However,

this goal was difficult to achieve because most lipid-anchored synaptobrevin-2 mutants we tested were mistargeted. For example, geranyl-geranylated versions of synaptobrevin-2 carrying the C-terminal sequence of Rab3A were ineffective even though Rab3A itself is a synaptic vesicle protein (Johnston et al., 1991). Only when we fused the cytoplasmic synaptobrevin-2 sequence to the C-terminal palmitoylated sequence of cysteine-string protein-α (CSPα) did we observe good targeting of lipid-anchored synaptobrevin-2 to synapses (Figure 4). In these experiments, we compared two synaptobrevin-CSPα Selleckchem trans-isomer fusion proteins that differed by two residues (Figure 4A; referred to as Syb2ΔTMR#1 and Syb2ΔTMR#2), and employed neurons from synaptobrevin-2 KO mice to express these proteins in the complete absence of endogenous synaptobrevin-2 (Schoch et al., 2001). Quantification of the levels and targeting of

lipid-anchored synaptobrevin-2 revealed that the concentration of both synaptobrevin-CSPα fusion proteins represented ∼35%–45% of wild-type synaptobrevin-2 rescue protein (expressed as MK0683 in vivo an mVenus fusion protein), and that they were targeted to synapses almost as effectively as wild-type synaptobrevin-2 (Figures 4B–4E). In these experiments, the longer version of lipid-anchored synaptobrevin-2 (Syb2ΔTMR#2) containing two extra residues was expressed at slightly lower levels and was targeted to synapses with a lower efficiency than the shorter version (Syb2ΔTMR#1). In the next set of experiments, we tested the function of lipid-anchored synaptobrevin-2. We found that the shorter lipid-anchored synaptobrevin-2 (Syb2ΔTMR#1) was as efficient as wild-type synaptobrevin-2 in rescuing spontaneous excitatory or inhibitory mini release in synaptobrevin-2 KO neurons, whereas the longer lipid-anchored synaptobrevin-2 (Syb2ΔTMR#2) was mafosfamide less efficient (Figure 5). This rescue was observed for both the frequency and the amplitude of spontaneous events; the latter is decreased in synaptobrevin-2 KO neurons probably because of

the role of synaptobrevin in AMPA-receptor exocytosis (Jurado et al., 2013). Strikingly, synaptobrevin-deficient neurons exhibited a significant increase in the rise times of mEPSCs and of mIPSCs, possibly because the remaining sporadic fusion events observed in these neurons are mediated by a noncognate SNARE protein (Figure 5; Schoch et al., 2001). This phenotype again was fully rescued by lipid-anchored synaptobrevin-2, providing further evidence that lipid-anchored synaptobrevin-2 is functional. Measurements of evoked release at different extracellular Ca2+-concentrations demonstrated that lipid-anchored synaptobrevin-2 also rescued this fusion reaction, but was approximately half as efficient as wild-type synaptobrevin-2 (Figures 6A and S5). Moreover, both lipid-anchored synaptobrevin-2 versions rescued the desynchronization of release in synaptobrevin-2 KO neurons (Figure 6B).