(Received for publication, December 29, 1995; and in revised form, January 25, 1996)
From the
P- and Q-type calcium channels, which trigger rapid
neurotransmitter release at many mammalian synapses, are blocked by
-conotoxin MVIIC.
I-
-Conotoxin MVIIC binding
to rat cerebellar synaptosomes was not displaced by
-conotoxins
GVIA or MVIIA (K
> 1 µM),
which are selective for N-type calcium channels. Solubilized
I-
-conotoxin MVIIC receptors were specifically
recognized by antibodies directed against
A calcium
channel subunits, proteins known to constitute a pore with P/Q-like
channel properties. Antibodies against syntaxin 1, SNAP 25, and VAMP 2
(synaptobrevin) each immunoprecipitated a similar fraction
(20-40%) of
-conotoxin MVIIC receptors. Immunoprecipitation
was not additive, suggesting that heterotrimeric (SNARE) complexes
containing these three proteins interact with P/Q-type calcium
channels. Immobilized monoclonal anti-syntaxin antibodies retained
A calcium channel subunits of 220, 180 and 160 kDa
monitored by immunoblotting with site directed antibodies.
Synaptotagmin was detected in channel-associated complexes, but not
synaptophysin, Rab 3A nor rat cysteine string protein. Trimeric SNARE
complexes are implicated in calcium-dependent exocytosis, a process
thought to be regulated by synaptotagmin. Our results indicate that
these proteins interact with P/Q-type calcium channels, which may
optimize their location within domains of calcium influx.
Neuronal calcium channels are heteromeric proteins constituted
by an subunit, which forms the voltage-gated
transmembrane pore, associated with auxiliary
and
subunits. Five genes encoding homologous
subunits (
A-E) with different channel
properties are expressed in the rat brain (reviewed by Snutch and
Reiner(1992) and Birnbaumer et al.(1994)).
C
and
D subunits each form 1,4-dihydropyridine-sensitive
L-type channels, whereas
B subunits constitute N-type
channels that are specifically blocked by
-conotoxins GVIA or
MVIIA (
GVIA,
MVIIA). (
)Heterologously expressed
A subunits induce currents that are inhibited by
-agatoxin IVA (
AgaIVA) and
-conotoxin MVIIC
(
MVIIC) (Sather et al., 1993; Stea et al.,
1994), and thus have similar properties to both P- and Q-type currents
described in cerebellar Pürkinje and granule
neurons (Mintz et al., 1992; Hillyard et al., 1992;
Randall et al., 1995), where
A transcripts
are strongly expressed (Mori et al., 1991; Starr et
al., 1991; Stea et al., 1994). For the sake of brevity,
we shall refer to the calcium channels that contain
A
subunits as P/Q-type channels.
Neurotransmitter release from axonal
terminals is triggered by calcium entry through voltage-gated channels
in the presynaptic plasma membrane. The sub-millisecond delay between
calcium influx and exocytosis of the contents of synaptic vesicles
implies that the calcium channels involved are in close proximity to
release sites (Llinas et al., 1981; Adler et al.,
1991). Synaptic transmission in mammals is generally insensitive to the
antagonists that act at L-type calcium channels, but is inhibited by
peptide neurotoxins that block N- or P/Q-type calcium channels
(Takahashi and Momiyama, 1993; Wheeler et al., 1994; Mintz et al., 1995; Turner and Dunlap, 1995). Pharmacological
evidence therefore suggests that channels containing A
or
B subunits are preferentially coupled to the
exocytosis of rapid neurotransmitters.
Recent biochemical evidence
suggests that direct interactions between N-type calcium channel
proteins and the exocytotic machinery may contribute to
excitation-secretion coupling. N-type calcium channels can associate
with syntaxin and synaptotagmin (Leveque et al., 1992; Yoshida et al., 1992; Bennett et al., 1992; Leveque et
al., 1994), two key proteins that contribute to the targeting and
calcium-dependent fusion of synaptic vesicles at the plasma membrane
(reviewed by Scheller and Bennett(1994) and
Südhof(1995)). This interaction involves binding of
syntaxin to a cytoplasmic loop of the B subunit (Sheng et al., 1994). In contrast, associations between syntaxin and
L-type channels have not been detected (Yoshida et al., 1992;
El Far et al., 1995).
AgaIVA and
MVIIC inhibit
a predominant component of transmitter release at most rapid mammalian
synapses, by blocking calcium influx through presynaptic P/Q-type
calcium channels (Takahashi and Momiyama, 1993; Wheeler et
al., 1994; Mintz et al., 1995). However, biochemical
evidence for an interaction between P/Q-type channels and the
exocytotic complex has not been reported. We have therefore used
I-
MVIIC to label calcium channels containing
A subunits solubilized from rat cerebellar
synaptosomes, and to explore their association with proteins of the
secretory pathway. Our results indicate that a population of calcium
channels containing
A subunits can interact with a
trimeric SNARE complex (Söllner et al.,
1993a) composed of syntaxin 1, SNAP 25, and VAMP 2 (synaptobrevin).
Anti-syntaxin antibodies (mAb 10H5) were covalently coupled to Protein A-Sepharose (Leveque et al., 1994). 4 ml of a CHAPS extract of cerebellar synaptosomes were batch-incubated overnight with 1 ml of immunoaffinity resin. Beads were loaded into a column, washed with 20 ml of 10 mM Tris, 0.2 M NaCl, 0.4% CHAPS adjusted to pH 7.5 with HCl, and eluted with 0.1 M triethylamine, 0.2 M NaCl, 0.4% CHAPS adjusted to pH10.5 with HCl. All steps were performed at 4 °C in buffers containing Complete protease inhibitor mixture (Boehringer Mannheim). SDS-polyacrylamide gel electrophoresis and Western blotting were performed as described previously (Martin-Moutot et al., 1995), using Protein A-peroxidase, and an ECL detection kit (Amersham Corp.).
-Conotoxins provide useful probes for the biochemical
assay of voltage-dependent calcium channels.
GVIA and
-conotoxin MVIIA (
MVIIA) are selective antagonists of
N-type channels in which the transmembrane pore is constituted by an
B subunit. In contrast
MVIIC, which is highly
homologous to
MVIIA, blocks P-, Q-, and N-type calcium channels
in neurons (Hillyard et al., 1992; Randall and Tsien, 1995),
and inhibits currents induced by the heterologous expression of calcium
channels containing
A or
B subunits
(Sather et al., 1993; Grantham et al., 1994).
Although micromolar concentrations of
MVIIC display little
selectivity and block both P/Q- and N-type channels, subnanomolar
concentrations of
I-
MVIIC reveal binding sites
pharmacologically distinct from those associated with N-type channels
(Hillyard et al., 1992; Kristipati et al., 1994), and
which are therefore likely to be constituted by P/Q-type channels
(Martin-Moutot et al., 1995).
I-
MVIIC
displayed specific high affinity binding to rat cerebellar synaptosomes (Fig. 1A), which was displaced by native
MVIIC
with a K
of about 1 nM, but not by the
selective N-type calcium channel antagonists
GVIA or
MVIIA (K
> 1 µM). Conversely, using
I-
GVIA as a probe, significant inhibition was only
obtained with relatively high concentrations of
MVIIC (K
> 30 nM; data not shown),
presumably due to
MVIIC competing with
GVIA at the same
binding site on N-type calcium channels. Nevertheless these data
suggest that at sub-nanomolar concentrations,
I-
MVIIC labels binding sites that are distinct from
those associated with
B subunits.
Figure 1:
I-
MVIIC
binding to P/Q-type calcium channels in rat cerebellar synaptosomes. A, synaptosomes were incubated for 1 h at 30 °C with 0.3
nM
I-
MVIIC, in the presence or absence of
MVIIC (closed squares),
MVIIA (closed
circles), or
GVIA (open circles). Membrane-bound
radioactivity was measured by filtration and
counting.
Nonspecific binding, defined as radioactivity bound in the presence of
0.3 µM
MVIIC, was subtracted (means ± S.D., n = 6). B, synaptosomes were labeled with
I-
MVIIC, in the presence or absence of 0.3
µM
MVIIC or
GVIA. CHAPS extracts containing
I-ligand/receptor complexes were incubated with
anti-
A subunit antibodies (rbA-1) in the presence or
absence of an excess of the
A peptide used to raise
the antibodies, and the radioactivity in immune complexes was counted
(means ± S.D., n =
6).
Cerebellar
membranes were labeled with I-
MVIIC, and
radioligand-receptor complexes were solubilized using CHAPS.
Approximately 50% of these complexes were adsorbed by antibodies
directed against a peptide specific to the
A sequence.
Only 8% were retained if antibodies were preincubated with the cognate
A peptide (Fig. 1B).
Anti-
A antibodies reacted with specific
I-
MVIIC binding sites as the amount of
immunoprecipitated radioactivity was strongly reduced if labeling was
performed in the presence of 0.3 µM
MVIIC but not
0.3 µM
GVIA. No significant immunoprecipitation of
I-
MVIIC binding sites was detected with antibodies
directed against
B (8 ± 1%) or
C (8 ± 1%) subunits, although these antibodies
recognize >50% of
I-
GVIA receptors (N-type
channels) or [
H]PN200-110 receptors (L-type
channels) respectively in analogous experiments (not shown).
High
affinity I-
MVIIC binding thus provides a specific
assay for calcium channels containing
A subunits, and
immunoprecipitation of solubilized receptors can be used to examine
whether proteins implicated in exocytosis are associated with these
subunits. Antibodies against three proteins that form the core SNARE
complex, syntaxin 1, SNAP 25, and VAMP2, each adsorbed 20-40% of
solubilized
I-
MVIIC receptors, whereas non-immune
IgG trapped <5% (Fig. 2A). Recovery of
I-
MVIIC receptors by antibodies against other
synaptic proteins implicated in the secretory pathway: synaptophysin,
Rab 3A (Fig. 2A), and cysteine string protein (data not
shown) was similar to that obtained with non-immune IgG, although these
antibodies did immunoprecipitate their respective antigens (not shown).
Figure 2:
Association of SNARE complexes with
P/Q-type calcium channels A, solubilized I-
MVIIC receptor complexes were incubated with
anti-syntaxin 1 (squares), anti-SNAP 25 (diamonds),
anti-VAMP 2 (circles), anti-Rab 3A (triangles), and
anti-synaptophysin (asterisks) antibodies or non-immune IgG (crosses), and the radioactivity recovered in immune complexes
was counted (means ± S.D., n = 3) B,
additivity was examined with 20 µg of each of the two indicated
antibodies combined in the same assay (means ± S.D., n = 6). C, immunoassays were performed with
I-
MVIIC receptors labeled in the absence of native
-conotoxin (shaded) or in the presence of 0.3 µM
MVIIC (open) or
GVIA (hatched)
(means ± S.D., n =
6).
Immunoadsorption with increasing concentrations of antibodies
against syntaxin, SNAP 25, or VAMP reached a plateau level, indicating
that a limited fraction of calcium channels is associated with each
SNARE protein (Fig. 2A). If each SNARE protein
interacts individually with a different fraction of calcium channels,
immunoprecipitation by two antibodies combined should be additive.
Alternatively, if all three SNARE proteins are associated with the same
population of calcium channels, immunoprecipitation should not be
additive. The results shown in Fig. 2B are consistent
with the second hypothesis. When saturating amounts (20 µg) of two
antibodies were combined in the same assay, the percentage of
immunoprecipitation was similar to that obtained when anti-syntaxin
antibodies were combined with control antibodies. These results suggest
that SNAP 25 and VAMP are only associated with calcium channels that
are bound to syntaxin, implying the interaction of a trimeric
syntaxin-SNAP 25-VAMP complex with a significant fraction of I-
MVIIC-labeled calcium channels.
The
experiments illustrated in Fig. 2C were performed to
verify that immunoprecipitation of I-
MVIIC binding
sites associated with N-type channels did not occur. In each case the
amount of radioactivity adsorbed by the antibodies was strongly reduced
by addition of 0.3 µM
MVIIC, but not significantly
affected by 0.3 µM
GVIA, indicating that the
detected interaction with SNARE complexes is specific for P/Q-type
channels that contain
A subunits. Synaptotagmin, a
synaptic vesicle transmembrane protein that is thought to function as a
calcium sensor in exocytosis, can associate with N-type calcium
channels (Leveque et al., 1992, 1994; Yoshida et al.,
1992) and SNARE complexes (Söllner et al.,
1993b), by binding to syntaxin (Li et al., 1995). An antibody
that recognizes synaptotagmin isoforms I and II also immunoprecipitated
P/Q-type channels (Fig. 2C).
In order to confirm the
association of A subunits with syntaxin, CHAPS
extracts of cerebellar membranes were loaded onto an immunoaffinity
matrix composed of a monoclonal anti-syntaxin antibody covalently
coupled to Protein A-Sepharose 4BCL. After extensive washing, the
column was eluted by a step to pH 10.5. Western blots of the recovered
proteins were probed with anti-
A antibodies, revealing
several immunoreactive bands (Fig. 3, lane 1). These
proteins were not detected when CHAPS extracts were loaded onto a
control column (Fig. 3, lane 2) or when the antibodies
were preincubated with an excess of the cognate
A
peptide (Fig. 3, lane 3). The 220-, 180-, and 160-kDa
proteins have been detected in cerebellar homogenates using
anti-
A (rbA-2) antibodies (Martin-Moutot et
al., 1995), and similar size forms have also been reported by
Westenbroek and colleagues(1995). Heterogeneity in the size of these
A polypeptides expressed in the brain may result from
alternative splicing or proteolytic processing. However, the 140-kDa
protein was not detected in homogenates and is likely to be an
A cleavage product, generated during affinity
chromatography in spite of the presence of protease inhibitors. These
results thus indicate that syntaxin interacts with
A
subunits of P/Q-type calcium channels. Sheng et al.(1994) did
not detect binding of fusion proteins containing part of the
cytoplasmic loop (residues 723-868) linking homologous domains
II-III of the
1A subunit to syntaxin 1A. Our data indicate that
there are alternative sites of interaction between SNARE complexes and
P/Q-type calcium channel subunits, although they have yet to be
identified at the sequence level.
Figure 3:
Interaction of syntaxin with calcium
channel A subunits. Cerebellar synaptosomes were
solubilized in CHAPS and applied to either monoclonal anti-syntaxin 1
antibodies (lanes 1 and 3) or non-immune mouse IgG (lanes 2 and 4) covalently coupled to Protein
A-Sepharose. After washing, bound proteins were eluted by a step to pH
10.5 and analyzed by Western blotting with anti-
A
peptide antibodies (rbA-2) in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of the
cognate
A peptide.
Current models of exocytosis are
based on sequential protein-protein interactions leading to the
assembly of a multi-molecular complex, located at the interface between
a synaptic vesicle and the presynaptic plasma membrane (reviewed by
Südhof(1995)). The core interaction concerns a
vesicle membrane protein VAMP, which binds to two proteins that are
predominantly located at the plasma membrane: syntaxin and SNAP 25. A
second vesicle protein synaptotagmin can bind to the trimeric SNARE
complex, and there is compelling evidence that synaptotagmin acts as a
calcium-dependent regulator of transmitter release (reviewed by
Littleton and Bellen(1995)). Our data indicate that a SNARE complex
associated with synaptotagmin interacts with native voltage-gated
calcium channels containing A subunits solubilized
from nerve terminals. Taken together with reports that N-type (Leveque et al., 1994; Sheng et al., 1994), but not L-type
(Yoshida et al., 1992; El Far et al., 1995) channels
bind to syntaxin, they are thus compatible with a predominant role for
P/Q- and N-type but not L-type channels in rapid synaptic transmission
in the mammals. Furthermore, our findings are consistent with recent
functional evidence that syntaxin modulates the gating properties of
calcium channels formed by the heterologous expression of
A or
B, but not
C
subunits in Xenopus oocytes (Bezprozvanny et al.,
1995). Molecular interactions that link calcium channels to proteins of
the exocytotic complex could contribute to the rapid kinetics of
excitation-secretion coupling by optimally locating the calcium sensors
that trigger vesicle fusion, and may also regulate the ability of
channels to open in response to depolarization.