Are neuronal SNARE proteins Ca2+ sensors?
The neuronal SNARE complex formed by synaptobrevin, syntaxin and SNAP-25 plays a central role in Ca2+-triggered neurotransmitter release. The SNARE complex contains several potential Ca2+-binding sites on the surface, suggesting that the SNAREs may be involved directly in Ca2+-binding during release. Indeed, overexpression of SNAP-25 bearing mutations in two putative Ca2+ ligands (E170A/Q177A) causes a decrease in the Ca2+-cooperativity of exocytosis in chromaffin cells. To test whether the SNARE complex might function in Ca2+-sensing, we analyzed its Ca2+-binding properties using transverse relaxation optimized spectroscopy (TROSY)-based NMR methods. Several Ca2+-binding sites are found on the surface of the SNARE complex, but most of them are not specific for Ca2+ and all have very low affinity. Moreover, we find that the E170A/Q177A SNAP-25 mutation does not alter interactions between the SNAREs and the Ca2+ sensor synaptotagmin 1, but severely impairs SNARE complex assembly. These results suggest that the SNAREs do not act directly as Ca2+ receptors but SNARE complex assembly is coupled tightly to Ca2+-sensing during neurotransmitter release.
Chen
Xiaocheng
X
Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA.
Tang
Jiong
J
Sudhof
Thomas C
TC
Rizo
Josep
J
D001665:Binding Sites; D015374:Biosensing Techniques; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050600:SNARE Proteins; D050863:Synaptotagmin I; D050857:Synaptotagmins; D033921:Vesicular Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; D050863:Synaptotagmin I; D033921:Vesicular Transport Proteins; D050857:Synaptotagmins; D002118:Calcium