Program in Molecular, Cellular, and Integrative Neuroscience, Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523, USA.
J Neurosci. 2011 Feb 9;31(6):2248-57. doi: 10.1523/JNEUROSCI.3153-09.2011.
The vesicle protein synaptotagmin I is the Ca(2+) sensor that triggers fast, synchronous release of neurotransmitter. Specifically, Ca(2+) binding by the C(2)B domain of synaptotagmin is required at intact synapses, yet the mechanism whereby Ca(2+) binding results in vesicle fusion remains controversial. Ca(2+)-dependent interactions between synaptotagmin and SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment receptor) complexes and/or anionic membranes are possible effector interactions. However, no effector-interaction mutations to date impact synaptic transmission as severely as mutation of the C(2)B Ca(2+)-binding motif, suggesting that these interactions are facilitatory rather than essential. Here we use Drosophila to show the functional role of a highly conserved, hydrophobic residue located at the tip of each of the two Ca(2+)-binding pockets of synaptotagmin. Mutation of this residue in the C(2)A domain (F286) resulted in a ∼50% decrease in evoked transmitter release at an intact synapse, again indicative of a facilitatory role. Mutation of this hydrophobic residue in the C(2)B domain (I420), on the other hand, blocked all locomotion, was embryonic lethal even in syt I heterozygotes, and resulted in less evoked transmitter release than that in syt(null) mutants, which is more severe than the phenotype of C(2)B Ca(2+)-binding mutants. Thus, mutation of a single, C(2)B hydrophobic residue required for Ca(2+)-dependent penetration of anionic membranes results in the most severe disruption of synaptotagmin function in vivo to date. Our results provide direct support for the hypothesis that plasma membrane penetration, specifically by the C(2)B domain of synaptotagmin, is the critical effector interaction for coupling Ca(2+) binding with vesicle fusion.
囊泡蛋白突触融合蛋白相关蛋白 I 是触发神经递质快速、同步释放的 Ca(2+) 传感器。具体而言，在完整的突触中，需要突触融合蛋白的 C(2)B 结构域与 Ca(2+) 结合，但 Ca(2+) 结合如何导致囊泡融合的机制仍存在争议。Ca(2+) 依赖的突触融合蛋白与 SNARE（可溶性 N-乙基马来酰亚胺敏感融合蛋白附着受体）复合物和/或阴离子膜之间的相互作用可能是效应器相互作用。然而，迄今为止，没有效应器相互作用突变会像 C(2)B Ca(2+) 结合模体的突变那样严重影响突触传递，这表明这些相互作用是促进性的，而不是必需的。在这里，我们使用果蝇来显示位于突触融合蛋白两个 Ca(2+) 结合口袋尖端的一个高度保守的疏水性残基的功能作用。该残基在 C(2)A 结构域（F286）中的突变导致完整突触中诱发的递质释放减少约 50%，再次表明其具有促进作用。另一方面，该疏水性残基在 C(2)B 结构域（I420）中的突变阻断了所有运动，即使在 syt I 杂合子中也是胚胎致死的，并导致比 syt(null) 突变体中更少的诱发递质释放，比 C(2)B Ca(2+) 结合突变体的表型更严重。因此，单个 C(2)B 疏水性残基的突变，对于 Ca(2+) 依赖的阴离子膜穿透是必需的，这导致了突触融合蛋白功能在体内迄今为止最严重的破坏。我们的结果为这样一种假设提供了直接支持，即质膜穿透，特别是通过突触融合蛋白的 C(2)B 结构域，是将 Ca(2+) 结合与囊泡融合偶联的关键效应器相互作用。
