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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2001 Nov 13;98(24):14038–14043. doi: 10.1073/pnas.251532398

SNAP-29: A general SNARE protein that inhibits SNARE disassembly and is implicated in synaptic transmission

Qingning Su *, Sumiko Mochida †, Jin-Hua Tian *, Rashi Mehta *, Zu-Hang Sheng *,‡
PMCID: PMC61163  PMID: 11707603

Abstract

Using the yeast two-hybrid system with syntaxin-1A as bait, we isolated soluble NSF attachment protein (SNAP)-29 from a human brain cDNA library. Synaptosomal fractionation and immunocytochemical staining of hippocampal neurons in culture showed that SNAP-29 is present at synapses and is predominantly associated with synaptic vesicles. The interaction of SNAP-29 with syntaxin-1 was further confirmed with immunoprecipitation analysis. Binding competition studies with SNAP-29 demonstrated that it could compete with α-SNAP for binding to synaptic SNAP receptors (SNAREs) and consequently inhibit disassembly of the SNARE complex. Introduction of SNAP-29 into presynaptic superior cervical ganglion neurons in culture significantly inhibited synaptic transmission in an activity-dependent manner. Although SNAP-29 has been suggested to be a general SNARE component in membrane trafficking, our findings suggest that it may function as a regulator of SNARE complex disassembly and modulate the process of postfusion recycling of the SNARE components.

Neurotransmitter release involves a series of interactions between the membranes of synaptic vesicles and presynaptic terminals (1–4). The synaptic vesicle-associated protein VAMP (synaptobrevin), as a v-SNAP receptor (SNARE), interacts with two target membrane-associated proteins (t-SNAREs), soluble NSF attachment protein (SNAP)-25 and syntaxin, to form a stable SNARE complex (5–12). Considerable evidence indicates that the SNARE complex is a biochemical intermediate essential for a late step in the membrane fusion process (13–18). Synaptic vesicle fusion with the plasma membrane results in the incorporation of the SNARE complex proteins into the plasma membrane. Postfusion recycling of the complex components after clathrin-mediated endocytosis requires that this very stable SNARE complex be dissociated by the action of α-SNAP and the ATPase N-ethylmaleimide-sensitive factor (NSF) (9). It is evident that the assembly and disassembly of the SNARE fusion complexes must be tightly and rapidly regulated at nerve terminals to mediate the Ca2+-triggered membrane fusion process. A number of molecules (including Munc18/n-Sec1/rbSec1, complexins, Munc-13, tomosyn, snapin, and syntaphilin that bind to individual SNARE proteins) have been suggested to regulate the availability and/or propensity of free SNARE proteins to form functional release machinery (19–25). However, the number and identity of the regulatory proteins involved in vesicle priming and recycling remain largely unclear. In the current study, we focused our search for these regulatory proteins by using the yeast two-hybrid selection technique. Using syntaxin-1A as bait, we isolated SNAP-29 from a human brain cDNA library. SNAP-29 was initially isolated by yeast two-hybrid selection by using syntaxin-3 and was localized predominantly in intracellular membrane structures in transiently transfected NRK cells (26). The sequence of SNAP-29 is 83% identical to its rat homologue, GS32 (27). Because of its intracellular membrane association in non-neuronal cells and its interactions with most members of syntaxin family, SNAP-29 has been considered to be a ubiquitous cytoplasmic SNARE protein involved in general membrane-trafficking steps. However, its exact function in SNARE-mediated membrane fusion remains elusive. Here, we report that SNAP-29 is present at synapses, interacts directly with syntaxin-1A, competes with α-SNAP for binding to the SNARE complex, and consequently modulates synaptic transmission by inhibiting disassembly of the SNARE complex.

Experimental Procedures

Isolation of Human SNAP-29.

The rat syntaxin-1A (181) cDNA was inserted into a pGBT9 bait vector containing the GAL4 DNA-binding domain (CLONTECH). Yeast two-hybrid screens of a human brain cDNA library in vector pACT1 (CLONTECH) were performed and evaluated according to the protocols described for the matchmaker yeast two-hybrid system (CLONTECH).

Fusion Protein Construction, Preparation, and in Vitro Binding.

SNAP-29, syntaxin-1A, SNAP-25, and VAMP-2 were subcloned into glutathione S-transferase (GST)-fusion vectors pGEX-4T (Amersham Pharmacia) or hexahistidine-tagged fusion protein vector pET28 (Novagen). Fusion proteins were prepared and purified as described (25). For pull-down experiments, glutathione-Sepharose beads coupled with GST-fusion proteins were added to the His-fusion protein or brain homogenates and incubated for 3 h at 4°C. The beads were then washed three times with TBS buffer (50 mM Tris⋅HCl, pH 7.5/140 mM NaCl/0.1% TX-100 plus protease inhibitors), and bound proteins were electrophoresed on a 10–20% SDS-tricine gradient gel followed by immunoblotting.

Synaptosome Preparation and Immunoprecipitation.

Rat brain synaptosomes were prepared by differential and discontinuous Percoll gradient centrifugation and solubilized as described (24, 25, 28). Synaptosome fractions were isolated as described (25, 29). Solubilized proteins from brain homogenate, synaptosomes, or transfected HEK293 T cells were incubated with 3 μg of anti-syntaxin-1 (10H5), anti-VAMP-2 antibodies, or 5 μl of affinity-purified polyclonal anti-SNAP-29 serum in 0.5 ml TBS at 4°C for 1 h. Protein A-Sepharose CL-4B resin (2.5 mg) (Amersham Pharmacia) was added to each sample, and the incubation continued for an additional 3 h, followed by three washes with TBS. Subsequent interaction assays of immunoprecipitated and immobilized protein complexes were performed as described above. For multiple detection with different antibodies, blots were first stripped in a solution of 62.5 mM Tris⋅HCl, pH 7.5/20 mM DTT/1% SDS for 30 min at 50°C and then washed with TBS/0.1% Tween-20 for 2 × 15 min.

Synaptic Transmission Between Superior Cervical Ganglion Neurons (SCGNs).

SCG cells from 7-day-old postnatal rats were prepared as described (30). After 4–5 weeks in culture, conventional intracellular recordings were made from two neighboring neurons by using microelectrodes filled with 1 M KAc (70–90 MΩ). Postsynaptic responses (EPSPs) were recorded from one of the neurons whereas action potentials were generated in the other neuron by passage of current through an intracellular recording electrode. Neurons were superfused with modified Krebs solution consisting of 136 mM NaCl, 5.9 mM KCl, 5.1 mM CaCl2, 1.2 mM MgCl2, 11 mM glucose, and 3 mM Na-Hepes, pH 7.4. Recombinant SNAP-29, α-SNAP, or synthetic fragment of SNAP-25 peptide was dissolved in 150 mM Kac/5 mM Mg2+-ATP/10 mM Hepes (pH 7.4) and introduced into the presynaptic cell body by diffusion from a suction glass pipette (17–20 MΩ tip resistance). Fast Green FCF (5%, Sigma) was included in the injection peptide solution to confirm entry into the presynaptic cell body. The injection pipette was removed 2–3 min after starting injection. Electrophysiological data were collected and analyzed by using software written by the late Ladislow Tauc (Centre National de la Recherche Scientifique, Paris). Peak amplitudes of EPSPs were measured and averaged (see Fig. 6 C–F). The resultant values were smoothed by an eight-point moving average algorithm and plotted against recording time, with t = 0 indicating the beginning of the presynaptic injection.

Figure 6.

Figure 6

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A synaptic activity-dependent effect of SNAP-29 on synaptic transmission of cultured SCGNs. (A) A typical trace of EPSPs with SNAP-29 injection into the presynaptic neuron. The pipette concentration of the protein was 170 μM. The presynaptic neuron was stimulated every 20 s (0.05 Hz). EPSPs from one representative experiment recorded 3 min before injection and 42 and 78 min after injection are illustrated. (B) A typical trace of EPSPs with 2.5 mM SNAP-25 (196) peptide injection. EPSPs from one representative experiment recorded 3 min before injection and 33 and 64 min after injection are illustrated. (C) Normalized EPSP amplitudes with SNAP-29 injection were averaged and plotted from experiments with presynaptic stimulation once every 20 s (0.05 Hz) (n = 6; ◊) or every 5 s (0.2 Hz) (n = 4; □). As a control, heat-denatured full-length SNAP-29 did not show any significant effects for 60 min after injection at a stimulation frequency of 0.2 Hz (n = 4; ●). (D) Normalized EPSP amplitudes with 2.5 mM SNAP-25 (196) or SNAP-25 (146) peptide injection were averaged and plotted from experiments with presynaptic stimulation as indicated. (E) Normalized EPSP amplitudes with 170 μM SNAP-29 injection at 0.02-Hz stimulation (●) and with 2.5 mM SNAP-25 (196) peptide injection at 0.01-Hz stimulation (○). (F) Normalized EPSP amplitudes with coinjection of SNAP-29 (170 μM) and α-SNAP (43 or 68 μM; □ or ◊; n = 4) at 0.2-Hz stimulation. As a control, α-SNAP (68 μM) was injected at 0.05-Hz stimulation (n = 7; ●).

Results

Isolation and Subcellular Localization of SNAP-29.

The yeast two-hybrid system was used to identify proteins that interact with syntaxin-1A and therefore might regulate synaptic vesicle exocytosis. A human brain cDNA library was screened by using a bait containing the carboxyl-terminal half (amino acids 181–288) of syntaxin-1A. Screening of ≈2 × 106 colonies led to the isolation of six classes of cDNAs encoding α-SNAP (10 clones), β-SNAP (13 clones), SNAP-25 (three clones), syntaphilin (two clones), syntaxin-1A (five clones), and two overlapping cDNAs encoding SNAP-29. We conducted two lines of experiments to address whether SNAP-29 is present at nerve terminals, a subcellular distribution that would allow its participation in synaptic vesicle exocytosis. First, we examined subcellular fractions from synaptosomal preparations. By sucrose density gradient centrifugation, rat crude cerebral synaptosomes were further fractionated into cytosol, synaptic vesicle, and synaptic plasma membrane fractions and then analyzed by sequential immunoblotting with various antibodies as indicated in Fig. 1A. Although SNAP-29 was associated with synaptosomal membranes, it was present predominantly in the synaptic vesicle fraction and to a lesser extent in the plasma membrane fraction (Fig. 1A). A detectable level of SNAP-29 was also found in synaptic cytosol. In contrast, immunoreactivity pattern corresponding to lactate dehydrogenase, a marker of soluble cytoplasmic proteins, was detected primarily in the cytosol fraction. Na+/K+-ATPase, a plasma membrane protein marker, was detected predominantly in the plasma membrane fraction, indicating the relative purity of these subcellular synaptosomal fractions. To eliminate the possibility that detected SNAP-29 was derived from other contaminated membrane organelles such as Golgi complex and endosomes, we further isolated synaptic vesicles by using anti-synaptotagmin-1-conjugated Sepharose beads. Synaptotagmin-I is a synaptic vesicle membrane protein with little association to other intracellular organelles. Immunoblot analysis shows that the affinity-purified synaptic vesicles contain significant amounts of VAMP-2 along with SNAP-29, syntaxin-1, and SNAP-25 but contain no markers for Golgi apparatus (syntaxin-6) (31), endosomes (transferrin), or plasma membrane (Na+/K+ ATPase) (Fig. 1B).

Figure 1.

Figure 1

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Subcellular distribution of SNAP-29 in synaptosomal fractions. (A) Crude synaptosomes (SS) were separated into fractions enriched in presynaptic cytosol, synaptic vesicles, and synaptosomal plasma membrane by sucrose gradient centrifugation. Ten micrograms of synaptosomal fractions on SDS/PAGE was sequentially immunoblotted with antibodies, as indicated, after stripping between antibody applications. (B) Synaptic vesicles purified by anti-synaptotagmin-conjugated affinity beads (anti-tagmin 1) were analyzed by SDS/PAGE and sequential immunoblotting with antibodies as indicated. Normal IgG-conjugated beads (IgG) were applied as a control.

To further determine the subcellular localization of SNAP-29, we stained low-density hippocampal neuron cultures by using polyclonal anti-SNAP-29 antibody. As shown in Fig. 2A, SNAP-29 was distributed widely with small punctate staining in the cell body and along neuronal processes. Specificity of the staining was confirmed by blocking with the antigen (data not shown). SNAP-29 and synaptophysin, a marker for synaptic vesicles, were to a large extent colocalized along the entire processes of hippocampal neurons (Fig. 2 B and C). Thus, our results suggest that SNAP-29 is present at synapses, possibly in association with synaptic vesicles.

Figure 2.

Figure 2

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SNAP-29 is present in the synapses of cultured hippocampal neurons. Hippocampal cultures were immunostained with antibodies against SNAP-29 (green) (A) and synaptophysin (red) (B). Double punctate staining on the neuronal processes is marked by arrowheads (C).

Association of SNAP-29 with Syntaxin-1 and Neuronal SNARE Complex.

To investigate whether SNAP-29 might play a role in synaptic vesicle exocytosis by binding directly to syntaxin-1A, we performed three lines of biochemical experiments. First, we conducted in vitro binding assays with recombinant proteins. Although both His-tagged SNAP-29 and α-SNAP selectively bound to GST-syntaxin-1A, no binding was detectable to SNAP-25, VAMP-2 under our experimental conditions (data not shown). Next, we sought to confirm the SNAP-29–syntaxin-1A interaction in a mammalian expression system. A cDNA-encoded His-tagged SNAP-29 was cotransfected into HEK293 T cells with cDNA-encoded syntaxin-1A. The association of SNAP-29 with syntaxin-1A was then confirmed by immunoprecipitation with anti-SNAP-29 antibodies (Fig. 3A). Finally, we examined whether SNAP-29 and syntaxin-1 exist in a complex in rat brain homogenates. Syntaxin-1 was immunoprecipitated by either anti-SNAP-29 or anti-VAMP-2 antibody but not by preimmune serum or normal control IgG (Fig. 3B). The results from our in vitro binding assays, immunoprecipitation studies, and yeast two-hybrid selection confirm previous finding of a direct binding of SNAP-29 to syntaxin-1 (26, 27) and suggest that these proteins interact in neurons.

Figure 3.

Figure 3

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Association of SNAP-29 with syntaxin-1 and SNARE complex. (A) Immunoprecipitation of syntaxin-1A with SNAP-29 from cotransfected HEK293 T cells. The syntaxin-1A–SNAP-29 complex was immunoprecipitated from cell extracts using anti-SNAP-29 antiserum (anti-29) followed by immunoblot with antibody to syntaxin-1. Normal rabbit IgG (IgG) was used as a control. (B) Immunoprecipitation of syntaxin-1 with SNAP-29 from solubilized rat brain homogenates. Syntaxin-1 was immunoprecipitated by anti-SNAP-29 antiserum and by anti-VAMP, but not by preimmune serum (preserum) or normal rabbit IgG (IgG). A 10% volume of brain homogenate (b.h.) was loaded (right lane). (C) Immobilized GST–SNAP-29 (GST-29) or GST alone on Sepharose beads was incubated with 100 μg of rat brain homogenates at 4°C for 3 h. Native SNARE complex captured was subjected to SDS/PAGE at temperatures as indicated and then analyzed by immunoblotting with the antibodies against syntaxin-1, SNAP-25, and VAMP-2. (Lower) Immunoblotting on the same membrane with anti-GST antibody.

To ascertain association of SNAP-29 with the SNARE complex consisting of neuronal syntaxin-1, SNAP-25, and VAMP-2, we performed pull-down experiments with GST–SNAP-29 Sepharose beads. The SNARE complex is stable during SDS/PAGE; thus, the appearance at 60°C of three bands ranging from 75 to 100 kDa and disappearance of these bands at 100°C are indicative of SDS-resistant SNARE complex when blotted with antibodies against syntaxin-1, SNAP-25, and VAMP-2. Although GST alone could not pull down significant amounts of SNARE protein, GST–SNAP-29 affinity beads efficiently isolated SNARE complexes resistant to SDS at 60°C (Fig. 3C), indicating that SNAP-29 is capable of binding to the native neuronal SNARE complex. Furthermore, our binding competition studies with both recombinant SNAREs demonstrated that SNAP-29 binds to syntaxin-1 in a manner noncompetitive with SNAP-25 (data not shown).

SNAP-29 Competes with α-SNAP for Binding to SNAREs and Inhibits Disassembly of the SNARE Complex.

Because SNAP-29 is capable of interacting with native SNARE complexes (Fig. 3C), it would be interesting to investigate its role in assembly and/or disassembly of the SNARE complex. First, we performed binding competition studies using recombinant SNAREs and α-SNAP. Immobilized GST–syntaxin-1A, preformed GST–syntaxin-1A–SNAP-25 heterodimers, or ternary GST–VAMP2–syntaxin-1A–SNAP-25 complexes were coincubated with equal amounts of α-SNAP in the presence or absence of His-SNAP-29. As shown in Fig. 4A, SNAP-29 inhibited binding of α-SNAP to GST–syntaxin-1A in its monomeric form, in a heterodimer with SNAP-25, and in a trimeric SNARE complex in a concentration-dependent manner (Fig. 4B). Our binding competition results suggest that SNAP-29 and α-SNAP bind to syntaxin or the SNARE complex in a mutually exclusive manner.

Figure 4.

Figure 4

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SNAP-29 competes with α-SNAP for binding to SNAREs. (A) Immobilized GST–syntaxin-1A (stx-1A) or GST–stx-1A–SNAP-25 heterodimers (stx 1A-SNAP-25) and ternary SNARE complex (SNARE) on glutathione-Sepharose were incubated with 1 μg of recombinant α-SNAP in the presence (5 μg) or absence of His-SNAP-29. Bound α-SNAP was visualized by anti-α-SNAP antibody. (B) Concentration-dependent competition of SNAP-29 with α-SNAP for binding to immobilized GST–stx-1A. (C) Immobilized GST–syntaxin-1A was incubated with ≈1 μmol of His-tagged SNAP-25 and VAMP-2 to form recombinant SNARE complex in vitro, and the beads were then washed extensively. The preformed SNARE complex was then incubated with His-tagged α-SNAP (0.16 μmol) in the presence or absence of 0.8 μmol of His-SNAP-29 or His-SNAP-25 as indicated. Protein complexes were analyzed by SDS/PAGE and visualized by sequential immunoblotting. (D) The relative levels of α-SNAP binding to the SNARE complex were calculated based on a linear standard curve of α-SNAP aliquots by using National Institutes of Health image scanning. Semiquantitative analysis revealed that normalized percentage of α-SNAP binding to SNARE complex in the presence of 0.8 μmol of His-SNAP-29 (n = 5) or His-SNAP-25 (n = 3) relative to control in the absence of both SNAP-29 and SNAP-25. Data are expressed as mean ± SEM. Result marked as * (P < 0.01) was considered significantly different.

We then tested whether excess SNAP-29 affects the assembly of the SNARE complex by competing with α-SNAP for binding to the SNARE complex. We incubated immobilized GST–syntaxin-1A with ≈1 μmol of His-tagged SNAP-25 and VAMP-2 to form recombinant SNARE complex in vitro in the presence or absence of 1 μmol of His-SNAP-29. SNAP-29 showed no significant effect on the assembly of recombinant SNARE complex in vitro (data not shown). Furthermore, we incubated the preformed SNARE complex with 0.16 μmol of His-α-SNAP in the absence or presence of 0.8 μmol of His-SNAP-25 or His-SNAP-29 as indicated (Fig. 4C). Although SNAP-25 had no significant effect on the interaction of α-SNAP with preformed SNARE complex (87 ± 15%, mean ± SEM, n = 3), SNAP-29 efficiently prevented α-SNAP from binding to the SNARE complex (45 ± 6%, mean ± SEM, n = 5, P < 0.01) relative to a control in the absence of SNAP-29 (Fig. 4 C and D).

Next, we investigated whether SNAP-29 influences the disassembly of preformed recombinant or native SNARE complexes by NSF and α-SNAP in vitro. Recombinant SNARE core complex was constituted with purified SNARE proteins and captured on GST–VAMP-2-linked glutathione-Sepharose beads in the absence or presence of SNAP-29 (Fig. 5A). Native SNARE complexes were isolated by incubation of anti-VAMP-2 antibody with solubilized synaptosomes and incubated in the presence or absence of SNAP-29 (Fig. 5B). The SNARE complex is thermally stable during SDS/PAGE (11, 32, 33). Thus, appearance at 80°C of an ≈100-kDa band of the recombinant SNARE complex—or of three bands ranging from 75 to 100 kDa of native SNARE complexes—and disappearance of these bands at 100°C are indicative of preformed SNARE complex when blotted with anti-SNAP-25 antibodies. To test disassembly, purified α-SNAP and NSF were added in the presence of either ATP-Mg2+ or EDTA as indicated. A reduced signal intensity of these high molecular weight bands at 80°C indicates disassembly of the core complex by α-SNAP and NSF. As shown in Fig. 5, a significant reduction in disassembly was observed when the SNARE complexes were incubated with SNAP-29 at 80°C, suggesting an inhibitory role of SNAP-29 on disassembly of both recombinant and native SNARE complexes. Because α-SNAP is known to bind to monomeric syntaxin and SNAP-25 weakly, it is reasonable to ask whether it also binds to SNAP-29 and thereby leaves insufficient amounts of unbound α-SNAP to bind to the ternary SNARE complex. However, under our experimental conditions, immobilized and preassembled SNARE–SNAP-29 complex was washed extensively to remove free SNAP-29 before further incubation with α-SNAP and NSF. Furthermore, our in vitro binding studies showed no significant interaction between SNAP-29 and α-SNAP under our experimental conditions (data not shown). Thus, it is unlikely that α-SNAP was removed from the active reaction pool by excess SNAP-29. Rather, the disassembly of SNARE complex by α-SNAP and NSF was inhibited by SNAP-29 bound to the SNARE complex.

Figure 5.

Figure 5

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SNAP-29 reduces disassembly of preformed SNARE core complex. (A) Recombinant SNARE core complex was constituted by incubation of GST–VAMP-2 (1 μg) bound glutathione-Sepharose beads with purified syntaxin-1A and SNAP-25 (5 μg each) at 4°C for 3 h. (B) Native SNARE complexes were isolated from solubilized synaptosomes (50 μg) by anti-VAMP-2 antibody. After washing to remove unbound SNARE proteins, purified His-SNAP-29 (10 μg) was added into the preformed SNARE complexes and further incubated at room temperature for 1 h. SNARE core complexes were disassembled by addition of recombinant NSF and α-SNAP (2 μg each) in buffer with 2 mM MgCl2/2.5 mM ATP/20 mM Tris, pH 8.4/100 mM NaCl for 30 min at 30°C. As a control, the ATPase activity of NSF was abolished by chelating MgCl2 with 10 mM EDTA. All samples were heated at either 80°C or 100°C for 5 min and analyzed by SDS/PAGE and sequential immunoblotting with antibodies against SNAP-25 and His tag.

The Functional Effect of SNAP-29 on Synaptic Transmission.

To investigate the physiological significance of SNAP-29 on the disassembly of preformed SNARE complexes, we examined the role of SNAP-29 in synaptic transmission at the well-characterized cholinergic synapses formed between SCGN in culture (24, 25, 30, 34, 35). After a stable period of control EPSPs recordings at 0.05 Hz, full-length recombinant SNAP-29 was diffused (at t = 0 for 2–3 min) into a presynaptic neuron from a suction pipette. SNAP-29 had no effect on synaptic transmission for 15–20 min after injection, but this period was followed by a gradual reduction in the EPSP amplitude (Fig. 6 A and C). The maximum decrease, 29 ± 6.6% (n = 6, mean ± SEM), was observed 45–60 min after starting injection. EPSP amplitude then gradually recovered. When the stimulation frequency was increased to 0.2 Hz, a pronounced and prolonged decrease in EPSP amplitude was observed. The reduction of averaged EPSP amplitude was 34 ± 13% and 55 ± 9.3% (n = 4) at 50 and 100 min after injection, respectively. In contrast, EPSPs recorded at 0.02 Hz were not significantly affected by SNAP-29 injection (4.5 ± 7.3%, n = 5, at 50 min) (Fig. 6E), suggesting an activity-dependent inhibitory effect of exogenously applied SNAP-29 on synaptic transmission. As a control, heat-denatured recombinant SNAP-29 produced no significant change in EPSP amplitude (5.4 ± 7.6%, n = 4, at 60 min) (Fig. 6C).

To eliminate a possibility that injected SNAP-29 functions as a Q-SNARE and competes with endogenous SNAP-25 for binding to other SNAREs, we introduced a synthetic peptide containing the carboxyl-terminal 13 aa of SNAP-25 (amino acids 194–206) into presynaptic SCGNs. This peptide contains a binding site for VAMP-2 and is capable of blocking the interaction of SNAP-25 with VAMP-2 in vitro (36). Injection of this SNAP-25 peptide into SCGNs should mimic the competitive effect of the peptide on SNARE complex formation observed in vitro. In contrast to the effect of SNAP-29 injection, the latency of effects on synaptic transmission after SNAP-25 peptide injection was much shorter. EPSP amplitude gradually decreased 5–10 min after injection (Fig. 6 C and D). The maximum EPSP decrease was observed 40–50 min and 40–60 min with frequencies of 0.05 and 0.2 Hz stimulation, respectively. At 50 min after injection, the reduction of averaged EPSPs was 20 ± 12% (0.02 Hz, n = 6) and 31 ± 9.3% (0.2 Hz, n = 5). As a control, a peptide containing 26 aa of SNAP-25 (amino acids 146–171), which does not interact with presynaptic SNAREs, showed no significant effect on synaptic transmission (Fig. 6D). In contrast to the insignificant effects of SNAP-29 on synaptic transmission with stimulation at 0.02 Hz, SNAP-25 reduced EPSP amplitude at the even lower frequency of 0.01 Hz stimulation (Fig. 6E). The reduction value, 13 ± 6.3% (n = 5) at 30 min, is significantly larger than that induced by SNAP-29 injection, 3.1 ± 7.1% (n = 5, at 30 min), with stimulation at 0.02 Hz (Fig. 6E). Similar to the SNAP-29 injection, no obvious change in the time course of the EPSPs was observed with SNAP-25 peptide injection (Fig. 6 A and B). These results suggest that, whereas both injected SNAP-29 protein and SNAP-25 peptide efficiently inhibit synaptic transmission, the effect of SNAP-29 apparently occurs in an activity-dependent manner, indicating a role for SNAP-29 in modulating the recycling of SNARE complex rather than as a competitor with SNAP-25 for SNARE complex assembly.

To confirm that α-SNAP could compete with SNAP-29 for binding to the SNARE complex and consequently reduce the inhibitory effect of SNAP-29 on synaptic transmission, we performed coinjection of SNAP-29 and α-SNAP into presynaptic SCGNs (Fig. 6F). Reduction of EPSP amplitude by 170 μM SNAP-29 along with 43 μM α-SNAP was 36 ± 5.0% (n = 4, at 50 min, 0.2 Hz), similar to the reduction of EPSP, 34 ± 13% (n = 4) by injection of 170 μM SNAP-29 alone (Fig. 6C). However, when the concentration of α-SNAP was increased to 68 μM, reduction of EPSP amplitude by injection of 170 μM SNAP-29 was reduced to 14 ± 7.5% (n = 4, at 50 min, 0.2 Hz), indicating that the activity-dependent inhibition of neurotransmitter release by SNAP-29 could be reversed by a coinjection of α-SNAP. As a control, injection of 68 μM α-SNAP alone showed no significant effects on synaptic transmission.

Discussion

The findings of our present study provide biochemical, morphological, and physiological evidence that SNAP-29 is involved in synaptic transmission and suggest a potential role for SNAP-29 as a regulator of SNARE complex disassembly. The current SNARE hypothesis proposes that the precise assembly, rearrangement, and disassembly of SNARE complex mediate synaptic vesicle exocytosis. In vivo, the v-SNARE and t-SNARE proteins anchored in membrane lipids can be in cis (on the same membrane) or in trans (on opposing membranes) conformations. Numerous studies have suggested that syntaxin-1 and VAMP-2 are anchored to opposing membranes through single transmembrane-spanning helices. Formation of a stable trans-SNARE complex brings these membranes into direct opposition and may result in membrane fusion or an activated docking state (11, 17, 36). After fusion, SNAREs are aligned in parallel within the same membrane in the form of a cis-complex. The cis-SNARE complex must be dissociated through the action of α-SNAP and the ATPase NSF to allow the SNARE proteins to be recycled (9, 14). Our functional studies demonstrated that introduction of SNAP-29 into presynaptic SCGNs in culture significantly inhibits synaptic transmission in an activity-dependent manner. We interpret our results to suggest that injected SNAP-29 competitively blocks the disassembly of postfusion cis-SNARE complexes, resulting in a decrease of free SNARE proteins available to form functional trans-SNARE complexes necessary for vesicle release. This interpretation is consistent with the results from our in vitro biochemical studies, which showed that SNAP-29 competes with α-SNAP for binding to the neuronal SNARE complex and consequently blocks disassembly of preformed SNARE complex in vitro. In contrast, the relatively rapid and activity-independent inhibition of synaptic transmission induced by injection of SNAP-25 C-terminal peptide may be the result of a competitive block of formation of the trans-SNARE complexes. The different effects of SNAP-29 and SNAP-25 on synaptic transmission further support our biochemical evidence that SNAP-29 is not functionally equivalent to SNAP-25 during the process of assembly/disassembly of the SNARE complex.

The sequence of SNAP-29 is only 23% identical to that of SNAP-25, and the conserved residues are confined mostly to the coiled-coil domains. Our biochemical observations showed that the binding properties of full-length SNAP-29 are not equivalent to SNAP-25 and suggest that the sequence and/or structure of full-length SNAP-29 has a significant impact on the formation of complexes with neuronal t-SNARE and v-SNARE. This finding is supported by previous rescue studies in cracked PC12 cells pretreated with botulinum neurotoxin-E (37). Whereas the resulting inhibition of fusion was effectively rescued by addition of the C-terminal coils of either SNAP-25 or SNAP-23, addition of SNAP-29 C-terminal coil was much less effective. However, the N- and C-terminal coiled-coil domains of SNAP-29 have been reported to be capable of forming less thermally stable core complexes with other interacting domains of t-SNARE and v-SNARE proteins in vitro (33, 37); the possibility that SNAP-29 could substitute for SNAP-25 during SNARE assembly and consequently affect exocytosis could not be excluded. In light of this possibility, an alternative explanation of our results would be that SNARE complexes containing SNAP-29 bind α-SNAP less effectively and are thus not readily dissociated by NSF. Therefore, at the SNAP-29-enriched synapses, the assembly–disassembly equilibrium of the SNARE complex would be shifted in favor of forming the SNAP-29-containing and NSF-resistant complexes, resulting in a reduced recycling of the SNARE components.

Our immunoblot analysis of detergent-extracted rat synaptosomes, standardized by each antibody against purified recombinant protein for quantification, demonstrated the relative amounts of SNAP-29 to be ≈45%, 20%, and 17% of that of α-SNAP, syntaxin-1, and SNAP-25, respectively (data not shown). Compared with the relative abundance of other SNARE regulators such as complexins and tomosyn (6% and 3% of syntaxin-1, respectively, in brain) (21, 23), the ratio of SNAP-29 demonstrates that it is relatively enriched in synapses. Furthermore, our immunocytochemical studies on cultured hippocampal neurons using anti-SNAP-29 antibody demonstrated punctate staining along neuronal processes, suggesting an even higher enrichment of SNAP-29 in some synapses. Thus, the SNAP-29-enriched synapses may represent negatively regulated or silent synapses, whereas those with lower or absent SNAP-29 expression may represent active synapses. Therefore, SNAP-29 may be present in sufficient quantities to serve as a SNARE disassembly regulator at some selective synapses.

The cis-SNARE complex present in recycling vesicles can be inferred from a number of published observations (38, 39). Although there are presently no reliable assays for measuring cis- or trans-complexes on which SNAP-29 may act, a SNARE complex in the cis-conformation probably resembles the fully assembled complex in vitro under solubilized conditions. SNAREs in a trans-complex might only loosely or partially interact because of the resistance posed by the opposing membranes, would likely not be SDS resistant or thermostable, and might not require α-SNAP and NSF for dissociation (40, 41). It is attractive, therefore, to speculate that SNAP-29 may control the disassembly of the cis-SNARE complex and regulate the availability of individual SNAREs to form new SNARE complexes between opposing membranes, consequently modulating the process of membrane trafficking. Further biochemical, genetic, and physiological characterization of SNAP-29 will shed light on the regulation of SNARE complex formation and dynamic recycling during exocytotic membrane fusion events.

Acknowledgments

We thank G. Lao for initiating yeast two-hybrid screening, C. Gerwin for experimental assistance, X. Zheng for synaptosome fractionation, J. McCallum and C. A. Winters for hippocampal neuron culture, J. W. Nagle for DNA sequencing, M. Takahashi and W. Hong for SNAP-25 peptides and antibodies, and S. Das and J. Coulombe for critical reading of this manuscript. This work was supported by the intramural research program of the National Institute of Neurological Disorders and Stroke, National Institutes of Health (to Z.-H.S.), and a Grant-in-Aid for Scientific Research on Priority Areas (A) and the Uehara Memorial Foundation (to S.M.).

Abbreviations

NSF

N-ethylmaleimide-sensitive factor

SNAP

soluble NSF attachment protein

SNARE

SNAP receptor

SCGN

superior cervical ganglion neuron

GST

glutathione S-transferase

EPSP

excitatory postsynaptic potential

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