Molecular Psychiatry (1998) 3, 293–297
 1998 Stockton Press All rights reserved 1359–4184/98 $12.00
NEWS & VIEWS
SNARE proteins and the timing of neurotransmitter
release
The SNARE complex proteins have been implicated in exocytotic neurotransmitter release
and other forms of membrane fusion. Recent work shows that NSF, the ATPase of the SNARE
complex, regulates the kinetics of neurotransmitter release and can thereby control the integrative properties of synapses.
Time is one of the most critical parameters in the functioning of the brain. Information transfer on the timescale of milliseconds (10−3 seconds) is typical throughout the brain and in certain brain regions, such as the
auditory brainstem, time differences on the order of
microseconds (10−6 seconds) are used to define the frequency and location of perceived sounds. Thus information processing not only depends on a fast underlying process but also on the precise timing of synaptic
activity. Such high temporal fidelity must rely upon
very finely-regulated molecular mechanisms. However,
until recently the identity of these mechanisms has
been remarkably elusive. We summarize here our
recent experiments that provide the first clues about
the identity of the molecular timers of synaptic transmission.
Synaptic transmission occurs when synaptic vesicles
containing neurotransmitters fuse with the plasma
membrane of a neuron, causing the neurotransmitters
to be released onto downstream neurons and other target cells. Biochemical and molecular biological
approaches have identified a group of presynaptic proteins that may be key players in neurotransmitter
release. A soluble ATPase, called NSF (N-ethylmaleimide Sensitive Factor) binds, via another protein called
SNAP (Soluble NSF Attachment Protein) to its membrane receptors, known as SNAREs (SNAP Receptors).
Two of these SNAREs—syntaxin and SNAP-25 (for
synaptosome-associated protein of 25-kDa molecular
weight)—are found on the plasma membrane and
another SNARE—synaptobrevin or VAMP (vesicleassociated membrane protein)—is in the membrane of
the synaptic vesicles. In vitro experiments indicate that
these proteins associate in various combinations1
(Figure 1). The SNAREs can bind together to form the
so-called 7S complex and addition of the soluble NSF
and SNAP leads to the formation of a larger, 20S complex. This 20S complex breaks apart when NSF
Correspondence: FE Schweizer, Dept Neurobiology, UCLA, Box
951763, Los Angeles, CA 90095-1763, USA. E-mail: felixs얀ucla.edu
hydrolyzes ATP. Because SNAREs are found on both
the synaptic vesicle membrane and the plasma membrane, it has been postulated that the various SNARE
complexes mediate the interaction between the two
membranes before fusion and thus may be necessary
for neurotransmitter release.2
Evidence for a role for SNARE proteins in neurotransmitter release has come from a variety of sources.
The most compelling indication of the central importance of the three membrane SNARE proteins is that
these proteins are remarkably specific targets of tetanus
and botulinum toxins, a group of potent neurotoxins
that completely paralyze neurotransmitter release.
These toxins act as proteases that cleave one of the
SNARE proteins, either syntaxin, SNAP-25 or synaptobrevin.3 These and other observations4,5 indicate that
the proteins of the 7S complex are necessary for neurotransmitter release. Is there similar experimental data
to support a role for the additional proteins of the 20S
complex, SNAP and NSF, in neurotransmitter release?
Evidence that SNAP is important comes from the finding that injection of SNAP protein into a presynaptic
terminal increases neurotransmitter release, while preventing SNAP function inhibits transmitter release.6
Although there are genetic hints that NSF might also
be important for neurotransmitter release,7 the specific
role of NSF has been left unresolved.
Our approach to determining the role of NSF in neurotransmitter release began with identification of parts
of the NSF molecule that are responsible for its function. We synthesized peptides derived from various
regions of NSF and found that two of these peptides,
termed NSF2 and NSF3, prevented NSF from
hydrolyzing ATP despite the presence of SNAP.8
Therefore, these peptides prevent some molecular
rearrangement, either within NSF or between NSF and
SNAP, that is needed for NSF to act as an enzyme to
catalyze the dissociation of the 20S SNARE complex.
We next microinjected these peptides into the giant
presynaptic terminal of a squid giant synapse to determine the effect of these peptides on neurotransmitter
release. The presynaptic terminal of this squid synapse
is about one million times larger than a typical synapse
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Figure 1 Complexes formed by SNARE proteins. The SNARE synaptobrevin is localized on synaptic vesicles (yellow) while syntaxin
and SNAP-25 are localized to the plasma membrane (green). When
these proteins interact in vitro, they form a high molecular weight 7S
complex (bottom right). Addition of the soluble proteins NSF and
SNAP leads to the formation of a larger, 20S complex (bottom left).
This 20S complex dissolves if NSF is able to hydrolyze ATP and
‘free’ SNAREs are recovered (top).
in the brain, making this an ideal system for such
microinjection studies.
Injection of the two NSF peptides led to a rapid inhibition of synaptic transmission, as determined by a
block of electrical activity in the postsynaptic neuron
following stimulation of its presynaptic neuron (Figure
2a). This inhibition occurred very quickly after beginning peptide injection. Synaptic transmission then
recovered after we stopped injecting the peptides,
because the peptides diffused away from the synapse
into the rest of the giant presynaptic neuron (Figure
2b). These results, together with appropriate controls,
are the first evidence that NSF is crucial for neurotransmitter release under physiological conditions.8
To our surprise, we found that both NSF peptides
not only reduced neurotransmitter release but also
slowed the time course of release from the presynaptic
terminal. This became clear when we measured the
postsynaptic currents resulting from neurotransmitter
release; the NSF peptides not only reduced the amplitude of these currents but also slowed both the onset
and the decay of the currents (Figure 2c).8 This observation indicates that NSF influences a reaction that
occurs during the release of neurotransmitters. This
was a surprise because neurotransmitter release at this
synapse lasts for only a millisecond or two, which is
quite fast relative to the speed of NSF and most other
enzymes.
Slowing of neurotransmitter release kinetics could
result from slowing the speed at which each vesicle
fuses with the plasma membrane and/or releases its
contents into the synaptic cleft. Alternatively, a slowing could be achieved by desynchronization of the time
at which individual vesicles fuse (Figure 3). We propose that more than one fusogenic protein complex
containing NSF exists for each vesicle and that any of
these complexes, or fusion particles, can initiate vesicle fusion.9 A reduction in the number of functional
fusion particles, caused by the NSF peptides, could
then lead to a slowing of the rate at which an individual vesicle releases its contents or a desynchronization
of vesicle fusion.
The fusion of synaptic vesicles is just one step in a
complex cycle of trafficking reactions that occurs
locally within the presynaptic terminal.10 Vesicles are
formed within the terminal, fill with neurotransmitter
and dock at the plasma membrane. The vesicles then
fuse and are subsequently retrieved from the plasma
membrane via the process of endocytosis. Endocytosis
Figure 3 Possible explanations for the kinetic actions of NSF peptides. The release of neurotransmitter from a single vesicle can be
detected as a small event, called a ‘mini’. Measured synaptic
responses (EPSC) are typically made up of many such release events.
In control (left), a simulation with 10 simultaneous release events is
illustrated. A slowing of the EPSC can be observed if all 10 vesicles
still fuse at the same time as in control, but the time course of the
mini is slowed (right, top). Alternatively, slowing can occur if the
time course of the mini is unchanged, but the fusion of the 10 vesicles
is desynchronized (right, bottom). Dashed lines on right-hand panel
indicate ‘control’ EPSC.
Figure 2 Effects of NSF peptides upon transmission at the squid giant synapse. (a) Presynaptic action potentials (Vpre) elicit an inward current
in the postsynaptic cell (EPSC) that is proportional to transmitter release. After injection of the peptide NSF-2 the postsynaptic current (thick
trace) is reduced in magnitude relative to control (thin trace). (b) Records as shown in (a) were obtained every 30 s. The amplitude of the
postsynaptic current was measured and plotted against time. The bar indicates time during which NSF-2 was injected into the presynaptic
terminal. Inhibition of transmitter release reaches a maximum soon after injection is stopped and is fully reversible. Traces in (a) were taken
right before injection and at the maximum of inhibition. Injection of more peptide leads to full inhibition.8 (c) The EPSCs in (a) were scaled
to the same amplitude to emphasize the change in time course of transmitter release. Not that both the onset and the decay of the EPSC are
slowed by the NSF peptide.
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a
b
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Figure 4 Two models for the role of NSF in neurotransmitter
release. (a) NSF acts before fusion. SNAREs on vesicles and plasma
membrane interact to form a 7S complex. SNAP and NSF are
recruited into the 20S complex. ATP hydrolysis by NSF then dissolves
the 20S complex and leaves the SNAREs in a ‘free’ conformation
suitable for fusion upon calcium entry. (b) NSF acts after fusion.
SNAREs on vesicles and plasma membrane interact to form a 7S
complex which allows the two membranes to fuse upon calcium entry.
After fusion, the 7S complex is localized to the plasma membrane.
In order to recycle the SNAREs into their respective membrane compartments, the complex needs to be dissolved. Thus the 20S complex
is formed and ATP hydrolysis by NSF breaks the SNAREs apart.
The vesicle membrane can now be retrieved with its correct proteins
via endocytosis.
thereby provides the precursors of new vesicles that
then repeat the cycle. There has recently been much
unresolved debate about where within this cycle NSF
acts.11,12 We found that the NSF peptides allowed synaptic vesicles to dock at the plasma membrane, yet
these vesicles could not fuse with the plasma membrane.8 Our results thus suggest that NSF regulates a
step that is essential after the initial interaction of the
two membranes and regulates fusion. One possibility
is that the hydrolysis of ATP by NSF acts to prime the
SNARE proteins before fusion, leaving the vesicle and
plasma membranes in a fusogenic state (Figure 4a).
Alternatively, it is possible that NSF breaks apart
SNARE complexes that are stuck in the plasma membrane following membrane fusion (Figure 4b). In either
case, the accumulation of undissociated 20S complexes could impair the speed of vesicle fusion reactions by reducing the number of fusogenic particles per
vesicle. Thus, a role for NSF in transmitter release
either before or after vesicle fusion is compatible with
the observed kinetic actions of the NSF peptides and
additional experiments are currently being carried out
to distinguish between these two possibilities.
Regardless of the specific mechanisms by which NSF
acts, our work makes clear that NSF is important for
brain function and serves as a molecular timer during
synaptic transmission. In this regard, it is worth noting
that the effects of NSF upon release kinetics have
important computational consequences regardless of
whether an action potential causes the release of the
contents of thousands of vesicles—as is the case for eg
the squid giant synapse—or on average fewer than one
vesicle, as is the case for many other synapses. While at
present the investigation of neuronal plasticity focuses
almost exclusively on the magnitude of synaptic transmission, it will be interesting to see whether and how
changes in transmission kinetics are used for information processing.13 As we learn more about the role
of NSF in brain function, it seems possible that this
enzyme will serve as a novel target for future therapies
for synaptic disorders of the brain.
FE Schweizer1 and GJ Augustine2
Dept Neurobiology, UCLA, Box 951763
Los Angeles, CA 90095-1763;
2
Dept Neurobiology, Duke Medical Center
PO Box 3209, Durham, NC 27710, USA
1
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