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. 2001 Dec 1;21(23):9142-50.
doi: 10.1523/JNEUROSCI.21-23-09142.2001.

Targeted mutations in the syntaxin H3 domain specifically disrupt SNARE complex function in synaptic transmission

T Fergestad  1 , M N Wu, K L Schulze, T E Lloyd, H J Bellen, K Broadie
Affiliations

Targeted mutations in the syntaxin H3 domain specifically disrupt SNARE complex function in synaptic transmission

T Fergestad et al. J Neurosci. .

Abstract

The cytoplasmic H3 helical domain of syntaxin is implicated in numerous protein-protein interactions required for the assembly and stability of the SNARE complex mediating vesicular fusion at the synapse. Two specific hydrophobic residues (Ala-240, Val-244) in H3 layers 4 and 5 of mammalian syntaxin1A have been suggested to be involved in SNARE complex stability and required for the inhibitory effects of syntaxin on N-type calcium channels. We have generated the equivalent double point mutations in Drosophila syntaxin1A (A243V, V247A; syx(4) mutant) to examine their significance in synaptic transmission in vivo. The syx(4) mutant animals are embryonic lethal and display severely impaired neuronal secretion, although non-neuronal secretion appears normal. Synaptic transmission is nearly abolished, with residual transmission delayed, highly variable, and nonsynchronous, strongly reminiscent of transmission in null synaptotagmin I mutants. However, the syx(4) mutants show no alterations in synaptic protein levels in vivo or syntaxin partner binding interactions in vitro. Rather, syx(4) mutant animals have severely impaired hypertonic saline response in vivo, an assay indicating loss of fusion-competent synaptic vesicles, and in vitro SNARE complexes containing Syx(4) protein have significantly compromised stability. These data suggest that the same residues required for syntaxin-mediated calcium channel inhibition are required for the generation of fusion-competent vesicles in a neuronal-specific mechanism acting at synapses.

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Figures

Fig. 1.
Fig. 1.
Syntaxin double point mutations lie in neighboring hydrophobic layers. a, Alignment of amino acids 229–267 of the H3 domains of Drosophila, C. elegans, rat, squid, and yeast syntaxin-1A homologs. The central ionic layer is indicated by the zero, and thenumbers indicate adjacent hydrophobic layers. These layer assignments are based on the crystal structure of the core complex (Sutton et al., 1998). The Syx4 protein is altered for the two boxed amino acids. b, A schematic showing the position of thesyx4 mutations in relation to the entire H3 domain of syntaxin. The solid bars indicate the syx4 mutations, and thehatched bars indicate the Ca2+effector domain (Wu et al., 1999).
Fig. 2.
Fig. 2.
syx4mutant animals exhibit normal levels of synaptic proteins. The levels of syntaxin, ROP, synaptotagmin I, and CSP are unchanged in syx4 mutants, compared with controls (syxwt). Westerns were performed on extract obtained from four embryos of the appropriate genotype, i.e., syxwt-1,syxwt-2,syx4–1, andsyx4–2. Bands shown for a given protein were taken from the same exposure of a single gel. The smaller synaptotagmin I band represents a degradation product (Littleton et al., 1993b).
Fig. 3.
Fig. 3.
Gross phenotypes ofsyx4 mutants suggest defects in neuronal but not non-neuronal secretion. a, Lethal phase and movement in the syx null (syx229) background. Spontaneous muscle contractions of embryos 22–24 hr after egg laying were observed for 5 min and quantified. Spontaneous peristaltic contractions of syx4 embryos are significantly reduced, compared with syxwt embryos. Evoked contractile responses, determined after a brisk tactile stimulation, were present in syx4although absent in the null allele. b, Cuticle secretion is not impaired in syx4 mutants. Cuticles from control, syx229, andsyx4 (insyx229 background) embryos were imaged using dark-field microscopy. Anterior is to the left. Cuticular structures, including denticle belts and mouth hooks, are absent in the null mutant but present in the control andsyx4 mutant.
Fig. 4.
Fig. 4.
syx4 mutants display a profound reduction in evoked neurotransmission. a, Representative excitatory junctional current (EJC) traces are shown forsyxwt;syx229 andsyx4;syx229 embryos. Recordings were performed at the embryonic muscle 6 NMJ (22–24 hr after egg laying). In addition to a severe reduction in the EJC amplitude insyx4, neurotransmitter release is clearly asynchronous. Four traces are superimposed;arrow indicates nerve stimulation artifact.b, Mean EJC amplitudes forsyxwt-1 (n = 5),syxwt-2 (n = 9),syxwt-3 (n = 5),syx4–1 (n = 3),syx4–2 (n = 2), and syx4–3 (n = 3) embryos. c, Mean latency (time to peak) data are pooled for syxwt(n = 21) and syx4(n = 8) embryos. d, Evoked neurotransmission is variable in syx4mutants, compared with syxwt. Coefficient of variation (EJC amplitude SD/EJC amplitude) is plotted for syxwt-1,syxwt-2,syxwt-3,syx4–1,syx4–2, andsyx4–3 embryos. e, Percentage of failures of evoked response after nerve stimulation in 1.8 mm Ca2+ forsyxwt andsyx4 embryos.syxwt transmission almost never fails, whereas syx4 fails 50% of all stimuli. Error bars signify SEM. **p < 0.01.
Fig. 5.
Fig. 5.
The frequency of spontaneous mEJCs is reduced insyx4 mutants. a, Representative mEJC traces are shown forsyxwt;syx229 andsyx4;syx229 animals. Recordings were performed in 0.5 mm Ca2+ + TTX as described in Materials and Methods. b, Mean mEJC frequency in syxwt(n = 11) and syx4(n = 13) animals. Data from individual transgenic lines were not statistically different and thus were pooled.syx4 mutants show a 50% reduction in mEJC frequency relative to controls. c, Mean mEJC amplitude in syxwt andsyx4 animals.syx4 mutants display a slight, but significant, increase in quantal amplitude. Error bars represent SEM. *p < 0.05; **p < 0.01.
Fig. 6.
Fig. 6.
Mutant Syx4 protein interacts normally with syntaxin binding partners. a, GST pull-down assays were performed by incubating GST, GST-Syxwt, or GST-Syx4 with recombinant target proteins or Drosophila head extract, and bands were detected by immunoblotting as described in Materials and Methods. Bands shown for a given target protein were taken from a single exposure of a single gel. b, Thesyx4 mutation does not significantly alter ternary complex formation. Ternary complex formation was assessed by incubating immobilized GST-Syxwt or GST-Syx4 with SNAP-25 and increasing amounts of n-synaptobrevin. Bound n-synaptobrevin was determined by immunoblotting and ECL, followed by densitometry with known standards.c, Dose–response binding for GST-Syxwt and GST-Syx4 to synaptotagmin I. Increasing amounts of synaptotagmin I were incubated with either GST-Syxwt or GST-Syx4, and bound synaptotagmin I was determined by immunoblotting and ECL. d, Syx4and Syxwt show similar dose–response binding to CSP.
Fig. 7.
Fig. 7.
Ternary complexes containing Syx4 mutant protein interact normally with synaptotagmin I. Binary and ternary complexes of n-synaptobrevin, syntaxin, Syx4, and SNAP-25 were compared for their ability to bind GST-synaptotagmin in the presence and absence of Ca2+. a, Complexes were detected by Western blotting with anti-synaptobrevin antibody. Lanes 1–6 are control proteins and binary combinations that do not bind GST-synaptotagmin. Lanes 7–14 show that core complexes formed with mutant Syx4 protein bind GST-synaptotagmin as readily as wild type (lanes 7–10are boiled and lanes 11–14 are unboiled).b, The graph shows the percentage binding to GST-Syt as quantified using 125I-labeled secondary antibody. Percentage binding was normalized, with the highest pixel value for each individual experiment being assigned 100%, from four independent experiments: syntaxin with EGTA, 59%; syntaxin with Ca2+, 99%; Syx4 with EGTA, 58%; Syx4 with Ca2+, 98%. Each bar represents the average of four independent experiments ± SEM. *p < 0.05.
Fig. 8.
Fig. 8.
Core complex stability in vitro is impaired by the syx4 mutation. The heat lability of SDS-resistant core complexes containing Syx4 is increased, compared with core complexes containing Syxwt. Complexes were formed by incubating His-tagged SNAP-25 and n-synaptobrevin overnight with GST-Syxwt or GST-Syx4 immobilized on glutathione-Sepharose beads. a, Syx4-containing complexes are resistant to 2% SDS at 25°C, similar to control complexes (Syxwt). b, Complexes were challenged in sample buffer for 5 min at the temperature shown. The lower molecular weight complexes (asterisk) correspond to the trimeric SNARE complex, whereas the higher molecular weight bands likely represent a dimeric form. Note the increased instability of complexes containing Syx4 relative to Syxwt protein.
Fig. 9.
Fig. 9.
Core complex function is impaired insyx4 mutant synapses.a, Representative current traces are shown forsyxwt;syx229,syx4;syx229,syx229, andsytAD4 animals in response to 3 sec application of hyperosmotic saline (1175 mOsm; diagonally striped bar). The syxwtsynapse responds with robust, high-frequency secretory events, whereas the syx null (syx229) displays no detectable response. Note the obvious response reduction with increased latencies in both syx4and sytAD4. b, Total charge induced by transmitter release was measured from the area of the current trace responses (nanoamperes times millisecond). Average responses are shown for syxwt(n = 6), syx4(n = 7), syx229(n = 5), andsytAD4 (n = 6) animals. Data from individual transgenic lines were not statistically different and thus were pooled. Error bars represent SEM. **p < 0.01; ***p < 0.001.
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