Photolysis of a caged peptide reveals rapid action of N -ethylmaleimide sensitive factor before neurotransmitter release

Abstract

The time at which the N-ethylmaleimide-sensitive factor (NSF) acts during synaptic vesicle (SV) trafficking was identified by timecontrolled perturbation of NSF function with a photoactivatable inhibitory peptide. Photolysis of this caged peptide in the squid giant presynaptic terminal caused an abrupt (0.2 s) slowing of the kinetics of the postsynaptic current (PSC) and a more gradual (2-3 s) reduction in PSC amplitude. Based on the rapid rate of these inhibitory effects relative to the speed of SV recycling, we conclude that NSF functions in reactions that immediately precede neurotransmitter release. Our results indicate the locus of SNARE protein recycling in presynaptic terminals and reveal NSF as a potential target for rapid regulation of transmitter release.caged probes ͉ exocytosis ͉ synaptic transmission ͉ synaptic vesicle cycle N eurotransmitter release relies on the precisely coordinated actions of many proteins that serve to recruit synaptic vesicles (SVs) to active zones, prepare SVs for Ca 2ϩ -dependent exocytosis, and recycle used components (1-5). At the core of these trafficking reactions lies the SNARE [soluble Nethylmaleimide sensitive factor (NSF) attachment protein receptor] complex, which consists of proteins present in SVs (v-SNAREs) and the plasma membrane (t-SNAREs) (6). It is thought that trans-SNARE complexes bridging the SV and plasma membranes bring these two membranes into close apposition and mediate membrane fusion (7,8). Because SNARE complexes are highly stable, hydrolysis of ATP by the molecular chaperone NSF (9, 10) is required to disassemble used SNARE complexes and, thereby, recycle SNARE proteins in preparation for future rounds of exocytosis (11-13). Although it is generally agreed that this action of NSF is important for neurotransmitter release, it is not clear whether NSF works before or after vesicle fusion. This distinction is critical for understanding the dynamic control of synaptic transmission by NSF and elucidating the life cycle of SNARE complexes during SV trafficking.Two models have been proposed for the timing of NSF action during neurotransmitter release (Fig. 1). SNAREs could be disassembled just before fusion, meaning that NSF is active only when needed for a fusion reaction (Fig. 1 A). This model is consistent with observations that NSF is required before vesicle fusion in several experimental systems (14-20). Alternatively, NSF could dissociate SNARE complexes immediately after neurotransmitter release (Fig. 1B). Such a postfusion action of NSF could provide an attractive mechanism for sorting of v-and t-SNAREs after fusion: in this case, newly separated v-SNAREs would be carried along as recycled SVs bud from the plasma membrane, whereas t-SNAREs would remain behind in the plasma membrane. Although experimental evidence supporting this conclusion is limited (21,22), the ability to explain SNARE sorting makes a postfusion action of NSF part of most current models of SV trafficking (8,(23)(24)(25)(26).One way to distinguish between these two alternatives ...

The Role of Mitochondrially Derived ATP in Synaptic Vesicle Recycling

“…In contrast to endocytosis, we show that vesicle release from the readily releasable pool requires little ATP, which is consistent with studies in permeabilized endocrine cells and goldfish retinal bipolar neurons, showing that once "primed," vesicle release is independent of ATP but depends on Ca 2ϩ (51). Although endocytosis is far more sensitive to decreased energy than exocytosis, some ATP is still needed to facilitate exocytosis, presumably in part due to the ATPase activity of the N-ethylmaleimide-sensitive factor (NSF), which is required before vesicle fusion (52).…”

Distinct Initial SNARE Configurations Underlying the Diversity of Exocytosis

“…Thus unitary-SNAREs are dynamically maintained and may be generated shortly before exocytosis is triggered (336). Unassembled syntaxin can be chaperoned by Munc18a, which bundle SNARE motifs and Habc for stabilization (715).…”

“…Priming is often assumed to follow vesicle docking in these preparations and in the active zone (446,451); however, priming can precede docking. For instance, ATP-dependent disassembly of SNAREs by N-ethylmaleimide-sensitive fusion protein (NSF) is necessary for exocytosis (336,392,705), and such priming reactions precede the docking of vesicles. In addition, there is a specific priming process called Ca 2ϩ priming (673,677), which is induced by moderate increases in [Ca 2ϩ ] i and markedly hastens exocytosis, presumably by inducing prestimulus docking and assembly of SNARE complexes in synapses and chromaffin cells (442).…”

Molecular mechanisms determining conserved properties of short-term synaptic depression revealed in NSF and SNAP-25 conditional mutants

“…Finally, an alternative view of NSF function favoring prefusion disassembly of SNARE complexes was reported in a study employing caged NSF peptides to acutely disrupt NSF function at the squid giant synapse (43). However, the activity-dependent effects of the peptide on EPSC amplitude are largely consistent with the working model presented here, in which the timing of the NSF requirement is not constrained by the total vesicle cycling time (43). Although the NSF peptide was also found to slow EPSC rise and decay times, no analogous effects were observed in the present study (Fig.…”