Major efforts have been directed at the elucidation of the functional role of SNARE recycling in regulated exocytosis. Genetic, pharmacological, and biochemical manipulations of the SNARE disassembly machinery have been performed, either targeting NSF or SNAPs.
1.9.1 Functional Studies on NSF
The most detailed information on the functional role of NSF in synaptic transmission originates from the analysis of the temperature-sensitive paralitic Comatose mutation in Drosophila (Pallanck et al., 1995; Kawasaki et al., 1998). The great advantage of the Comatose mutant is that at the restrictive temperature an acute and fast inactivation of NSF is achieved, which provides a useful system to study its acute function in synaptic transmission. Synaptic transmission was analysed at the neuromuscular junction (Kawasaki et al., 1998) and at a central synapse within the giant fiber patway (Kawasaki et al., 1999). It was shown that at the restrictive temperature, single AP evoked transmission at synapses was normal. On the other
strongly and faster in mutants as compared to controls. Furthermore, accumulation of SNARE complexes and of physically-docked vesicles at presynaptic sites was observed in mutants at the restrictive temperature. These results were taken as evidence for a role of NSF in the activity-dependent maintenance of the RRP, i.e. in the priming process of synaptic vesicles.
Several other studies have examined the role of NSF in regulated secretion and seem to corroborate the hypothesis of NSF activity being required for priming the release process. One study examined the role of NSF using injection for NSF peptides into the presynaptic terminal of the squid giant axon (Schweitzer et al., 1998). Two peptides were found to inhibit release, probably by inhibiting SNAP-mediated stimulation of the ATPase activity of NSF. In this microinjection study, NSF was shown to reduce the degree and to slow down the kinetics of neurotransmitter release. However, in a subsequent study the same inhibitory peptides were tested for their effect in neurotransmitter release at the crayfish neuromuscular junction (Parnas et al., 2006). In this case, the peptides inhibited release but had no effect on its time course. A related study on PC12 cells (Banerjee et al., 1996) indicated that NSF functions in a priming step but is not directly involved in fusion.
Taken together, above studies suggest a role for NSF in the maintenance of the RRP. Inhibition of its activity resulted in a reduction in Ca2+ regulated exocytosis, which was shown to be activity-dependent. These experiments were taken as evidence for a priming defect after NSF perturbation that arises from a limited supply of free SNAREs for subsequent rounds of fusion.
1.9.2 Functional Studies on SNAP isoforms
Functional studies to assess the role of SNAPs in Ca2+ regulated exocytosis were performed on different model systems. In chromaffin cells, catecholamine release in the presence of exogenous α-SNAP (either included in the patch pipette or applied to permeabilised cells) was shown to be increased (Kibble et al., 1996; Xu et al., 1999;
Morgan and Burgoyne, 1995). High-resolution capacitance measurements showed that α-SNAP increases the amplitude of the exocytotic burst and the slow release component without changing their kinetics.
Effects of α-SNAP on neurotransmitter exocytosis were so far analysed in three different synapses, (1) the squid giant synapse (DeBello et al., 1995), (2) the crayfish
neuromuscular junction (He et al., 1999), and (3) the Drosophila neuromuscular junction (Babcock et al., 2004). In the first two studies, recombinant α-SNAP protein was microinjected into presynaptic terminals. In both synapses, exogenous α-SNAP increased neurotransmitter release without significantly affecting the kinetics of the release process. At the Drosophila neuromuscular junction, α-SNAP levels were increased via a transgenic approach. Intriguingly, this study showed that increased α-SNAP levels resulted in reduced neurotransmitter release. To date, however, this is not the only indication for a negative function of α-SNAP in Ca2+ regulated exocytosis. Several other studies also showed that excess α-SNAP might indeed result in inhibition of exocytosis. Addition of α-SNAP was shown to inhibit cell-free fusion reactions, including sperm acrosome exocytosis (Tomes et al., 2005), yeast vacuole fusion (Wang et al., 2000), secretory granule fusion in PC12 cells, and liposome fusion (Barszczewski et al., 2007). In the latter study, it was proposed that α-SNAP inhibits exocytosis by binding directly to free Syntaxin-1, thereby preventing its interaction with the other SNARE partners.
α-SNAP has been the isoform of choice in all of the studies mentioned above.
The β-SNAP isoform, on the other hand, has not been investigated systematically. To date, in fact, only two functional studies compared the role of α- and β-SNAP isoforms in Ca2+ regulated exocytosis in chromaffin cells (Suldow et al., 1996; Xu et al., 2002). While in the first study both isoforms were found to stimulate exocytosis to the same extent, in the second study, β-SNAP was far less efficient than α-SNAP in stimulating exocytosis.
On aggregate, multiple functional studies indicated that an oversupply of α-SNAP might enhance exocytosis in various cellular systems, including bovine chromaffin cells, the squid giant synapse, the crayfish neuromuscular junction, and pancreatic β-cells. However, a significant number of studies indicated that excess of α-SNAP can inhibit fusion. As regards the β-SNAP isoform, its functional role has been investigated only superficially. Although several biochemical findings indicate that α- and β-SNAP may be functionally equivalent, two comparative functional studies yielded contradictory results (Suldow et al., 1996; Xu et al., 2002).
1.9.3 A New Function of α-SNAP in Regulating Apical Membrane Trafficking - HYH Mutant Mice
Recently, an unexpected role for α-SNAP in apical membrane trafficking has been proposed, based on the analysis of the "Hydrocephaly with Hop-gait" (HYH) mutant mouse. HYH is a recessive mutation that arose spontaneously in the C75L/10J mouse strain, and two independent studies identified it as a missense mutation (M105I) in the α-SNAP protein (Chae et al., 2004; Hong et al., 2004). Both studies showed that this mutation results in a drastic reduction of α-SNAP levels (approximately 50 % of wild-type levels) as a consequence of either mRNA (Chae et al., 2004) or protein (Hong et al.,, 2004) instability. Chae et al. (2004) showed that the single aminoacid change did not alter protein folding, and in vitro disassembly of the SNARE core complex was comparable in the presence of HYH mutant or wild-type α-SNAP (2004). Based on these data, it was concluded that HYH is a hypomorphic mutation that results in low levels of protein but does not affect protein function per se.
HYH mice suffer from progressive hydrocephalous. They show a remarkably small cerebral cortex at birth and die postnatally from progressive enlargement of the ventricular system. Chae et al. (2004) suggested that the small HYH cortex may result from altered cell fate. Neural progenitor cells displayed altered cell fate, accompanied by altered localisation of many apical proteins implicated in regulation of neural cell fate (e.g. E-cadherin, β-catenin). Therefore, it was concluded that α-SNAP may be essential for apical protein localisation and cell fate determination in neuroepithelial cells (Chae et al., 2004).