 |
 Previous Article | Next Article 
The Journal of Neuroscience, May 1, 2001, 21(9):2939-2948
Distinct Molecular Determinants Govern Syntaxin 1A-Mediated
Inactivation and G-Protein Inhibition of N-Type Calcium Channels
Scott E.
Jarvis and
Gerald W.
Zamponi
Departments of Physiology and Biophysics and Pharmacology and
Therapeutics, Neuroscience and Smooth Muscle Research Groups,
University of Calgary, Calgary, T2N 4N1, Canada
 |
ABSTRACT |
We have reported recently that syntaxin 1A mediates two
effects on N-type channels transiently expressed in tsA-201 cells: a
hyperpolarizing shift in the steady-state inactivation curve as well as
a tonic inhibition of the channel by G-protein   subunits (Jarvis
et al., 2000 ). Here we have examined some of the molecular determinants
and factors that modulate the action of syntaxin 1A on N-type calcium
channels. With the additional coexpression of SNAP25, the syntaxin
1A-induced G-protein modulation of the channel became reduced in
magnitude by ~50% but nonetheless remained significantly higher than
the low levels of background inhibition seen with N-type channels
alone. In contrast, coexpression of nSec-1 did not reduce the syntaxin
1A-mediated G-protein inhibition; however, interestingly, nSec-1 was
able to induce tonic G-protein inhibition even in the absence of
syntaxin 1A. Both SNAP25 and nSec-1 blocked the negative shift in
half-inactivation potential that was induced by syntaxin 1A. Activation
of protein kinase C via phorbol esters or site-directed mutagenesis of
three putative PKC consensus sites in the syntaxin 1A binding region of
the channel (S802, S896, S898) to glutamic acid (to mimic a permanently
phosphorylated state) did not affect the syntaxin 1A-mediated G-protein
modulation of the channel. However, in the S896E and S898E mutants, or
after PKC-dependent phosphorylation of the wild-type channels, the
susceptibility of the channel to undergo shifts in half-inactivation
potential was removed. Thus, separate molecular determinants govern the ability of syntaxin 1A to affect N-type channel gating and its modulation by G-proteins.
Key words:
SNARE proteins; protein kinase C; G  ; phosphorylation; calcium
channels; site-directed mutagenesis
| |
INTRODUCTION |
The interaction of presynaptic
calcium channels with the vesicle release core complex is of
fundamental importance for neurotransmitter release (Pevsner et al.,
1994a; Mochida et al., 1996; Stanley, 1997) (for review, see Linial and
Bledi, 2000). In the presynaptic nerve terminal, calcium currents are
generated predominantly by P/Q-type and N-type calcium channels
(Westenbroek et al., 1992, 1995). They are colocalized with synaptic
vesicles by calcium-dependent binding of the t-SNARE proteins syntaxin
1A and SNAP25 to the domain II/III linker region of the N-type and
P/Q-type calcium channel  1 subunits [termed
synaptic protein interaction (synprint) site; residues 718-963 of the
1B subunit] and by direct interaction of
these two proteins with the v-SNARE proteins synaptotagmin and
synaptobrevin (Sheng et al., 1994, 1996; Rettig et al., 1996, 1997;
Walker and De Waard, 1998) (for review, see Lin and Scheller, 2000).
The amount of calcium entry via the channel and, thus, neurotransmitter
release is modulated with the activation of certain second messenger
systems, including protein kinase C (PKC) and G-protein subunits
(Dunlap and Fischbach, 1981; Swartz, 1993; Swartz et al., 1993; Zamponi
et al., 1997; Hamid et al., 1999). Moreover, the interactions between
the channels and the SNARE protein complex can be modulated by
phosphorylation of the syntaxin 1A binding site on the channel
(Yokoyama et al., 1997) and by other types of presynaptic proteins such
as cysteine string protein (Umbach and Gunderson, 1997; Wu et al.,
1999).
The interaction between syntaxin 1A and the N-type calcium channel
1 subunit has two major functional
consequences. First, with the coexpression of syntaxin 1A and
1B N-type calcium channels, the channels
undergo a 15-20 mV negative shift in half-inactivation potential
(Bezprozvanny et al., 1995, 2001; Jarvis et al., 2000) as well as
enhanced slow inactivation (Degtiar et al., 2000). Furthermore, in the
presence of syntaxin 1A the channels become subject to a large tonic
inhibition by G-protein subunits, which can be relieved with the
application of strong depolarizing prepulses (Jarvis et al., 2000).
Although the basis of the syntaxin 1A effect on channel inactivation is
not yet understood, G-protein inhibition may occur via a syntaxin
1A-mediated colocalization of the calcium channel
1 subunit and endogenous
G (Jarvis et al.,
2000).
Here we identify novel factors that affect the ability of
syntaxin 1A to promote G-protein modulation and voltage-dependent inactivation of N-type calcium channels. We show that coexpression of
either SNAP25 or neuronal Sec-1 (nSec-1) antagonizes the ability of
syntaxin 1A to inactivate N-type calcium channels, whereas the ability
of syntaxin 1A to enhance tonic G-protein inhibition is retained in the
presence of SNAP25 and nSec-1. PKC-dependent phosphorylation of the
channel induced by the application of phorbol esters or point mutations
mimicking the effects of protein kinase C-dependent phosphorylation of
the synprint region also preclude the inactivation effect of syntaxin
1A while leaving the G-protein effects intact. Thus, the molecular
determinants that govern the two syntaxin 1A-mediated phenomena are
distinct in nature. Moreover, our data may suggest that syntaxin 1A
might have only a transient effect on N-type channel inactivation
in vivo, depending on the presence of other SNARE proteins
and the activity of PKC.
| |
MATERIALS AND METHODS |
Molecular biology
Scoring of PKC consensus sites
The likelihood of PKC-dependent phosphorylation of PKC consensus
sites was obtained by using the probability values reported by Kennelly
and Krebs (1991). Scores were assigned depending on the proximity of
basic (i.e., arginine or lysine) residues in positions  1 to 3 and
+1 to +3 around threonine and serine residues as follows:
R/K 3, 25; R/K2, 31;
R/K1, 9; R/K+1, 5;
R/K+2, 34; R/K+3, 24. For
S/T residues bracketed on either side by basic residues on either side,
the scores were added. For S/T sites flanked by more than one basic residue on the same side, the higher score of the two was assigned. CaMKII sites were assigned on the basis of the sequence R-X-X-S/T (see
Kennelly and Krebs, 1991).
Mutagenesis of 1B synprint
phosphorylation sites
The pSL1180 vector was digested with
StuI and SmaI to remove a 151 bp section of the polycloning site (PCS), creating
pSL1180( Stu-Sma). The full-length
1B calcium channel clone (Dubel et al., 1992) in a cytomegalovirus (CMV) mammalian expression vector, kindly provided
by Dr. T. P. Snutch (University of British Columbia, Vancouver,
Canada), was digested with SpeI and BsiWI,
liberating a fragment coding for the first 969 amino acids of
1B plus 700 bp of CMV vector sequence, which
was ligated into pSL1180(Stu-Sma) to give
pSL1180(1B SpeI, BsiWI). This
construct was digested by AgeI to liberate a fragment coding
for amino acids 438-951, which was inserted into the PCS of pSL1180 to
give pSL1180(1B AgeI). This construct
consequently was used as the template in mutagenesis reactions.
Mutagenesis was performed with QuikChange Site-Directed Mutagenesis
(Stratagene, La Jolla, CA). Reaction conditions in a volume of 50 µl
consisted of 5 µl of 10× reaction buffer, 1 µl dNTP mix, 2.5 U of
PfuTurbo DNA polymerase, 5 ng of pSL1180-AgeI
(or subsequent mutated constructs), and 125 ng of each forward and
reverse primer (University of Calgary Core DNA services). Using a
PTC-100HB thermal cycler (MJ Research, Watertown, MA), we
hot-started the reaction and held it at 95°C for 30 sec. We
conducted 16 cycles, which consisted of denaturation for 30 sec at
95°C, annealing for 1 min at 55°C, and extension for 16 min at
68°C. The reaction product was cooled to 37°C and digested with
DpnI for 2 hr. Then the DpnI-digested reaction
was transformed into DH5 supercompetent cells (Life Technologies,
Burlington, Ontario, Canada). DNA was isolated from colonies with a
QIAprep Spin Miniprep (Qiagen, Chatsworth, CA) and sequenced. The
mutated pSL1180(1B AgeI) construct was
digested with AgeI, and the 1513 bp fragment was isolated
and ligated into the original AgeI-digested
pSL1180(1B SpeI, BsiWI)
construct. Correct orientation was confirmed by checking the
SacI digestion pattern. Then the construct was digested with
SpeI and BsiWI, and the fragment containing the
mutations was ligated into the original CMV 1B construct.
All restriction enzymes were purchased from New England Biolabs
(Mississauga, Ontario, Canada), unless otherwise stated. All digested
DNA was run on 0.8% agarose gels, extracted, and purified with
QIAquick Gel Extraction columns (Qiagen). T4 DNA ligase
(Roche-Boehringer Mannheim, Laval, Quebec, Canada) was used for
all ligations. Bacterial alkaline phosphatase (BAP) and all PCR
reagents and primers were purchased from Life Technologies, unless
otherwise stated. Rat brain cDNA (oligo-dT-primed) was kindly provided
by Bob Winkfein (University of Clagary, Calgary, Canada). PCR and
mutagenesis reactions were performed in a PTC-100HB thermal cycler (MJ Research).
Cloning of SNARE proteins
The syntaxin 1A construct used here was the same as that
described in Jarvis et al. (2000). The cloning of syntaxin 1B was performed via RT-PCR, as described (Lu et al., 2001).
Cloning of SNAP25. A forward primer containing a
NotI restriction site and an ideal Kozak sequence and a
reverse primer containing a ClaI restriction site downstream
of the stop codon were used to clone SNAP25 via PCR from rat brain
cDNA. The primer sequences were as follows: forward
5'-GAGCGGCCGCCATGGCCGAGGACGCAGACATGC-3' and reverse
5'-GGAGAGCACAGCAGAAGGATCGATTTAACCACTTCCCAGC-3'.
The PCR reaction solution, in a 50 µl volume, consisted of (in
mM) 20 Tris-HCl, pH 8.4, 50 KCl, dNTPs (0.2 each), and 1.5 MgCl2 plus 2.5 U of platinum Taq DNA
polymerase, 20 pmol of each primer, and 50 ng of cDNA. The reaction was
hot-started and held at 94°C for 2 min. We conducted 34 cycles, which
consisted of denaturation for 30 sec at 94°C, annealing for 45 sec at
60°C, and extension for 1.15 min at 72°C. The resultant SNAP25b DNA product was ligated into a pGEM T-Easy vector (Promega, Madison, WI)
and sequenced. The SNAP25-T-Easy construct was digested with NotI and ClaI; the liberated SNAP25 fragment was
ligated with T4 ligase (Roche-Boehringer Mannheim) into pMT2sx for
subsequent expression studies.
Cloning of nSec-1. A forward primer containing a
SpeI restriction site and an ideal Kozak sequence and a
reverse primer containing a ClaI restriction site downstream
of the stop codon were used to clone nSec-1 from rat cDNA with the
following primer sequences: forward
5'-CGGAGACTAGTAGGCCGCCATGGCCC-3' and reverse
5'-GCCAGGGTTTTGGAGGATCGATATTTTAACTGCTTATTTCTTCG-3'.
The PCR reaction solution, in a 50 µl volume, consisted of (in
mM) 20 Tris-HCl, pH 8.8, 10 KCl, 1.5 MgCl2, 2 MgSO4, and 10 (NH4)2SO4
plus 0.1% Triton X-100, 5 µg BSA, dNTPs (0.2 mM each), 2.5 U of PfuTurbo (Stratagene), 20 pmol of each primer, and
50 ng of cDNA. The reaction was hot-started and held at 94°C for 2 min. We conducted 30 cycles, which consisted of denaturation for 30 sec
at 94°C, annealing for 45 sec at 60°C, and extension for 5 min at
74°C. The resultant blunt-ended DNA product was ligated into
pCR-Blunt II-TOPO (Invitrogen, San Diego, CA) and sequenced. The
nSec-1-pCR-Blunt II construct was digested with SpeI and
ClaI; the liberated nSec-1 fragment was ligated into pMT2sx
for expression studies.
Biochemistry
Generation and preparation of fusion proteins
6xHis synprint fusion proteins were generated and prepared as
described previously (Jarvis et al., 2000). Briefly, residues 718-963
from the II/III linker of the 1B calcium
channel (wild type and mutants) were amplified and subcloned into
pTrcHisC (Invitrogen), followed by transformation into
Escherichia coli TOP10. Induction and preparation of
purified fusion proteins were performed by using conditions adapted
from the manufacturer and described in detail in Jarvis et al.
(2000).
Immunoblots for SNARE proteins in human embryonic kidney
(HEK) cells
Primary antibodies were purchased from StressGen (Victoria,
British Columbia). Secondary antibodies were purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). tsA-201 cells were transfected with SHAM, SNAP-25, nSec1, or syntaxin 1A as described below and prepared for Western blotting as described previously in Jarvis et al.
(2000). Anti-syntaxin 1A and anti-SNAP25 monoclonal antibodies were
used at 1:1000 and 1:2000, respectively; anti-nSec1 polyclonal was used
at 1:1000 in a 5% solution of skim milk powder in PBS supplemented
with 0.1% Triton X-100 (PBST), all for 2 hr at room temperature.
Secondary antibodies were used at 1:2000 in a 2% solution of skim milk
powder in PBST for 1 hr at room temperature.
In vitro phosphorylation of the II/III synprint
peptide and syntaxin binding assay
6xHis-tagged synprint peptides (wild type and EEE) were prepared
as described previously (Jarvis et al., 2000). Purified synprint peptides were phosphorylated by 0.25 µg of PKC (Sigma, Oakville, Ontario, Canada) for 3 hr at 30°C in a solution containing (in mM) 20 HEPES, pH 7.4, 10 MgCl2, 10 DTT, 0.15 CaCl2, and 0.4 ATP plus 20% mixed
micelles (PS and 1,2-DG). After phosphorylation the peptides were
washed three times with TBS and quantified by a Bradford assay.
Syntaxin 1A-glutathione S-transferase (GST) was purified as described
previously (Jarvis et al., 2000). The GST tag was removed by cleavage
with thrombin (Sigma), and the purified syntaxin 1A was dialyzed
overnight against Tris-buffered saline (TBS; 10 mM
Tris-HCl, pH 7.4, and 150 mM NaCl). Syntaxin 1A and the
synprint peptides were quantified by using a Bradford assay against a
standard BSA curve.
Synprint peptides were immobilized on
Ni2+-NTA beads, and syntaxin 1A was added
in ratios ranging from 1:8 to 1:1. The proteins were incubated in TBS
supplemented with 0.1% Triton X-100 (TBST) at 4°C with end-over-end
rotation for 3 hr. After incubation the beads were washed with 30 bed
volumes of TBST. Laemmli sample buffer (2×) was added to the washed
beads, which then were boiled for 2 min at 90°C and shaken at 4°C
for 2 hr to ensure the dissociation of the complexes. The protein
samples subsequently were subjected to SDS-PAGE; Western blots were
performed as described previously (Jarvis et al., 2000).
| |
Tissue culture and transient transfection of tsA-201 cells |
HEK cells were grown to 80% confluence in DMEM medium
supplemented with 10% fetal bovine serum and 1%
penicillin/streptomycin (v/v). Cells were split and plated on glass
coverslips at 10% confluence 12 hr before transfection. Immediately
before transfection the medium was renewed, and a calcium phosphate
transfection procedure was used to transfect cDNAs encoding for
wild-type or mutant 1B, 2- , and 1b
subunits and the reporter gene EGFP (Clontech, Cambridge, UK). Cells
were washed after 12 hr and maintained at 37°C for an additional 12 hr before being moved into a CO2 incubator set at
28°C. The cells were maintained under those conditions for 24-72 hr
before recording.
| |
Patch-clamp recordings |
Glass coverslips carrying transfected cells were transferred to
a 3 cm culture dish and bathed in recording solution consisting of (in
mM) 20 BaCl2, 1 MgCl2, 10 HEPES, 40 tetraethylammonium chloride
(TEA-Cl), 10 glucose, and 65 CsCl (pH 7.2 with TEA-OH). Whole-cell
patch-clamp recordings were performed with an Axopatch 200B amplifier
(Axon Instruments, Foster City, CA) linked to a personal computer
equipped with pClamp version 6.0. Patch pipettes (Sutter borosilicate
glass, BF150-86-15) were pulled with a Sutter P-87 microelectrode
puller and fire-polished; they showed typical resistances of 3-4 M .
The internal pipette solution contained (in mM) 108 cesium
methane sulfonate, 4 MgCl2, 9 EGTA, and 9 HEPES, pH 7.2. Data were filtered at 1 kHz and recorded directly onto the hard
drive of the computer. Unless stated otherwise, currents were evoked by
stepping from 100 mV to a test potential of +20 mV. G-protein
inhibition was assessed by the application of a strong depolarizing
(+150 mV) prepulse (PP) of 50 msec duration preceding the test
depolarization by 10 msec. Steady-state inactivation curves were
obtained by maintaining the cells at various holding potentials for 5 sec before stepping to a test depolarization of +10 mV.
The raw data were analyzed with Clampfit software. Steady-state
inactivation curves were fit with standard Boltzmann relations, using
Sigmaplot software (Jandel Scientific, San Rafael, CA). All figures
were generated in Sigmaplot. The numbers in parentheses reflect
individual experiments that typically were obtained in multiple
transfections. The error bars that are given reflect SE; p
values reflect Student's t tests, except in Figure
2C in which we used a one-way ANOVA test
(Student-Newman-Keuls method).
| |
RESULTS |
Syntaxin 1A exerts dual effects on N-type channel function
We have shown previously that syntaxin 1A promotes G-protein
inhibition of N-type channels, which may occur by a syntaxin 1A-mediated colocalization of
G and the N-type
channel 1 subunit (Jarvis et al., 2000). This
effect of syntaxin 1A is illustrated in Figure
1A in the form of
current records. With the coexpression of syntaxin 1A, N-type
(1B+1b+2-) currents can be enhanced dramatically after the application of a strong
depolarizing prepulse (PP), consistent with tonic G-protein inhibition.
To provide additional support for the involvement of G-protein
subunits, we coexpressed syntaxin 1A and the channel with the
C-terminal fragment of the -adrenergic receptor kinase (-ARKct,
residues 495-689; see Magga et al., 2000) to create a
G sink (Koch et al.,
1994). As shown in Figure 1A, in the presence of the
fragment the effect of syntaxin 1A became greatly diminished from
86 ± 10 to 29 ± 3% PP relief (<0.05) (Fig.
1B), further supporting a role of
G in the PP effect.
To demonstrate that the effect of syntaxin 1A-mediated G-protein
inhibition was dependent on a physical interaction between the channel
and syntaxin 1A, we overexpressed a peptide of the synprint region on
the N-type channel (residues 718-963; see Magga et al., 2000) to
inhibit syntaxin 1A binding competitively to the channel. As seen in
Figure 1B, coexpression with the synprint peptide
resulted in a significant reduction in the degree of PP relief (28 ± 4%), indicating that the effect of syntaxin 1A on G-protein
inhibition of the channel is indeed dependent on syntaxin 1A binding to
the channel rather than on a diffuse activation of G-protein signaling
in the tsA-201 cells. If the syntaxin 1A-mediated enhancement of
G-protein inhibition instead had been attributable to a syntaxin
1A-mediated transport of
G to the plasma
membrane, the synprint peptide should not have been able to interfere
with such a process because syntaxin 1A can interact concomitantly with
G and the synprint
region (Jarvis et al., 2000).
View larger version (32K):
[in this window]
[in a new window]
|
Figure 1.
Effect of syntaxin 1A on N-type channel function.
A, Current records obtained from N-type
(1B + 1b + 2-) calcium
channels coexpressed with either syntaxin 1A (top) or
syntaxin 1A plus the C-terminal fragment of the -adrenergic receptor
kinase (-ARKct;
bottom). In the presence of syntaxin 1A the channels
undergo a pronounced tonic inhibition, which is reversed with the
application of a strong depolarizing prepulse (PP). In
the presence of -ARKct the PP effect is attenuated, consistent with
the involvement of G. B,
Comparison of the degree of PP relief obtained in the absence of
syntaxin 1A, in the presence of syntaxin 1A alone, or in combination
with -ARKct or a peptide fragment of the synprint region of the
channel. C, Steady-state inactivation curves obtained
from transiently expressed N-type channels in the absence and presence
of syntaxin 1A. The data were fit with the Boltzmann relation; the
half-inactivation potentials obtained from the fits were 44.2 and
59.7 mV in the absence and presence of syntaxin 1A, respectively,
with respective slope factors of 3.9 and 3.0. Inset,
Half-inactivation potentials obtained in the absence of syntaxin 1A, in
the presence of syntaxin 1A alone, or with syntaxin 1A plus either
-ARKct or the synprint peptide. The numbers in
parentheses indicate the numbers of experiments; error
bars denote SE.
|
|
A second action of syntaxin 1A is illustrated in Figure 1C.
With the coexpression of syntaxin 1A the half-inactivation potential observed with wild-type 1B
(1b+2-) calcium
channels was shifted toward more hyperpolarized potentials by ~15 mV,
which is consistent with previous results obtained in
Xenopus oocytes (Bezprozvanny et al., 1995, 2001). Unlike in
the case of the G-protein effect, the coexpression of the -ARKct
fragment did not affect the syntaxin 1A-mediated shift in
half-inactivation potential significantly (p > 0.05), indicating that the effect of syntaxin 1A on channel inactivation occurs independently of
G. As expected, the
coexpression of the synprint peptide completely blocked the shift in
half-inactivation potential. Overall, the data presented in Figure 1
illustrate two separate actions of syntaxin 1A on N-type channel gating
and modulation by G-proteins and show that the two effects are not
coupled to each other.
Coexpression of nSec-1 and SNAP25 affects syntaxin 1A action
Besides syntaxin 1A, the N-type calcium channel domain II/III
linker also interacts with SNAP25 (Yokoyama et al., 1997). In addition,
syntaxin 1A tightly binds to nSec-1 (Hata et al., 1993; Pevsner et al.,
1994b). This raises the possibility that the effects of syntaxin 1A on
calcium channel function may be affected by these two proteins. To test
this possibility, we cloned nSec-1 and SNAP25 from rat brain,
coexpressed them individually with the N-type calcium channel and
syntaxin 1A in tsA-201 cells, and assessed the level of tonic G-protein
inhibition and inactivation properties of the channel.
To ensure that our SNAP25 and nSec-1 constructs were expressed in
tsA-201 cells, we performed Western blot analysis of the transfected
cells. As seen in Figure
2A, whereas tsA-201
cells did not express the syntaxin 1A, nSec-1, or SNAP25 endogenously, transient transfection of the appropriate cDNA constructs resulted in
bands that were detected by specific antibodies to the three proteins.
Hence, we conclude that our cDNA constructs are expressed effectively
in our experimental system and only when exogenously transfected.
View larger version (32K):
[in this window]
[in a new window]
|
Figure 2.
Effect of SNAP25 and nSec-1 on syntaxin
1A-mediated effects on N-type calcium channels. A,
Immunoblots from tsA-201 cell lysates obtained from sham-transfected
cells () or cells transfected with syntaxin 1A, SNAP25, or nSec-1
(+). Note that the three proteins are detected only when exogenously
expressed. B, Degree of PP relief obtained in various
combinations of syntaxin 1A, SNAP25, and nSec-1. Note that SNAP25
reduces the degree of syntaxin 1A-mediated G-protein inhibition and
that coexpression of the channel with nSec-1 per se results in
pronounced PP relief. C, Effect of SNAP25 and nSec-1 on
the syntaxin 1A-mediated shift in half-inactivation potential. Similar
to what is observed with syntaxin 1A, SNAP25 mediates a hyperpolarizing
shift in half-inactivation potential; however, in the presence of both
syntaxin 1A and SNAP25, or syntaxin 1A and nSec-1, the
half-inactivation potential returns to control levels. The data
obtained with SNAP25 plus syntaxin 1A differed from those obtained with
either syntaxin 1A or SNAP25 alone, whereas no statistical difference
was found in conditions in which the two proteins were present
individually (one-way ANOVA). Error bars denote SE; the
numbers in parentheses indicate the
numbers of experiments. The control and syntaxin 1A data in
B and C are the same as in Figure
1.
|
|
Figure 2B examines the degree of prepulse relief that
is obtained with transiently expressed N-type calcium channels under several experimental conditions. As already shown in Figure 1, in the
absence of any exogenously expressed SNARE proteins the N-type calcium
channels showed only a low degree of tonic G-protein inhibition (9 ± 5% relief) that was enhanced dramatically after the coexpression of
syntaxin 1A. Coexpression of the channel with SNAP25 alone did not
induce G-protein inhibition (15 ± 4% PP relief; p > 0.05 vs control) but was able to reduce the
syntaxin 1A-mediated effect significantly, although substantial
modulation (44 ± 6% relief) remained. nSec-1 did not appear to
affect the syntaxin 1A-mediated G-protein inhibition; however,
surprisingly, nSec-1 alone produced a large tonic inhibition of the
channel, thus precluding us from ruling out the possibility that nSec-1
could interfere functionally with the syntaxin 1A-mediated inhibition.
Commonly, it is thought that nSec-1 does not interact directly with the channel but rather with syntaxin 1A (Misura et al., 2000); hence, unlike syntaxin 1A, nSec-1 by itself is unlikely to be able to mediate
a colocalization of the channel and
G. Furthermore,
Western blots indicate that nSec-1 expression does not induce the
expression of syntaxin 1A (data not shown). One possible explanation of
this effect could be a direct interaction between nSec-1 and
G , thereby creating a
G sink and making endogenous
G available for modulating the channel, but additional biochemical work will be needed
to substantiate this possibility. Nonetheless, our data show that
syntaxin 1A-mediated tonic G-protein inhibition of the channel is
maintained in the presence of other SNARE proteins.
To test whether the presence of SNAP25 or nSec-1 could affect the
ability of syntaxin 1A to promote voltage-dependent inactivation of the
channel, we recorded steady-state inactivation curves from transiently
expressed N-type channels either in the absence of or in the presence
of various combinations of SNARE proteins. As shown in Figure
2C, qualitatively similar to syntaxin 1A, SNAP25 significantly shifted the half-inactivation potential of the channel toward more depolarized potentials (from 44.9 ± 1.8 to
53 ± 1.6 mV). In contrast, the coexpression of nSec-1 did not
affect channel gating per se but ablated the syntaxin 1A-mediated shift in half-inactivation potential. Interestingly, despite the finding that
syntaxin 1A and SNAP25 both affected inactivation when coexpressed with
the channels individually, no shift was observed when both proteins
were present concomitantly (Vh = 42.5 ± 6 mV). This may be consistent with the idea that SNAP25
and syntaxin 1A form a highly stable complex (Söllner et al.,
1993; Hayashi et al., 1994) in which these proteins may adopt a
different conformation that is not conducive to inducing shifts in
half-inactivation potentials. Thus, the full effects of syntaxin 1A on
N-type channel inactivation are dependent on the absence of other types
of SNARE proteins and thus may occur in intact neurons only under
certain specific conditions. Furthermore, the data presented in Figure 2 indicate that separate determinants govern the effects of syntaxin 1A
on channel inactivation and on G-protein modulation.
PKC-dependent phosphorylation affects syntaxin 1A action
Work by Yokoyama et al. (1997) indicates that phosphorylation of
the synprint site (residues 718-963 of the 1B
subunit) of the N-type calcium channel domain II/III linker region by
either PKC or CaMKII completely abolishes syntaxin 1A binding. In
contrast, neither PKA nor PKG was effective (Yokoyama et al., 1997). In view of this evidence, we hypothesized that PKC-dependent
phosphorylation of the appropriate sites in the synprint region should
result in the removal of the syntaxin 1A-mediated effect on
inactivation and G-protein inhibition of the channel.
To test this hypothesis, we coexpressed N-type calcium channels with
syntaxin 1A and then applied a 30 nM concentration of the
phorbol ester phorbol-12-myristate 13-acetate (PMA) to activate protein
kinase C during whole-cell patch-clamp recordings (see Hamid et al.,
1999) and to assess any putative effects on steady-state inactivation
of the channel. As shown in Figure
3A, the syntaxin 1A-mediated
negative shift in half-activation potential was abolished after PMA
treatment (Vh = 40.4 ± 2 mV;
n = 6), indicating that protein kinase C-dependent
phosphorylation of the channel interferes with the functional
interaction between syntaxin 1A and the N-type channel. Application of
PMA per se did not affect the half-inactivation potential of N-type
channels that were expressed in the absence of syntaxin 1A
(Vh = 45.9 ± 1.6 mV;
n = 5), suggesting that PKC-dependent phosphorylation
in itself does not affect steady-state inactivation of N-type
channels.
View larger version (29K):
[in this window]
[in a new window]
|
Figure 3.
Effect of protein kinase C-dependent
phosphorylation on the ability of syntaxin 1A to modulate steady-state
inactivation and G-protein inhibition of N-type calcium channels.
A, Effect of the application of 30 nM PMA on
half-inactivation potentials obtained with N-type calcium channels in
the presence and absence of syntaxin 1A. Note that PMA treatment
precludes the syntaxin 1A-mediated shift in half-inactivation potential
but does not affect the inactivation of N-type calcium channels per se.
The half-inactivation potentials obtained with PMA plus syntaxin 1A did
not differ significantly from those obtained in the presence of PMA
alone (p = 0.08). B, Protein
kinase C-dependent phosphorylation does not affect G-protein inhibition
of mutant N-type calcium channels in which the I/II linker residue T422
has been replaced with alanine (to prevent PKC-G-protein crosstalk)
(Hamid et al., 1999). In this case, data in the absence and presence of
PMA (30 nM for 90 sec) (Cooper et al., 2000) were obtained
from the same cell. C, The effect of phosphorylation on
the in vitro binding of syntaxin 1A to the
1B synprint peptide. Protein kinase C-dependent
phosphorylation of the synprint peptide does not inhibit the binding of
syntaxin 1A under our experimental conditions. In all, 1 nmol SHAM or
PKC-treated synprint peptide (wild type and mutated S802, S896, S898E)
immobilized on Ni2+-NTA agarose was incubated with
increasing amounts of purified syntaxin 1A. Syntaxin was added in
increasing ratios from 1:8 to 1:1. The two bands in the
doublet represent nonphosphorylated and phosphorylated syntaxin 1A,
which is a result of bacterial post-translational modification. Note
that syntaxin 1A phosphorylation does not interfere with binding to the
synprint motif. The control lanes show 10, 25, and 50 pmol of purified
syntaxin 1A, which allows for comparisons between the WT and EEE
blots.
|
|
It is not possible to use wild-type N-type channels to determine
whether PKC-dependent phosphorylation is able to antagonize the
syntaxin 1A-mediated G-protein inhibition of the channel because of the
well documented antagonistic effects of PKC-dependent phosphorylation on G-protein inhibition of the channel (Swartz, 1993; Zamponi et al.,
1997), which occurs via phosphorylation of residue T422 in the
G binding region of
the N-type calcium channel domain I/II linker (Hamid et al., 1999;
Cooper et al., 2000). We have, however, shown previously that
replacement of residue 422 with alanine renders the channels
insensitive to this PKC-G-protein crosstalk (Hamid et al., 1999).
Hence, to isolate any putative effects of PKC-dependent phosphorylation
on the ability of syntaxin 1A to induce tonic G-protein inhibition of
the channel, we coexpressed the 1B(T422A)
mutant with 1b,
2-, and syntaxin 1A and assessed the
ability of syntaxin 1A to induce G-protein inhibition before and after
the stimulation of PKC with 30 nM PMA. In the absence of
PMA the T422A mutant displayed robust G-protein inhibition after
coexpression with syntaxin 1A (55 ± 7% PP relief), albeit to a
somewhat smaller degree as compared with the wild-type channels, which
is consistent with our previous observations that used a somatostatin
receptor-mediated inhibition of this mutant (Hamid et al., 1999). More
importantly, as shown in Figure 3B, PMA application did not
affect the syntaxin 1A-mediated G-protein inhibition of the channel
significantly (54 ± 7% PP relief; p = 0.48, paired t test). Hence, PKC-dependent phosphorylation
selectively interferes with the ability of syntaxin 1A to affect N-type
channel inactivation while leaving the syntaxin 1A-induced G-protein
inhibition intact.
PKC-dependent phosphorylation does not abolish syntaxin 1A binding
to the synprint motif
We have suggested previously that the effect of syntaxin 1A on
G-protein inhibition might be attributable to a syntaxin 1A-mediated colocalization of G
and the channel (Jarvis et al., 2000). Within the framework of this
model a retention of the syntaxin 1A effect on G-protein inhibition of
the channel after PKC-dependent phosphorylation would imply that
syntaxin 1A should maintain some ability to bind to the channel. To
test this hypothesis, we performed an in vitro binding assay
involving recombinant syntaxin 1A and fusion proteins of the N-type
calcium channel synprint motif. As shown in Figure 3C,
syntaxin 1A binds to the synprint motif in a dose-dependent manner,
consistent with previous work (Sheng et al., 1994; Jarvis et al.,
2000). Interestingly, however, under our conditions PKC-dependent
phosphorylation of the synprint fusion proteins did not affect their
ability to interact with syntaxin 1A. This was observed in seven
experiments, whereas no binding of syntaxin 1A to histidine beads could
be detected (data not shown). We note that, in the experiments shown in
Figure 3C, immobilized synprint fusion proteins were
phosphorylated with commercially available purified PKC and recombinant
syntaxin 1A subsequently was allowed to bind, whereas Yokoyama et al.
(1997) phosphorylated the synprint peptides in solution (i.e., after elution) and then bound them to immobilized GST-syntaxin 1A. In two
additional experiments in which we followed a reverse paradigm (i.e.,
binding of phosphorylated synprint peptide to immobilized syntaxin 1A),
synprint binding was reduced, albeit not eliminated, after
PKC-dependent phosphorylation (data not shown). Hence, it appears that
the previously reported PKC-mediated ablation of the syntaxin
1A-synprint interaction in vitro perhaps may be highly dependent on the experimental paradigm that is used. Nonetheless, under
physiological conditions syntaxin 1A binding to the domain II/III
linker of N-type calcium channels likely occurs even after PKC-dependent phosphorylation of this region because PMA treatment did
not appear to affect syntaxin 1A-induced G-protein inhibition of the
channel. Yet, the data shown in Figure 3A clearly support a
role of protein kinase C in the modulation of at least some of the
effects of syntaxin 1A on N-type calcium channels, consistent with the
work of Yokoyama et al. (1997).
The effect of PKC on syntaxin 1A action is mimicked by point
mutations in the synprint motif
To identify the molecular basis of the effects of PKC on
syntaxin 1A-mediated inactivation of N-type channels, we considered a
mutagenesis strategy of possible PKC sites in the synprint motif of
N-type channels. This region contains a total of 18 serines and 13 threonine residues (Fig. 4). Of those,
nine residues were not flanked by basic residues in positions 1 to
3 or +1 to +3, leaving 22 possible phosphorylation sites. To help
identify the best candidate sites, we estimated the likelihood of PKC-
and CaMKII-dependent phosphorylation of each of the remaining residues according to Kennelly and Krebs (1991). As shown in Figure 4, we
identified three sites that scored well above the other sites (S774,
S802, S896), of which S802 was also a substrate for CaMKII and S896 was
located immediately adjacent to another CaMKII site, S898. For the
purposes of mutagenesis, we therefore first considered serines in
positions 774, 802, 896, and 898 while being aware that this may not
encompass all of the possible residues in the synprint region that
might contribute to the antagonistic effect of phosphorylation on the
actions of syntaxin 1A.
View larger version (71K):
[in this window]
[in a new window]
|
Figure 4.
Likelihood of PKC-dependent phosphorylation of
serine and threonine residues in the synprint region of the N-type
calcium channel 1 subunit according to Kennelly and
Krebs (1991). Asterisks indicate substrates for CaMKII.
Of the three best PKC consensus sites, only S802 forms a CaMKII site,
but S896 is immediately adjacent to a CaMKII consensus motif. S921 is
the only PKA consensus site within the synprint region.
|
|
To mimic the effects of phosphorylation permanently, we substituted
these serine residues for glutamic acid either individually or in
combination and assessed their effects on the ability of syntaxin 1A to
shift the steady-state inactivation curve and to promote G-protein
inhibition. The S774E construct yielded current levels that were too
low to permit meaningful recordings. Circular dichroism spectral
analysis on wild-type and the S774E mutant synprint fusion proteins
suggests that this mutation may result in the disruption of an
-helix (data not shown); hence, this residue was not considered
further. However, mutant channels in which residues 802, 896, and 898 were replaced individually or simultaneously with glutamic acid
exhibited robust expression. As shown in Figure
5A, after individual or even
concomitant substitution of S802, S896, and S898 the channels underwent
a large tonic G-protein inhibition in the presence of syntaxin 1A,
which did not differ significantly from that observed with the
wild-type channel, consistent with the data shown in Figure 3,
B and C.
View larger version (47K):
[in this window]
[in a new window]
|
Figure 5.
Effect of point mutations in PKC consensus sites
of the synprint motif on syntaxin 1A-mediated changes in channel
function. A, Effect of point mutations on the degree of
syntaxin 1A-induced PP facilitation of the channel. Note that, even in
the triple S802,896,898E mutant, the G-protein effect remains evident.
B, Effect of the mutations on the ability of syntaxin 1A
to mediate shifts in half-inactivation potential. Note that the
syntaxin 1A-mediated shifts in the half-inactivation potential were
abolished with the mutagenesis of residues 896 and 898, but not with
the substitution of residue 802. The numbers in
parentheses denote the numbers of experiments; error
bars are SE. The control and syntaxin 1A data are the same as in Figure
1.
|
|
Figure 5B examines the effect of the triple mutation on the
position of the steady-state inactivation curve along the voltage axis
in the absence and presence of syntaxin 1A. As seen from Figure
5B, the triple mutant exhibited a slightly more negative half-inactivation potential (49.7 ± 2 mV) as compared with the wild-type channel but was no longer susceptible to further negative shifts in Vh with the coexpression of
syntaxin 1A. Site-directed mutagenesis of the individual serine
residues reveals that the syntaxin 1A-induced shift in
Vh was blocked by "permanent
phosphorylation" of either residue 896 or 898, but not by residue
802. We also created a channel in which residues 896 and 898 were
replaced concomitantly by alanine to test whether the removal of these two PKC consensus sites could block the effects of PMA on N-type channel inactivation, but this construct could not be expressed functionally in tsA-201 cells. Thus, we cannot rule out the possibility that phosphorylation of one of the remaining serine/threonine residues
within the synprint region perhaps also might be able to interfere with
the syntaxin 1A-mediated inactivation of the channel.
Nonetheless, the data shown in Figure 5 are consistent with a mechanism
by which phosphorylation of residues 896 and/or 898 by protein kinase C
is sufficient to prevent the effects of syntaxin 1A on
voltage-dependent inactivation of N-type calcium channels. Additionally, these observations further support the notion that distinct structural determinants govern the ability of syntaxin 1A to
modulate G-protein inhibition and inactivation of the N-type calcium channel.
Syntaxin 1B selectively affects steady-state inactivation of
N-type channels
We have shown recently that, in contrast with syntaxin 1A, the
expression of syntaxin 1B does not result in a tonic G-protein inhibition of transiently expressed N-type calcium channels (Lu et al.,
2001) To assess whether syntaxin 1B was able to shift the
half-inactivation potential of N-type calcium channels, we coexpressed
syntaxin 1B with 1B
(+1b+2-) calcium
channels and recorded a series of steady-state inactivation curves. As shown in Figure 6, syntaxin 1B was able
to induce a negative shift in half-inactivation potential
(Vh = 55.7 ± 2.4 mV), which
was similar in magnitude to that observed with syntaxin 1A. Hence, syntaxin 1B affects steady-state inactivation of the channels without
promoting their inhibition by G-proteins, indicating that separate
syntaxin structural requirements underlie these two phenomena.
View larger version (19K):
[in this window]
[in a new window]
|
Figure 6.
Syntaxin 1B induces a negative shift in
half-inactivation potential. Shown is an ensemble of seven steady-state
inactivation curves recorded from N-type calcium channels in the
presence of syntaxin 1B. The data were fit with the Boltzmann equation.
The half-inactivation potential obtained from the fit was 55.6 mV.
Inset, Comparison of the half-inactivation potentials
obtained in the absence and in the presence of syntaxin 1B. Individual
inactivation curves were fit with the Boltzmann equation, and the means
plus SEM values are plotted in the form of bar graphs. The
numbers in parentheses denote the numbers
of experiments.
|
|
| |
DISCUSSION |
Modulation of syntaxin 1A action by other SNARE proteins
Both syntaxin 1A and SNAP25 have been shown to interact with the
domain II/III linker regions of P/Q-type and N-type calcium channels,
and both proteins can bind to this region simultaneously (Sheng et al.,
1994; Rettig et al., 1996; Yokoyama et al., 1997). The bound proteins
appear to participate in the interaction with v-SNARES, and it is known
that syntaxin 1A forms tight interactions with nSec-1 (Hata et al.,
1993; Pevsner et al., 1994b). One therefore might expect that the
changes in calcium channel function observed with the coexpression of
the channel with only syntaxin 1A in transient expression systems
(Bezprozvanny et al., 1995; Jarvis et al., 2000) may not be truly
indicative of the events occurring in neurons. Indeed, coexpression of
either nSec-1 or SNAP25 removed the ability of syntaxin 1A to mediate a
negative shift in Vh while leaving a
significant portion (~50%) of its effect on G-protein modulation
intact, which is consistent with the notion that the interactions of
syntaxin 1A with SNAP25/nSec-1 and the synprint region are not mutually
exclusive. Thus, depending on which proteins are associated with the
channel at a given point in time, calcium channels would have altered
inactivation properties. Although our experiments were designed
primarily to elucidate molecular determinants that govern the two
distinct effects of syntaxin 1A on N-type calcium channel function, it
is tempting to speculate on possible physiological implications of our
results. For example, in the presence of only syntaxin 1A or SNAP25,
vesicles cannot dock to the channel; thus, such a channel would be
unlikely to participate in fast neurotransmitter release. The negative
shift in half-inactivation potential and the concomitant tonic
G-protein inhibition perhaps could reduce calcium entry through this
channel and thus prevent unnecessary calcium overload (Bezprozvanny et al., 1995). With the binding of both SNAP25 and syntaxin 1A, the docking of the presynaptic vesicle would become possible, and concomitantly the level of tonic G-protein inhibition would become reduced and the availability for opening increased because of the
removal of the syntaxin 1A-induced hyperpolarizing shift in Vh. In principle, such a mechanism
perhaps could provide for selective calcium entry through channels
bound to docked vesicles. However, unlike tsA-201 cells an intact
synapse likely expresses a number of other binding partners for SNARE
proteins, which could influence the effects of syntaxin 1A on both
G-protein modulation and voltage-dependent inactivation of the channel
plus the regulation of these effects by PKC. Thus, our findings should
not be extrapolated firmly to what might occur in intact neurons.
Indeed, our observations with nSec-1 and the recently reported effects
of cysteine string proteins on N-type channel regulation by G-proteins
(Magga et al., 2000) may be indicative of such added complexity.
Phosphorylation dependence of syntaxin 1A effects
The interaction between SNARE proteins and presynaptic calcium
channels is considered a key step in the fast release of
neurotransmitter, and factors that modulate either the entry of calcium
or the association of the SNARE protein/calcium channel complex have
the propensity to regulate neurotransmission (Wheeler et al., 1994;
Dunlap et al., 1995; Mochida et al., 1996; Stanley, 1997). It is well
established that G
subunits and activation of PKC are important determinants of the
regulation of calcium channel activity (Swartz et al., 1993; Stea et
al., 1995; Herlitze et al., 1996; Ikeda, 1996; De Waard et al., 1997;
Page et al., 1997; Zamponi et al., 1997; Hamid et al., 1999; Arnot et
al., 2000). In addition, there appears to be a complex interplay
between calcium channel modulation by second messengers and the
association of the channel with SNARE proteins (Stanley and Mirotznik,
1997; Yokoyama et al., 1997; Jarvis et al., 2000). At least within the confines of a recombinant system, our present work adds further intricacy to these interactions.
Our functional electrophysiological data show that activation of
protein kinase C with PMA continued to result in the syntaxin 1A-mediated tonic G-protein inhibition of the channel. We note that
disruption of syntaxin 1A binding to the channel by other means (i.e.,
overexpression of synprint peptides) was able to depress the extent of
G-protein inhibition dramatically. It therefore seems unlikely that
syntaxin 1A would be dislodged fully from the channel after
PKC-dependent phosphorylation under physiological conditions, although
we must acknowledge the possibility that PKC-dependent phosphorylation
of synprint peptides in vitro may be capable of abolishing
their interactions with syntaxin 1A completely under certain
circumstances (Yokoyama et al., 1997). Under physiological conditions,
however, syntaxin 1A well may associate more tightly with the channel
because of the presence of the plasma membrane and possibly weak
interactions with other channel regions that may contribute to the
maintained G-protein effect despite PKC-dependent phosphorylation.
In contrast, the syntaxin 1A-mediated shift in half-inactivation
potential was removed after activation of PKC or by site-directed mutagenesis of putative PKC consensus sites. Together with the observation that coexpression of either SNAP25 or nSec-1 also prevented
the ability of syntaxin 1A to shift the half-inactivation potential,
this may indicate that the conformation of the domain II/III linker,
which results in the negative shifts in
Vh, is disrupted easily. Although
individual substitutions of residues 896 and 898 with glutamic acid
were sufficient to mimic the effect of protein kinase C-dependent
phosphorylation on voltage-dependent inactivation of the channel, we
cannot rule out the possibility that phosphorylation of one or more of
the other serine/threonine residues contained in the N-type channel
domain II/III linker perhaps could result in a similar loss of the
effects of syntaxin 1A on N-type channel inactivation.
We have shown previously that activation of PKC results in two effects
on N-type calcium channels (Zamponi et al., 1997; Hamid et al., 1999).
First, PKC-dependent phosphorylation of either residues T422 or S425 in
the N-type calcium channel domain I/II linker region results in an
upregulation of current activity. In addition, selective PKC-dependent
phosphorylation of T422 antagonizes G-protein binding to the channel
(see also Swartz et al., 1993; Barrett and Rittenhouse, 2000; Cooper et
al., 2000). The observation that PKC-dependent phosphorylation of
residues S896 or S898 is sufficient to block the negative shift in
half-inactivation potential would suggest a further increase in the
availability of the channels despite the presence of syntaxin 1A. This
built-in redundancy, together with the notion that the activity of
N-type calcium channel can be increased via multiple avenues after
phosphorylation, may support a critical role of the phosphorylation
process in the control of calcium entry.
Possible mechanism underlying the modulation of syntaxin
1A action
One common theme emerging from the present study is the notion
that the molecular determinants that govern the effect of syntaxin 1A
on channel inactivation differ from those underlying the syntaxin 1A-induced G-protein inhibition. Neither of the point mutations of PKC
consensus sites in the synprint motif nor activation of PKC with PMA
nor the coexpression of SNAP25 or nSec-1 eliminated the tonic G-protein
inhibition mediated by syntaxin 1A, whereas either one of these factors
could abolish the effects of syntaxin 1A on inactivation of the
channel. In contrast, syntaxin 1B promoted N-type channel inactivation
without mediating a G-protein effect (Lu et al., 2001), which suggests
that syntaxin 1B is capable of interacting with the channel, but
perhaps not with
G.
Our observation that PKC-dependent phosphorylation did not appear to be
capable of removing syntaxin 1A from the synprint site under
physiological conditions would be consistent with the presence of
multiple syntaxin micro-binding sites within the synprint region.
PKC-dependent phosphorylation or site-directed mutagenesis of S896 and
S898 might result in the loss of one of the contact points between
syntaxin 1A and the channel, but syntaxin 1A nonetheless could remain
attached to other subregions of the channel that are not affected by
phosphorylation. The possibility of a weak interaction between syntaxin
1A and a portion of the synprint motif that is linked to calcium
channel inactivation perhaps could explain the observation that the H3
domain of syntaxin 1A, which appears to be required for the
inactivation effect, does not exhibit detectable binding to the domain
II/III linker of the N-type channel in vitro (Bezprozvanny
et al., 2001). Along these lines, the coexpression of nSec-1 or SNAP25
either could hinder the interaction of syntaxin 1A with the regions
responsible for the inactivation effect sterically or perhaps could
allosterically preclude the translation of syntaxin 1A binding into a
change in channel gating. However, ultimately for these possibilities
to be substantiated, more detailed structural information, such as a
crystal structure of the synprint region in the presence and absence of
SNARE proteins and G-proteins, will be required.
Overall, our data provide novel insights into the interactions among
N-type calcium channels, SNARE proteins, and cytoplasmic messengers at
the molecular level. To appreciate fully the significance of these
interactions for neurotransmission is a daunting task, particularly in
light of recent evidence that the cysteine string protein also may
interact with the synprint region to produce functional consequences on
G-protein modulation (Leveque et al., 1998; Magga et al., 2000) and
that the expression of P/Q-type calcium channels can mediate
calcium-dependent gene transcription of syntaxin 1A (Sutton et al.,
1999). The sheer complexity of these interactions suggests that the
mammalian brain has devised multiple avenues for the precise control of
events resulting in calcium homeostasis and vesicle release in the
presynaptic nerve terminal.
| |
FOOTNOTES |
Received Dec. 7, 2000; revised Jan. 18, 2001; accepted Jan. 23, 2001.
This work was supported by a grant from the Canadian Institutes of
Health Research (CIHR). G.W.Z. holds faculty scholarships from the
CIHR, the Alberta Heritage Foundation for Medical Research (AHFMR), and
The EJLB Foundation; he is the Novartis Investigator in
Schizophrenia Research. S.E.J. is the recipient of studentship awards
from the AHFMR and the Health Research Foundation of Canada. We thank
Dr. Terry Snutch for providing cDNA encoding for wild-type calcium
channel subunits and Dr. William Catterall for discussion. We also
thank Dr. Janice Braun for helpful discussion and for donating a
syntaxin 1A-GST construct and antibodies to SNARE proteins. Finally,
we thank Dr. Jawed Hamid for providing the T422A mutant, Bob Winkfein
for providing rat brain cDNA, and Clinton Doering for technical assistance.
Correspondence should be addressed to Dr. Gerald W. Zamponi, Department
of Physiology and Biophysics, University of Calgary, 3330 Hospital
Drive NW, Calgary, T2N 4N1, Canada. E-mail: Zamponi{at}ucalgary.ca.
| |
REFERENCES |
-
Arnot MI,
Stotz SC,
Jarvis SE,
Zamponi GW
(2000)
Differential modulation of N-type a1B and P/Q-type a1A calcium channels by different G-protein -subunit isoforms.
J Physiol (Lond)
527:203-212[Abstract/Free Full Text].
-
Barrett CF,
Rittenhouse AR
(2000)
Modulation of N-type calcium channel activity by G-proteins and protein kinase C.
J Gen Physiol
115:277-286[Abstract/Free Full Text].
-
Bezprozvanny I,
Scheller RH,
Tsien RW
(1995)
Functional impact of syntaxin on gating of N-type and Q-type calcium channels.
Nature
378:623-626[Medline].
-
Bezprozvanny I,
Zhong P,
Scheller RH,
Tsien RW
(2001)
Molecular determinants of the functional interaction between syntaxin and N-type Ca2+ channel gating.
Proc Natl Acad Sci USA
97:13943-13948[Abstract/Free Full Text].
-
Cooper CB,
Arnot MI,
Feng Z-P,
Jarvis SE,
Hamid J,
Zamponi GW
(2000)
Crosstalk between G-protein and PKC modulation of N-type calcium channels is dependent on the G-protein -subunit isoform.
J Biol Chem
275:40777-40781[Abstract/Free Full Text].
-
Degtiar VE,
Scheller RH,
Tsien RW
(2000)
Syntaxin modulation of slow inactivation of N-type calcium channels.
J Neurosci
20:4355-4367[Abstract/Free Full Text].
-
De Waard M,
Liu H,
Walker D,
Scott VE,
Gurnett CA,
Campbell KP
(1997)
Direct binding of G-protein complex to voltage-dependent calcium channels.
Nature
385:446-450[Medline].
-
Dubel SJ,
Starr TVB,
Hell J,
Ahlijanian MK,
Enyeart JJ,
Catterall WA,
Snutch TP
(1992)
Molecular cloning of the 1 subunit of an
 -conotoxin-sensitive calcium channel.
Proc Natl Acad Sci USA
89:5058-5062[Abstract/Free Full Text]. -
Dunlap K,
Fischbach GD
(1981)
Neurotransmitters decrease the calcium conductance activated by depolarization of embryonic chick sensory neurones.
J Physiol (Lond)
317:519-535[Abstract/Free Full Text].
-
Dunlap K,
Luebke JL,
Turner TJ
(1995)
Exocytotic Ca2+ channels in the mammalian central nervous system.
Trends Neurosci
18:89-98[Web of Science][Medline].
-
Hamid J,
Nelson D,
Spaetgens R,
Dubel SJ,
Snutch TP,
Zamponi GW
(1999)
Identification of an integration center for crosstalk between PKC and G-protein modulation of N-type calcium channels.
J Biol Chem
278:6195-6202.
-
Hata Y,
Slaughter CA,
Sudhof TC
(1993)
Synaptic vesicle fusion complex contains unc-18 homologue bound to syntaxin.
Nature
366:347-351[Medline].
-
Hayashi T,
McMahon H,
Yamasaki S,
Binz T,
Hata Y,
Sudhof TC,
Niemann H
(1994)
Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly.
EMBO J
13:5051-5061[Web of Science][Medline].
-
Herlitze S,
Garcia DE,
Mackie K,
Hille B,
Scheuer T,
Catterall WA
(1996)
Modulation of Ca2+ channels by G-protein subunits.
Nature
380:258-262[Medline].
-
Ikeda SR
(1996)
Voltage-dependent modulation of N-type calcium channels by G-protein subunits.
Nature
380:255-258[Medline].
-
Jarvis SE,
Magga J,
Beedle A,
Braun J,
Zamponi GW
(2000)
G-protein modulation of N-type calcium channels is facilitated by physical interactions between syntaxin 1A and G.
J Biol Chem
275:6388-6394[Abstract/Free Full Text].
-
Kennelly PJ,
Krebs EG
(1991)
Consensus sequences as substrate specificity determinants for protein kinases and protein phosphatases.
J Biol Chem
266:15555-15558[Free Full Text].
-
Koch WJ,
Hawes BE,
Inglese J,
Luttrell LM,
Lefkowitz RJ
(1994)
Cellular expression of the carboxyl terminus of a G-protein-coupled receptor kinase attenuates G-mediated signaling.
J Biol Chem
269:6193-6197[Abstract/Free Full Text].
-
Leveque C,
Pupier S,
Marqueze B,
Geslin L,
Kataoka M,
Takahashi M,
De Waard M,
Seagar M
(1998)
Interaction of cysteine string proteins with the 1A subunit of the P/Q-type calcium channel.
J Biol Chem
273:13488-13492[Abstract/Free Full Text].
-
Lin RC,
Scheller RH
(2000)
Mechanisms of synaptic vesicle exocytosis.
Annu Rev Cell Dev Biol
16:19-49[Web of Science][Medline].
-
Linial M,
Bledi Y
(2000)
Syntaxin the pillar of secretion.
Modulator
12:11-13.
-
Lu Q,
Atkisson MS,
Jarvis SE,
Feng Z-P,
Zamponi GW,
Dunlap KD
(2001)
Syntaxin 1A supports voltage-dependent inhibition of 1B Ca2+ channels by G in chick sensory neurons.
J Neurosci
21:2949-2957[Abstract/Free Full Text].
-
Magga JM,
Jarvis SE,
Arnot MI,
Zamponi GW,
Braun JEA
(2000)
Cysteine string protein regulates G-protein modulation of N-type calcium channels.
Neuron
28:195-204[Web of Science][Medline].
-
Misura KMS,
Scheller RH,
Weis WI
(2000)
Three-dimensional structure of the neuronal Sec1-syntaxin 1A complex.
Nature
404:355-362[Medline].
-
Mochida S,
Sheng Z-H,
Baker C,
Kobayashi H,
Catterall WA
(1996)
Inhibition of neurotransmission by peptides containing the synaptic protein interaction site of N-type Ca2+ channels.
Neuron
17:781-788[Web of Science][Medline].
-
Page KM,
Stephens GJ,
Berrow NS,
Dolphin AC
(1997)
The intracellular loop between domains I and II of the B-type calcium channel confers aspects of G-protein sensitivity to the E-type calcium channel.
J Neurosci
17:1330-1338[Abstract/Free Full Text].
-
Pevsner J,
Hsu SC,
Braun JE,
Calakos N,
Ting AE,
Bennett M,
Scheller RH
(1994a)
Specificity and regulation of a synaptic vesicle docking complex.
Neuron
13:353-361[Web of Science][Medline].
-
Pevsner J,
Hsu SC,
Scheller RH
(1994b)
nSec-1: a neuronal-specific syntaxin binding protein.
Proc Natl Acad Sci USA
91:1445-1449[Abstract/Free Full Text].
-
Rettig J,
Sheng ZH,
Kim DK,
Hodson CD,
Snutch TP,
Catterall WA
(1996)
Isoform-specific interaction of the 1A subunits of brain Ca2+ channels with the presynaptic proteins syntaxin and SNAP25.
Proc Natl Acad Sci USA
93:7363-7368[Abstract/Free Full Text].
-
Rettig J,
Heinemann C,
Ashery U,
Sheng ZH,
Yokoyama CT,
Catterall WA,
Neher E
(1997)
Alteration of Ca2+ dependence of neurotransmitter release by disruption of Ca2+channel/syntaxin interaction.
J Neurosci
17:6647-6656[Abstract/Free Full Text].
-
Sheng Z,
Rettig T,
Takahashi M,
Catterall WA
(1994)
Identification of a syntaxin-binding site on N-type calcium channels.
Neuron
13:1303-1313[Web of Science][Medline].
-
Sheng Z-H,
Rettig J,
Cook T,
Catterall WA
(1996)
Calcium-dependent interaction of N-type calcium channels with the synaptic core complex.
Nature
379:451-454[Medline].
-
Söllner T,
Whiteheart SW,
Brunner M,
Erdjument-Bromage H,
Geromanos S,
Tempst P,
Rothman JE
(1993)
SNAP receptors implicated in vesicle targeting and fusion.
Nature
362:318-324[Medline].
-
Stanley EF
(1997)
The calcium channel and the organization of the presynaptic transmitter release face.
Trends Neurosci
20:404-409[Web of Science][Medline].
-
Stanley EF,
Mirotznik RR
(1997)
Cleavage of syntaxin prevents G-protein regulation of presynaptic calcium channels.
Nature
385:340-343[Medline].
-
Stea A,
Soong TW,
Snutch TP
(1995)
Determinants of PKC-dependent modulation of a family of neuronal calcium channels.
Neuron
5:929-940.
-
Sutton KG,
McRory JE,
Guthrie H,
Murphy TH,
Snutch TP
(1999)
P/Q-type calcium channels mediate the activity-dependent feedback of syntaxin 1A.
Nature
401:800-804[Medline].
-
Swartz KJ
(1993)
Modulation of Ca2+ channels by protein kinase C in rat central and peripheral neurons: disruption of G-protein-mediated inhibition.
Neuron
11:305-320[Web of Science][Medline].
-
Swartz KJ,
Merrit A,
Bean BP,
Lovinger DM
(1993)
Protein kinase C modulates glutamate receptor inhibition of Ca2+ channels and synaptic transmission.
Nature
361:165-168[Medline].
-
Umbach JA,
Gunderson CB
(1997)
Evidence that cysteine string proteins regulate an early step in Ca2+-dependent secretion of neurotransmitter at Drosophila neuromuscular junctions.
J Neurosci
17:7203-7209[Abstract/Free Full Text].
-
Walker D,
De Waard M
(1998)
Subunit interaction sites in voltage-dependent Ca2+ channels: role in channel function.
Trends Neurosci
21:148-154[Web of Science][Medline].
-
Westenbroek RE,
Hell JW,
Warberm C,
Dubel SJ,
Snutch TP,
Catterall WA
(1992)
Biochemical properties and subcellular distribution of an N-type calcium channel 1 subunit.
Neuron
9:1099-1115[Web of Science][Medline].
-
Westenbroek RE,
Sakurai T,
Elliott EM,
Hell JW,
Starr TV,
Snutch TP,
Catterall WA
(1995)
Immunochemical identification and subcellular distribution of the 1A subunits of brain calcium channels.
J Neurosci
15:6403-6418[Abstract/Free Full Text].
-
Wheeler DB,
Randall A,
Tsien RW
(1994)
Roles of N-type and Q-type channels in supporting hippocampal synaptic transmission.
Science
264:107-111[Abstract/Free Full Text].
-
Wu MN,
Fergestad T,
Lloyd TE,
He Y,
Broadie K,
Bellen HJ
(1999)
Syntaxin 1A interacts with multiple exocytotic proteins to regulate neurotransmitter release in vivo.
Neuron
23:593-605[Web of Science][Medline].
-
Yokoyama CT,
Sheng Z,
Catterall WA
(1997)
Phosphorylation of the synaptic protein interaction site on N-type calcium channels inhibits interactions with SNARE proteins.
J Neurosci
17:6929-6938[Abstract/Free Full Text].
-
Zamponi GW,
Bourinet E,
Nelson D,
Nargeot J,
Snutch TP
(1997)
Crosstalk between G-proteins and protein kinase C mediated by the calcium channel 1 subunit.
Nature
385:242-246.
Copyright © 2001 Society for Neuroscience 0270-6474/01/2192939-10$05.00/0
This article has been cited by other articles:
|
|
|
|
 
B. Ng, Y. Kang, H. Xie, H. Sun, and H. Y. Gaisano
Syntaxin-1A inhibition of P-1075, cromakalim, and diazoxide actions on mouse cardiac ATP-sensitive potassium channel
Cardiovasc Res,
December 1, 2008;
80(3):
365 - 374.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. W. Tedford and G. W. Zamponi
Direct G Protein Modulation of Cav2 Calcium Channels
Pharmacol. Rev.,
December 1, 2006;
58(4):
837 - 862.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Parnas, I. Slutsky, G. Rashkovan, I. Silman, J. Wess, and I. Parnas
Depolarization Initiates Phasic Acetylcholine Release by Relief of a Tonic Block Imposed by Presynaptic M2 Muscarinic Receptors
J Neurophysiol,
June 1, 2005;
93(6):
3257 - 3269.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. M. Cibulsky, H. Fei, and I. B. Levitan
Syntaxin-1A Binds to and Modulates the Slo Calcium-Activated Potassium Channel via an Interaction That Excludes Syntaxin Binding to Calcium Channels
J Neurophysiol,
March 1, 2005;
93(3):
1393 - 1405.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Li, F. Wang, M. Lai, Y. Chen, and J.-f. Zhang
A Protein Phosphatase 2c{alpha}-Ca2+ Channel Complex for Dephosphorylation of Neuronal Ca2+ Channels Phosphorylated by Protein Kinase C
J. Neurosci.,
February 23, 2005;
25(8):
1914 - 1923.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Tsuk, I. Michaelevski, G. N. Bentley, R. H. Joho, D. Chikvashvili, and I. Lotan
Kv2.1 Channel Activation and Inactivation Is Influenced by Physical Interactions of Both Syntaxin 1A and the Syntaxin 1A/Soluble N-Ethylmaleimide-Sensitive Factor-25 (t-SNARE) Complex with the C Terminus of the Channel
Mol. Pharmacol.,
February 1, 2005;
67(2):
480 - 488.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. K. Sandberg and P. Low
Altered Interaction and Expression of Proteins Involved in Neurosecretion in Scrapie-infected GT1-1 Cells
J. Biol. Chem.,
January 14, 2005;
280(2):
1264 - 1271.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Liu, S. A. Ernst, S. E. Gladycheva, Y. Y. F. Lee, S. I. Lentz, C. S. Ho, Q. Li, and E. L. Stuenkel
Fluorescence Resonance Energy Transfer Reports Properties of Syntaxin1A Interaction with Munc18-1 in Vivo
J. Biol. Chem.,
December 31, 2004;
279(53):
55924 - 55936.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. B. Harkins, A. L. Cahill, J. F. Powers, A. S. Tischler, and A. P. Fox
Deletion of the synaptic protein interaction site of the N-type (CaV2.2) calcium channel inhibits secretion in mouse pheochromocytoma cells
PNAS,
October 19, 2004;
101(42):
15219 - 15224.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. W. Tedford, N. Gilles, A. Menez, C. J. Doering, G. W. Zamponi, and G. F. King
Scanning Mutagenesis of {omega}-Atracotoxin-Hv1a Reveals a Spatially Restricted Epitope That Confers Selective Activity against Insect Calcium Channels
J. Biol. Chem.,
October 15, 2004;
279(42):
44133 - 44140.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. H Hurley, A. L Cahill, M. Wang, and A. P Fox
Syntaxin 1A regulation of weakly inactivating N-type Ca2+ channels
J. Physiol.,
October 15, 2004;
560(2):
351 - 363.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. E. Gladycheva, C. S. Ho, Y. Y. F. Lee, and E. L. Stuenkel
Regulation of syntaxin1A-munc18 complex for SNARE pairing in HEK293 cells
J. Physiol.,
August 1, 2004;
558(3):
857 - 871.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Q. Li, A. Lau, T. J. Morris, L. Guo, C. B. Fordyce, and E. F. Stanley
A Syntaxin 1, G{alpha}o, and N-Type Calcium Channel Complex at a Presynaptic Nerve Terminal: Analysis by Quantitative Immunocolocalization
J. Neurosci.,
April 21, 2004;
24(16):
4070 - 4081.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I. Michaelevski, D. Chikvashvili, S. Tsuk, D. Singer-Lahat, Y. Kang, M. Linial, H. Y. Gaisano, O. Fili, and I. Lotan
Direct Interaction of Target SNAREs with the Kv2.1 Channel: MODAL REGULATION OF CHANNEL ACTIVATION AND INACTIVATION GATING
J. Biol. Chem.,
September 5, 2003;
278(36):
34320 - 34330.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Arien, O. Wiser, I. T. Arkin, H. Leonov, and D. Atlas
Syntaxin 1A Modulates the Voltage-gated L-type Calcium Channel (Cav1.2) in a Cooperative Manner
J. Biol. Chem.,
August 1, 2003;
278(31):
29231 - 29239.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. C. Miller, L. A. Swayne, J. G. Kay, Z.-P. Feng, S. E. Jarvis, G. W. Zamponi, and J. E. A. Braun
Molecular determinants of cysteine string protein modulation of N-type calcium channels
J. Cell Sci.,
July 15, 2003;
116(14):
2967 - 2974.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. D. Spafford, L. Chen, Z.-P. Feng, A. B. Smit, and G. W. Zamponi
Expression and Modulation of an Invertebrate Presynaptic Calcium Channel {alpha}1 Subunit Homolog
J. Biol. Chem.,
June 6, 2003;
278(23):
21178 - 21187.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. M. Leung, Y. Kang, X. Gao, F. Xia, H. Xie, L. Sheu, S. Tsuk, I. Lotan, R. G. Tsushima, and H. Y. Gaisano
Syntaxin 1A Binds to the Cytoplasmic C Terminus of Kv2.1 to Regulate Channel Gating and Trafficking
J. Biol. Chem.,
May 2, 2003;
278(19):
17532 - 17538.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I. Slutsky, J. Wess, J. Gomeza, J. Dudel, I. Parnas, and H. Parnas
Use of Knockout Mice Reveals Involvement of M2-Muscarinic Receptors in Control of the Kinetics of Acetylcholine Release
J Neurophysiol,
April 1, 2003;
89(4):
1954 - 1967.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. D. Spafford, D. W. Munno, P. van Nierop, Z.-P. Feng, S. E. Jarvis, W. J. Gallin, A. B. Smit, G. W. Zamponi, and N. I. Syed
Calcium Channel Structural Determinants of Synaptic Transmission between Identified Invertebrate Neurons
J. Biol. Chem.,
January 31, 2003;
278(6):
4258 - 4267.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. W. Zamponi and T. P. Snutch
Modulating Modulation: Crosstalk Between Regulatory Pathways of Presynaptic Calcium Channels
Mol. Interv.,
December 1, 2002;
2(8):
476 - 478.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
S. E. Jarvis, W. Barr, Z.-P. Feng, J. Hamid, and G. W. Zamponi
Molecular Determinants of Syntaxin 1 Modulation of N-type Calcium Channels
J. Biol. Chem.,
November 8, 2002;
277(46):
44399 - 44407.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I. Michaelevski, D. Chikvashvili, S. Tsuk, O. Fili, M. J. Lohse, D. Singer-Lahat, and I. Lotan
Modulation of a Brain Voltage-gated K+ Channel by Syntaxin 1A Requires the Physical Interaction of Gbeta gamma with the Channel
J. Biol. Chem.,
September 13, 2002;
277(38):
34909 - 34917.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Kawasaki, S. C. Collins, and R. W. Ordway
Synaptic Calcium-Channel Function in Drosophila: Analysis and Transformation Rescue of Temperature-Sensitive Paralytic and Lethal Mutations of Cacophony
J. Neurosci.,
July 15, 2002;
22(14):
5856 - 5864.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Chen, X. Zheng, K. L Schulze, T. Morris, H. Bellen, and E. F Stanley
Enhancement of presynaptic calcium current by cysteine string protein
J. Physiol.,
January 15, 2002;
538(2):
383 - 389.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Kaneko, C. B. Cooper, N. Nishioka, H. Yamasaki, A. Suzuki, S. E. Jarvis, A. Akaike, M. Satoh, and G. W. Zamponi
Identification and Characterization of Novel Human Cav2.2 (alpha 1B) Calcium Channel Variants Lacking the Synaptic Protein Interaction Site
J. Neurosci.,
January 1, 2002;
22(1):
82 - 92.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Q. Lu, M. S. AtKisson, S. E. Jarvis, Z.-P. Feng, G. W. Zamponi, and K. Dunlap
Syntaxin 1A Supports Voltage-Dependent Inhibition of {alpha}1B Ca2+ Channels by G{beta}{gamma} in Chick Sensory Neurons
J. Neurosci.,
May 1, 2001;
21(9):
2949 - 2957.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Z.-P. Feng, M. I. Arnot, C. J. Doering, and G. W. Zamponi
Calcium Channel beta Subunits Differentially Regulate the Inhibition of N-type Channels by Individual Gbeta Isoforms
J. Biol. Chem.,
November 21, 2001;
276(48):
45051 - 45058.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Chen, X. Zheng, K. L. Schulze, T. Morris, H. Bellen, and E. F. Stanley
Enhancement of presynaptic calcium current by cysteine string protein
J. Physiol.,
December 19, 2001;
(2001)
200101339.
[Abstract]
[PDF]
|
|
|
|
TOP
ABSTRACT
INTRODUCTION
