1 Neuroscience Research Group, Department of Physiology and Biophysics,
University of Calgary, Calgary, Alberta, T2N 4N1, Canada
2 NeuroMed Technologies Inc., Suite 301, Don Rix Building, 2389 Health Sciences
Mall, UBC, Vancouver, BC, V6T 1Z4, Canada
Author for correspondence (e-mail:
braunj{at}ucalgary.ca)
Accepted 3 April 2003
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Summary |
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Key words: Cysteine string protein, Chaperones, G proteins, N-type-calcium channels, Synaptic transmission, J domain
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Introduction |
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We have recently shown that CSP is capable of binding to both the N-type
calcium channel and to Gß in vitro and that the
interaction between CSP and the N-type calcium channel results in a robust
tonic inhibition of channel activity by G protein
ß subunits
(Magga et al., 2000
). The
CSP/G protein interaction was confirmed by co-immunoprecipitation, GST
pull-down assays, crosslinking in intact brain slices as well as evaluation of
functional proteins in HEK cells (Magga et
al., 2000
) and has recently been confirmed by others
(Evans et al., 2001
). Numerous
synaptic proteins were absent from the CSP immunoprecipitations and GST
pull-down assays, demonstrating the specificity of the CSP/G protein
interaction. Interestingly, CSP and G proteins have been shown to co-enrich in
detergent-insoluble lipid raft fractions from rat hippocampus
(Magga et al., 2002
). Binding
of G proteins appears to involve two separate regions of CSP, such that
G
interacts with the J domain of CSP in an ATP-dependent
manner, whereas G
ß associates with full-length CSP but
not the J domain of CSP in an ATP-independent fashion. Although CSP interacts
with G proteins, it is not clear exactly how CSP affects G protein function.
In particular, it is unclear exactly which regions of CSP associate with G
protein subunits. Furthermore, it is unknown whether CSP's interaction with
G
proteins is direct or requires an additional component.
Understanding the nature of the interaction between CSP, G proteins and
calcium channels is crucial in understanding the molecular role of CSP. In the
present study we have therefore analyzed the association of CSP with G
proteins and N-type calcium channels.
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Materials and Methods |
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Preparation of fusion proteins
GST fusion proteins of CSP and the J domain were prepared as described
previously (Braun et al., 1996;
Braun and Scheller, 1995
). CSP
deletion mutants were prepared by subcloning CSP PCR fragments into pGEX-KG
(Guan and Dixon, 1991
) and
expressed as GST fusion proteins in AB1899 cells. His6
1B calcium channel II-III linker synprint motif fusion
protein (amino acids 718-963) was prepared as described previously
(Jarvis et al., 2000
). The
sequences of all constructs were verified by sequencing both strands using the
dideoxynucleotide chain termination method. After induction of expression with
100 µM isopropyl-ß-D-thiogalactopyranside for 5 hours, the bacteria
were suspended in phosphate buffer saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM
Na2HPO4, 2 mM KH2PO4) supplemented
with 0.05% Tween 20, 2 mM EDTA and 0.1% ß-mercaptoethanol and lysed by
two passages through a French Press (Spectronic Instruments, Rochester, NY).
The fusion proteins were recovered by binding to glutathione agarose beads
(Sigma) or Ni2+NTA agarose (Qiagen). The fusion protein beads were
washed extensively and finally resuspended in 0.2% TritonX100, 20 mM MOPS (pH
7.0), 4.5 mM Mg(CH3COO)2, 150 mM KCl and 0.5 mM PMSF.
Recombinant CSP or CSP truncation mutants were purified from the GST fusion
protein by cleavage with 0.2 µM thrombin in 50 mM Tris pH 8, 150 mM NaCl,
2.5 mM CaCl2, followed by incubation in 0.3 mM PMSF. Synprint was
eluted from Ni2+NTA agarose with 500 mM imidazole. The protein
concentration of recombinant proteins was estimated by Coomassie Blue staining
of protein bands after SDS-polyacrylamide gel electrophoresis using bovine
serum albumin as a standard.
Immunoblotting
Proteins were transferred eletrophoretically at constant voltage from
polyacrylamide gels to nitrocellulose (0.45 µm or 0.2 µm) in 20 mM Tris,
150 mM glycine, 12% methanol. Transferred proteins were visualized by staining
with Ponceau S. Nitrocellulose membranes were blocked for non-specific binding
using 5% milk, 0.1% Tween 20, PBS solution [137 mM NaCl, 2.7 mM KCl, 4.3 mM
Na2HPO4, 1.4 mM KH2PO4 (pH 7.3)]
and incubated overnight at 4°C or 2 hours at room temperature with primary
antibody. The membranes were washed three to four times in the above
milk/Tween/PBS solution and incubated for 30 minutes with goat anti-rabbit or
goat anti-mouse IgG-coupled horseradish peroxidase. Antigen was detected using
chemiluminescent horseradish peroxidase substrate (ECL, Amersham).
Immunoreactive bands were visualized following exposure of the membranes to
Amersham Hyperfilm-MP.
Transient transfection of HEK cells
N-type calcium channel subunits, G protein subunits, the
1B II-III linker (corresponding to residues 718-963) and the
C-terminus of ßARK (corresponding to residues 495-689) were prepared as
described previously (Magga et al.,
2000
). cDNAs encoding the entire open reading frame of CSP,
CSP1-82, CSP1-112, or CSP83-198 were obtained
by polymerase chain reaction (CSP accession number U39320). Sequences were
verified and subcloned into pmt2-SX for expression. Human embryonic kidney
tsa-201 cells were grown in standard DMEM (Dulbecco's modified Eagle's
medium), supplemented with 10% fetal bovine serum and penicillin-streptomycin.
The cells were grown to 85% confluency, split with trypsin-EDTA and plated on
glass coverslips at 10% confluency 12 hours before transfection. Immediately
prior to transfection, the medium was exchanged, and a standard
Ca2+ phosphate protocol was used to transfect the cells with cDNAs
encoding Ca2+ channel subunits (
1B +
2-
+ ß1b; accession numbers
1B: M92905,
2-
:
NM000722, ß1b: AB054985) and, as appropriate green
fluorescent protein (EGFP; Clontech, CA) CSP, CSP1-82 or
CSP83-198. After 12 hours, the cells were washed with fresh DMEM
and allowed to recover for 12 hours.
Patch clamp recordings
Immediately prior to recording, individual coverslips were transferred to a
3 cm culture dish containing recording solution comprised of 20 mM
BaCl2, 1 mM MgCl2, 10 mM HEPES, 40 mM Tetraethylammonium
chloride (TEA-Cl), 10 mM glucose and 65 mM CsCl, (pH 7.2 with TEA-OH). Whole
cell patch clamp recordings were performed using an Axopatch 200B amplifier
(Axon Instruments, Foster City, CA) linked to a personal computer equipped
with pCLAMP v 6.0. Patch pipettes (Sutter borosilicate glass, BF150-86-15)
were pulled using a Sutter P-87 microelectrode puller, fire polished using a
Narashige microforge, and showed typical resistances of 3 to 4 M. The
internal pipette solution contained 108 mM CsMS, 4 mM MgCl2, 9 mM
EGTA, 9 mM HEPES (pH 7.2). Data were filtered at 1 kHz and recorded directly
onto the hard drive of the computer. Series resistance and capacitance were
compensated. Currents were evoked by stepping from -100 mV to a test potential
of +20 mV. The degree of tonic voltage-dependent G protein inhibition was
assessed by the degree of current facilitation that occurred after application
of a 50 ms depolarizing prepulse to +150 mV, 5 ms prior to the test
depolarization. The degree of prepulse relief of the G-protein inhibition was
determined by the ratio of the peak current obtained in the absence and
presence of the depolarizing prepulse (Ipeak (+pp/-pp)). The raw
data were analyzed using Clampfit and Sigmaplot (Jandel Scientific) software.
All figures were generated using Sigmaplot v 4.0. Unless stated otherwise, all
error bars are standard errors, and numbers in parentheses displayed in the
figures reflect numbers of experiments, Statistical analysis was carried out
using SigmaStat 2.0 (Jandel Scientific). Differences between mean values from
each group were tested using ANOVAS followed by a Tukey post-ANOVA test for
multiple comparisions. Differences were considered significant if
P<0.05.
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Results |
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Next we examined the nucleotide dependence of the association of
G with the J domain (residues 1-82) of CSP.
CSP1-82-GST was immobilized on glutathione agarose beads and
incubated with equal amounts of hippocampal homogenate in the presence of
various nucleotides. Unbound protein was washed away, and the presence of
G
, Gß and Hsc70 were evaluated by western
blot analysis. Fig. 2 shows
that the association of G
with immobilized recombinant J
domain-GST in vitro was dependent on ATP and that ATP
S, ADP, GTP,
GTP
S and GDP did not support J-domain-G-protein association. The
presence of Gß was analyzed by western blotting with an
anti-Gß monoclonal. Gß did not associate with
the J domain under any of the conditions examined. We have previously shown
that CSP interacts with and activates the ATPase activity of Hsc70
(Braun et al., 1996
). Hsc70 is
an abundant neural protein with coupled protein binding and ATPase activities.
Although the function of the CSP-Hsc70 complex is unknown, regulation of the
assembly/disassembly of multimeric complexes, such as presynaptic complexes,
is typical of this family of chaperone proteins. In contrast to the
J-domain-G
association, ATP was not essential for the
J-domain-Hsc70 interaction (Fig.
2). However, ATP, ADP and GTP increased the association of Hsc70
with CSP in agreement with previous reports
(Magga et al., 2000
). Overall,
these results indicate that the indirect interaction between
G
and the J domain of CSP is dependent on ATP binding and
that the J domain cannot bind Gß.
|
To investigate the stability of the association between G proteins and CSP,
we examined their in vitro association over a range of pH. GST fusion proteins
consisting of full-length CSP or the N-terminus of CSP (amino acids 1-82,
including the J domain) coupled to glutathione agarose beads were used in our
in vitro binding assay. Fig. 3
shows that G and Gß associated with
full-length CSP. However deletion of amino acids 83-198 resulted in a loss of
direct G
and Gß binding under all pH
conditions examined (in the absence of ATP). Note that at a pH of 3.0 or 10
the recombinant fusion proteins only partially remained immobilized to the
agarose beads. The G-protein-CSP complex was detected over the full pH range
examined. These observations emphasize that the nucleotide-independent direct
interaction between CSP and G proteins is a very stable complex.
|
CSP binding regions for G proteins and N-type calcium channels
To begin to understand the structural requirements for CSP association with
G proteins, a series of CSP deletion mutants were constructed, expressed and
purified (Fig. 4). The regions
of CSP required for interaction with G proteins were determined through
binding experiments with the CSP deletion mutants.
Fig. 4C shows the Coomassie
staining profile of the CSP-GST truncation mutants. Each of the CSP constructs
produced a protein that migrated at the expected molecular weight when
analyzed by SDS PAGE with the exception of CSP83-198 and CSP137-198. The
anomalous migration of these regions of CSP by SDS-PAGE is in agreement with
other reports of specific CSP truncations
(Evans et al., 2001). To assay
G protein binding, the CSP-GST fusion proteins were immobilized on glutathione
beads and incubated with equal amounts of hippocampal homogenate. Unbound
proteins were washed away and bound proteins were then denatured and
fractionated by SDS-PAGE and detected by western blotting. An interesting
pattern of binding was revealed through this analysis. Full-length CSP,
CSP83-198, CSP1-190, CSP1-180, CSP1-165 and CSP1-146, were observed to bind to
Gß in the absence of ATP. In contrast, CSP137-198 and
CSP1-82 did not associate with Gß. Addition of
residues 83-112 to the J domain of CSP dramatically increased binding of
Gß, indicating that Gß binds to CSP in a
region between amino acids 83 and 112. Some association of Gß
was also observed with the cysteine string region of CSP that is thought to be
post-translationally modified in vivo; however, such modifications may make G
protein association with this region less significant.
|
The ability of the CSP constructs to support G binding
was also examined. The association of G
with the CSP
truncation constructs was not robust; however, weak associations were
identified with full-length CSP, CSP83-198, CSP1-190, CSP1-180, CSP1-165 and
CSP1-146. In addition to the identification of hippocampal
G
, anti-G
polyclonal (Calbiochem) was
observed to crossreact nonspecifically with abundant proteins, especially
CSP83-198 and CSP137-198 (Fig.
4B). However, anti-G
i polyclonal
(Santa Cruz Inc) confirmed the association of G
with CSP but
it did not crossreact with the recombinant proteins, suggesting that the
G
that interacts with CSP in the rat hippocampus is a
G
i isoform. The finding that two independent
antibodies detect association of G
with CSP confirms the
G
-CSP interaction. Relatively minor amounts of
G
or Gß were observed to associate with GST
alone compared to CSP, demonstrating the specificity of the CSP-G protein
interaction. Taken together our data demonstrate that the N-terminal binding
site of CSP, which includes the J domain, binds to G
subunits but not G
ß subunits, whereas the C terminal
binding site of CSP associates with either free G
ß
subunits or G
ß in complex with G
.
The CSP deletion mutants were also evaluated for their association with
1B His6 synprint region (amino acids 718-963) of
the N-type calcium channel (Fig.
5). To assay synprint binding, the CSP-GST deletion mutants were
immobilized on glutathione agarose beads and then incubated with soluble
His6 synprint followed by several washes to remove unbound protein.
The proteins were then denatured and fractionated by SDS-PAGE, and bound
synprint was detected by western blotting. Full-length CSP, CSP83-198,
CSP1-190, CSP1-180, CSP1-165, CSP1-146 and CSP1-112 were observed to bind
synprint. Several His6 fusion proteins in addition to synprint were
evaluated and observed not to interact with CSP (data not shown), which
demonstrates the specificity of the CSP-synprint association. Synprint did not
associate with CSP1-82 or CSP137-198. Deletion of CSP residues
83-112 corresponding to the `linker' region significantly reduced synprint's
association with CSP, indicating the importance of amino acids 83-112 for the
CSP-synprint interaction. Like that found for G
and
Gß, some association of synprint was observed with the
cysteine string region of CSP that is thought to be post-translationally
modified in vivo. Overall, the binding profile indicates that amino acids
between 83-112 of CSP are important for binding synprint, G
and Gß subunits.
|
Two distinct domains of CSP trigger G protein inhibition of N-type
channels
We have previously shown that co-expression of Cav.2.2 N-type
(1B +
2 -
+ ß1b)
calcium channels with CSP in tsa-201 cells results in a tonic inhibition of
channel activity by G protein
ß subunits, which can be reversed by
application of strong depolarizing prepulses
(Magga et al., 2000
). This
effect was antagonized by the C-terminal fragment of the ß adrenergic
receptor kinase, a known G
ß-binding protein and by
overexpression of the CSP-binding region on the N-type calcium channel. On the
basis of this evidence, we proposed that CSP acts as a chaperone to promote
N-type calcium channel-G=protein interactions. Since the cysteine string
region appears to interact with both the N-type calcium channel domain II-III
linker and Gß (i.e. Figs
4 and
5), we hypothesized that
co-expression of this region with N-type calcium channels should mimic the
ability of full-length CSP to promote G protein inhibition of N-type calcium
channels. As shown in Fig. 6,
this is indeed the case. Whereas N-type channels undergo little prepulse
facilitation in the absence of coexpressed CSP, coexpression of the cysteine
string (CSP83-198) induces tonic modulation that can be reversed by
application of a +150 mV voltage pulse, resulting in a 65% increase in peak
current amplitude (Fig. 6).
These data are consistent with our previous model suggesting that CSP may
anchor and chaperone G protein
ß subunits to the N-type calcium
channel (Magga et al.,
2000
).
|
By contrast, based on the inability of the J domain (CSP1-82) to
interact with the channel or ß subunits, one might expect
co-expression of this region to be ineffective in mediating N-type channel
inhibition. To our surprise, however, co-expression of the channel with the J
domain resulted in robust prepulse relief, independently of the presence of
ATP in the patch pipette (Fig.
6). In addition to CSP1-82, co-expression of the
channel with CSP1-112 also resulted in robust prepulse relief (data not
shown). To assess whether the CSP1-82 and CSP83-198 inhibition of the N-type
channel both involved G proteins, we co-expressed N-type channels together
with CSP and the C-terminal fragment of the ß-adrenergic receptor kinase
(ßARK), a known G
ß-binding protein
(Fig. 6). The degree of PP
relief was greatly attenuated in the presence of the ßARK fragment,
indicating that the CSP-induced N-type calcium channel inhibition is G protein
mediated.
If our hypothesis that the cysteine string region serves to colocalize the
channel and Gß is correct, overexpression of the
CSP-binding region on the channel molecule (i.e. the synprint region) should
uncouple CSP from the channel and thereby eliminate the G protein effect. As
expected, this did indeed occur. As shown in
Fig. 7, the ability of the
cysteine string domain to induce G protein inhibition of the channel was
virtually abolished following coexpression of the synprint motif. In contrast,
the synprint region did not interfere with the J-domain-mediated effect
(Fig. 7), indicating that the
J-domain-mediated effect occurs independently of the N-type channel domain
II-III linker. Hence, separate portions of the CSP molecule can independently
promote G protein inhibition of transiently expressed N-type calcium channels,
and they can do this by distinct molecular mechanisms.
|
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Discussion |
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The observation that the J domain (CSP1-82) independently
triggers G protein inhibition of the channel is somewhat unexpected
(Fig. 6). Neither
Gß nor synprint were observed to associate with the J domain
under any conditions examined (Figs
2,
3,
4,
5)
(Magga et al., 2000). However,
G
subunits were observed to associate with the J domain of
CSP in an indirect and ATP-dependent manner. In contrast to that observed for
CSP83-198, co-expression of synprint with CSP1-82 and N-type calcium channels
did not affect the G protein inhibition of the J domain
(Fig. 7). This raises the
possibility that the J domain might either stimulate the dissociation of the
G
ß
trimer or, alternatively, that the J domain
may prevent the assembly of the trimer. In each case, this would result in
free G
ß subunits that would then be available to
produce a tonic inhibition of the channel. To discriminate between the
alternatives, we perfused J domain peptides into tsa-201 cells expressing
N-type channels, but did not observe the development of G protein inhibition
over a 10 minute time course (data not shown), indicating that the J domain
cannot acutely trigger the activation of the G protein complex. Hence, we
favor a model in which the J domain inhibits the assembly of
G
to G
ß subunits. This would also be
consistent with the observation that the physiological effects of the J domain
did not depend on the presence of ATP in the patch pipette
(Fig. 6), since the
J-domain-G
interactions would probably already occur before
recordings/patch rupture.
The question remains as to whether two separate actions occur with the
full-length CSP construct. As we showed previously
(Magga et al., 2000), the
effect of CSP on G protein inhibition of N-type calcium channels was
antagonized by overexpression of the synprint motif, indicating that a large
part of the action of CSP was due to the interaction of the cysteine string
region with the calcium channel II-III linker region. Nonetheless, unlike in
the case of the CSP83-198, this inhibition was partial, leaving a
statistically significant portion of the G protein effect intact
(Magga et al., 2000
). It is
conceivable that the remaining modulation was mediated by the interaction of
the J domain with G
.
The identification of specific Hsc70/DnaJ chaperone machines and the
molecular events they regulate in vivo remains a central biological question.
Compelling evidence indicates that CSP interacts with and activates the ATPase
activity of members of the heat-shock family Hsp70
(Braun et al., 1996;
Bronk et al., 2001
;
Chamberlain and Burgoyne, 1997
;
Stahl et al., 1999
). Recently,
SGT (small glutamine rich tetratricopeptide repeat) has been shown to be a key
component of the CSP-Hsc70 chaperone machine
(Tobaben et al., 2001
).
Formation of the trimeric CSP-SGT-Hsc70 complex strongly activates the ATPase
activity of Hsc70 (Tobaben et al.,
2001
). Given the number of proteins and protein complexes
participating in neurotransmitter release, it is likely that CSP chaperone
activity is important in supervising specific transitions and interactions
among synaptic proteins, such as G proteins, during the rapid
exocytosis/endocytosis synaptic vesicle cycle.
CSP was originally proposed to promote the activity of presynaptic calcium
channels (Mastrogiacomo et al.,
1994; Gundersen and Umbach,
1992
). However, additional lines of evidence have suggested
alternative CSP/calcium channel models. Several studies conclude that CSP is
important in exocytosis rather than the regulation of calcium transmembrane
fluxes (Brown et al., 1998
;
Chamberlain and Burgoyne, 1998
;
Zhang et al., 1999
;
Zhang et al., 1998
;
Cribbs et al., 1998
;
Graham and Burgoyne, 2000
). In
contrast, the synprint sites of P/Q calcium channels
(Leveque et al., 1998
) and N
type calcium channels (Magga et al.,
2000
) have been shown to bind CSP. Although CSP has been proposed
to play a role in the recruitment of calcium channels
(Chen et al., 2002
), CSP has
also been proposed to play an inhibitory role in depolarization dependent
calcium entry (Dawson-Scully et al.,
2002
), which is consistent with our results
(Magga et al., 2000
). In
addition to G proteins (Magga et al.,
2000
) and calcium channels
(Chen et al., 2002
;
Leveque et al., 1998
;
Wu et al., 1999
;
Magga et al., 2000
), several
other targets of CSP chaperone activity have been proposed including syntaxin
(Nie et al., 1999
;
Evans et al., 2001
;
Wu et al., 1999
), VAMP (also
called synaptobrevin) (Leveque et al.,
1998
), synaptotagmin I (Evans
and Morgan, 2002
),
GDI
(Sakisaka et al., 2002
) and
CFTR (Zhang et al., 2002
).
Further experimentation is required to establish the role of CSP in
controlling the aggregation state and conformation of these possible target
proteins and the nature of the conflicting results regarding the role of CSP
in exocytosis.
In conclusion, our results reveal that two separate motifs of CSP bind to G
proteins and regulate G protein inhibition of N-type calcium channels. The
multitude of G-protein-triggered cascades and the distinct mechanisms of
action by CSP N- and C-terminal domains may, in part, explain the paradoxical
findings regarding CSP's role in exocytosis. Chaperone-assisted protein
folding of signaling components has been shown for other signal transduction
cascades (e.g. transcription factors and glucocorticoid receptors) (reviewed
in Hartl and Hayer-Hartl,
2002). The regulation of G protein function by chaperones such as
CSP represents an important concept with regard to the control of
neurotransmitter release and synaptic efficacy.
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Acknowledgments |
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![]() |
Footnotes |
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References |
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