SNARE proteins mediate membrane-membrane fusion events between distinct intracellular organelles in all cell types2-4
. Many different SNAREs have been identified, and in some cases a role for a particular SNARE in a defined membrane fusion event has been defined. For example, SNAP-25 is a neuron-specific SNARE that regulates synaptic vesicle fusion with the presynaptic plasma membrane2-4,6,30
. Curiously, there is a ubiquitously-expressed homolog of SNAP-25, termed SNAP-23, that is present in the brain but whose neuronal role in membrane fusion events has not been elucidated. We now show that SNAP-23 and SNAP-25 have unique and non-overlapping distributions and functions in neurons. Our own immunofluorescence studies, together with previous electron microscopy studies, show that SNAP-25 is localized on the presynaptic plasma membrane as well as on synaptic vesicles recycled from this membrane5-7
. In marked contrast, SNAP-23 expression is somato-dendritic. Furthermore, immunofluorescence microscopy, electron microscopy, and subcellular fractionation studies show that SNAP-23 is localized at synaptic spines, and particularly enriched at the PSD.
The exclusive localization of SNAP-23 on dendrites supports a role for SNAP-23 in postsynaptic membrane trafficking events. Whereas previous studies have shown that ablation of SNAP-25 prevents stimulus-evoked neurotransmission8
, our studies using knock-down of SNAP-25 expression with shRNA revealed no postsynaptic role for SNAP-25 in regulating glutamate receptors. By contrast, whole cell patch clamp recording in CA1 pyramidal neurons clearly shows that postsynaptic knock-down of SNAP-23, but not SNAP-25, reduced the size of NMDA-evoked currents, suggesting the number of NMDA receptors on the neuronal surface is regulated by SNAP-23. Furthermore, examination of NMDA EPSCs revealed that SNAP-23 regulates synaptic NMDA receptors, supporting our biochemical analyses and highlighting the physiological relevance of this work.
Our data reveal that SNAP-23 knock-down suppressed plasma membrane expression of NR2B by inhibiting the recycling of internalized receptors. This finding is consistent with previous studies showing that SNAP-23 is required for transferrin recycling in polarized epithelial cells31
and suggests that SNAP-23 may be a general regulator of membrane protein recycling. Our findings that NMDA receptors do not interact directly with SNAP-23 (Supplementary Fig. 6
) are consistent with a more general role for SNAP-23 acting as a component of the membrane fusion machinery. Our hypothesis is that recycling endosomal compartments containing postsynaptic glutamate receptors are delivered to the plasma membrane and that membrane-bound SNARE proteins promote membrane fusion. While the precise nature of this postsynaptic plasma membrane SNARE complex remains to be determined, these data have identified SNAP-23 as a key player in this process.
SNARE-dependent exocytosis of glutamate receptors has been implicated in several studies. For example, the surface expression of AMPA receptors is regulated by the interaction of the AMPA receptor GluR2 subunit with NSF (N-ethylmaleimide-sensitive factor) and SNAPs (soluble NSF-attachment proteins), proteins that play a role in SNARE complex disassembly32-35
. In addition, surface expression of NMDA receptors is increased upon activation of protein kinase C or mGluR1 and this effect is dependent on SNAP-2536,37
. Finally, it has been demonstrated that SNARE cleavage by exogenously added clostridial neurotoxins can affect glutamate receptor expression24,32,36-39
. However, SNARE-dependent trafficking of postsynaptic glutamate receptors that is attributed to SNAP-25 should be carefully evaluated. First, SNAP-25 is expressed at very low levels (if at all) on postsynaptic spines5-7
, whereas the SNAP-25 homolog SNAP-23 is enriched at postsynaptic sites. Second, SNAP-25 has a much higher affinity than SNAP-23 for other SNAREs13
, and thus overexpression of wild-type (or mutant) forms of SNAP-25 will also inhibit SNAP-23-dependent SNARE interactions. Third, infusion of small SNAP-25-derived blocking peptides to inhibit the formation of SNARE complexes will also likely inhibit SNAP-23 function because these two proteins share considerable amino acid identity9,27
. Finally, even the use of clostridial neurotoxins to identify a role for particular SNARE proteins in neuronal function must be interpreted with caution, since these toxins generally cleave only free, and not complexed, SNAREs40
. It is quite possible, therefore, that studies reporting the regulation of postsynaptic glutamate receptor expression by SNAP-25 may actually reflect effects on endogenous SNAP-23.
In conclusion, our findings reveal a distinct molecular composition of SNARE complexes at pre- and postsynaptic sites in neurons. The effects of SNAP-23 depletion on postsynaptic, but not presynaptic, glutamate receptor expression is consistent with the highly enriched localization of SNAP-23 to the PSD along dendritic spines. The reason for such a distinction between axonal and dendritic membrane fusion is fascinating and important topic for future study. Our work reveals a clear division of labor between SNAP-25 and neuronal SNAP-23 and supports a central role for SNAP-23 in postsynaptic trafficking events.