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 inte- grative properties of synapses. Time is one of the most critical parameters in the func- hydrolyzes ATP. Because SNAREs are found on both tioning of the brain. Information transfer on the time- the synaptic vesicle membrane and the plasma mem- scale of milliseconds (10−3 seconds) is typical through- brane, it has been postulated that the various SNARE out the brain and in certain brain regions, such as the complexes mediate the interaction between the two auditory brainstem, time differences on the order of membranes before fusion and thus may be necessary microseconds (10−6 seconds) are used to define the fre- for neurotransmitter release.2 quency and location of perceived sounds. Thus infor- Evidence for a role for SNARE proteins in neuro- mation processing not only depends on a fast underly- transmitter release has come from a variety of sources. ing process but also on the precise timing of synaptic The most compelling indication of the central impor- activity. Such high temporal fidelity must rely upon tance of the three membrane SNARE proteins is that very finely-regulated molecular mechanisms. However, these proteins are remarkably specific targets of tetanus until recently the identity of these mechanisms has and botulinum toxins, a group of potent neurotoxins been remarkably elusive. We summarize here our that completely paralyze neurotransmitter release. recent experiments that provide the first clues about These toxins act as proteases that cleave one of the the identity of the molecular timers of synaptic trans- SNARE proteins, either syntaxin, SNAP-25 or synapto- mission. brevin.3 These and other observations4,5 indicate that Synaptic transmission occurs when synaptic vesicles the proteins of the 7S complex are necessary for neuro- containing neurotransmitters fuse with the plasma transmitter release. Is there similar experimental data membrane of a neuron, causing the neurotransmitters to support a role for the additional proteins of the 20S to be released onto downstream neurons and other tar- complex, SNAP and NSF, in neurotransmitter release? get cells. Biochemical and molecular biological Evidence that SNAP is important comes from the find- approaches have identified a group of presynaptic pro- ing that injection of SNAP protein into a presynaptic teins that may be key players in neurotransmitter terminal increases neurotransmitter release, while pre- release. A soluble ATPase, called NSF (N-ethylmalei- venting SNAP function inhibits transmitter release.6 mide Sensitive Factor) binds, via another protein called Although there are genetic hints that NSF might also SNAP (Soluble NSF Attachment Protein) to its mem- be important for neurotransmitter release,7 the specific brane receptors, known as SNAREs (SNAP Receptors). role of NSF has been left unresolved. Two of these SNAREs—syntaxin and SNAP-25 (for Our approach to determining the role of NSF in neur- synaptosome-associated protein of 25-kDa molecular otransmitter release began with identification of parts weight)—are found on the plasma membrane and of the NSF molecule that are responsible for its func- another SNARE—synaptobrevin or VAMP (vesicle- tion. We synthesized peptides derived from various associated membrane protein)—is in the membrane of regions of NSF and found that two of these peptides, the synaptic vesicles. In vitro experiments indicate that termed NSF2 and NSF3, prevented NSF from these proteins associate in various combinations1 hydrolyzing ATP despite the presence of SNAP.8 (Figure 1). The SNAREs can bind together to form the Therefore, these peptides prevent some molecular so-called 7S complex and addition of the soluble NSF rearrangement, either within NSF or between NSF and and SNAP leads to the formation of a larger, 20S com- SNAP, that is needed for NSF to act as an enzyme to plex. This 20S complex breaks apart when NSF catalyze the dissociation of the 20S SNARE complex. We next microinjected these peptides into the giant presynaptic terminal of a squid giant synapse to deter- Correspondence: FE Schweizer, Dept Neurobiology, UCLA, Box mine the effect of these peptides on neurotransmitter 951763, Los Angeles, CA 90095-1763, USA. E-mail: felixsȰ- release. The presynaptic terminal of this squid synapse ucla.edu is about one million times larger than a typical synapse News & Views 294 News & Views 295 Figure 1 Complexes formed by SNARE proteins. The SNARE syn- at which individual vesicles fuse (Figure 3). We pro- aptobrevin is localized on synaptic vesicles (yellow) while syntaxin pose that more than one fusogenic protein complex and SNAP-25 are localized to the plasma membrane (green). When containing NSF exists for each vesicle and that any of these proteins interact in vitro, they form a high molecular weight 7S these complexes, or fusion particles, can initiate ves- complex (bottom right). Addition of the soluble proteins NSF and icle fusion.9 A reduction in the number of functional SNAP leads to the formation of a larger, 20S complex (bottom left). fusion particles, caused by the NSF peptides, could This 20S complex dissolves if NSF is able to hydrolyze ATP and then lead to a slowing of the rate at which an individ- ‘free’ SNAREs are recovered (top). ual vesicle releases its contents or a desynchronization of vesicle fusion. in the brain, making this an ideal system for such The fusion of synaptic vesicles is just one step in a complex cycle of trafficking reactions that occurs microinjection studies. 10 Injection of the two NSF peptides led to a rapid inhi- locally within the presynaptic terminal. Vesicles are bition of synaptic transmission, as determined by a formed within the terminal, fill with neurotransmitter block of electrical activity in the postsynaptic neuron and dock at the plasma membrane. The vesicles then following stimulation of its presynaptic neuron (Figure fuse and are subsequently retrieved from the plasma 2a). This inhibition occurred very quickly after begin- membrane via the process of endocytosis. Endocytosis ning 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 neuro- transmitter 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 ampli- tude of these currents but also slowed both the onset and the decay of the currents (Figure 2c).8 This obser- vation indicates that NSF influences a reaction that occurs during the release of neurotransmitters. This Figure 3 Possible explanations for the kinetic actions of NSF pep- was a surprise because neurotransmitter release at this tides. The release of neurotransmitter from a single vesicle can be synapse lasts for only a millisecond or two, which is detected as a small event, called a ‘mini’. Measured synaptic quite fast relative to the speed of NSF and most other responses (EPSC) are typically made up of many such release events. In control (left), a simulation with 10 simultaneous release events is enzymes. illustrated. A slowing of the EPSC can be observed if all 10 vesicles Slowing of neurotransmitter release kinetics could still fuse at the same time as in control, but the time course of the result from slowing the speed at which each vesicle mini is slowed (right, top). Alternatively, slowing can occur if the fuses with the plasma membrane and/or releases its time course of the mini is unchanged, but the fusion of the 10 vesicles contents into the synaptic cleft. Alternatively, a slow- is desynchronized (right, bottom). Dashed lines on right-hand panel ing could be achieved by desynchronization of the time 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. News & Views 296 a b News & Views 297 Figure 4 Two models for the role of NSF in neurotransmitter almost exclusively on the magnitude of synaptic trans- release. (a) NSF acts before fusion. SNAREs on vesicles and plasma mission, it will be interesting to see whether and how membrane interact to form a 7S complex. SNAP and NSF are changes in transmission kinetics are used for infor- recruited into the 20S complex. ATP hydrolysis by NSF then dissolves mation processing.13 As we learn more about the role the 20S complex and leaves the SNAREs in a ‘free’ conformation of NSF in brain function, it seems possible that this suitable for fusion upon calcium entry.