Synaptic vesicle docking and fusion in ribbon synapses of the retina

Purpose: Ribbon synapses of photoreceptors and bipolar cells release neurotransmitter tonically as opposed to phasic release in conventional synapses. In conventional synapses syntaxin 1, SNAP-25, and synaptobrevin are proteins essential for synaptic vesicle exocytosis. We compared the distributions of these proteins in ribbon and conventional synapses of the retina. We used a biochemical approach to identify a ribbon synapse form of syntaxin. Methods: Syntaxin 1, SNAP-25, and synaptobrevin were localized in rat retina using immunofluorescence and electron microscopy. To identify a ribbon synapse form of syntaxin, detergent extracts of retina and brain were depleted of syntaxin 1 by successive immunoprecipitations with a syntaxin 1 antibody. The immuno-depleted extracts were then immunoprecipitated with a SNAP-25 antibody. The immunoprecipitated material was analyzed by SDS-PAGE followed by either Coomassie blue staining or transfer to nitrocellulose and immunoblotting with SNAP-25 and syntaxin antibodies. Identification of the immunoprecipitated proteins was achieved by amino acid microsequencing. Results: We identified synaptobrevin and SNAP-25 in both ribbon and conventional synapses of the retina by immunofluorescence microscopy. In contrast, syntaxin 1 was present only in conventional synapses. Immunoelectron microscopy confirmed the absence of syntaxin 1 from ribbon synapses. Immunoelectron microscopy revealed that SNAP-25 was present not only on the plasma membrane of all retinal neurons but also on synaptic vesicles and the golgi apparatus of ganglion cells and photoreceptors. Immunoprecipitation of syntaxin 1-depleted retina extract with a SNAP-25 antibody recovered a complex of three proteins of 35, 25 and 18 kD. Sequencing the polypeptides revealed that the 35 kD protein is syntaxin 3, the 25 kD protein is SNAP-25, and that the 18kD band contained a mixture of complexin and a novel protein with sequence homology to synaptobrevin. Conclusions: Ribbon synapses of the retina contain a synaptic vesicle fusion complex consisting of syntaxin 3, SNAP-25 and a novel synaptobrevin.