The C2A Domain of Synaptotagmin Alters the Kinetics of
Voltage-gated Ca2+ Channels Cav1.2 (Lc-type)
and Cav2.3 (R-type)*
Roy
Cohen
,
Lisa A.
Elferink§, and
Daphne
Atlas
¶
From the
Department of Biological Chemistry, The
Hebrew University of Jerusalem, Jerusalem 91904, Israel and the
§ Department of Physiology & Biophysics and the Marine and
Biomedical Institute, University of Texas Medical Branch,
Galveston, Texas 77555-1069
Received for publication, October 8, 2002, and in revised form, December 18, 2002
 |
ABSTRACT |
Biochemical and genetic studies implicate
synaptotagmin (Syt 1) as a Ca2+ sensor for neuronal
and neuroendocrine neurosecretion. Calcium binding to Syt 1 occurs
through two cytoplasmic repeats termed the C2A and C2B domains. In
addition, the C2A domain of Syt 1 has calcium-independent properties
required for neurotransmitter release. For example, mutation of a
polylysine motif (residues 189-192) reverses the inhibitory effect of
injected recombinant Syt 1 C2A fragment on neurotransmitter release
from PC12 cells. Here we examined the requirement of the C2A polylysine
motif for Syt 1 interaction with the cardiac Cav1.2
(L-type) and the neuronal Cav2.3 (R-type) voltage-gated
Ca2+ channels, two channels required for neurotransmission.
We find that the C2A polylysine motif presents a critical interaction surface with Cav1.2 and Cav2.3 since truncated
Syt 1 containing a mutated motif (Syt 1*1-264) was
ineffective at modifying the channel kinetics. Mutating the
polylysine motif also abolished C2A binding to Lc753-893,
the cytosolic interacting domain of Syt 1 at Cav1.2
1
subunit. Syt 1 and Syt 1* harboring the mutation at the KKKK motif
modified channel activation, while Syt 1* only partially reversed the
syntaxin 1A effects on channel activity. This mutation would interfere
with the assembly of Syt 1/channel/syntaxin into an exocytotic unit.
The functional interaction of the C2A polylysine domain with
Cav1.2 and Cav2.3 is consistent with tethering
of the secretory vesicle to the Ca2+ channel. It indicates
that calcium-independent properties of Syt 1 regulate voltage-gated
Ca2+ channels and contribute to the molecular events
underlying transmitter release.
 |
INTRODUCTION |
The synaptic vesicle protein Synaptotagmin I (Syt
1),1 is proposed to function
as a Ca2+ sensor for neurotransmitter release (1, 2).
Consistent with its proposed role as a calcium sensor protein, Syt 1 binds calcium via two repeating structures termed C2A and C2B domains (3).
A role for the C2A and C2B domains of Syt 1 in calcium-triggered
neurosecretion is well established (4-7). For example,
Ca2+ binding to the C2A domain enhances the association of
Syt 1 with several proteins required for neurotransmission including
syntaxin 1A (8, 9), SNAP-25 (10, 11), and AP2 (9, 12). Furthermore, Ca2+ binding to the C2A domain promotes its insertion into
membranes via an interaction with the acidic phospholipids (8, 9, 13,
14) consistent with the Ca2+ requirements of
neurosecretion. Microinjection of recombinant C2A domains and
antibodies specific for this region impair neurotransmitter release
from neuroendorine PC12 cells (15) and giant squid axons (16).
Interestingly, the inhibitory effect of recombinant C2A fragments in
PC12 cells occurs independently of its calcium binding properties and
is mediated through a novel polybasic motif (17). Thus, the Syt 1 C2A
domain contains calcium-dependent and -independent activities, which mediate Syt 1 function during neurotransmitter release. Furthermore, Syt 1 and Syt 4 were recently shown to promote transmitter release independently of Ca2+ binding to the
C2A domain (18).
Interactions through the Syt 1 C2B domain are also functionally
important for neurosecretion (19-26). Several studies have demonstrated that the activity of Ca2+ channels is modified
by syntaxin 1A, Syt 1, and SNAP-25 (27-30). The syntaxin 1A or SNAP-25
inhibitory effects of Cav1.2, Cav2.2, and
Cav2.3 activity are reversed by co-expression of Syt 1 (31-34). Recovery of channel activity by Syt 1 was directly
proportional to the ratio of Syt 1 and syntaxin 1A, indicating that Syt
1 and syntaxin 1A regulate the Ca2+ channel directly (32,
33). Consistent with this, recombinant proteins comprising the C2A and
C2B domains of Syt I bind to the II-III cytosolic domain of the
a11.2, a12.1, and
12.2 channel subunits (31-33, 35, 36).
Here we studied the functional interaction of Syt 1 with
Cav1.2 and Cav2.3 by examining the relative
contribution of the C2A polylysine motif on channel activity and
binding to the cytosolic domain Lc753-893 of the
11.2 of Cav1.2. Our data indicate that the
C2A domain of Syt 1 modulates the activation kinetics of
Cav1.2 and Cav2.3. Mutation of the C2A
polylysine motif abolished the binding to the cytosolic interaction
domains of the channel. Moreover, this mutation altered the modulatory
effect of Syt 1 on Cav1.2 and Cav2.3 activity,
impairing the ability of Syt 1 to reverse the syntaxin 1A inhibition of
channel activity.
 |
EXPERIMENTAL PROCEDURES |
11.2 (dN60-del1773; X15539) rat
2A (m80545);
12.3 subunit cloned into pHBE239 (L27745) were obtained
from Dr. L. Birnbaumer;
2/
rabbit skeletal (M86621) from
A. Schwartz . Rat Syt 1 was obtained from M. L Bennett. The in
vitro transcription kit was from Stratagene. Anti-syntaxin
antibody was a kind gift of M. Takahashi and was prepared by us;
anti-Syt 1 antibody was from Sigma; Anti-Lc753-893
antibody (32). CM5 sensor chip, N-hydroxysuccinimide,
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), and ethanolamine-HCl were purchased from Biacore, AB (Uppsala, Sweden). Glutathione-agarose 4B beads were from Amersham Biosciences.
Syt 1 Mutants--
Syt 1* (K189A/K190A/K191A/K192A) was
prepared by insertion of the Eco47III-PflM
fragment into Syt 1. Syt 11-264* (K189A/K190A/K191A/K192A) was prepared by excising the C2B domain (amino acids 265-421) and
religating the PflMI-XbaI fragment in directional cloning.
cRNA Injection and Protein Expression in Xenopus
Oocytes--
Stage V-VI oocytes were removed and defolliculated by
collagenase (type I) treatment as described (37). Oocytes were injected with cRNA of
11.2 or
12.3 (5 ng/oocyte),
2
1 (5 ng/oocyte),
2a (10 ng/oocyte), and a day later with Syt
1, Syt 1*, Syt 11-264, or Syt-1*1-264 (5 ng/oocyte). After cRNA injection, oocytes were maintained for 6 days at
19 °C in ND96 solution (mM): 96 NaCl/2 KCl/1
MgCl2/1.8 CaCl2/2.5 sodium pyruvate/5 HEPES, pH
7.4, with antibiotics. Plasmid DNA for the channel subunits,
11.2 (
*1C dN60-del1773),
12.3 (
1E),
2
1
,
2a, syntaxin 1A, Syt 1, Syt 1*, and Syt
11-264, were linearized and transcribed in
vitro using T7 or T3 polymerase (Stratagene kit) in the presence of the cap analog G (5') ppp (5') G (Amersham Biosciences). The in vitro transcribed capped cRNAs were injected into oocytes
at a final volume of 40 nl per oocyte. Channel subunits
11.2 or
12.3,
2
1, and
2A were
injected 1 day prior to injection of synaptic proteins.
Electrophysiological Assays--
Whole cell currents were
recorded at room temperature (20-24 °C) by applying a standard
two-microelectrode voltage clamp using a Dagan 8500 amplifier. Voltage
and current agar-cushioned electrodes (0.3-0.6 M
) were filled with
3 M KCl (32). Current-voltage relationships were determined
by voltage steps as indicated in the legend to figures, in
Ba2+ solution (mM): 5 Ba(OH)2/50
N-methyl-D-glucamine/1 KOH/40
tetraethylammonium/5 HEPES, pH 7.5 and titrated to pH 7.5 with
(CH3)2SO4. The activation kinetics
was determined from leak-subtracted current traces by a
mono-exponential fit of the pClamp8 software (Axon Inst.). The activation time constants were determined by fitting the raw current data with the equation: I(t) = Imax
[1-exp(t/
act)], where
I(t) indicates the amplitude of current at time
t, Imax is the maximum amplitude, and
act is the time constant for activation. Each trace was
fitted separately according to Boltzmann, and the averaged values were
plotted. Cav1.2 activation was fitted to single exponential function, while a two exponential function nicely described the data of
Cav2.3 time course. Data presentation was done using Origin 6 software (Microcal). All quantitative results are given as the mean ± S.E. (n = 6-10)
Protein Expression--
Protein expression in oocytes was tested
for by Western analysis 5-7 days after cRNA injection. Oocytes were
homogenized in buffer containing (in mM): 1 EDTA/250
sucrose/10 Tris-HCl, pH 7.0, and addition of a mixture of
protease:aprotinin, phenylmethylsulfonyl fluoride,
iodoacetamide, pepstatin A, and leupeptin at 4 °C. Homogenates were
centrifuged (12,000 × g, 10 min); the pellet was
discarded and supernatant was collected. Protein was determined by a
micro-Bradford assay in enzyme-linked immunosorbent assay-reader plate
using bovine serum albumin as standard (38). Protein samples (30 µg) mixed with 100 mM dithiothreitol and 2% SDS, boiled 3 min,
applied to 10% SDS-PAGE, transferred to nitrocellulose, and probed
using affinity-purified monoclonal anti-syntaxin 1A (Sigma) followed by
a horseradish peroxidase-conjugated anti-mouse antibody. Syntaxin 1A
expression was detected by enhanced chemiluminescence (ECL system).
Affinity Determination Using the Surface Plasmon Resonance
Spectroscopy and GST Binding Assays--
The affinity of
Lc753-893, the II-III loop that links domains II-III of
the CaV1.2
1 subunit (32) and GST-C2A wt (31) or
GST-C2A* mutant (17) was determined using (i) Biacore 3000 system
(Biacore AB) based on surface plasmon resonance methodology and (ii)
glutathione-S-transferase (GST) binding assay using
GST-agarose beads.
Purified His6-tagged Lc753-893
was immobilized on a research-grade CM5 sensor chip in a flow cell
coated with carboxyl-methyl dextran as the surface matrix using
activated carboxyl groups and EDC coupling in HBS-EP buffer (150 mM NaCl, 3.4 mM EDTA, and 0.005% (v/v) 10 mM Hepes, pH 7.4, and surfactant P20) at a flow rate of 10 µl/min. The surface was activated for 7 min with a mixture of
N-hydroxysuccinimide (0.05 M) and EDC (0.02 M). His6-tagged Lc753-893 was
injected at a concentration of 20 µg/ml in 10 mM sodium
acetate, pH 3.5, until the desired level of binding was achieved.
Ethanolamine (1 M, pH 8.5) was injected for 7 min to block
the remaining activated groups. Control flow-cell surface was prepared
by activating and then deactivating (blocking) the carboxyl groups as
mentioned above. His6Lc753-893 binding studies
to wild type and mutant C2A domains were initiated by passing the
recombinant fusion proteins GST-C2A wild type, GST C2A* mutant, and GST
alone at increasing concentrations as indicated through the flow cells
at a rate of 20 µl/min in HBS-EP running buffer. Surface regeneration
was carried out after each binding assay by a 10-µl pulse of 1 M NaCl in 10 mM NaOH. The data were analyzed
using the Kinetics Wizard of the Biacore control software with
automatic corrections for nonspecific binding by subtraction of the
responses obtained for the control surface from the data obtained. The
kinetics of binding and affinity constants were calculated using the
Biaevaluation software.
Binding of the cytoplasmic domain of Lc753-893 (100 nmol)
to GST fusion proteins, Syt 1, C2A, C2B, C2A*, and GST alone (100 pmol)
using glutathione-agarose 4B beads (25 µl) was performed as described
(31, 32). Immunoblots were probed using affinity-purified anti-Lc753-893 antibody and visualized by enhanced
chemiluminescence (ECL system).
 |
RESULTS |
The C2A Domain of Syt 1 Is Required for Functional Interactions
with the Voltage-gated Ca2+ Channels,
Cav1.3--
Functional interactions of voltage-gated
Ca2+ channels with the full-length Syt 1 have been
previously described using the Xenopus oocytes expression
system. To assess the role of the C2A polylysine motif (amino acids
189-192) on Syt 1 interactions with the Ca2+ channel, the
C2A polylysine motif was substituted with alanine residues in
full-length Syt 1 (Syt 1*) or a truncated form of Syt 1 (Syt
11-264) lacking the C2B domain (Fig.
1).

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 1.
Schematic presentation of Syt 1 and the Syt 1 mutants. Mutations at the C2A polylysine motif of synaptotagmin
(Syt 1) were generated by substituting 189KKKK with Ala
residues (Syt 1*) as indicated. The two truncated Syt mutants aa
1-164, Syt 11-264, and the truncated mutant bearing the
same mutation at 189KKKK to Ala, Syt
1*1-264, are shown.
|
|
Cav1.2 currents were elicited in oocytes co-expressing the
three-channel subunits
11.2/
2a/
2
1 with Syt 1 or
Syt 1* from a holding potential of
80 mV to test potentials between
30 and +45 mV in response to 160-ms test pulse (Fig.
2). Peak current amplitudes were not
affected by Syt 1 (35) or Syt 1, as demonstrated by
current-voltage relationship (Fig. 2A). The activation
component of Cav1.2 current was measured at each test pulse
and was fitted with a single exponential function between the lines
marked by asterisks (Fig. 2B). Under these experimental
conditions both Syt 1 and Syt 1* slightly reduced activation rate at
voltage range of
15 to
5 mV, while at more positive potentials
act approached control values (Fig.
2C; Table I). Lysates of
oocytes co-injected with Syt 1, Syt 1*, and Cav1.2 were
prepared and analyzed for Syt 1/Syt *1 expression by Western analysis
using anti-Syt 1 antibody (see "Experimental Procedures"). As shown
in Fig. 2D no significant difference in the expression of
Syt 1 and Syt 1* in injected oocytes was observed.

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 2.
Syt 1 and Syt 1* interact with
Cav1.2 (Lc-channel). Oocytes injected with
cRNA of 11.2 (5 ng/oocyte), 2 1 (5 ng/oocyte),
2a (10 ng/oocyte), and a day later with Syt 1 (5 ng/oocyte) or Syt
1* (5 ng/oocyte) are shown. Inward Ba2+ currents were
elicited from a holding potential of 80 mV in response to a 160-ms
pulse by voltage steps to potentials between 30 and +45 mV in 5-mV
increments. A, leak-subtracted peak current-voltage
relationship: collected data from oocytes expressing the three channel
subunits ( ) together with Syt 1 ( ) or Syt 1* ( ). The data
points correspond to the mean ± S.E. of current amplitude
(n = 8). B, the activation component of a
typical current produced at each test pulse was fitted with a single
exponential function between the lines marked by asterisks.
C, the averaged time constants of activation
( act, mean ± S.E., n = 6) are
plotted against test pulses in the absence ( ) and in the presence of
Syt 1 ( ) and Syt 1* ( ). Two sample Student's t tests
were applied, and p values <0.05 were obtained from the
two-tailed tests. D, protein expression of Syt 1 and Syt 1*
was determined at day 5 following cRNA (5 ng/oocyte) injection into
Xenopus oocytes. The proteins were separated on 10%
SDS-PAGE, transferred to nitrocellulose membrane, and subjected to
Western analysis using anti-Syt 1 antibody and ECL detection.
|
|
View this table:
[in this window]
[in a new window]
|
Table I
Differential effect of syntaxin 1A Syt 1 wild type and Syt 1 mutants on
the kinetic parameters of Cav 1.2 and Cav 2.3
Whole cell Ba2+ currents were evoked from a holding potential
of 80 mV by a single voltage step to test potentials as indicated by
the superscripts. Values are mean ± S.D. for (n = 6-10). Test pulse duration for Cav1.2 was 160 ms and 80 ms for
Cav2.3. peak, peak current; act, time
constant of activation. Test potentials are depicted by superscripts
a, b, c, d.
|
|
Syt 1 and Syntaxin 1A Are Functionally Coupled to
Cav1.2 Activity--
We previously demonstrated that
Cav1.2 as well as Cav2.2 (neuronal N-type
channel) activities are inhibited with co-expression of syntaxin 1A
(28, 32, 37). Since the inhibitory effect of syntaxin 1A on these
channels is reversed by Syt 1 (31, 32, 33) we next examined the Syt 1*
mutant for reversal of syntaxin 1A inhibitory effects on channel
activity. Fig. 3 shows the results of
co-expressing Cav1.2 and syntaxin 1A with Syt 1 and Syt*1
in Xenopus oocytes. Superimposed traces of macroscopic whole
cell Ba2+ currents showed an 80% inhibition of current
amplitude by syntaxin 1A, which was fully reversed in the presence of
Syt 1 and partially by Syt 1* (Fig. 3A; Table I).
Furthermore, peak current amplitudes normalized to maximum current
(I/Imax) showed a large voltage shift
in the half-maximal voltage (V1/2) induced by
syntaxin 1A from V1/2 =
21 ± 1.2 mV to
V1/2 =
7.5 ± 1.8 mV. This voltage shift
was reverted to V1/2 =
18.6 ± 2 mV by
Syt 1 and
19 ± 2.2 mV by Syt 1* (Fig. 3B).
Similarly, a complete reversal of the syntaxin 1A effect on
Cav1.2 activation was observed with co-expression of Syt 1 (Fig. 3C), and only partial reversal by Syt 1* (Fig. 3D). The mutation at the polylysine motif impaired Syt 1*
capacity to reverse the inhibitory effects of syntaxin 1A on
Cav1.2 current amplitude and activation kinetics.

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 3.
Syt 1 and Syt 1* interact with
Cav1.2 in the presence of syntaxin 1A.
A, superimposition of macroscopic 11.2,
2 1, and 2a currents evoked from a holding potential 80 mV by
a single voltage step (160 ms) to +20 mV in oocytes expressing the
three-channel subunits in various combinations as indicated.
B, peak-current amplitudes (data not shown) normalized to
maximum current (I/Imax) plotted
against test potentials were fitted according to Boltzmann; channel
subunits ( ) with syntaxin 1A ( ), syntaxin 1A and Syt 1 ( ), or
syntaxin 1A and Syt 1* ( ). The mid-point of activation
(V1/2) and Boltzmann slope (k) of
11.2/ 2 1/ 2a were V1/2 = 21.6 ± 1.2 mV, k = 2.9 ± 1.9; with
syntaxin 1A, V1/2 = 7.5 ± 1.75 mV,
k = 7.2 ± 1.44; with syntaxin 1A and Syt 1 V1/2 = 18.6 ± 2.1 mV, k = 2.4 ± 0.8; and with syntaxin 1A and Syt 1*,
V1/2 = 19.0 ± 2.2 mV and
k = 3.9 ± 2.1. C, the activation time
constants ( act, mean ± S.E., n = 8) are plotted against test pulses between 10 and +30 mV; the channel
alone ( ), with syntaxin 1A ( ), with syntaxin 1A and Syt 1 ( )
and D, with syntaxin 1A and Syt 1* ( ). Two sample
Student's t tests were applied, and p values
<0.05 were obtained from the two-tailed tests. See Fig. 2 for
cRNA/oocyte of channel subunits, syntaxin 1A (2 ng/oocyte).
|
|
Cav1.2 Interacts with Syntaxin 1A and the Truncated
Syt 11-264 and Syt 1*1-264
Mutants--
The partial reversion of the syntaxin 1A effect on
Cav1.2 activation by Syt 1* compared with Syt 1 suggests
that the polylysine C2A motif couples Syt 1 to channel activation (Fig.
3, C and D). To isolate the contribution of C2A
domain we co-expressed truncated Syt 1 lacking the C2B domain
(Syt 11-264) with Cav1.2 and syntaxin 1A. Cav1.2 whole cell currents were activated from
a holding potential of
80 to 0 mV test pulse (Fig.
4A). Both the superimposed
traces as well as the current-voltage relationships (Fig. 4,
A-C) showed diminished current amplitudes by syntaxin 1A
that were only partially reversed by Syt 11-264
and Syt 1*1-264. Syt 1*1-264 was
significantly less effective than Syt 11-264. Furthermore
the large shift in the half-maximal voltage induced by syntaxin 1A,
(see above) was shifted back to V1/2 =
20.6 ± 2.3 mV by Syt 11-264 and only to
12.9 ± 3 mV by Syt 1*1-264 (Fig.
4D).

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 4.
Syt 11-264 and Syt
1*1-264 interact with Cav1.2 in the
presence of syntaxin 1A. Inward Ba2+ currents were
elicited in oocytes co-expressing 11.2, 2 1, 2a,
syntaxin 1A, and syntaxin 1A with Syt 11-264 and syntaxin
1A with Syt 1*1-264 from a holding potential of 80 mV by
voltage steps of 160 ms applied in 5-mV increments at potentials
between 30 and +45 mV. A, superposition of macroscopic
11.2, 2 1, and 2a currents activated from a
holding potential 80 by a single voltage step of 160 ms to a test
potential of 0 mV in various combinations as indicated. B,
leak-subtracted peak current-voltage relationship: collected data from
oocytes expressing the three-channel subunits ( ) with syntaxin 1A
( ), syntaxin 1A and Syt 11-264 ( ), or C,
syntaxin 1A and Syt 1*1-264 ( ). The data points
correspond to the mean ± S.E. of current amplitude
(n = 7). D, peak current amplitudes
normalized to maximum current
(I/Imax) are plotted against test
potentials (data from B and C) and were fitted
according to Boltzmann equation. The mid-point of activation
(V1/2) and the Boltzmann slope (k) of
11.2/ 2 1/ 2a were V1/2 = 21.6 ± 1.2 mV, k = 2.9 ± 1.9, with Syt
11-264 V1/2 = 20.6 ± 2.3 mV, k = 5.1 ± 2.89, and with Syt
1*1-264 V1/2 = 12.9 ± 3.1 mV, k = 5.4 ± 2.1. E, activation time
constants of the channel ( act, mean ± S.E.,
n = 6) are plotted against test potentials ( ) with
syntaxin 1A ( ), syntaxin 1A and Syt 11-264 ( ) and
F, syntaxin 1A and Syt 1*1-264 ( ).
Two sample Student's t tests were applied, and p
values <0.05 were obtained from the two-tailed tests. (See legend to
Fig. 2 for cRNA injected per oocyte,)
|
|
A more striking difference between Syt 11-264 and Syt
1*1-264 was observed on channel activation (Fig. 4,
E and F). The marked slowing effect of activation
kinetics by syntaxin 1A was fully reversed by Syt 11-264,
(Fig. 4C; Table I). In contrast, Syt 1*1-264
was completely ineffective (Fig. 4E). Together, these
results suggest the involvement of the C2A polylysine motif in the
interaction with the channel.
K189-192A Mutations Abolish Lc753-893 Binding to the
C2A Domain of Syt 1--
The polylysine motif (189-192) at the C2A
domain is exposed on the surface of the
-sandwich of Syt 1 where
they are accessible for interacting with potential effector molecules
(7). To determine whether the loss of functional interaction with the
channel is related to impaired binding to Lc743-893,
the II-III linker of the Cav1.2
1 subunit (32)
. Two types of binding studies of C2A and mutant C2A* domains were
preformed. (i) Recombinant GST-C2A, GST-C2A*, GST-Syt 1, and GST
proteins were immobilized to GSH-agarose beads and incubated with
equimolar concentrations of recombinant
His6Lc753-893 (0.5 µM; 2.5 µg). As shown by Western analysis using anti-Lc753-893
antibody (32), C2A, C2B, and Syt 1 bind Lc753-893, but no
binding of C2A* was observed (Fig.
5A). (ii) The affinity of C2A
and C2A* to Lc753-893 was tested using the Biacore
technology (Biacore; see "Experimental Procedures").
His6Lc753-893 was immobilized on a sensor chip
surface. Recombinant samples of GST-C2A, GST-C2A* at the indicated
concentrations were injected into the flow cell of the system
(Biacore), and changes in resonance units were recorded as a function
of time to yield sensorgrams as shown in Fig. 5B. The C2A
binding to Lc753-893 is manifested as large amplitude of
the surface plasmon resonance signal while no resonance signal was obtained by C2A*. GST alone showed no binding (data not shown). The
calculated affinity of C2A was O.213 µM (
2 = 4.06; Fig. 5B). Hence the C2A mutant does not bind to the
intracellular domain of the channel that comprises the Syt 1 interaction (31, 32, 35, 40, 47).

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 5.
Direct interaction of C2A and C2A* with
Cav1.2 cytosolic domain
Lc753-893. Lc753-893 binding to Syt 1 and C2 domains. A, GST fusion proteins (100 pmol) were
immobilized on GSH-agarose beads and incubated for 60 min with
His6Lc753-893 (250 pmol). After extensive
washing, the remaining bound proteins were eluted with 15 mM GSH, separated on 10% SDS-PAGE, transferred to
nitrocellulose, and subjected to Western analysis by using
anti-Lc753-893 antibody and detected by ECL.
His6Lc753-893 (30 ng) was used as a marker.
B, sensorgram of C2A and C2A* interaction with
Lc753-893 obtained by surface plasmon resonance
spectroscopy. The solution of recombinant GST C2A, GST C2A*, and GST at
concentrations as indicated was flowed over immobilized
His6Lc753-893, and the interaction at the
surface was recorded. The apparent equilibrium dissociation constant
for C2A (Kd = 0.213 µM;
2 = 4.06) was calculated from the ratio of the
dissociation and association rate constants
(koff/kon).
RU, resonance units.
|
|
The C2A Domain of Syt 1 Is Required for Functional Interactions
with Cav2.3 (R-channel)--
Cav2.3 currents
were elicited in oocytes co-expressing
12.3/
2a/
2
1 subunits (41) and Syt 1 or Syt 1*
(Fig. 6). Syt 1 or Syt 1* modified
neither Cav2.3 current-voltage relationship nor
peak-current amplitude (Fig. 6A). Conversely, Syt 1 strongly accelerated Cav2.3 activation in the range of
20 to +5
mV, converging at more depolarized values (>5 mV) (Fig. 6B;
Table I; Ref. 30). Interestingly, Syt 1 was previously shown to
accelerate the activation kinetic of Cav2.2 (N-type
channel; Ref. 28). The effect of Syt 1* on Cav2.3 was more
complex, showing mixed effects of this mutant on channel activation
(Fig. 6C). At negative potentials between
20 and
10 mV,
the rate was accelerated by Syt 1* similar to Syt 1, but between
5
and 0 mV an abrupt decrease in the rate was observed, which was slower
than the channel (Fig. 6D). At more positive potentials, in
the range of +5 to +30 mV,
act approached control values
(Fig. 6, C and D).

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 6.
Syt 1 and Syt 1* interact with
Cav2.3 (R-channel). Oocytes were injected with cRNA of
12.3 (5 ng/oocyte), 2 1 (5 ng/oocyte), 2a (10 ng/oocyte), and a day later, with Syt 1 (5 ng/oocyte) or Syt 1* (5 ng/oocyte). Inward Ba2+ currents were elicited from a
holding potential of 80 mV in response to an 80-ms pulse to various
test potentials between 30 and +45 mV in 5-mV increments.
A, leak-subtracted peak current-voltage relationship:
collected data from oocytes expressing the three channel subunits ( )
together with Syt 1 ( ) or Syt 1* ( ). The data points correspond
to the mean ± S.E. of current (n = 8).
B, the activation component of a typical current produced
by a test pulse was fitted with a single exponential function between
the lines marked by asterisks and was applied to determine
the time constant of activation ( act). C,
activation time constants ( act, mean ± S.E.,
n = 6) are plotted against potentials between 20 and
+30 mV in the absence ( ) and in the presence of Syt 1 ( ) or D,
Syt 1* ( ). Two sample Student's t test were applied, and
p values <0.05 were obtained from the two tailed
tests
|
|
Activation of Cav2.3 Requires the C2A Polylysine
Motif--
We next examined the requirement of the Syt 1 C2A
polylysine motif on channel activation using the truncated Syt 1 mutants. Cav2.3 currents were elicited in oocytes
co-expressing the three channel subunits along with Syt
11-264 and Syt 1*1-264. In both mutants the
C2B domain is missing, and in Syt 1*1-264 the polylysine
motif was substituted with alanine residues. The effects of the
truncated mutants on channel activity are shown in Fig.
7, A and B.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 7.
Syt 11-264 and Syt
1*1-264 interact with Cav2.3. Inward
Ba2+ currents were elicited in oocytes co-expressing
12.3, 2 1, and 2a along with Syt
11-264 and Syt 1*1-264 in response to voltage
steps of 80 ms to test potentials between 30 and +40 mV in 5-mV
increments. Holding potential was 80 mV. A,
superimposition of 12.3, 2 1, and 2a current
traces, either alone or in combination with Syt 11-264 and
Syt 1*1-264, activated from a holding potential of 80 mV
in response to a single voltage step to a 0-mV test pulse.
B, leak-subtracted peak current-voltage relationship:
collected data from oocytes expressing the three channel subunits alone
( ), with Syt 11-264 ( ) or C, with Syt
1*1-264 ( ). The data points correspond to the mean ± S.E. of current (n = 8). D, peak
current-amplitudes normalized to maximum current
(I/Imax) are plotted against test
potentials (data from B and C) were fitted according to Boltzmann. The
mid-point of activation (V1/2) and the Boltzmann
slope (k) of 12.3/ 2 1/ 2a were
V1/2 = 8.8 ± 0.1 mV, k = 2.3 ± 0.1; with Syt 11-264
V1/2 = 0.5 ± 0.1 mV, k = 2.9 ± 0.1; and with Syt 1*1-264
V1/2 = 6.2 ± 0.1 mV and
k = 2.5 ± 0.1. E, activation time
constants ( act, mean ± S.E., n = 6) are plotted against test potentials in the absence ( ) and in the
presence of Syt 11-264 ( ) and F, Syt
1*1-264 ( ). Two sample Student's t tests
were applied, and p values <0.05 were obtained from the
two-tailed tests. cRNA of channel subunits injected per oocyte, see
Fig. 5; Syt 1*1-264 (5 ng/oocyte); Syt 11-264
(5 ng/oocyte).
|
|
Superimposed traces of whole cell current were activated from a
holding potential of
80 mV by a single voltage step to 0-mV test
pulse (Fig. 7A). Syt 11-264 appeared to inhibit
current amplitude by 60% at 0 mV, while Syt 1*1-264
displayed no effect on current amplitude but significantly slowed
channel inactivation (inactivation kinetics were not explored in the
present study). Current-voltage relationships were significantly
shifted in the presence of Syt 11-264 but not Syt
1*1-264 (Fig. 7, B and C). Peak
current amplitudes normalized to maximum current (I/Imax) show that the half-maximal
voltage activation (V1/2) was significantly
displaced by Syt 11-264 from
8.8 ± 0.1 mV to
0.5 ± 0.1 mV and only marginally to
6.2 ± 0.1 mV by Syt 1*1-264, (Fig. 7D). This voltage shift can
account for the apparent reduction in current amplitude. The slope
factors were directly comparable between control conditions and those
expressing Syt 11-264 or Syt 1*1-264 (Fig.
6D). Syt 11-264 was also efficient at
accelerating Cav2.3 activation in the
20 to
5 mV range
similar to full-length Syt 1 (Fig. 7E; Fig. 6C). In contrast, the acceleration of activation by Syt 1*1-264 was smaller and was detected only in
20 to
10 mV range (Fig. 6F). The effects of Syt 11-264 and Syt
1*1-264 on Cav2.3 kinetics were specific for
this channel as co-expression of these proteins result in no effect on
Cav1.2 activation kinetics (see Table I). Together, these
data suggest that Cav2.3 activation involves the C2A domain
of Syt 1. Moreover, mutation of the polylysine motif modifies the
interaction of the C2A domain with the channel.
A Cross-interaction of Syntaxin 1A with Syt 1 Mutants and
Cav2.3--
Superimposed traces of macroscopic whole cell
Cav2.3 current elicited from a holding potential of
80 mV
by a single voltage step to a 0-mV test pulse showed a partial reversal
by Syt 1 (from 50% to 18%) of the syntaxin 1A-mediated current
inhibition but not by Syt 1* (Fig.
8A). Current-voltage
relationships obtained in the presence of syntaxin 1A or syntaxin 1A
with either one of the Syt 1 mutants indicated a shift toward more
positive potentials by Syt 1* (Fig. 8, B and C)
that could account for the reduction in current amplitude at 0 mV (Fig.
7A). Peak current amplitudes normalized to maximum current
(I/Imax) showed no shift in the half-maximal voltage of Cav2.3 (V1/2 =
8.8 ± 0.1 mV) by syntaxin 1A (V1/2 =
8.0 ± 2.1 mV) (Fig. 8D), unlike the large shift
induced by syntaxin 1A in Cav1.2 (Fig. 3B). In
the presence of syntaxin 1A, V1/2 was
shifted toward more positive potentials to
4.3 ± 1.2 mV by Syt
1 and to
0.4 ± 0.7 mV by Syt 1*, (Fig. 8D). Hence, Syt 1 and Syt 1* differently modify the syntaxin-associated
channel.

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 8.
Syt 1 and Syt 1* modify Cav2.3
properties in the presence of syntaxin 1A. A, superposition
of macroscopic 12.3, 2 1, and 2a current traces
evoked in response to an 80-ms pulse from a holding potential of 80
mV by a single voltage step to a 0-mV test pulse in oocytes
co-expressing the three-channel subunits alone and together with either
Syt 1 or Syt 1*. B, leak-subtracted peak current-voltage
relationship: collected data from oocytes expressing the three-channel
subunits ( ) with syntaxin 1A ( ), syntaxin 1A and Syt 1 ( -),
and C, syntaxin 1A and Syt 1* ( ). The data points
correspond to the mean ± S.E. of current (n = 8).
D, Peak current amplitudes normalized to maximum current
(I/Imax) (data from B and
C) are plotted against test potentials displayed with a
Boltzmann fit. The mid-point of activation
(V1/2) and the Boltzmann slope (k) of
12.3/ 2 1/ 2a were V1/2 = 8.8 ± 0.1 mV, k = 2.3 ± 0.1; with Syt 1 V1/2 = 2.8 ± 0.8 mV, k = 3.9 ± 0.25, and with Syt 1*1-264
V1/2 = 2.7 ± 1.38 mV, k = 4.2 ± 0.4. E, the activation time constants
( act, mean ± S.E., n = 6) are
plotted against test potentials between 20 and +25 mV: the channel
alone ( ), with syntaxin 1A ( ), syntaxin 1A and Syt 1 ( ), or
F, syntaxin 1A and Syt 1* ( ). Two sample Student's
t tests were applied, and p values <0.05 were
obtained from the two-tailed tests. cRNA/oocyte, see Fig. 3; Syntaxin
1A (2 ng/oocyte).
|
|
Cav2.3 activation was accelerated in cells expressing
syntaxin 1A and was not modified further by Syt 1 (Fig. 8E).
In contrast, Syt 1* slowed the activation kinetics in the presence of
syntaxin 1A, in particular in potentials between 0-15 mV (Fig.
8F). Thus, mutation of the polylysine motif increased the
current voltage shift of Cav2.3 and was less effective than
Syt 1 at reversing the syntaxin 1A inhibition. In addition, the
mutation appeared to affect the interaction of the channel with
syntaxin 1A, slowing the activation kinetics. Together, these data
suggest that the Syt 1 C2A polylysine motif participates in the
syntaxin 1A modulation of Cav2.3 activation.
Since Syt 1 and Syt 1 mutants affect syntaxin 1A modulation
of the channel, their effect on syntaxin 1A expression in oocytes was
tested (Fig. 9). Oocytes were injected
with syntaxin 1A cRNA (5 ng/oocyte) and cRNA encoding the various Syt 1 mutants (5 ng/oocyte) as indicated. At day five after injection,
oocytes were lysed and proteins were separated on SDS-PAGE and analyzed
using monoclonal anti-syntaxin 1A antibody (Fig. 9). As shown by the
Western blot analysis there were no significant changes in syntaxin 1A
expression in the presence of either one of the four Syt 1 mutants
(Fig. 9).

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 9.
The effect of Syt 1 wild type and mutants on
syntaxin 1A expression in oocytes. Xenopus oocytes were
injected with cRNA of 12.3 (5 ng/oocyte), 2 1 (5 ng/oocyte), 2a (10 ng/oocyte), and 11.2 (5 ng/oocyte)
with the corresponding subunits, syntaxin 1A (5 ng/oocyte), and with
Syt 1, Syt 1*, Syt 11-264, or Syt 1*1-264 (5 ng/oocyte) as indicated. Five days later, oocytes were lysed and
proteins were separated on 10% SDS-PAGE and transferred to a
nitrocellulose membrane. The level of syntaxin 1A expressed in the
oocytes was determined in a Western analysis by using monoclonal
anti-syntaxin 1A antibody and detection by ECL.
|
|
The contribution of the KKKK motif to the channel interaction with
syntaxin 1A was examined by using the two truncated Syt 1 mutants
lacking the C2B domain. Currents were evoked from a holding potential
of
80 mV to 0 mV test pulse (Fig.
10A). As shown current
amplitude was reduced by syntaxin 1A and was partially reversed by the
two mutants. Current-voltage relationships showed that Syt
11-264 and Syt 1*1-264 were equally
effective at reverting syntaxin 1A inhibition of Cav2.3
current amplitude (Fig. 10B). The half-maximal voltage
(V1/2) of the channel was not affected by
syntaxin 1A (see Fig. 8D), but a small shift toward more
positive potentials was observed by Syt 11-264 to 2.7 ± 1.4 mV and to
2.8 ± 0.8 mV by Syt 1*1-264(Fig.
10C). Syntaxin 1A accelerated Cav2.3 activation
(Fig. 10D), which was further increased in the presence of
Syt 11-264 (Fig. 10E). In contrast, Syt
1*1-264 lost the ability to accelerate
Cav2.3/syntaxin 1A activation (Fig. 10F).
Together, these data show that the mutant failed to modify
Cav2.3 interaction with syntaxin and suggest that
the C2A polylysine motif participates in the syntaxin 1A cross-talk
with Cav2.3.

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 10.
Syt 11-264 and Syt
1*1-264 modify Cav2.3 kinetics in the presence
of syntaxin 1A. A, superposition of macroscopic
12.3, 2 1, and 2a current traces evoked in
response to an 80-ms pulse from a holding potential 80 mV by a single
voltage step to a 0-mV test pulse in oocytes co-expressing the
three-channel subunits alone and together with Syt 11-264
or Syt 1*1-264 as indicated. B, leak-subtracted
peak current-voltage relationship: collected data from oocytes
expressing the three-channel subunits ( ), syntaxin 1A ( ), or with
syntaxin 1A and Syt 11-264 ( ) or syntaxin 1A and Syt
1*1-264 ( ). The data points correspond to the mean ± S.E. of current amplitude (n = 9). C,
peak current amplitudes normalized to maximum current
(I/Imax) (data from B) are
plotted against test potentials and displayed with a Boltzmann fit. The
mid-point of activation (V1/2) and the Boltzmann
slope (k) of 12.3/ 2 1/ 2a were
V1/2 = 8.8 ± 0.01 mV, k = 2.3 ± 0.1; with syntaxin 1A, V1/2 = 8 ± 2.1mV, k = 3.9 ± 0.7; with syntaxin
1A and Syt 11-264, V1/2 = 2.8 ± 0.8 mV, k = 3.9 ± 0.25; and with
syntaxin 1A and Syt 1*1-264 V1/2 = 2.7 ± 1.4 mV, k = 4.2 ± 0.4. D, the activation time constants ( act,
mean ± S.E., n = 6-8) of the channel ( ) or
with syntaxin 1A ( -) were plotted against test potentials between
20 and +30 mV as indicated. E, the activation time
constants were measured in oocytes expressing both syntaxin 1A and Syt
11-264 ( ) and F, syntaxin 1A and Syt
1*1-264 ( ). Two sample Student's t tests
were applied, and p values <0.05 were obtained from the
two-tailed tests. cRNA (ng/oocyte) injected, see Figs. 6 and. 7.
|
|
 |
DISCUSSION |
A role for the C2A domain of Syt 1 in calcium-triggered
neurotransmitter release has been well established in neurons and neuroendocrine cells (15-17, 42). Mutation of a polylysine motif distal to the calcium coordination site reverses the inhibitory effect
of injected Syt C2A fragments on calcium-regulated secretion (17, 43).
Since mutation of the polylysine motif does not affect the overall
structure or Ca2+ binding properties of the C2A domain,
calcium-independent properties involving the polylysine motif are
important for the Syt-mediated steps leading to neurotransmitter
release (17). However the nature of these interactions remained unknown.
We addressed the possibility that the voltage-gated Ca2+
channel (Cav1.2, Cav2.1, Cav2.2,
and Cav2.3), an established effector for Syt 1 (30-32, 34,
40), may be functionally coupled through the polylysine C2A domain.
Using the Xenopus oocytes expression system we examined the
functional consequences of mutating the C2A polylysine motif on
Cav1.2 (Lc-type) the channel that supports evoked secretion
in PC12 cells and the neuronal Cav2.3 (R-type) channel. The
changes induced in the activation kinetics and current amplitude of
voltage-sensitive Ca2+ channels demonstrate that the C2A
polylysine motif participates in the interaction of Syt 1 with both
Cav1.2 and Cav2.3.
Modulation of Cav1.2--
Since the Syt 1/syntaxin 1A
interaction occurs independently of the C2A polylysine motif (17), the
observed differences in syntaxin 1A modulation of channel activity in
the presence of Syt 1 may result from either a direct interaction with
the channel or with a new site formed by the association of syntaxin 1A
with the channel. The full-length Syt 1 reversed syntaxin 1A inhibition
of Cav1.2 activity, while Syt 1* was significantly less
effective. The marked slowing of activation kinetics of
Cav1.2 by syntaxin 1A was reversed by Syt
11-264 but not by Syt 1*1-264. The mutation
of the polylysine motif in the Syt 11-264 protein lacking
a C2B domain (Syt 1*1-264) results in a complete loss of
function. These results were further substantiated when no binding of
His6Lc753-893 to the isolated C2A domain were
observed. Lc753-893, the intracellular domain of
Cav1.2
1 subunit, was previously shown to be the site of
interaction of Syt 1, C2A, and C2B (31, 32). The four mutated KKKK
residues abolished GST-C2A* binding to
His6Lc753-893 in two methods, GSH-agarose
beads and plasmon resonance spectroscopy. Therefore, these results, in
part, could provide an explanation to why unlike the intact C2A,
polylysine-mutated C2A peptide when injected into PC12 was unable to
interfere with transmitter release (17).
Interestingly, the effect of a mutated C2A polylysine motif in
full-length Syt 1*, appeared to be partially attenuated by C2B domain,
consistent with a functional relationship between the two C2 domains of
Syt 1 and the calcium channel (24). Interactions through the Syt 1 C2B
domain are also functionally important for neurosecretion (19, 20, 22,
24). More recent studies using a genetic rescue approach in
Drosophila reveals a role for the polylysine motif of the
C2B domain in evoked release (25). Moreover, the C2B domain promotes
the Ca2+-dependent binding of syntaxin 1A to
C2A, suggesting a level of functional synergy between the two C2
domains of Syt 1 (44). Interestingly, secretion from PC12 cells carried
out using the cracked cell method showed that dense-core vesicle
exocytosis does not require vesicular synaptotagmin 1 but may use
instead the plasma membrane synaptotagmins 3 and 7 as Ca2+
sensors (45).
Modulation of Cav2.3--
Previously, induction of
faster activation by Syt 1 was observed for the neuronal
Ca2+ channels, Cav2.2 and Cav2.3,
in contrast to no effect on Cav1.2 (28-30). Here we show
that truncated Syt 1 (Syt 11-264) accelerated Cav2.3 activation suggesting that C2A and not C2B domain is
responsible for the observed effects. Mutation of the polylysine motif
in C2A abolished the stimulatory effect on Cav2.3
activation, indicating the role of this motif in the interaction of the
vesicular protein with the channel as well as with Cav2.3
associated with syntaxin 1A.
The truncated mutant Syt 1*1-264 partially restored
(~75%) current amplitude but did not reverse the syntaxin 1A
effect on activation. Thus modulation of the
syntaxin/Cav2.3 kinetics was affected by Syt
11-264 but was lost in Syt 11-264*. In
contrast, Syt 1 and Syt 1* effectively reversed the syntaxin 1A
inhibition of Cav2.3 current amplitude. These results
propose that the C2B domain partially compensates for the mutation in the C2A polylysine motif.
Together, the data indicate that the C2A polylysine motif affects the
activation of the channel and modulates the kinetics of syntaxin
1A-associated channel. In Fig. 11 we
showed a schematic model of putative interactions of the channel,
syntaxin, and Syt 1.

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 11.
A schematic model illustrating putative
cross-talk interfaces of the voltage-gated calcium channel, syntaxin 1A
(SX), and the C2A domains of synaptotagmin (Syt
1) and synaptotagmin mutated at the C2A* polylysine motif
(Syt 1*). Transmembrane II6 and
III1 are the boundary of Lc753-893, the
cytosolic domain of Cav1.2.
|
|
In summary, our studies provide compelling evidence that the Syt 1 C2A
domain is involved in a functional coupling of the vesicle with the
voltage-gated Ca2+-channels, Cav1.2 and
Cav2.3. The C2A polylysine motif appears to participate in
this interaction and likely functions independently of the Syt 1 Ca2+-mediated interactions with phospholipids or syntaxin
1A (17). The effects of C2A polylysine motif on transmitter release in PC 12 cells as previously reported, may result from a direct
modification of the activation kinetics of the Ca2+ channel
or function indirectly by competing with endogenous Syt 1 for
interactions with the channel. The ability of Syt 1, syntaxin 1A, and
the Ca2+ channel to interact is consistent with the
formation of a functional exocytotic unit, the excitosome (32). The
excitosome complex composed of the Ca2+ channel, syntaxin
1A, SNAP-25, and Syt 1 displays distinct kinetic properties required
for calcium-triggered secretion (28, 29, 32, 33, 39, 46). Therefore,
inhibition of neurotransmitter release by C2A domain might occur by
interfering with generating the excitosome complex and the ensuing
propagation of the signal from the channel to the fusion/docking
machinery rather than Ca2+ binding to Syt 1 (30). The
physiological relevance and the consequences of the different
modulation of neuroendocrine (Cav1.2) and neuronal
(Cav2.3) Ca2+ channels by Syt 1 during the
steps leading to transmitter release will require further studies.
 |
ACKNOWLEDGEMENTS |
D. Atlas thanks the H. L. Lauterbach fund.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed. Tel.:
972-2-658-5406; Fax: 972-2-658-5413; E-mail:
datlas@vms.huji.ac.il.
Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M210270200
 |
ABBREVIATIONS |
The abbreviations used are:
Syt, synaptotagmin;
EDC, N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide
hydrochloride;
GST, glutathione S-transferase.
 |
REFERENCES |
1.
|
Brose, N.,
Petrenko, A. G.,
Südhof, T. C.,
and Jahn, R.
(1992)
Science
256,
1021-1025[Medline]
[Order article via Infotrieve]
|
2.
|
Littleton, J. T.,
Stern, M.,
Schulze, K.,
Perin, M.,
and Bellen, H. J.
(1993)
Cell
74,
1125-1134[Medline]
[Order article via Infotrieve]
|
3.
|
Perin, M. S.,
Fried, V. A.,
Mignery, G. A.,
Jahn, R.,
and Südhof, T. C.
(1990)
Nature
345,
260-263[CrossRef][Medline]
[Order article via Infotrieve]
|
4.
|
Shao, X.,
Fernandez, I.,
Südhof, T. C.,
and Rizo, J.
(1998)
Biochemistry
37,
16106-16115[CrossRef][Medline]
[Order article via Infotrieve]
|
5.
|
Rizo, J.,
and Südhof, T.
(1998)
J. Biol. Chem.
273,
15879-15882[Free Full Text]
|
6.
|
Ubach, J.,
Lao, Y.,
Fernandez, I.,
Arac, D.,
Südhof, T. C.,
and Rizo, J.
(2001)
Biochemistry
40,
5854-5860[CrossRef][Medline]
[Order article via Infotrieve]
|
7.
|
Sutton, R. B.,
Ernst, J. A.,
and Brunger, A. T.
(1999)
J. Cell Biol.
147,
589-598[Abstract/Free Full Text]
|
8.
|
Chapman, E. R.,
Hanson, P. I,
An, S.,
and Jahn, R.
(1995)
J. Biol. Chem.
270,
23667-23671[Abstract/Free Full Text]
|
9.
|
Li, C.,
Ullrich, B.,
Zhang, J. Z.,
Anderson, R. G.,
Brose, N.,
and Südhof, T. C.
(1995)
Nature
375,
594-599[CrossRef][Medline]
[Order article via Infotrieve]
|
10.
|
Schiavo, G.,
Stenbeck, G.,
Rothman, J. E.,
and Sollner, T. H.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
997-1001[Abstract/Free Full Text]
|
11.
|
Gerona, R. R.,
Larsen, E. C.,
Kowalchyk, J. A.,
and Martin, T. F.
(2000)
J. Biol. Chem.
275,
6328-6336[Abstract/Free Full Text]
|
12.
|
Zhang, J. Z,
Davletov, B. A.,
Südhof, T. C.,
and Anderson, R. G.
(1994)
Cell
78,
751-760[Medline]
[Order article via Infotrieve]
|
13.
|
Niinobe, M.,
Yamaguchi, Y.,
Fukuda, M.,
and Mikoshiba, K.
(1994)
Biochem. Biophys. Res. Commun.
205,
1036-1042[CrossRef][Medline]
[Order article via Infotrieve]
|
14.
|
Fukuda, M.,
Moreira, J. E.,
Lewis, F. M.,
Sugimori, M.,
Niinobe, M.,
Mikoshiba, K.,
and Llinas, R.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
10708-10712[Abstract]
|
15.
|
Elferink, L. A.,
Peterson, M. R.,
and Scheller, R. H.
(1993)
Cell
72,
153-159[Medline]
[Order article via Infotrieve]
|
16.
|
Mikoshiba, K.,
Fukuda, M.,
Moreira, J. E.,
Lewis, F. M.,
Sugimori, M.,
Niinobe, M.,
and Llinas, R.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
10703-10707[Abstract]
|
17.
|
Thomas, D. M.,
and Elferink, L. A.
(1998)
J. Neurosci.
18,
3511-3520[Abstract/Free Full Text]
|
18.
|
Robinson, I. M.,
Ranjan, R.,
and Schwarz, T. L.
(2002)
Nature
418,
336-340[CrossRef][Medline]
[Order article via Infotrieve]
|
19.
|
DiAntonio, A.,
and Schwarz, T. L.
(1994)
Neuron
12,
909-920[Medline]
[Order article via Infotrieve]
|
20.
|
Littleton, J. T.,
Stern, M.,
Perin, M.,
and Bellen, H. J.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
10888-10892[Abstract/Free Full Text]
|
21.
|
Littleton, J. T.,
Bai, J.,
Vyas, B.,
Desai, R.,
Baltus, A. E.,
Garment, M. B.,
Carlson, S. D.,
Ganetzky, B.,
and Chapman, E. R.
(2001)
J. Neurosci.
21,
1421-1433[Abstract/Free Full Text]
|
22.
|
Bommert, K.,
Charlton, M. P.,
DeBello, W. M.,
Chin, G. J.,
Betz, H.,
and Augustine, G. J.
(1993)
Nature
363,
163-165[CrossRef][Medline]
[Order article via Infotrieve]
|
23.
|
Lang, J.,
Fukuda, M.,
Zhang, H.,
Mikoshiba, K.,
and Wollheim, C. B.
(1997)
EMBO J
16,
5837-5846[Abstract/Free Full Text]
|
24.
|
Desai, R. C.,
Vyas, B.,
Earles, C. A.,
Littleton, J. T.,
Kowalchyck, J. A.,
Martin, T. F.,
and Chapman, E. R.
(2000)
J. Cell Biol.
150,
1125-1136[Abstract/Free Full Text]
|
25.
|
Mackler, J. M.,
and Reist, N. E.
(2001)
J. Comp. Neurol.
436,
4-16[CrossRef][Medline]
[Order article via Infotrieve]
|
26.
|
Mackler, J. M.,
Drummond, J. A.,
Loewen, C. A.,
Robinson, I. M.,
and Reist, N. E.
(2002)
Nature
418,
340-344[CrossRef][Medline]
[Order article via Infotrieve]
|
27.
|
Catterall, W. A.
(2000)
Annu. Rev. Cell Dev. Biol.
16,
521-555[CrossRef][Medline]
[Order article via Infotrieve]
|
28.
|
Atlas, D.
(2001)
J. Neurochem.
77,
972-985[CrossRef][Medline]
[Order article via Infotrieve]
|
29.
|
Atlas, D.,
Wiser, O.,
and Trus, M.
(2001)
Cell. Mol. Neurobiol.
21,
717-731[CrossRef][Medline]
[Order article via Infotrieve]
|
30.
|
Wiser, O.,
Cohen, R.,
and Atlas, D.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
3968-3973[Abstract/Free Full Text]
|
31.
|
Wiser, O.,
Tobi, D.,
Trus, M.,
and Atlas, D.
(1997)
FEBS Lett.
404,
203-207[CrossRef][Medline]
[Order article via Infotrieve]
|
32.
|
Wiser, O.,
Trus, M.,
Hernandez, A.,
Renström, E.,
Barg, S.,
Rorsman, P.,
and Atlas, D.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
248-253[Abstract/Free Full Text]
|
33.
|
Tobi, D.,
Wiser, O.,
Trus, M.,
and Atlas, D.
(1998)
Receptors Channels
6,
89-98[Medline]
[Order article via Infotrieve]
|
34.
|
Zhong, H.,
Yokoyama, C. T.,
Scheuer, T.,
and Catterall, W. A.
(1999)
Nat. Neurosci.
2,
939-941[CrossRef][Medline]
[Order article via Infotrieve]
|
35.
|
Sheng, Zu-H.,
Yokoyama, C. T.,
and Catterall, W. A.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
5405-5410[Abstract/Free Full Text]
|
36.
|
Charvin, N.,
L'eveque, C.,
Walker, D.,
Berton, F.,
Raymond, C.,
Kataoka, M.,
Shoji-Kasai, Y.,
Takahashi, M.,
De Waard, M.,
and Seagar, M. J.
(1997)
EMBO J.
16,
4591-4596[Abstract/Free Full Text]
|
37.
|
Wiser, O.,
Bennett, M. K.,
and Atlas, D.
(1996)
EMBO J.
15,
4100-4110[Abstract]
|
38.
|
Zor, T.,
and Selinger, Z.
(1996)
Anal. Biochem.
236,
302-308[CrossRef][Medline]
[Order article via Infotrieve]
|
39.
|
Fergestad, T.,
Wu, M. N.,
Schulze, K. L.,
Lloyd, T. E.,
Bellen, H. J.,
and Broadie, K.
(2001)
J. Neurosci.
21,
9142-9150[Abstract/Free Full Text]
|
40.
|
Kim, D. K.,
and Catterall, W. A.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
14782-14786[Abstract/Free Full Text]
|
41.
|
Schneider, T.,
Wei, X.,
Olcese, R.,
Costantin, J. L.,
Neely, A.,
Palade, P.,
Perez-Reyes, E.,
Qin, N.,
Zhou, J.,
Crawford, G. D.,
Smith, G. R.,
Appel, S. H.,
Stefani, E.,
and Birnbaumer, L.
(1994)
Receptors Channels
2,
255-270[Medline]
[Order article via Infotrieve]
|
42.
|
Fernandez-Chacon, R.,
Konigstorfer, A.,
Gerber, S. H.,
Garcia, J.,
Matos, M. F.,
Stevens, C. F.,
Brose, N.,
Rizo, J.,
Rosenmund, C.,
and Südhof, T. C.
(2001)
Nature
410,
41-49[CrossRef][Medline]
[Order article via Infotrieve]
|
43.
|
Thomas, D. M.,
Ferguson, G. D.,
Herschman, H. R.,
and Elferink, L. A.
(1999)
Mol. Biol. Cell
10,
2285-2295[Abstract/Free Full Text]
|
44.
|
Davis, A. F.,
Bai, J.,
Fasshauer, D.,
Wolowick, M. J.,
Lewis, J. L.,
and Chapman, E. R.
(1999)
Neuron
24,
363-376[Medline]
[Order article via Infotrieve]
|
45.
|
Shin, O. H.,
Rizo, J.,
and Sudhof, T. C.
(2002)
Nat. Neurosci.
5,
649-656[Medline]
[Order article via Infotrieve]
|
46.
|
Barg, S.,
Ma, X.,
Elliasson, L.,
Galvanovskis, J.,
Gopel, S. O.,
Obermuller, S.,
Platzer, J.,
Renstrom, E.,
Trus, M.,
Atlas, D.,
Streissnig, G.,
and Rorsman, P.
(2001)
Biophys. J.
81,
3308-3323[Abstract/Free Full Text]
|
47.
|
Mochida, S.
(2000)
Neurosci. Res.
36,
175-182[CrossRef][Medline]
[Order article via Infotrieve]
|
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.