Reinvestigation of the role of snapin in neurotransmitter release.
Snapin, a 15-kDa protein, has been identified recently as a binding partner of SNAP-25. Moreover, snapin is regulated by phosphorylation and enhances synaptotagmin binding to SNAREs. Furthermore, snapin and C-terminal snapin fragments have been effective in changing the release properties of neurons and chromaffin cells. Here we have reinvestigated the role of snapin using both biochemical and electrophysiological approaches. Snapin is ubiquitously expressed at low levels with no detectable enrichment in the brain or in synaptic vesicles. Using non-equilibrium and equilibrium assays including pulldown experiments, co-immunoprecipitations, and CD and fluorescence anisotropy spectroscopy, we were unable to detect any specific interaction between snapin and SNAP-25. Similarly, overexpression of a C-terminal snapin fragment in hippocampal neurons failed to influence any of the analyzed parameters of neurotransmitter release. Initial biochemical characterization of recombinant snapin revealed that the protein is a stable dimer with a predominantly alpha-helical secondary structure. We conclude that the postulated role of snapin as a SNARE regulator in neurotransmitter release needs reconsideration, leaving the true function of this evolutionarily conserved protein to be discovered.
Vites
Olga
O
Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Gottingen, Germany.
Rhee
Jeong-Seop
JS
Schwarz
Martin
M
Rosenmund
Christian
C
Jahn
Reinhard
R
D000818:Animals; D016880:Anisotropy; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003902:Detergents / Q000494:pharmacology; D019281:Dimerization; D004594:Electrophysiology; D019143:Evolution, Molecular; D005982:Glutathione Transferase / Q000378:metabolism; D006624:Hippocampus / Q000378:metabolism; D008027:Light; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D018377:Neurotransmitter Agents / Q000378:metabolism*; D017830:Octoxynol / Q000494:pharmacology; D016716:PC12 Cells; D011233:Precipitin Tests; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D050600:SNARE Proteins; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013347:Subcellular Fractions / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25; D013997:Time Factors; D014018:Tissue Distribution; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D003902:Detergents; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents; D011994:Recombinant Proteins; D050600:SNARE Proteins; C494210:Snap25 protein, rat; C498452:Snapin protein, rat; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; D017830:Octoxynol; D005982:Glutathione Transferase