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 Ca ²⁺-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.