Syntaxin 3b is a t-SNARE specific for ribbon synapses of the retina

Abstract

Previous studies have demonstrated that ribbon synapses in the retina do not contain the t-SNARE (target-soluble N-ethylmaleimide-sensitive factor attachment protein receptor) syntaxin 1A that is found in conventional synapses of the nervous system. In contrast, ribbon synapses of the retina contain the related isoform syntaxin 3. In addition to its localization in ribbon synapses, syntaxin 3 is also found in nonneuronal cells, where it has been implicated in the trafficking of transport vesicles to the apical plasma membrane of polarized cells. The syntaxin 3 gene codes for four different splice forms, syntaxins 3A, 3B, 3C, and 3D. We demonstrate here by using analysis of EST databases, RT-PCR, in situ hybridization, and Northern blot analysis that cells in the mouse retina express only syntaxin 3B. In contrast, nonneuronal tissues, such as kidney, express only syntaxin 3A. The two major syntaxin isoforms (3A and 3B) have an identical N-terminal domain but differ in the C-terminal half of the SNARE domain and the C-terminal transmembrane domain. These two domains are thought to be directly involved in synaptic vesicle fusion. The interaction of syntaxin 1A and syntaxin 3B with other synaptic proteins was examined. We found that both proteins bind Munc18/N-sec1 with similar affinity. In contrast, syntaxin 3B had a much lower binding affinity for the t-SNARE SNAP25 compared with syntaxin 1A. By using an in vitro fusion assay, we could demonstrate that vesicles containing syntaxin 3B and SNAP25 could fuse with vesicles containing synaptobrevin2/VAMP2, demonstrating that syntaxin 3B can function as a t-SNARE.

Fig. 1. Different syntaxin 3 isoforms are generated by differential splicing of the syntaxin3 gene

A. The exon/intron structure of the gene is depicted with exons labeled by numbers. Differentially spliced exons are depicted in different colors that correspond to the differential spliced mRNAs depicted below: exon 3AB, yellow; exon 3C, green, exon 9A, 10A, 11A, blue; exon 9B, 10B, 11B, red. B. Structure of the mRNAs of the different isoforms of syntaxin 3. The position of the different exons in the mRNA is depicted. The same colors as in A. are used to label the different exons. The position of the stop codons at the end of the translated regions are marked by an asterisk. The domain structure of the syntaxin3A protein is shown underneath the corresponding mRNA. The different domains are of the protein are marked: HA, HB and HC domains (HA, HB, HC), SNARE domain (SNARE), transmembrane domain (TM).

Fig. 2. Syntaxin 3B is the syntaxin 3 isoform expressed in the retina

A. RT-PCR analysis of the expression of different syntaxin isoforms in different tissues. Arrows mark the position of the specific bands generated by the indicated primers (P2, P4, P8, P10A). Asterisks mark nonspecific PCR products. B. The location of the PCR primers in relation to the mRNAs of the different syntaxin 3 isoforms is indicated. The different exons are marked and the predicted coding region is shaded.

Fig. 3. Analysis of the tissue distribution of syntaxin3B mRNA

Top panel: Expression of syntaxin 3B mRNA in different tissues was analyzed by Northern blot using a probe specific for syntaxin 3B. Bottom panel: Total RNA separated on denaturing/formaldehyde gel and stained with ethidium bromide. The presence of distinct ribosomal 28S and 18S bands demonstrates that the RNA is not degraded.

Fig. 4. Syntaxin 3B is expressed in ribbon synapse- forming neurons in the retina

Antisense riboprobes (left panel) specific for the syntaxin 3A (top panel) and syntaxin 3B (bottom panel) were used to analyze the mRNA distribution in the mouse retina by in situ hybridization. The different layers of the retina are labeled on the left [outer nuclear layer (ONL), outer plexiform layer (OPL), Inner nuclear layer (INL), inner plexiform layer (IPL) and ganglion cell layer (GCL)]. Sense probes were used as negative controls (right panel). Antisense probes for syntaxin 3B label the cell bodies of neurons in the ONL and the INL. Scale bar = 100 μm

Fig. 5. Sequence alignement of the different syntaxin isoforms

The protein sequences of mouse syntaxin 3A, syntaxin 3B and syntaxin 1A have been aligned for maximal homology. Sequences are identified on the left and residues numbered on the right. Residues that are conserved in all three isoforms are labeled with green background. Residues conserved between two of the syntaxin isoforms are labeled with yellow background. Conservative exchanged residues are labeled with blue background. The position of the differential spliced exons is marked above the sequence. The positions of the hydrophobic interacting layers are numbered in relation to the glutamine (Q) of the central 0 layer.

Fig. 6. Munc-18/n-sec1 binds to both syntaxin 1A and syntaxin 3B

Lane 1: Retina extract alone. Lanes 2-3: For a negative control, GST bound to glutathione-sepharose beads was incubated with extract that had either 150 μM calcium or 2 mM EGTA added to it. Lanes 4-7: Retina extract with either 150 μM calcium or 2 mM EGTA was incubated with the indicated GST-fusion proteins (GST-syntaxin 1A and GST-syntaxin 3B). Membrane was probed with an antibody to GST to ensure that the amount of fusion protein in each pulldown was comparable (results below Munc-18/n-sec1 blot).

Fig. 7. SNAP-25 binds weakly to syntaxin 3B

Lane 1: Retina extract alone. Lanes 2-3: For a negative control, GST bound to glutathione sepharose beads was incubated with extract that had either 150 μM calcium or 2 mM EGTA added to it. Lanes 4-7: Retina extract with either 150 μM calcium or 2 mM EGTA was incubated with the indicated GST-fusion proteins (GST-syntaxin 1A and GST-syntaxin 3B). Membrane was probed with antibody to GST to ensure that the amount of fusion protein in each pulldown was comparable (results below SNAP-25 blot).

Fig. 8. Syntaxin 3B/SNAP-25 forms a functional SNARE complex

A. Liposomes containing either syntaxin 1A/SNAP-25 (filled circles) or syntaxin 3B/SNAP-25 (open circles) t-SNARE complexes were mixed with v-SNARE liposomes containing synaptobrevin/VAMP2. Background levels of fusion were determined by including soluble synaptobrevin/VAMP2 (VAMP2 w/o TMD, filled box) to reactions containing syntaxin 3B/SNAP-25. Lipid mixing was monitored as an increase in NBD fluorescence for 120 minutes. Fusion is represented as percent maximum fluorescence obtained following detergent solubilization of the liposomes. B. Histogram showing initial fusion rate from 4 to 14 minutes of the t-SNARE liposomes containing either syntaxin1A/SNAP-25 or syntaxin 3B/SNAP-25. C. Five microliters of liposomes containing each complex were run on a 12% SDS-PAGE gel and stained with Coomassie blue.