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Munc18-dependent regulation of synaptic vesicle exocytosis by syntaxin-1A in hippocampal neurons
Neuropharmacology 48 (2005) 372–380 www.elsevier.com/locate/neuropharm
Munc18-dependent regulation of synaptic vesicle exocytosis by syntaxin-1A in hippocampal neurons Simon J. Mitchell1, Timothy A. Ryan) Department of Biochemistry, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA Received 25 June 2004; received in revised form 10 September 2004; accepted 15 October 2004
Abstract The fusion of secretory vesicles with the plasma membrane requires the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes between the vesicle-SNARE vesicle-associated membrane protein present on the vesicular membrane and the target-SNAREs SNAP-25 and syntaxin-1A. Syntaxin-1A fluctuates between an open and closed form allowing it to selectively bind to different biological effectors in different conformations. In the open form, it can participate in SNARE complex formation, however, in the closed form it negatively regulates N- and P/Q-type voltage-dependent calcium channels, and is capable of inhibiting calcium influx. Thus paradoxically, syntaxin appears to have both positive and negative roles in controlling calcium-driven synaptic vesicle fusion at synaptic terminals. We show here that overexpression of syntaxin-1A inhibited exocytosis, in a manner that could be rescued by either elevating or reducing external calcium, or increasing action potential firing frequency. Elevating the level of Munc18 by coexpression with syntaxin-1A also abolished this inhibition, suggesting that Munc18 serves to limit the negative regulatory role of syntaxin by binding to, and thereby buffering, its closed form. Our results also indicate that syntaxin can control the frequency–response characteristics of the presynaptic fusion machinery. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Syntaxin; Munc18; Vesicle; Calcium; Exocytosis; SNARE protein
1. Introduction Docking and fusion of synaptic vesicles to the plasma membrane are mediated by SNAREs, which form a stable complex between vesicle-SNAREs present on vesicular membrane and target-SNAREs on the target plasma membrane (Sutton et al., 1998). Synaptic vesicle exocytosis occurs in proximity to calcium microdomains produced by clusters of voltage-gated calcium channels (Pumplin et al., 1981; Sugimori et al., 1994; DiGregorio et al., 1999). Binding of syntaxin-1A to a synaptic ) Corresponding author. Tel.: C1 212 746 6403; fax: C1 212 746 8875. E-mail address: [email protected] (T.A. Ryan). 1 Present address: Neurobiology Division, MRC Laboratory of Molecular Biology, Cambridge CB2 2QH, UK. 0028-3908/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2004.10.017
protein interaction (synprint) site within the N- and P/Q-type voltage-gated calcium channel a1 subunit (Sheng et al., 1994; Bezprozvanny et al., 2000) may therefore localize docked vesicles near calcium microdomains. Paradoxically, syntaxin-1A inhibits calcium influx by promotion of slow inactivation of calcium channels (Bezprozvanny et al., 1995; Wiser et al., 1996; Degtiar et al., 2000). There is evidence that calcium channel modulation is conferred by the transmembrane region and a section of the H3 domain of syntaxin, which are at distinct loci from the synprint site (Bezprozvanny et al., 2000; Trus et al., 2001). Calcium influx and evoked neurotransmission are enhanced by introduction of syntaxin-1 antibodies or application of botulinum toxin C1 (Yamaguchi et al., 1997; Sugimori et al., 1998; Blasi et al., 1993; Stanley, 2003). Furthermore, synchronous release is reduced, whereas
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asynchronous release is enhanced, by the introduction of peptides containing the synprint site (Mochida et al., 1996), an effect that could be due to an interruption of the binding of syntaxin, SNAP-25 and/or synaptotagmin to the synprint site on calcium channels (Sheng et al., 1997). Coordination of synaptic transmission involves many synaptic proteins, including Munc18, the mammalian Sec1/UNC-18/ROP homologue (Rizo and Sudhof, 2002). Munc18 has been proposed to serve several possible roles in SNARE function, including trafficking of syntaxin to the plasma membrane, vesicle priming and docking (Weimer and Jorgensen, 2003). Munc18 is required for neurotransmission since both evoked and spontaneous release are abolished in neurons from knockout mice (Verhage et al., 2000) and severely reduced in UNC-18 mutants in Caenorhabditis elegans (Weimer et al., 2003). Syntaxin continuously switches between an open conformation, which is a constituent of the core complex, and a closed form (Margittai et al., 2003). Munc18 binds only the closed form of syntaxin, as constitutively open forms of syntaxin fail to bind Munc18 (Dulubova et al., 1999). Since the Munc18binding domain on syntaxin-1A includes the synprint binding sites of syntaxin-1A (Sheng et al., 1994; Jarvis et al., 2002) and only the closed form of syntaxin modulates calcium channels (Jarvis et al., 2002), there is a possibility that Munc18 might promote exocytosis by decoupling closed syntaxin from voltage-gated channels. Although, ROP has been shown to prevent the inhibition of evoked EPSPs at the neuromuscular junction of Drosophila (Wu et al., 1998), it is unclear whether Munc18 performs an analogous role in mammalian neurons. We investigated whether transiently overexpressed syntaxin-1A regulates synaptic vesicle exocytosis in hippocampal neurons in culture, by monitoring activitydependent staining and destaining of synaptic vesicles using the optical probe FM 4-64. We find that syntaxin-1A slows the rate of synaptic vesicle exocytosis during trains of stimuli, in a manner that depends on external calcium concentration as well as stimulus frequency. Coexpression of Munc18-1 reversed this inhibition, without affecting the surface fraction of expressed syntaxin-1A.
2. Methods 2.1. Cell culture and transfection Hippocampal CA3–CA1 regions were dissected from postnatal 2- to 4-day-old rats, and neurons were dissociated and plated on coverslips coated with 10 mg/ml poly-L-ornithine, and cultured and maintained as described previously (Ryan, 1999). All experiments
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were performed on cultures of between 2 and 3 weeks of age. Syntaxin-1A fusion protein was generated by subcloning the rat syntaxin-1A cDNA into a vector encoding super-ecliptic GFP (pHluorin; Miesenbock et al., 1998). Munc18-1-FLAG fusion protein was generated by subcloning the rat Munc18-1 cDNA into a vector encoding a FLAG epitope (amino acid sequence of DYKDDDDK). Transfection of syntaxin-1A and Munc18-1-FLAG was performed using calcium phosphate precipitation as described (Threadgill et al., 1997). Overexpression of Munc18-1 did not impair the overexpression of syntaxin-1A, but rather it increased the level of overexpression 3.7-fold, in agreement with a previous study showing that the syntaxin level is suppressed in UNC-18 mutant flies (Weimer et al., 2003). Unless otherwise stated, all reagents were obtained from Sigma (St. Louis, MO). All animal experiments and use were approved by the Institutional Animal Care and Use Committee of the Weill Medical College of Cornell University. 2.2. Experimental conditions Coverslips of neuronal cell cultures were mounted in a superfusion chamber equipped with field stimulation electrodes on the stage of a custom-built laser-scanning confocal microscope as described (Ryan, 1999). Field stimulation was applied by passing 1 ms current pulses across the chamber to produce an electric field of 10 V/cm using platinum–iridium electrodes. Unless otherwise indicated, cells were superfused at room temperature in a saline solution consisting of 119 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 5 mM HEPES (pH 7.4), 30 mM glucose, 10 mM CNQX (an AMPA receptor antagonist), and 50 mM APV (an NMDA receptor antagonist). Synaptic vesicle pools were labeled by fieldstimulating cultures for 30 s at 20 Hz in the presence of 15 mM FM 4-64 in normal saline. An additional 60 s of dye exposure was allowed to ensure complete labeling of all recycling vesicles. The cultures were subsequently rinsed in dye-free solution for 10 min prior to dye destaining. The external solution contained 3.5 mM CaCl2 and 0.5 mM MgCl2 for experiments requiring an elevated calcium/magnesium concentration ratio and 0.5 mM CaCl2 and 3.5 mM MgCl2 for experiments requiring a lower calcium/magnesium concentration ratio. Ammonium chloride solution (pH 7.4) was prepared by substituting 50 mM NaCl in the above saline with NH4Cl. Acidic solution at pH 5.5 was prepared by replacing HEPES in the standard saline with 2-[N-morpholino]ethane sulphonic acid (pK Z 6.1). 2.3. Immunocytochemistry Neurons were processed for immunofluorescence for either syntaxin-1A or Munc18-1-FLAG after fixation in
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3. Results
Although a number of reports have suggested that dye destaining rates are not a faithful report of exocytosis due to possible dye retention during endocytosis (Pyle et al., 2000), we find little evidence for that in our system (Ferna´ndez-Alfonso and Ryan, 2004). The destaining time course was 38 G 10% (n Z 25; P ! 0.001) slower in syntaxin-1A transfected cells than non-transfected control neurons in the same culture dish (Fig. 1B and C). The level of FM loading (recycling vesicle pool size) was quantified by measuring the total amount of destaining for two successive rounds of stimulation at 10 Hz for 90 s and 20 Hz for 60 s, a protocol that maximally destains the recycling vesicle pool. The vesicle pool size in syntaxin overexpressing cells was 25 G 6% smaller than in controls (Fig. 1D; n Z 23; P ! 0.001). As syntaxin is known to inhibit voltage-dependent calcium channels involved in synaptic vesicle release, we investigated whether the inhibition of FM 4-64 destaining might be dependent on external calcium concentration. In non-transfected control cells the destaining time constant was only 8 G 5% (n Z 3) faster when the calcium/magnesium ion concentration ratio was elevated from unity to seven, suggesting that the destaining rate was near maximal under control conditions for steadystate release during sustained stimulation. With an elevated calcium/magnesium ratio, the destaining rate of syntaxin-1A-positive neurons was now similar to control cells (107 G 3% of control; n Z 5; P O 0.05; Fig. 2A and C). In non-transfected controls, when the calcium/magnesium ion concentrations were 7-fold lower than unity the destaining rate was 76 G 20% (n Z 3) slower than under control conditions. However, at a lower calcium/magnesium ratio, as at an elevated calcium/magnesium ratio, the inhibition in destaining rate in syntaxin-1A positive cells was also relieved (101 G 5% of control; n Z 3; P O 0.05; Fig. 2B and C). These data show that changing extracellular calcium/ magnesium ratio, and thus the level of calcium influx during an AP, modulates the inhibition of exocytosis by excess syntaxin-1A.
3.1. Overexpression of syntaxin-1A reduces synaptic vesicle exocytosis rate
3.2. Munc18-1 reverses syntaxin-mediated reduction of exocytosis
We investigated whether overexpression of syntaxin1A affected synaptic vesicle exocytosis by monitoring the release of the styryl dye FM 4-64, which labels synaptic vesicle membranes in an activity-dependent manner. Synaptic vesicles in cultured hippocampal neurons were labeled using 600 action potentials (APs) at 20 Hz in the presence of 15 mM FM 4-64 (Fig. 1A), which labels w90% of the recycling pool of synaptic vesicles in control cells (Ryan and Smith, 1995). The rate of exocytosis was estimated from a single exponential fit to the decay in FM 4-64 fluorescence during electrical stimulation of 900 action potentials at 10 Hz (Fig. 1B).
Munc18-1 has been shown to bind to the closed conformation of syntaxin-1A (Dulubova et al., 1999; Misura et al., 2000). To examine whether enhanced levels of Munc18-1 might reverse the impaired exocytosis in syntaxin-1A positive neurons, we coexpressed Munc18-1 with syntaxin-1A. The distribution of Munc18-1 as detected by retrospective immunofluorescence with an anti-FLAG antibody, was similar to that of syntaxin-1A (Fig. 3A), consistent with a previous report (Garcia et al., 1995). The rate of FM destaining in syntaxin-1A and Munc18-1-FLAG cotransfected terminals was similar to non-transfected terminals
4% paraformaldehyde (Electron Microscopy Science, Washington, PA) and 4% sucrose in PBS for 20 min. Cells were then permeabilized in the same fixative plus 0.25% Triton for 10 min, blocked in 10% bovine serum albumin (BSA) for 2 h at 37 C and incubated overnight with either affinity-purified monoclonal anti-syntaxin-1 antibody (Synaptic Systems, Go¨ttingen, Germany; 1:1000 dilution) or purified monoclonal anti-FLAG M2 antibody (Sigma, St. Louis, MO; 10 mg/ml). All antibodies were diluted in 1% BSA in PBS. Cells were then incubated with either an anti-mouse or an antirabbit Alexa-546-conjugated goat IgG secondary antibody (Molecular Probes, Eugene, OR; 1:2000 dilution), as appropriate, for 3 h and mounted for laser-scanning microscopic analysis. Using these analyses the level of expression of syntaxin-1 was w2-fold higher in cells expressing exogenous syntaxin-1A (Mitchell and Ryan, 2004). 2.4. Fluorescence microscopy and analysis Laser-scanning fluorescence images were acquired as described (Sankaranarayanan and Ryan, 2000). Quantitative measurements of fluorescence intensity at individual boutons were obtained by averaging a 4 ! 4 area of pixel intensities using custom written software. All subsequent analysis was performed in Igor Pro (Wavemetrics, OR). FM 4-64 destaining data were normalized to the total loss of fluorescence during two subsequent rounds of stimulation at 10 Hz for 90 s, then 20 Hz for 60 s after a 2-min rest. Time constants for FM 4-64 destaining were obtained by fitting the destaining curves to single exponential decays. The release rates in transfected cells were compared to those non-transfected control cells located in the same dish. Means are presented with their standard error. Data groups were compared using a two-tailed Student’s t test.
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Fig. 1. Overexpression of syntaxin-1A inhibits synaptic vesicle exocytosis. (A) Laser-scanning fluorescence image of an axon of a cultured hippocampal neuron expressing syntaxin-1A-pHluorin fusion protein, which was loaded with FM 4-64 for 30 s at 20 Hz. Exogenous syntaxin-1A expression (pHluorin tag fluorescence, green) is shown with synaptic vesicles labeled with FM 4-64 (red). Regions of colocalization are shown in yellow. FM 4-64 puncta in non-transfected and transfected cells were similar in size. (B) Semi-log plots of simultaneous destaining of FM 4-64 from synaptic vesicles of non-transfected control cells (filled circles) and syntaxin-1A overexpressing cells (open circles) in the same culture dish, during electrical stimulation at 10 Hz. Dashed and solid lines show single exponential fits of destaining rates for control and syntaxin-1A overexpressing cells, respectively. (C) Mean destaining time constant (tau) for control and syntaxin-1A overexpressing cells. (D) Mean total destaining (recycling pool size) for control and syntaxin-1A overexpressing cells (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
(104 G 18% of control; n Z 5; P O 0.05; Fig. 3B and C), consistent with the possibility that Munc18 reduces the ability of syntaxin-1A to inhibit calcium influx. However, FM uptake in cells overexpressing syntaxin and Munc18-1 cells was reduced by 29 G 4% compared to control terminals (n Z 5; P ! 0.01; Fig. 3D), which is similar to level of inhibition for terminals transfected with only syntaxin-1A (Fig. 3D). These data show that the balance of Munc18-1 and syntaxin-1A can regulate the ability of syntaxin to inhibit exocytosis rates. 3.3. Munc18-1 coexpression does not alter the surface fraction of syntaxin-1A In endothelial cells, overexpressed syntaxin-1A is retained intracellularly unless Munc18 is coexpressed (Rowe et al., 2001). A change in surface expression of syntaxin-1A caused by Munc18-1 might therefore underlie the ability of Munc18 to reverse the modulation of vesicle exocytosis rates of neurons by syntaxin. We analyzed whether coexpression of Munc18-1-FLAG altered the proportion of syntaxin-1A on the surface relative to internal compartments (surface fraction) at
functional synaptic boutons, previously identified by FM 4-64 loading and unloading (Mitchell and Ryan, 2004). To do this, we monitored fluorescence changes in syntaxin-1A during application of extracellular solution at pH 5.5, to quench surface fluorescence (Fig. 4A), or a solution that contained ammonium chloride to clamp internal compartments to pH 7.4 (Fig. 4B; Sankaranarayanan et al., 2000; Mitchell and Ryan, 2004). The change in fluorescence normalized to the initial fluorescence (DF/F ) during application of acidic extracellular solution was ÿ96 G 1% (n Z 6), indicating a large surface component of syntaxin-1A. The increase in fluorescence during application of ammonium chloride solution at pH 7.4 was 18 G 4% (n Z 6), showing a fraction of syntaxin-1A was present in an acidic intracellular compartment. Using these data and the known pK of pHluorin (pH 7.18), we calculated the surface fraction of syntaxin-1A as a function of the pH of the internal compartment as described in Mitchell and Ryan (2004). The functions for the acid quench and ammonium chloride data intersected at pH 5.5 (Supplementary Fig. 1), which is similar to the pH of the compartment that exogenous syntaxin-1A is localized to
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(13%; Mitchell and Ryan, 2004). Similar analyses revealed that the fraction of syntaxin-1A on nonsynaptic axonal plasma membrane was 98 G 2%, (n Z 6; 98% for cells transfected with syntaxin-1A only; Mitchell and Ryan, 2004). These data show that in cells cotransfected with Munc18-1 and syntaxin-1A, syntaxin-1A is mainly located on the plasma membrane, but that a small fraction is located in an acidic compartment at synaptic boutons. Stimulation of 600 APs at 20 Hz did not increase or decrease the pHluorin signal (Fig. 4C). The same stimulation protocol also failed to induce a change in fluorescence in the presence of bafilomycin-A1, which increases the signal by blocking synaptic vesicle reacidification, showing that syntaxin-1A was not present in recycling synaptic vesicles (data not shown). These findings are similar to those found in terminals transfected with syntaxin-1A alone (Mitchell and Ryan, 2004), indicating that overexpression of Munc18-1 did not alter the surface fraction of syntaxin-1A. 3.4. Frequency-dependent modulation of exocytosis rate by syntaxin-1A
Fig. 2. Magnitude of inhibition of release rate kinetics depends on external calcium/magnesium concentration. (A, B) Semi-log plots of simultaneous destaining of FM 4-64 from synaptic vesicles of nontransfected control cells (filled circles) and syntaxin-1A overexpressing cells (open circles) in the same culture dish, during electrical stimulation at 10 Hz, at (in mM): 3.5 Ca/0.5 Mg (High [Ca]/[Mg]; A); 0.5 Ca/3.5 Mg (Low [Ca]/[Mg]; B). (C) Mean inhibition of destaining rate relative to control cells at (in mM): 0.5 Ca/3.5 Mg (Low); 2 Ca/2 Mg (Mid); 3.5 Ca/0.5 Mg (High). Loading of FM 4-64 was performed at 2 mM Ca/ 2 mM Mg for all experiments.
when expressed without Munc18-1 (pH 5.3; Mitchell and Ryan, 2004), and the same as the synaptic vesicle lumen (pH 5.5; Miesenbock et al., 1998; Sankaranarayanan et al., 2000). This analysis showed that the fraction of syntaxin-1A in the internal compartment in these cotransfected terminals (15 G 3%; n Z 6) was similar to cells transfected with syntaxin-1A alone
We have previously shown that exocytosis rate was unaffected by the overexpression of syntaxin-1A when APs were stimulated at 20 Hz (Mitchell and Ryan, 2004). To investigate the frequency-dependence of the inhibition reported here, we stimulated APs between 5 Hz and 50 Hz. In non-transfected cells, the rate of destaining increased as the frequency was increased from 5 Hz to 20 Hz, with no further increase in rate observed at 50 Hz (Fig. 5). In transfected cells, the destaining rate was inhibited by 52 G 17% compared to non-transfected cells at a stimulation frequency of 5 Hz (n Z 5; P Z 0.02) and by 29 G 7% at 10 Hz in the same group of cells (n Z 5; P Z 0.01). However, at 20 Hz and 50 Hz no modulation of destaining rate was observed (n Z 6 and 4, respectively; P O 0.13). In this way, overexpression of syntaxin-1A modulated the frequency–response characteristics of release (Fig. 5).
4. Discussion Our results demonstrate that overexpression of syntaxin can inhibit synaptic vesicle exocytosis, in a manner that is consistent with its known inhibition of voltage-dependent calcium channels, and that this inhibition is eliminated when syntaxin is coexpressed with Munc18. Our findings suggest that the balance of Munc18-1 and syntaxin-1A expression regulates neurotransmission by modulating synaptic vesicle exocytosis. Furthermore, the inhibition of exocytosis rate by syntaxin-1A only occurs at stimulation frequencies of 10 Hz and lower, suggesting that the level of syntaxin-1A
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Fig. 3. Coexpression of syntaxin-1A and Munc18-1 prevents inhibition of exocytosis by syntaxin. (A) Laser-scanning fluorescence images of an axon of the same cultured hippocampal neuron coexpressing exogenous syntaxin-1A (live cell imaging of pHluorin tag fluorescence, top image) and Munc18-1 (fixed then stained with anti-FLAG primary and Alexa-546 secondary antibodies, bottom image). The expression of exogenous syntaxin-1A, as reported by pHluorin fluorescence, was not impaired in cells coexpressing Munc18-1 compared to cells overexpressing syntaxin-1A alone. (B) Semi-log plots of simultaneous destaining of FM 4-64 from synaptic vesicles of control cells (filled circles) and syntaxin-1A/Munc18-1 coexpressing cells (open circles), during electrical stimulation at 10 Hz. (C) Mean inhibition of destaining rate for syntaxin-1A overexpressing cells and syntaxin-1A/ Munc18-1 coexpressing cells. (D) Mean inhibition of recycling pool size for syntaxin-1A overexpressing cells and syntaxin-1A/Munc18-1 coexpressing cells (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
may help set the frequency–response characteristics of the synapse. 4.1. Calcium-dependent modulation of exocytosis Our study shows that the inhibition of exocytosis by syntaxin-1A is dependent on the ratio of extracellular calcium and magnesium concentration. Our data showing that conditions that lower calcium influx removed the inhibitory effects of syntaxin-1A overexpression are consistent with data showing that the binding of syntaxin and calcium channels is decreased at lower calcium concentration (Sheng et al., 1996). It is possible that reduced association of syntaxin and calcium channels at lower calcium levels may reduce the ability of syntaxin to modulate either the calcium influx or the coupling between calcium influx and exocytosis. Previous evidence has indicated that syntaxin-1A can modulate N- and P/Q-type calcium channels (Bezprozvanny et al., 1995; Wiser et al., 1996; Degtiar et al., 2000). Our results showing that syntaxin-1A inhibits exocytosis rates are consistent with this, since neurotransmission is mediated by N- and P/Q-type channels in cultured hippocampal neurons (Luebke
et al., 1993; Wheeler et al., 1994; Wu and Saggau, 1994; Scholz and Miller, 1995; Reid et al., 1997). The lack of effect at high external calcium concentrations was likely the effect of release rate saturation, such that the decrease in calcium influx caused by syntaxin was insufficient to affect exocytosis. Moreover, the fact that the inhibition can be overcome by elevation or reduction in calcium suggests that neither syntaxin-1A overexpression nor tagging of syntaxin with pHluorin produced a non-specific deficit in exocytosis. Overexpression of syntaxin in C. elegans did not modulate evoked excitatory currents at the neuromuscular junction at an external calcium/magnesium ratio of 5, at which the release rate appears to be approaching saturation in wild type preparations (Weimer et al., 2003). 4.2. Munc18-1 removes syntaxin-1A-mediated inhibition of exocytosis Munc18-1 binds and stabilizes the closed conformation of syntaxin-1A, which appears a necessary step for proper SNARE complex assembly (Dulubova et al., 1999; Yang et al., 2000). The ability of Munc18
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Munc18 effectively ‘‘buffers’’ the levels of closed syntaxin that might otherwise be free to interact with, and inhibit, voltage-dependent calcium channels. If this model is correct, our data indicate that the balance of syntaxin-1A and Munc18-1 levels is crucial for efficient synaptic vesicle release. These data compliment the finding in a C. elegans mutant that the absence of UNC18 inhibits evoked neuromuscular junction end plate potentials by affecting vesicle docking (Weimer et al., 2003). Interestingly, overexpression of ROP in Drosophila inhibited neurotransmission (Schulze et al., 1994; Wu et al., 1998). We were unable to produce viable cultured hippocampal neurons that overexpressed exogenous Munc18-1. We find no evidence that the ability of coexpressed Munc18 to abolish the inhibitory effects of syntaxin overexpression could be attributed to altered syntaxin trafficking, since the surface fraction of expressed syntaxin under these conditions and for cells overexpressing syntaxin alone was similar (Mitchell and Ryan, 2004). This is consistent with the finding that UNC-18 is not required for syntaxin trafficking in C. elegans (Weimer et al., 2003). 4.3. Syntaxin-1A preferentially inhibits release rate at lower stimulation frequencies Previous studies demonstrating that syntaxin-1A inhibits AP-dependent exocytosis used low-frequency stimulation (Wu et al., 1998). We have demonstrated in this study that the degree of inhibition of release rate by syntaxin-1A decreases from w50% at a stimulation frequency of 5 Hz to no inhibition at 20 Hz and higher. The increase in residual calcium that accumulates during high-frequency trains may underlie the reduced dependence of release rate on syntaxin-1A level. Alternatively, a release rate saturation effect may take place at higher frequencies. These findings indicate that syntaxin-1A can regulate the frequency–response properties of the synapse. Fig. 4. Subcellular localization of exogenous syntaxin-1A is not altered by coexpression with Munc18-1. (A) Quenching of surface syntaxin1A-pHluorin fluorescence by application of external solution at pH 5.5. (B) Increase in fluorescence of syntaxin-1A-pHluorin present in an acidic internal compartment during application of membrane permeant NH4Cl solution at pH 7.4. (C) Syntaxin-1A-pHluorin fluorescence during electrical stimulation at 20 Hz for 30 s.
coexpression to suppress inhibition of exocytosis by syntaxin-1A is consistent with previous studies in Drosophila, where a mutation of ROP, the Drosophila Munc18 homologue, or overexpression of syntaxin-1A both inhibited synaptic transmission (Wu et al., 1998). The simplest interpretation of our results is that in addition to its potential role in driving correct assembly,
4.4. Mechanisms for varying syntaxin-1-mediated inhibition of transmitter release Our results suggest that syntaxin expression levels, in particular with respect to that of Munc18, regulate important properties of synaptic transmission. Previous studies have shown that syntaxin levels might be dynamically modulated in an activity-dependent manner. High-frequency stimulation of the perforant path in the hippocampus induces a transient increase syntaxin-1B mRNA levels, which could be detected after 2 h (Hicks et al., 1997), and increases in syntaxin-1B mRNA and protein levels were detected 5 h after the induction of the long-term potentiation in the dentate gyrus (Hicks et al., 1997; Helme-Guizon et al., 1998). Calcium influx through
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Fig. 5. Magnitude of inhibition of release rate kinetics depends on frequency of stimulation. Destaining rates from control cells (filled circles) and cells overexpressing syntaxin-1A-pHluorin (open circles) located in the same culture dish were measured during AP stimulation at the indicated frequencies and normalized to the rate at 10 Hz stimulation in control cells.
P/Q-type calcium channels modulates syntaxin-1A mRNA level in HEK293 cells within 36 h, as well as regulating the endogenous syntaxin-1A mRNA level in cultured cerebellar granule cells (Sutton et al., 1999). Thus, given the results presented here, activity-dependent regulation of the level of syntaxin might provide a mechanism to tune the frequency- and/or calciumdependence of synaptic transmission at central synapses, which are important determinants for how information is processed in the nervous system.
Acknowledgments We thank W. Yan for technical assistance, K. Nicholson Tomishima for assistance with the syntaxin-1A-pHluorin and Munc18-1-FLAG constructs and members of the Ryan lab for discussions. This work was supported by grants from the NIH (NS24692 and GM61925) and the Hirschl Trust to T.A.R. S.J.M. is in receipt of an International Research Fellowship from the Wellcome Trust.
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Ryan, T.A., 1999. Inhibitors of myosin light chain kinase block synaptic vesicle pool mobilization during action potential firing. J. Neurosci. 19, 1317–1323. Ryan, T.A., Smith, S.J., 1995. Vesicle pool mobilization during action potential firing at hippocampal synapses. Neuron 14 (5), 983–989. Sankaranarayanan, S., Ryan, T.A., 2000. Real-time measurements of vesicle-SNARE recycling in synapses of the central nervous system. Nat. Cell Biol. 2, 197–204. Sankaranarayanan, S., De Angelis, D., Rothman, J.E., Ryan, T.A., 2000. The use of pHluorins for optical measurements of presynaptic activity. Biophys. J. 79, 2199–2208. Sheng, Z.H., Rettig, J., Takahashi, M., Catterall, W.A., 1994 Dec. Identification of a syntaxin-binding site on N-type calcium channels. Neuron 13 (6), 1303–1313. Sheng, Z.H., Rettig, J., Cook, T., Catterall, W.A., 1996 Feb 1. Calcium-dependent interaction of N-type calcium channels with the synaptic core complex. Nature 379 (6564), 451–454. Sheng, Z.H., Yokoyama, C.T., Catterall, W.A., 1997 May 13. Interaction of the synprint site of N-type Ca2C channels with the C2B domain of synaptotagmin I. Proc. Natl. Acad. Sci. U.S.A. 94 (10), 5405–5410. Scholz, K.P., Miller, R.J., 1995 Jun. Developmental changes in presynaptic calcium channels coupled to glutamate release in cultured rat hippocampal neurons. J. Neurosci. 15 (6), 4612–4617. Schulze, K.L., Littleton, J.T., Salzberg, A., Halachmi, N., Stern, M., Lev, Z., Bellen, H.J., 1994 Nov. rop, a Drosophila homolog of yeast Sec1 and vertebrate n-Sec1/Munc-18 proteins, is a negative regulator of neurotransmitter release in vivo. Neuron 13 (5), 1099–1108. Stanley, E.F., 2003 Mar. Syntaxin I modulation of presynaptic calcium channel inactivation revealed by botulinum toxin C1. Eur. J. Neurosci. 17 (6), 1303–1305. Sugimori, M., Lang, E.J., Silver, R.B., Llinas, R., 1994. Highresolution measurement of the time course of calcium-concentration microdomains at squid presynaptic terminals. Biol. Bull. 187, 300–303. Sugimori, M., Tong, C.K., Fukuda, M., Moreira, J.E., Kojima, T., Mikoshiba, K., Llinas, R., 1998 Sep. Presynaptic injection of syntaxin-specific antibodies blocks transmission in the squid giant synapse. Neuroscience 86 (1), 39–51.
Sutton, K.G., McRory, J.E., Guthrie, H., Murphy, T.H., Snutch, T.P., 1999 Oct 21. P/Q-type calcium channels mediate the activitydependent feedback of syntaxin-1A. Nature 401 (6755), 800–804. Sutton, R.B., Fasshauer, D., Jahn, R., Brunger, A.T., 1998 Sep 24. Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 A resolution. Nature 395 (6700), 347–353. Threadgill, R., Bobb, K., Ghosh, A., 1997 Sep. Regulation of dendritic growth and remodeling by Rho, Rac, and Cdc42. Neuron 19 (3), 625–634. Trus, M., Wiser, O., Goodnough, M.C., Atlas, D., 2001. The transmembrane domain of syntaxin 1A negatively regulates voltage-sensitive Ca(2C) channels. Neuroscience 104 (2), 599–607. Verhage, M., Maia, A.S., Plomp, J.J., Brussaard, A.B., Heeroma, J.H., Vermeer, H., Toonen, R.F., Hammer, R.E., van den Berg, T.K., Missler, M., Geuze, H.J., Sudhof, T.C., 2000 Feb 4. Synaptic assembly of the brain in the absence of neurotransmitter secretion. Science 287 (5454), 864–869. Weimer, R.M., Jorgensen, E.M., 2003 Sep 15. Controversies in synaptic vesicle exocytosis. J. Cell Sci. 116 (Pt 18), 3661–3666. Weimer, R.M., Richmond, J.E., Davis, W.S., Hadwiger, G., Nonet, M.L., Jorgensen, E.M., 2003 Oct. Defects in synaptic vesicle docking in unc-18 mutants. Nat. Neurosci. 6 (10), 1023–1030. Wheeler, D.B., Randall, A., Tsien, R.W., 1994 Apr 1. Roles of N-type and Q-type Ca2C channels in supporting hippocampal synaptic transmission. Science 264 (5155), 107–111. Wiser, O., Bennett, M.K., Atlas, D., 1996 Aug 15. Functional interaction of syntaxin and SNAP-25 with voltage-sensitive L- and N-type Ca2C channels. EMBO J. 15 (16), 4100–4110. Wu, M.N., Littleton, J.T., Bhat, M.A., Prokop, A., Bellen, H.J., 1998 Jan 2. ROP, the Drosophila sec1 homolog, interacts with syntaxin and regulates neurotransmitter release in a dosage-dependent manner. EMBO J. 17 (1), 127–139. Wu, L.G., Saggau, P., 1994 Sep. Pharmacological identification of two types of presynaptic voltage-dependent calcium channels at CA3– CA1 synapses of the hippocampus. J. Neurosci. 14 (9), 5613–5622. Yamaguchi, K., Takada, M., Fujimori, K., Tsuchimoto, Y., Kushima, Y., Sanada, M., Fujiwara, T., Akagawa, K., 1997 Nov 10. Enhancement of synaptic transmission by HPC-1 antibody in the cultured hippocampal neuron. Neuroreport 8 (16), 3641–3644. Yang, B., Steegmaier, M., Gonzalez Jr., L.C., Scheller, R.H., 2000 Jan 24. nSec1 binds a closed conformation of syntaxin1A. J. Cell Biol. 148 (2), 247–252.
Munc18-dependent regulation of synaptic vesicle exocytosis by syntaxin-1A in hippocampal neurons Simon J. Mitchell1, Timothy A. Ryan) Department of Biochemistry, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA Received 25 June 2004; received in revised form 10 September 2004; accepted 15 October 2004
Abstract The fusion of secretory vesicles with the plasma membrane requires the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes between the vesicle-SNARE vesicle-associated membrane protein present on the vesicular membrane and the target-SNAREs SNAP-25 and syntaxin-1A. Syntaxin-1A fluctuates between an open and closed form allowing it to selectively bind to different biological effectors in different conformations. In the open form, it can participate in SNARE complex formation, however, in the closed form it negatively regulates N- and P/Q-type voltage-dependent calcium channels, and is capable of inhibiting calcium influx. Thus paradoxically, syntaxin appears to have both positive and negative roles in controlling calcium-driven synaptic vesicle fusion at synaptic terminals. We show here that overexpression of syntaxin-1A inhibited exocytosis, in a manner that could be rescued by either elevating or reducing external calcium, or increasing action potential firing frequency. Elevating the level of Munc18 by coexpression with syntaxin-1A also abolished this inhibition, suggesting that Munc18 serves to limit the negative regulatory role of syntaxin by binding to, and thereby buffering, its closed form. Our results also indicate that syntaxin can control the frequency–response characteristics of the presynaptic fusion machinery. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Syntaxin; Munc18; Vesicle; Calcium; Exocytosis; SNARE protein
1. Introduction Docking and fusion of synaptic vesicles to the plasma membrane are mediated by SNAREs, which form a stable complex between vesicle-SNAREs present on vesicular membrane and target-SNAREs on the target plasma membrane (Sutton et al., 1998). Synaptic vesicle exocytosis occurs in proximity to calcium microdomains produced by clusters of voltage-gated calcium channels (Pumplin et al., 1981; Sugimori et al., 1994; DiGregorio et al., 1999). Binding of syntaxin-1A to a synaptic ) Corresponding author. Tel.: C1 212 746 6403; fax: C1 212 746 8875. E-mail address: [email protected] (T.A. Ryan). 1 Present address: Neurobiology Division, MRC Laboratory of Molecular Biology, Cambridge CB2 2QH, UK. 0028-3908/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2004.10.017
protein interaction (synprint) site within the N- and P/Q-type voltage-gated calcium channel a1 subunit (Sheng et al., 1994; Bezprozvanny et al., 2000) may therefore localize docked vesicles near calcium microdomains. Paradoxically, syntaxin-1A inhibits calcium influx by promotion of slow inactivation of calcium channels (Bezprozvanny et al., 1995; Wiser et al., 1996; Degtiar et al., 2000). There is evidence that calcium channel modulation is conferred by the transmembrane region and a section of the H3 domain of syntaxin, which are at distinct loci from the synprint site (Bezprozvanny et al., 2000; Trus et al., 2001). Calcium influx and evoked neurotransmission are enhanced by introduction of syntaxin-1 antibodies or application of botulinum toxin C1 (Yamaguchi et al., 1997; Sugimori et al., 1998; Blasi et al., 1993; Stanley, 2003). Furthermore, synchronous release is reduced, whereas
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asynchronous release is enhanced, by the introduction of peptides containing the synprint site (Mochida et al., 1996), an effect that could be due to an interruption of the binding of syntaxin, SNAP-25 and/or synaptotagmin to the synprint site on calcium channels (Sheng et al., 1997). Coordination of synaptic transmission involves many synaptic proteins, including Munc18, the mammalian Sec1/UNC-18/ROP homologue (Rizo and Sudhof, 2002). Munc18 has been proposed to serve several possible roles in SNARE function, including trafficking of syntaxin to the plasma membrane, vesicle priming and docking (Weimer and Jorgensen, 2003). Munc18 is required for neurotransmission since both evoked and spontaneous release are abolished in neurons from knockout mice (Verhage et al., 2000) and severely reduced in UNC-18 mutants in Caenorhabditis elegans (Weimer et al., 2003). Syntaxin continuously switches between an open conformation, which is a constituent of the core complex, and a closed form (Margittai et al., 2003). Munc18 binds only the closed form of syntaxin, as constitutively open forms of syntaxin fail to bind Munc18 (Dulubova et al., 1999). Since the Munc18binding domain on syntaxin-1A includes the synprint binding sites of syntaxin-1A (Sheng et al., 1994; Jarvis et al., 2002) and only the closed form of syntaxin modulates calcium channels (Jarvis et al., 2002), there is a possibility that Munc18 might promote exocytosis by decoupling closed syntaxin from voltage-gated channels. Although, ROP has been shown to prevent the inhibition of evoked EPSPs at the neuromuscular junction of Drosophila (Wu et al., 1998), it is unclear whether Munc18 performs an analogous role in mammalian neurons. We investigated whether transiently overexpressed syntaxin-1A regulates synaptic vesicle exocytosis in hippocampal neurons in culture, by monitoring activitydependent staining and destaining of synaptic vesicles using the optical probe FM 4-64. We find that syntaxin-1A slows the rate of synaptic vesicle exocytosis during trains of stimuli, in a manner that depends on external calcium concentration as well as stimulus frequency. Coexpression of Munc18-1 reversed this inhibition, without affecting the surface fraction of expressed syntaxin-1A.
2. Methods 2.1. Cell culture and transfection Hippocampal CA3–CA1 regions were dissected from postnatal 2- to 4-day-old rats, and neurons were dissociated and plated on coverslips coated with 10 mg/ml poly-L-ornithine, and cultured and maintained as described previously (Ryan, 1999). All experiments
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were performed on cultures of between 2 and 3 weeks of age. Syntaxin-1A fusion protein was generated by subcloning the rat syntaxin-1A cDNA into a vector encoding super-ecliptic GFP (pHluorin; Miesenbock et al., 1998). Munc18-1-FLAG fusion protein was generated by subcloning the rat Munc18-1 cDNA into a vector encoding a FLAG epitope (amino acid sequence of DYKDDDDK). Transfection of syntaxin-1A and Munc18-1-FLAG was performed using calcium phosphate precipitation as described (Threadgill et al., 1997). Overexpression of Munc18-1 did not impair the overexpression of syntaxin-1A, but rather it increased the level of overexpression 3.7-fold, in agreement with a previous study showing that the syntaxin level is suppressed in UNC-18 mutant flies (Weimer et al., 2003). Unless otherwise stated, all reagents were obtained from Sigma (St. Louis, MO). All animal experiments and use were approved by the Institutional Animal Care and Use Committee of the Weill Medical College of Cornell University. 2.2. Experimental conditions Coverslips of neuronal cell cultures were mounted in a superfusion chamber equipped with field stimulation electrodes on the stage of a custom-built laser-scanning confocal microscope as described (Ryan, 1999). Field stimulation was applied by passing 1 ms current pulses across the chamber to produce an electric field of 10 V/cm using platinum–iridium electrodes. Unless otherwise indicated, cells were superfused at room temperature in a saline solution consisting of 119 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 5 mM HEPES (pH 7.4), 30 mM glucose, 10 mM CNQX (an AMPA receptor antagonist), and 50 mM APV (an NMDA receptor antagonist). Synaptic vesicle pools were labeled by fieldstimulating cultures for 30 s at 20 Hz in the presence of 15 mM FM 4-64 in normal saline. An additional 60 s of dye exposure was allowed to ensure complete labeling of all recycling vesicles. The cultures were subsequently rinsed in dye-free solution for 10 min prior to dye destaining. The external solution contained 3.5 mM CaCl2 and 0.5 mM MgCl2 for experiments requiring an elevated calcium/magnesium concentration ratio and 0.5 mM CaCl2 and 3.5 mM MgCl2 for experiments requiring a lower calcium/magnesium concentration ratio. Ammonium chloride solution (pH 7.4) was prepared by substituting 50 mM NaCl in the above saline with NH4Cl. Acidic solution at pH 5.5 was prepared by replacing HEPES in the standard saline with 2-[N-morpholino]ethane sulphonic acid (pK Z 6.1). 2.3. Immunocytochemistry Neurons were processed for immunofluorescence for either syntaxin-1A or Munc18-1-FLAG after fixation in
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3. Results
Although a number of reports have suggested that dye destaining rates are not a faithful report of exocytosis due to possible dye retention during endocytosis (Pyle et al., 2000), we find little evidence for that in our system (Ferna´ndez-Alfonso and Ryan, 2004). The destaining time course was 38 G 10% (n Z 25; P ! 0.001) slower in syntaxin-1A transfected cells than non-transfected control neurons in the same culture dish (Fig. 1B and C). The level of FM loading (recycling vesicle pool size) was quantified by measuring the total amount of destaining for two successive rounds of stimulation at 10 Hz for 90 s and 20 Hz for 60 s, a protocol that maximally destains the recycling vesicle pool. The vesicle pool size in syntaxin overexpressing cells was 25 G 6% smaller than in controls (Fig. 1D; n Z 23; P ! 0.001). As syntaxin is known to inhibit voltage-dependent calcium channels involved in synaptic vesicle release, we investigated whether the inhibition of FM 4-64 destaining might be dependent on external calcium concentration. In non-transfected control cells the destaining time constant was only 8 G 5% (n Z 3) faster when the calcium/magnesium ion concentration ratio was elevated from unity to seven, suggesting that the destaining rate was near maximal under control conditions for steadystate release during sustained stimulation. With an elevated calcium/magnesium ratio, the destaining rate of syntaxin-1A-positive neurons was now similar to control cells (107 G 3% of control; n Z 5; P O 0.05; Fig. 2A and C). In non-transfected controls, when the calcium/magnesium ion concentrations were 7-fold lower than unity the destaining rate was 76 G 20% (n Z 3) slower than under control conditions. However, at a lower calcium/magnesium ratio, as at an elevated calcium/magnesium ratio, the inhibition in destaining rate in syntaxin-1A positive cells was also relieved (101 G 5% of control; n Z 3; P O 0.05; Fig. 2B and C). These data show that changing extracellular calcium/ magnesium ratio, and thus the level of calcium influx during an AP, modulates the inhibition of exocytosis by excess syntaxin-1A.
3.1. Overexpression of syntaxin-1A reduces synaptic vesicle exocytosis rate
3.2. Munc18-1 reverses syntaxin-mediated reduction of exocytosis
We investigated whether overexpression of syntaxin1A affected synaptic vesicle exocytosis by monitoring the release of the styryl dye FM 4-64, which labels synaptic vesicle membranes in an activity-dependent manner. Synaptic vesicles in cultured hippocampal neurons were labeled using 600 action potentials (APs) at 20 Hz in the presence of 15 mM FM 4-64 (Fig. 1A), which labels w90% of the recycling pool of synaptic vesicles in control cells (Ryan and Smith, 1995). The rate of exocytosis was estimated from a single exponential fit to the decay in FM 4-64 fluorescence during electrical stimulation of 900 action potentials at 10 Hz (Fig. 1B).
Munc18-1 has been shown to bind to the closed conformation of syntaxin-1A (Dulubova et al., 1999; Misura et al., 2000). To examine whether enhanced levels of Munc18-1 might reverse the impaired exocytosis in syntaxin-1A positive neurons, we coexpressed Munc18-1 with syntaxin-1A. The distribution of Munc18-1 as detected by retrospective immunofluorescence with an anti-FLAG antibody, was similar to that of syntaxin-1A (Fig. 3A), consistent with a previous report (Garcia et al., 1995). The rate of FM destaining in syntaxin-1A and Munc18-1-FLAG cotransfected terminals was similar to non-transfected terminals
4% paraformaldehyde (Electron Microscopy Science, Washington, PA) and 4% sucrose in PBS for 20 min. Cells were then permeabilized in the same fixative plus 0.25% Triton for 10 min, blocked in 10% bovine serum albumin (BSA) for 2 h at 37 C and incubated overnight with either affinity-purified monoclonal anti-syntaxin-1 antibody (Synaptic Systems, Go¨ttingen, Germany; 1:1000 dilution) or purified monoclonal anti-FLAG M2 antibody (Sigma, St. Louis, MO; 10 mg/ml). All antibodies were diluted in 1% BSA in PBS. Cells were then incubated with either an anti-mouse or an antirabbit Alexa-546-conjugated goat IgG secondary antibody (Molecular Probes, Eugene, OR; 1:2000 dilution), as appropriate, for 3 h and mounted for laser-scanning microscopic analysis. Using these analyses the level of expression of syntaxin-1 was w2-fold higher in cells expressing exogenous syntaxin-1A (Mitchell and Ryan, 2004). 2.4. Fluorescence microscopy and analysis Laser-scanning fluorescence images were acquired as described (Sankaranarayanan and Ryan, 2000). Quantitative measurements of fluorescence intensity at individual boutons were obtained by averaging a 4 ! 4 area of pixel intensities using custom written software. All subsequent analysis was performed in Igor Pro (Wavemetrics, OR). FM 4-64 destaining data were normalized to the total loss of fluorescence during two subsequent rounds of stimulation at 10 Hz for 90 s, then 20 Hz for 60 s after a 2-min rest. Time constants for FM 4-64 destaining were obtained by fitting the destaining curves to single exponential decays. The release rates in transfected cells were compared to those non-transfected control cells located in the same dish. Means are presented with their standard error. Data groups were compared using a two-tailed Student’s t test.
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Fig. 1. Overexpression of syntaxin-1A inhibits synaptic vesicle exocytosis. (A) Laser-scanning fluorescence image of an axon of a cultured hippocampal neuron expressing syntaxin-1A-pHluorin fusion protein, which was loaded with FM 4-64 for 30 s at 20 Hz. Exogenous syntaxin-1A expression (pHluorin tag fluorescence, green) is shown with synaptic vesicles labeled with FM 4-64 (red). Regions of colocalization are shown in yellow. FM 4-64 puncta in non-transfected and transfected cells were similar in size. (B) Semi-log plots of simultaneous destaining of FM 4-64 from synaptic vesicles of non-transfected control cells (filled circles) and syntaxin-1A overexpressing cells (open circles) in the same culture dish, during electrical stimulation at 10 Hz. Dashed and solid lines show single exponential fits of destaining rates for control and syntaxin-1A overexpressing cells, respectively. (C) Mean destaining time constant (tau) for control and syntaxin-1A overexpressing cells. (D) Mean total destaining (recycling pool size) for control and syntaxin-1A overexpressing cells (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
(104 G 18% of control; n Z 5; P O 0.05; Fig. 3B and C), consistent with the possibility that Munc18 reduces the ability of syntaxin-1A to inhibit calcium influx. However, FM uptake in cells overexpressing syntaxin and Munc18-1 cells was reduced by 29 G 4% compared to control terminals (n Z 5; P ! 0.01; Fig. 3D), which is similar to level of inhibition for terminals transfected with only syntaxin-1A (Fig. 3D). These data show that the balance of Munc18-1 and syntaxin-1A can regulate the ability of syntaxin to inhibit exocytosis rates. 3.3. Munc18-1 coexpression does not alter the surface fraction of syntaxin-1A In endothelial cells, overexpressed syntaxin-1A is retained intracellularly unless Munc18 is coexpressed (Rowe et al., 2001). A change in surface expression of syntaxin-1A caused by Munc18-1 might therefore underlie the ability of Munc18 to reverse the modulation of vesicle exocytosis rates of neurons by syntaxin. We analyzed whether coexpression of Munc18-1-FLAG altered the proportion of syntaxin-1A on the surface relative to internal compartments (surface fraction) at
functional synaptic boutons, previously identified by FM 4-64 loading and unloading (Mitchell and Ryan, 2004). To do this, we monitored fluorescence changes in syntaxin-1A during application of extracellular solution at pH 5.5, to quench surface fluorescence (Fig. 4A), or a solution that contained ammonium chloride to clamp internal compartments to pH 7.4 (Fig. 4B; Sankaranarayanan et al., 2000; Mitchell and Ryan, 2004). The change in fluorescence normalized to the initial fluorescence (DF/F ) during application of acidic extracellular solution was ÿ96 G 1% (n Z 6), indicating a large surface component of syntaxin-1A. The increase in fluorescence during application of ammonium chloride solution at pH 7.4 was 18 G 4% (n Z 6), showing a fraction of syntaxin-1A was present in an acidic intracellular compartment. Using these data and the known pK of pHluorin (pH 7.18), we calculated the surface fraction of syntaxin-1A as a function of the pH of the internal compartment as described in Mitchell and Ryan (2004). The functions for the acid quench and ammonium chloride data intersected at pH 5.5 (Supplementary Fig. 1), which is similar to the pH of the compartment that exogenous syntaxin-1A is localized to
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(13%; Mitchell and Ryan, 2004). Similar analyses revealed that the fraction of syntaxin-1A on nonsynaptic axonal plasma membrane was 98 G 2%, (n Z 6; 98% for cells transfected with syntaxin-1A only; Mitchell and Ryan, 2004). These data show that in cells cotransfected with Munc18-1 and syntaxin-1A, syntaxin-1A is mainly located on the plasma membrane, but that a small fraction is located in an acidic compartment at synaptic boutons. Stimulation of 600 APs at 20 Hz did not increase or decrease the pHluorin signal (Fig. 4C). The same stimulation protocol also failed to induce a change in fluorescence in the presence of bafilomycin-A1, which increases the signal by blocking synaptic vesicle reacidification, showing that syntaxin-1A was not present in recycling synaptic vesicles (data not shown). These findings are similar to those found in terminals transfected with syntaxin-1A alone (Mitchell and Ryan, 2004), indicating that overexpression of Munc18-1 did not alter the surface fraction of syntaxin-1A. 3.4. Frequency-dependent modulation of exocytosis rate by syntaxin-1A
Fig. 2. Magnitude of inhibition of release rate kinetics depends on external calcium/magnesium concentration. (A, B) Semi-log plots of simultaneous destaining of FM 4-64 from synaptic vesicles of nontransfected control cells (filled circles) and syntaxin-1A overexpressing cells (open circles) in the same culture dish, during electrical stimulation at 10 Hz, at (in mM): 3.5 Ca/0.5 Mg (High [Ca]/[Mg]; A); 0.5 Ca/3.5 Mg (Low [Ca]/[Mg]; B). (C) Mean inhibition of destaining rate relative to control cells at (in mM): 0.5 Ca/3.5 Mg (Low); 2 Ca/2 Mg (Mid); 3.5 Ca/0.5 Mg (High). Loading of FM 4-64 was performed at 2 mM Ca/ 2 mM Mg for all experiments.
when expressed without Munc18-1 (pH 5.3; Mitchell and Ryan, 2004), and the same as the synaptic vesicle lumen (pH 5.5; Miesenbock et al., 1998; Sankaranarayanan et al., 2000). This analysis showed that the fraction of syntaxin-1A in the internal compartment in these cotransfected terminals (15 G 3%; n Z 6) was similar to cells transfected with syntaxin-1A alone
We have previously shown that exocytosis rate was unaffected by the overexpression of syntaxin-1A when APs were stimulated at 20 Hz (Mitchell and Ryan, 2004). To investigate the frequency-dependence of the inhibition reported here, we stimulated APs between 5 Hz and 50 Hz. In non-transfected cells, the rate of destaining increased as the frequency was increased from 5 Hz to 20 Hz, with no further increase in rate observed at 50 Hz (Fig. 5). In transfected cells, the destaining rate was inhibited by 52 G 17% compared to non-transfected cells at a stimulation frequency of 5 Hz (n Z 5; P Z 0.02) and by 29 G 7% at 10 Hz in the same group of cells (n Z 5; P Z 0.01). However, at 20 Hz and 50 Hz no modulation of destaining rate was observed (n Z 6 and 4, respectively; P O 0.13). In this way, overexpression of syntaxin-1A modulated the frequency–response characteristics of release (Fig. 5).
4. Discussion Our results demonstrate that overexpression of syntaxin can inhibit synaptic vesicle exocytosis, in a manner that is consistent with its known inhibition of voltage-dependent calcium channels, and that this inhibition is eliminated when syntaxin is coexpressed with Munc18. Our findings suggest that the balance of Munc18-1 and syntaxin-1A expression regulates neurotransmission by modulating synaptic vesicle exocytosis. Furthermore, the inhibition of exocytosis rate by syntaxin-1A only occurs at stimulation frequencies of 10 Hz and lower, suggesting that the level of syntaxin-1A
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Fig. 3. Coexpression of syntaxin-1A and Munc18-1 prevents inhibition of exocytosis by syntaxin. (A) Laser-scanning fluorescence images of an axon of the same cultured hippocampal neuron coexpressing exogenous syntaxin-1A (live cell imaging of pHluorin tag fluorescence, top image) and Munc18-1 (fixed then stained with anti-FLAG primary and Alexa-546 secondary antibodies, bottom image). The expression of exogenous syntaxin-1A, as reported by pHluorin fluorescence, was not impaired in cells coexpressing Munc18-1 compared to cells overexpressing syntaxin-1A alone. (B) Semi-log plots of simultaneous destaining of FM 4-64 from synaptic vesicles of control cells (filled circles) and syntaxin-1A/Munc18-1 coexpressing cells (open circles), during electrical stimulation at 10 Hz. (C) Mean inhibition of destaining rate for syntaxin-1A overexpressing cells and syntaxin-1A/ Munc18-1 coexpressing cells. (D) Mean inhibition of recycling pool size for syntaxin-1A overexpressing cells and syntaxin-1A/Munc18-1 coexpressing cells (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
may help set the frequency–response characteristics of the synapse. 4.1. Calcium-dependent modulation of exocytosis Our study shows that the inhibition of exocytosis by syntaxin-1A is dependent on the ratio of extracellular calcium and magnesium concentration. Our data showing that conditions that lower calcium influx removed the inhibitory effects of syntaxin-1A overexpression are consistent with data showing that the binding of syntaxin and calcium channels is decreased at lower calcium concentration (Sheng et al., 1996). It is possible that reduced association of syntaxin and calcium channels at lower calcium levels may reduce the ability of syntaxin to modulate either the calcium influx or the coupling between calcium influx and exocytosis. Previous evidence has indicated that syntaxin-1A can modulate N- and P/Q-type calcium channels (Bezprozvanny et al., 1995; Wiser et al., 1996; Degtiar et al., 2000). Our results showing that syntaxin-1A inhibits exocytosis rates are consistent with this, since neurotransmission is mediated by N- and P/Q-type channels in cultured hippocampal neurons (Luebke
et al., 1993; Wheeler et al., 1994; Wu and Saggau, 1994; Scholz and Miller, 1995; Reid et al., 1997). The lack of effect at high external calcium concentrations was likely the effect of release rate saturation, such that the decrease in calcium influx caused by syntaxin was insufficient to affect exocytosis. Moreover, the fact that the inhibition can be overcome by elevation or reduction in calcium suggests that neither syntaxin-1A overexpression nor tagging of syntaxin with pHluorin produced a non-specific deficit in exocytosis. Overexpression of syntaxin in C. elegans did not modulate evoked excitatory currents at the neuromuscular junction at an external calcium/magnesium ratio of 5, at which the release rate appears to be approaching saturation in wild type preparations (Weimer et al., 2003). 4.2. Munc18-1 removes syntaxin-1A-mediated inhibition of exocytosis Munc18-1 binds and stabilizes the closed conformation of syntaxin-1A, which appears a necessary step for proper SNARE complex assembly (Dulubova et al., 1999; Yang et al., 2000). The ability of Munc18
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Munc18 effectively ‘‘buffers’’ the levels of closed syntaxin that might otherwise be free to interact with, and inhibit, voltage-dependent calcium channels. If this model is correct, our data indicate that the balance of syntaxin-1A and Munc18-1 levels is crucial for efficient synaptic vesicle release. These data compliment the finding in a C. elegans mutant that the absence of UNC18 inhibits evoked neuromuscular junction end plate potentials by affecting vesicle docking (Weimer et al., 2003). Interestingly, overexpression of ROP in Drosophila inhibited neurotransmission (Schulze et al., 1994; Wu et al., 1998). We were unable to produce viable cultured hippocampal neurons that overexpressed exogenous Munc18-1. We find no evidence that the ability of coexpressed Munc18 to abolish the inhibitory effects of syntaxin overexpression could be attributed to altered syntaxin trafficking, since the surface fraction of expressed syntaxin under these conditions and for cells overexpressing syntaxin alone was similar (Mitchell and Ryan, 2004). This is consistent with the finding that UNC-18 is not required for syntaxin trafficking in C. elegans (Weimer et al., 2003). 4.3. Syntaxin-1A preferentially inhibits release rate at lower stimulation frequencies Previous studies demonstrating that syntaxin-1A inhibits AP-dependent exocytosis used low-frequency stimulation (Wu et al., 1998). We have demonstrated in this study that the degree of inhibition of release rate by syntaxin-1A decreases from w50% at a stimulation frequency of 5 Hz to no inhibition at 20 Hz and higher. The increase in residual calcium that accumulates during high-frequency trains may underlie the reduced dependence of release rate on syntaxin-1A level. Alternatively, a release rate saturation effect may take place at higher frequencies. These findings indicate that syntaxin-1A can regulate the frequency–response properties of the synapse. Fig. 4. Subcellular localization of exogenous syntaxin-1A is not altered by coexpression with Munc18-1. (A) Quenching of surface syntaxin1A-pHluorin fluorescence by application of external solution at pH 5.5. (B) Increase in fluorescence of syntaxin-1A-pHluorin present in an acidic internal compartment during application of membrane permeant NH4Cl solution at pH 7.4. (C) Syntaxin-1A-pHluorin fluorescence during electrical stimulation at 20 Hz for 30 s.
coexpression to suppress inhibition of exocytosis by syntaxin-1A is consistent with previous studies in Drosophila, where a mutation of ROP, the Drosophila Munc18 homologue, or overexpression of syntaxin-1A both inhibited synaptic transmission (Wu et al., 1998). The simplest interpretation of our results is that in addition to its potential role in driving correct assembly,
4.4. Mechanisms for varying syntaxin-1-mediated inhibition of transmitter release Our results suggest that syntaxin expression levels, in particular with respect to that of Munc18, regulate important properties of synaptic transmission. Previous studies have shown that syntaxin levels might be dynamically modulated in an activity-dependent manner. High-frequency stimulation of the perforant path in the hippocampus induces a transient increase syntaxin-1B mRNA levels, which could be detected after 2 h (Hicks et al., 1997), and increases in syntaxin-1B mRNA and protein levels were detected 5 h after the induction of the long-term potentiation in the dentate gyrus (Hicks et al., 1997; Helme-Guizon et al., 1998). Calcium influx through
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Fig. 5. Magnitude of inhibition of release rate kinetics depends on frequency of stimulation. Destaining rates from control cells (filled circles) and cells overexpressing syntaxin-1A-pHluorin (open circles) located in the same culture dish were measured during AP stimulation at the indicated frequencies and normalized to the rate at 10 Hz stimulation in control cells.
P/Q-type calcium channels modulates syntaxin-1A mRNA level in HEK293 cells within 36 h, as well as regulating the endogenous syntaxin-1A mRNA level in cultured cerebellar granule cells (Sutton et al., 1999). Thus, given the results presented here, activity-dependent regulation of the level of syntaxin might provide a mechanism to tune the frequency- and/or calciumdependence of synaptic transmission at central synapses, which are important determinants for how information is processed in the nervous system.
Acknowledgments We thank W. Yan for technical assistance, K. Nicholson Tomishima for assistance with the syntaxin-1A-pHluorin and Munc18-1-FLAG constructs and members of the Ryan lab for discussions. This work was supported by grants from the NIH (NS24692 and GM61925) and the Hirschl Trust to T.A.R. S.J.M. is in receipt of an International Research Fellowship from the Wellcome Trust.
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