Neuroscience Research 36 (2000) 175 182
www.elsevier.com/locate/neures
Update article
Protein protein interactions in neurotransmitter release
Sumiko Mochida *
Department of Physiology, Tokyo Medical Uni ersity, 1-1 Shinjuku-6-chome, Shinjuku-ku, Tokyo 160-8402, Japan
Received 1 November 1999; accepted 10 December 1999
Abstract
The arrival of a nerve impulse at a nerve terminal leads to the opening of voltage-gated Ca2 + channels and a rapid influx of
Ca2 +. The increase in Ca2 + concentration at the active zone from the basal level of 100 200 mM triggers the fusion of docked
synaptic vesicles, resulting in neurotransmitter release. A large number of proteins have been identified at nerve terminals and a
cascade of protein protein interactions has been suggested to be involved in the cycling of synaptic vesicle states. Functional
studies in last half decade on synaptic-terminal proteins, including Ca2 + channels, have revealed that the SNARE core complex,
consisting of synaptobrevin VAMP, a synaptic vesicle-associated protein, syntaxin and SNAP-25, synaptic membrane-associated
proteins, acts as the membrane fusion machinery and that proteins interacting with the SNARE complex play essential roles in
synaptic vesicle exocytosis by regulating assembly and disassembly of the SNARE complex. © 2000 Elsevier Science Ireland Ltd.
All rights reserved.
Keywords: Neurotransmitter release; Synaptic vesicle; Exocytosis; Snare proteins; Synaptic terminal proteins; Ca2 + channels
tetanus and bolutinum neurotoxins, a group of eight
1. Introduction
related paralytic neurotoxins produced by Clostridia,
block neuronal exocytosis by selectively proteolyzing
Exocytosis in neurons requires proteins known as
the individual SNARE proteins (Niemann et al., 1994;
SNAREs A set of three synaptic membrane proteins,
Monteccuco and Schiavo, 1995; Fig. 1). Furthermore,
the synaptic vesicle protein synaptobrevin (also known
proteins related to syntaxin, SNAP-25 and VAMP are
as VAMP, vesicle-associated membrane protein) and
essential for a variety of other membrane transport
the plasma membrane proteins syntaxin and SNAP-25
reactions. This has been best characterized in yeast,
(synaptosome-associated protein of 25 kDa), were orig-
where proteins homologous to the SNAREs have been
inally identified as membrane receptors for NSF (N-
shown to be important in trafficking throughout the
ethylmaleimide-sensitive factor) and SNAPs (soluble
secretory pathway (Bennett and Scheller, 1993; Ferro-
NSF attachment proteins) and were therefore, desig-
Novick and Jahn, 1994). These lines of evidence consid-
nated as SNAREs(SNAP receptors) (Söllner et al.,
ered together implicate the SNAREs in neuronal
1993a). This finding directly linked these proteins to
exocytosis. SNARE-associated proteins, including small
exocytosis, as NSF and SNAPs are soluble proteins
GTP-binding proteins and Ca2 +-binding proteins, have
known to be essential for many intracellular vesicle
been identified in nerve terminals and their regulatory
fusion reactions. A second line of evidence linking these
roles in exocytosis have been discussed in a number of
proteins to exocytosis came from the discovery that
excellent reviews (Südhof, 1995; Augustine et al., 1996;
Bean and Scheller, 1997; Hanson et al., 1997). This
article will focus on recent findings concerning synaptic
protein protein interactions implicated in nerve im-
* Tel.: [phone] ext. 248; fax: [phone].
E-mail address: [e-mail] (S. Mochida)
pulse-evoked exocytosis.
0168-0102/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0168-0102(99)00128-5
176 S. Mochida / Neuroscience Research 36 (2000) 175 182
2. SNARE complexes SNARE in Drosophila had profound effects on the
function of secretory pathways, with complete loss of
SNAREs assemble with a 1:1:1 stoichiometry into synaptic transmission (Broadie et al., 1995; Schulze et
stable ternary complexes that are disassembled by NSF, al., 1995; Sweeney et al., 1995; Deitcher et al., 1998).
an ATPase, working together with -SNAP (Söllner et These results indicate that SNAREs do not play an
al., 1993b; Hayashi et al., 1995). Are SNAREs involved essential role in docking.
in vesicle docking or fusion? SNAREs represented by Recent in vitro experiments revealed that SNARE
synaptobrevin are vesicle-membrane, or  v-SNAREs , complexes are the minimal machinery required for
whereas SNAREs represented by syntaxin and SNAP- membrane fusion (Weber et al., 1998). Weber et al.
25 are target-membrane, or  t-SNAREs (Söllner et al., (1998) demonstrated that recombinant synaptobrevin
1993a; Rothman, 1994). As v and t-SNAREs are pre- and the syntaxin/SNAP-25 complex, reconstituted into
dominantly located on the vesicles and target mem- separate lipid vesicles assemble into trans SNARE com-
branes, respectively, it has been proposed that the plexes, designated as  SNAREpins , linking two mem-
formation of SNARE complexes may play a critical branes. This leads to spontaneous lipid mixing,
role in establishing and stabilizing membrane docking considered to be an index of fusion between the docked
(Söllner et al., 1993a; Rothman, 1994). However, func- membranes at physiological temperature. SNARE com-
tional experiments using clostridial neurotoxins have plexes are formed by coiled-coil interactions of the
shown that disruption of the v-SNARE, synaptobrevin a-helices of syntaxin, SNAP-25 and synaptobrevin
(Hunt et al., 1994; Sweeney et al., 1995), or that of the (Chapman et al., 1995a) immediately before fusion
t-SNARE, SNAP-25 (Banerjee et al., 1996), does not (Sutton et al., 1998). Electrostatic calculations show a
affect the docking or priming of synaptic vesicles. Ge- pronounced charge distribution of the synaptic fusion
netic deletion of the t-SNARE, syntaxin or the v- complex, the cumulative electrostatic potential may
Fig. 1. SNAREs. SNAREs, consisting of three synaptic membrane proteins, namely synaptobrevin/VAMP, a synaptic vesicle protein and syntaxin
and SNAP-25 which are plasma membrane proteins, form the four -helix bundles of the synaptic fusion complex (Sutton et al., 1998). SNAREs
are targets of clostridial neurotoxins that block neurotransmitter release. Synaptobrevin is cleaved by the botulinum neurotoxins (BoNT) B, D,
G and F and tetanus toxin (TeNT). Syntaxin is cleaved by BoNT C1. SNAP-25 is cleaved by BoNTA and E.
S. Mochida / Neuroscience Research 36 (2000) 175 182 177
Fig. 2. Hypothetical model of exocytosis regulated by the SNAREs and SNARE-interacting proteins. The simplest model for exocytosis is that
SNARE assembly, triggered by the binding of Ca2 + to synaptotagmin, induces the fusion of synaptic vesicles docked at the active zone close to
Ca2 + channels. Most of the SNARE-interacting proteins shown in this figure regulate SNARE assembly and disassembly. Arrows indicate
protein protein interactions related to the cascade of synaptic vesicle states shown below. Each color of arrows corresponds to the color of arrows
in the diagram of the synaptic vesicle cascade. More complicated models have, however, been suggested based on a number of studies on synaptic
proteins. Ca2 + (red circles) may act as a signal for Ca2 +-binding proteins to promote progression of synaptic vesicles to the next stage. Moreover,
the SNARE-interacting proteins, such as snapin, complexin and rab3A, could modulate SNARE complex formation and may thus regulate the
strength of synaptic transmission underlying synaptic plasticity.
promote membrane fusion by affecting by membrane the other hand, Xu et al. (1998) suggested that there is
surface (Sutton et al., 1998). It remains to be deter- an equilibrium between the assembly and disassembly
of SNAREs in the absence of exocytosis, based on the
mined how the SNARE complexes are formed and
finding of a blockade of exocytosis of chromaffin cells
whether the free energy released by the assembly of the
by botulinum toxins, which are known only to act on
synaptic fusion complex is sufficient to induce lipid
SNARE proteins in the disassembled state. Accord-
mixing. Chen et al. (1999) suggested that rapid signal-
ingly, it remains to be clarified when and how SNARE-
ing in neurons is achieved by organizing the SNARE
protein disassembly occurs.
complexes for very rapid Ca2+-triggered assembly. A
model for Ca2 +-triggered synaptic vesicle fusion pro-
poses that synaptotagmin, a Ca2 +-binding protein (see
Ca2 +-binding proteins), acts as an electrostatic switch,
3. Proteins interacting with SNAREs
promoting a structural rearrangement in the fusion
machinery (Shao et al., 1997).
A multitude of proteins control the SNAREs (Fig.
According to the above mechanism of fusion, disas-
2). Complexin binds to SNARE complexes and regu-
sembly of SNARE complexes by an ATPase, NSF,
lates the functions of SNAREs in competition with
together with -SNAP may occur after the fusion of
-SNAP (McMahon et al., 1995). The inhibitory roles
synaptic vesicles. In support of this, Littleton et al.
of complexin II on neurotransmitter release, have been
(1998), using syntaxin and NSF mutants of Drosophila,
suggested by functional studies in Aplysia buccal gan-
found evidence that NSF disassembles SNAREs resid- glia (Ono et al., 1998). Injection of recombinant com-
ing in the presynaptic membrane after the fusion. On plexin II and -SNAP into presynaptic neurons, caused
178 S. Mochida / Neuroscience Research 36 (2000) 175 182
depression and facilitation of neurotransmitter release, binding domains (Touchot et al., 1987; Matsui et al.,
respectively. The effect of complexin II was reversed by 1988). The rab3 homologue Ypt1p is known to regulate
a subsequent injection of recombinant -SNAP and the formation of SNARE complexes in yeast via tran-
vice versa. Enhancement of synaptic transmission by sient interactions (Lian et al., 1994; SÅ‚gaard, et al.,
recombinant SNAPs at giant synapses of the squid has 1994; Lupashin and Waters, 1997). At nerve terminals,
also been reported (DeBello et al., 1995). A recent rab3A and its binding proteins, rabphilin3A (Shirataki
study in complexin II-deficient mice reported that ordi- et al., 1993) and Rim (Wang et al., 1997) are involved
nary synaptic transmission and short-term plasticity are in exocytosis via hydrolysis of GTP (Bean and Scheller,
normal but long-term potentiation (LTP) in hippocam- 1997). Using hippocampal neurons from rab3A-mutant
pus is impaired (Takahashi et al., 1999). Complexin mice, Geppert et al. (1994a, 1997) demonstrated a
appears to be a multi-functional modulator of neuro- function for rab3A in limiting exocytosis. rab3A has
transmitter release, regulating the formation of the also been shown to be essential for generation of LTP
SNARE complex. at mossy fiber synapses in the hippocampal CA3 region
Snapin is a protein exclusively located on synaptic (Castillo et al., 1997). Rabphgilin-3A a synaptic vesicle
vesicle membranes that associates with the SNARE protein, is proposed to act as a rab3A effector protein
complex through direct interaction with SNAP-25, by binding to rab3A in a GTP-dependent manner.
modulating sequential interactions between the Injection of recombinant rabphilin-3A protein into
SNAREs and synaptotagmin, a Ca2 + sensor (see cal- squid giant synapses inhibited exocytosis (Burns et al.,
cium-binding proteins). Recombinant snapin injected 1998); however, studies on rabphilin-knockout mice
into presynaptic neurons reversibly inhibited neuro- revealed that this protein is not required for rab3A to
transmitter release at synapses between rat superior regulate neurotransmitter release (Schülter et al., 1999).
cervical ganglion neurons (SCGNs) in culture (Ilardi et Rim is a protein associated with the plasma membrane
al., 1999). SNARE complex formation was also regu- at the active zone and binds to rab3A complexed with
lated by cytoplasmic syntaxin-interacting proteins, such GTP, suggesting that Rim serves as a regulator (a
as Munc-18 and tomosyn. In vitro binding studies promoter) of synaptic vesicle fusion by inducing the
showed that Munc-18 interacts with syntaxin prevent- formation of a GTP-dependent complex between
ing its binding to SNAP-25 or synaptobrevin and synaptic vesicles and plasma membranes (Wang et al.,
thereby precluding formation of the SNARE complex 1997). However, until now, no functional studies of
(Pevsner, et al., 1994), while tomosyn promotes Rim have been performed in neurons.
SNARE complex assembly (Fujita et al., 1998a). Func-
tional studies on squid giant synapses, demonstrated
that peptides corresponding to a partial sequence of 4. Ca2 + channels
munc-18, inhibited exocytosis, indicating that the inter-
action of munc-18 with syntaxin is essential for the The SNARE complex interacts with N-type and P/Q-
fusion of docked vesicles (Dresbach et al., 1998). type Ca2 + channels that provide Ca2 + for triggering
Syntaphilin, a plasma membrane-associated protein, exocytosis in the peripheral and central nervous system
competes with SNAP-25 for binding to syntaxin and (Sheng et al., 1994a,b; Rettig et al., 1996). Disruption
inhibits the SNARE complex formation by binding to of Ca2 + channel interactions with SNAREs by a pep-
syntaxin at nerve terminals. Transient overexpression of tide sequence of the syntaxin-binding site of N-type
syntaphilin in cultured hippocampal neurons, signifi- Ca2 + channels altered the Ca2 +-dependence of neuro-
cantly reduces neurotransmitter release. Furthermore, transmitter release at neuromuscular junctions of Xeno-
introduction of the syntaphilin coiled-coil domain into pus (Rettig et al., 1997). This interaction was found to
presynaptic neurons of the SCGNs synapse inhibits be essential for synchronous neurotransmitter release.
synaptic transmission. Syntaphilin may function as a The peptide sequence of the syntaxin-binding site of
molecular clamp that controls the availability of free N-type Ca2 + channels inhibited synchronous transmit-
syntaxin for the assembly of the SNARE complex and ter release, while it increased the asynchronous trans-
thereby regulates synaptic vesicle exocytosis (Lao et al., mitter release that follows a train of action potentials at
in press). Septin CDCrel-1 a GTPase associated with synapses formed by SCGNs (Mochida et al., 1996). In
synaptic vesicles, also binds to syntaxin via the SNARE addition to mediating Ca2 +entry, N-type Ca2 + chan-
interaction domain and inhibits exocytosis by prevent- nels may have direct effects on the transmitter release
ing vesicle docking (Beites et al., 1999). process via interaction with SNARE proteins. Introduc-
tion of the peptide sequence of the syntaxin-binding site
3.1. GTP-binding proteins and associated proteins of N-type Ca2 + channels decreased the voltage-depen-
dent enhancement of Ca2 +-independent transmitter re-
A GTP-binding protein, rab3, which is a vesicle-asso- lease, suggesting that the N-type Ca2 + channel serves
ciated protein has a GTPase motif and GTP/GDP as a voltage sensor that enhances the docking and/or
S. Mochida / Neuroscience Research 36 (2000) 175 182 179
exocytosis of synaptic vesicles via its interaction with Other Ca2 +-binding proteins containing C2 domains,
SNARE proteins (Mochida et al., 1998b). The interac- such as rabphilin-3A, Munc-13 and Doc2, are thought
tion appears to tether SNARE complexes to Ca2 + to participate in vesicle trafficking or translocation to a
channels, thereby localizing the fusion machinery near readily releasable pool prior to docking/fusion at the
the site of Ca2 + influx and potentiating synaptic trans- active zone (Burns et al., 1998; Mochida et al., 1998a).
mission. However, studies in which syntaxin was ex- Rabphlin-3A and doc2 are synaptic vesicle-associated
pressed in Xenopus oocytes (Bezprozvanny et al., 1995; proteins (Shirataki et al., 1993; Orita et al., 1995) and
Wiser et al., 1996) and in which mutant syntaxin lack- Munc-13 is a membrane-associated protein (Brose et
ing the Ca2 + channel binding site was expressed in al., 1995). Doc2 interacts with Munc-13 (Orita et al.,
Drosophila (Wu et al., 1999), suggest that syntaxin also 1997) which, in turn, interacts directly with syntaxin in
functions to inhibit Ca2 + channels. a different state (Betz et al., 1997). These protein
protein interactions may regulate the progression of
synaptic vesicles to the docked and primed states (Süd-
hof, 1995) in Ca2 +-dependent manner. Rabphilin-3A
5. Calcium-binding proteins interacts with rab3A, which interacts with rim in a
GTP-dependent manner (see GTP-binding proteins).
Calcium-binding proteins containing two C2 do- Rim is also a Ca2 +-binding protein containing C2
mains, homologous to the Ca2+-binding regulatory domains (Wang et al., 1997).
region of PKC, are considered to act as Ca2 + sensors
in nerve terminals. Synaptotagmin a synaptic vesicle 5.1. Protein phosphorylation
protein which binds to Ca2 + and phospholipids via its
C2 domains, has been best characterized as a Ca2 + There are several lines of evidence suggesting that the
sensor in exocytosis (Brose et al., 1992; Chapman et al., proteins involved in exocytosis are targets for modula-
1995b). Twelve synaptotagmin isoforms have been tion by second messenger systems. Exocytosis from
identified. Synaptotagmin I (and II) interacts directly chromaffin cells is enhanced by protein kinase C (PKC)
with syntaxin. This interaction is regulated by Ca2 + via an increase in the size of the readily releasable pool
but requires more than 200 M for half-maximal bind- of secretory granules (Gillis et al., 1996). This could be
ing (Li et al., 1994). This approximates the Ca2 + attributable to phosphorylation of SNAREs or/and
requirement for synaptic vesicle exocytosis and suggests SNARE-interacting proteins. Munc-13, which interacts
a mechanism whereby Ca2 + triggers exocytosis by reg- with the Munc-18-syntaxin complex, has phorbol ester-
ulating synaptotagmin I (and II) interaction with syn- and diacylglycerol-binding domains (Maruyama and
taxin (Li et al., 1994) and other SNAREs (Schiavo et Brenner, 1991; Brose et al., 1995). Overexpression of
al., 1997). This idea was supported not only by bio- Munc-13 at the neuromuscular junctions of Xenopus
chemical evidence that synaptotagmin undergoes a increased the facilitatory actions of phorbol ester on
Ca2 +-dependent conformational change (Brose et al., transmitter release, suggesting that this protein is a
1992; Shao et al., 1997), but also by the following target for the diacylglycerol second messenger pathway
functional evidence. Ca2 +-dependent neurotransmitter (Betz et al., 1998). Nitric oxide, which stimulates Ca2 +-
release was severely impaired in synaptotagmin I- independent transmitter release from synaptosomes, en-
knockout mice (Geppert et al., 1994b) and at synapses hances the formation of the SNARE complex and
following injection of C2 domain peptides (Bommert et inhibits the binding of Munc-18 to syntaxin (Meffert et
al., 1993) or antibodies against the C2A domain al., 1996). PKC or cyclin-dependent kinase 5 phospho-
(Mikoshiba et al., 1995; Mochida et al., 1997). A recent rylates Munc-18 (Fujita et al., 1998b; Fletcher et al.,
study demonstrated that Ca2 + triggers the penetration 1999). Phosphorylation of Munc-18 increases the level
of synaptotagmin I into membranes and simultaneously of v-SNARE interaction with syntaxin and the secre-
enhances the binding of synaptotagmin I to the tory response. These lines of evidence indicate that
SNARE complex, supporting the molecular model in phosphorylation of SNAREs or synaptic terminal
which synaptotagmin triggers exocytosis via its interac- proteins that interact with SNAREs modulate the effi-
tions with membranes and SNARE complexes (Davis ciency of synaptic transmission. Phosphorylation of
et al., 1999). In addition, synaptic efficacy may be SNAP-25 decreased the amount of syntaxin co-im-
modulated by changes in the ratio of synaptotagmin munoprecipitated with SNAP-25 (Shimazaki et al.,
isomers at the synaptic vesicles (Littleton et al., 1999). 1996), suggesting that SNARE complex formation is
Synaptotagmin IV forms hetero-oligomers with synap- inhibited by phosphorylation of SNAP-25 Following
totagmin I, resulting in the formation of synaptotagmin phosphorylation by PKA, the binding of -SNAP to
clusters that cannot effectively penetrate into the mem- the SNARE complex is 10 times weaker than that of
brane, thereby changing the Ca2 + sensitivity of vesicle the dephosphorylated form (Hirling and Scheller, 1996).
fusion and decreasing evoked neurotransmission. These data suggest that phosphorylation of SNAREs
180 S. Mochida / Neuroscience Research 36 (2000) 175 182
Table 1
SNARES and SNARE-associated proteins implicated in exocytosisa
Classification Proteins Localization Speculated functions in neurotransmitter release
SNARES (SNARE core complex) Synaptobrevin/ Synaptic vesicles Fusion machinery
VAMP
Syntaxin Plasma membranes
SNAP-25 Plasma membranes
SNARE core complex-interacting NSF Cytoplasm SNARES disassembly
proteins (an ATPase)
-SNAP Cytoplasm SNAREs disassembly
Complexin Cytoplasm Modulation of SNAREs assembly
Snapin Synaptic vesicles Modulation of SNAREs-synaptotagmin interaction
N-(and P/Q- Plasma membranes (1) Synchronous neurotransmitter release
type) Ca2+
channels (2) Transmission of voltage signal to SNARE complexes
Syntaxin-interacting proteins Tomosyn Cytoplasm and plasma Stimulation of SNAREs assembly
membranes
Munc-18 Cytoplasmic face of (1) Inhibition of SNAREs assembly
plasma membranes (2) Essential for synaptic vesicle fusion
Syntaphilin Plasma membranes Inhibition of SNAREs assembly
Munc-13 Cytoplasmic face of (1) Enhancement of SNAREs assembly by diacylglycelol
plasma membranes (2) Promotion of synaptic vesicle trafficking by interac-
tion with Doc2
GTP-binding proteins and associ- Rab3A Synaptic vesicles (1) Limiting fusion machinery
ated proteins (a GTPase) (2) Generation of LTP
(3) Recruitment of synaptic vesicles to the active zone
Rabphilin-3A Synaptic vesicles (1) Modulation of synaptic vesicle fusion
(2) Modulation of synaptic vesicle trafficking
Rim Active zone Promotion of synaptic vesicle fusion
Ca2+-binding proteins containing Synaptotagmin Synaptic vesicles Trigger of synaptic vesicle fusion
two C2 domains I
Doc 2 Synaptic vesicles Promotion of synaptic vesicle trafficking by interaction
with Munc-13
Munc-13 Cytoplasmic face of (1) Enhancement of snares assembly by diacylglycelol
plasma membranes (2) Promotion of synaptic vesicle trafficking by interac-
tion with Doc2
Rabphilin-3A Synaptic vesicles (1) Modulation of synaptic vesicle fusion
(2) Modulation of synaptic vesicle trafficking
Rim Active zone Promotion of synaptic vesicle fusion
a
Although several isoforms of proteins have been detected, the best-characterized protein is listed on the table.
and related proteins may also be responsible for synap- or disassembly of SNAREs. Moreover, the SNARE-in-
tic depression. teracting proteins may regulate the efficiency and
strength of synaptic transmission underlying synaptic
plasticity and memory by modulating the SNARE com-
plex formation. Ca2 +-binding proteins could act as key
6. Conclusions
proteins that induce the progression of synaptic vesicles
to the next stage along the maturation pathway.
The hypothetical functions of SNAREs and SNARE-
interacting proteins in synaptic vesicle exocytosis are
summarized in Fig. 2 and Table 1. The protein protein
interactions appear to be very complicated. However, Acknowledgements
SNAREs are probably essential components for synap-
tic vesicle fusion machinery and most of the other Fig. 1 is printed by permission from Nature, 395,
proteins described in this article regulate the assembly 347 353, 1998 (Copyright: Macmillan Magazines),
S. Mochida / Neuroscience Research 36 (2000) 175 182 181
Deitcher, D.L., Ueda, A., Stewart, B.A., et al., 1998. Distinct require-
with kind agreement of Dr Reinhard Jahn (Department
ments for evoked and spontaneous release of neurotransmitter are
of Neurobiology, Max Planck-Institute for Biophysical
revealed by mutations in the Drosophila gene neuronal-VAMP. J.
Chemistry, Göttingen, Germany). I thank Dr Michael
Neurosci. 18, 2028 2039.
J. Seager (Neurobiologie des Canaux Ioniques, IN-
Dresbach, T., Burns, M.E., O Conner, V., et al., 1998. A neuronal
SERM, Marseille, France) for critical reading of the
Sec1 homolog regulates neurotransmitter release at the squid
giant synapse. J. Neurosci. 18, 2923 2932.
manuscript.
Ferro-Novick, S., Jahn, R., 1994. Vesicle fusion from yeast to man.
Nature 370, 191 193.
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