Abstract
Synaptosomal associated protein of 25 kDa (SNAP-25) is a member of the SNARE protein complex that has been implicated in synaptic vesicle docking and fusion. In this report, we have generated SNAP-25 mutants and assayed their functions in SNARE complex formation and glutamate release from cultured rat cerebellar granule cells. In vitro binding studies show that a deletion mutant lacking the C-terminal 181-206 amino acid sequence inhibits the formation of the SNARE core complex. Additional deletion of an N-terminal 1-31 amino acid sequence abolished this inhibitory activity. Adenovirus-mediated gene transfer is used to overexpress wild-type and mutant SNAP-25 in cerebellar granule cells. Neurons overexpressing the wild-type protein show slight reductions in glutamate release, ranging from 10 to 15% in both the developing and mature granule cells. A 30-35% inhibition is obtained with the C-terminal deletion mutant, and the inhibitory effect is abolished in the N- and C-terminal double deletion mutant. These results demonstrate that the SNARE core complex exists in a dynamic and reversible state, and the formation of the core complex is necessary for neurotransmitter release in neurons.
Highlights
The crystal structure of the SNARE core complex was solved [13]
To determine the effect of the Synaptosomal associated protein of 25 kDa (SNAP-25) and Neurotransmitter Release mutations on syntaxin binding in vivo, syntaxin 1A was coexpressed with wild-type or mutant SNAP-25, and the cells were lysed for co-immunoprecipitation
These results demonstrated that cytosolic SNAP-25 still retained the ability to interact with syntaxin, whereas the Nterminal deletion mutant no longer bound syntaxin effectively
Summary
Mutagenesis and Preparation of Recombinant Adenovirus—A mouse cDNA encoding synaptobrevin-2 was purchased from the I.M.A.G.E. Immunoprecipitation was performed using a mouse monoclonal antibody against syntaxin (clone HPC-1, Sigma) and protein G-plus agarose (Oncogene Science). Glutamate Release Assays—Granule cell cultures were washed 7– 8 times with 20 mM Hepes, pH 7.5, 100 mM NaCl, 5 mM KCl, 2 mM MgCl2, 1.3 mM CaCl2, and 5 mM glucose for a total of 60 min at 37 °C. Aliquots of 20 l of glutathione-agarose beads absorbed with 0.05 nmol of GST1⁄7synaptobrevin were incubated with 0.05 nmol of syntaxin, 0.05 nmol of SNAP-25, and 0.05 nmol of mutant SNAP-25, in 300 l of binding buffer containing 10 mM Hepes, pH 7.4, 150 mM NaCl, 1 mM EGTA, and 1% Triton X-100 overnight at 4 °C. Wild-type, and mutant SNAP-25 were expressed in COS-7 cells by rAd infection, and the solubilized total cellular membrane proteins were used. Samples were separated by 12% SDS-polyacrylamide gel electrophoresis and electrotransferred onto nitrocellulose membranes for immunoblot analysis
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