 429-445[CrossRef][Medline] [Order article via Infotrieve] |
49. | Mery, L., Magnino, F., Schmidt, K.-H., Krause, K., and Dufour, J.-F. (2001) FEBS Lett. 487, 377-383[CrossRef][Medline] [Order article via Infotrieve] |
50. | Zhang, S., Ehlers, M. D., Bernhardt, J. P., Su, C. T., and Huganir, R. L. (1998) Neuron 21, 443-453[Medline] [Order article via Infotrieve] |
51. |
Krupp, J. J.,
Vissel, B.,
Thomas, C. G.,
Heinemann, S. F.,
and Westbrook, G. L.
(1999)
J. Neurosci.
19,
1165-1178 |
52. |
Varnum, M. D.,
and Zagotta, W. N.
(1997)
Science
278,
110-113 |
53. |
Zubov, A. I.,
Kaznacheeva, E. V.,
Nikolaev, A. V.,
Alexeenko, V. A.,
Kiselyov, K.,
Muallem, S,
and Mozhayeva, G. N.
(1999)
J. Biol. Chem.
274,
25983-25985 |
54. | Vaca, L. (1996) FEBS Lett. 390, 289-293[CrossRef][Medline] [Order article via Infotrieve] |
55. | Zweifach, A., and Lewis, R. S. (1995) J. Gen. Physiol. 105, 209-226[Abstract] |
56. |
Liu, X.,
O'Connell, A.,
and Ambudkar, I. S.
(1998)
J. Biol. Chem.
273,
33295-33304 |