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Miao Tian, C. Shan Xu, Rachel Montpetit, Richard Kramer; The Role of Rab3a in Exocytosis at Ribbon Synapses. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4130.
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© ARVO (1962-2015); The Authors (2016-present)
Rab3a is a small neuronal GTP-binding protein specifically localized to synaptic vesicles. Studies in the Rab3a -/- mice showed that Rab3a regulates a late step in vesicle fusion at conventional synapses. However, the role of Rab3a at ribbon synapses remains unknown. Here we investigate whether Rab3a mediates the binding of vesicles to ribbons and/or regulates vesicle release.
Recombinant wild-type (WT) Rab3a and Rab3aQ81L (a GTP-hydrolysis deficient mutant) were expressed and purified from E.coli and chemically conjugated to Alexa Fluor 488. In both constructs, the membrane anchoring motif was deleted to keep the proteins cytosolic. Salamander retinal slices were prepared and the fluorescently-tagged proteins (F-Rab3a & F-Rab3aQ81L) were introduced into rod photoreceptors through the patch pipette during whole-cell recording and simultaneously imaged by 2-photon microscopy. Paired recordings from synaptically-coupled rods and OFF bipolar cells were performed to evaluate synaptic transmission.
The fluorescent Rab3a proteins showed a single band in the gel with a molecular weight of 25 kDa. The hydrolysis deficient mutant Rab3a (Rab3aQ81L) displayed much lower GTPase activity (0.003/min) than WT Rab3a (0.023/min). When rods were loaded with F-Rab3a or F-Rab3aQ81L, bright spots began to appear at the terminal within several minutes. The co-localization of either F-Rab3a or F-Rab3aQ81L with F-RBP (a well-known ribbon binding peptide) indicates that both bind to ribbons. Rab3a binding was abolished when GTP was replaced by GDP. Fluorescence recovery after photobleaching (FRAP) was different between WT (46±4%) and Rab3aQ81L (18±5%), consistent with the different GTP hydrolysis rates. When rods were loaded with 15 uM F-Rab3aQ81L and given voltage pulses every 30s, the amplitude of EPSCs recorded from OFF bipolar cells was significantly suppressed (40±3%) compared to the EPSC recorded immediately after rod break-in. Paired pulse depression was not affected. These results indicate that exogenous Rab3a may compete with vesicles to bind to ribbons.
Rab3a binds to ribbons in a GTP-dependent manner and competes with synaptic vesicles. These findings suggest that Rab3a is part of the protein machinery involved in vesicle replenishment at ribbon synapses.
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