SNAP-25 functional domains in SNARE core complex assembly and glutamate release of cerebellar granule cells.
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.
Yang
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Y
Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190, USA.
Xia
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Z
Liu
Y
Y
D000818:Animals; D002478:Cells, Cultured; D002531:Cerebellum / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D051381:Rats; D050600:SNARE Proteins; D013570:Synaptic Membranes / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050600:SNARE Proteins; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; D018698:Glutamic Acid