Paddock Brie E, Striegel Amelia R, Hui Enfu, Chapman Edwin R, Reist Noreen E
Department of Biomedical Sciences, Molecular, Cellular, and Integrative Neuroscience Program, Colorado State University, Fort Collins, Colorado 80523-1617, USA.
J Neurosci. 2008 Jul 23;28(30):7458-66. doi: 10.1523/JNEUROSCI.0197-08.2008.
Synaptotagmin I is the Ca(2+) sensor for fast, synchronous release of neurotransmitter; however, the molecular interactions that couple Ca(2+) binding to membrane fusion remain unclear. The structure of synaptotagmin is dominated by two C(2) domains that interact with negatively charged membranes after binding Ca(2+). In vitro work has implicated a conserved basic residue at the tip of loop 3 of the Ca(2+)-binding pocket in both C(2) domains in coordinating this electrostatic interaction with anionic membranes. Although results from cultured cells suggest that the basic residue of the C(2)A domain is functionally significant, such studies provide contradictory results regarding the importance of the C(2)B basic residue during vesicle fusion. To directly test the functional significance of each of these residues at an intact synapse in vivo, we neutralized either the C(2)A or the C(2)B basic residue and assessed synaptic transmission at the Drosophila neuromuscular junction. The conserved basic residues at the tip of the Ca(2+)-binding pocket of both the C(2)A and C(2)B domains mediate Ca(2+)-dependent interactions with anionic membranes and are required for efficient evoked transmitter release. Our results directly support the hypothesis that the interactions between synaptotagmin and the presynaptic membrane, which are mediated by the basic residues at the tip of both the C(2)A and C(2)B Ca(2+)-binding pockets, are critical for coupling Ca(2+) influx with vesicle fusion during synaptic transmission in vivo. Our model for synaptotagmin's direct role in coupling Ca(2+) binding to vesicle fusion incorporates this finding with results from multiple in vitro and in vivo studies.
突触结合蛋白I是神经递质快速同步释放的钙离子传感器；然而，将钙离子结合与膜融合偶联的分子相互作用仍不清楚。突触结合蛋白的结构主要由两个C2结构域组成，它们在结合钙离子后与带负电荷的膜相互作用。体外研究表明，两个C2结构域中钙离子结合口袋环3末端的一个保守碱性残基在协调与阴离子膜的这种静电相互作用中起作用。尽管来自培养细胞的结果表明C2A结构域的碱性残基具有功能重要性，但关于C2B碱性残基在囊泡融合过程中的重要性，此类研究提供了相互矛盾的结果。为了在体内完整突触处直接测试这些残基各自的功能重要性，我们中和了C2A或C2B碱性残基，并评估了果蝇神经肌肉接头处的突触传递。C2A和C2B结构域钙离子结合口袋末端的保守碱性残基介导了与阴离子膜的钙离子依赖性相互作用，并且是有效诱发递质释放所必需的。我们的结果直接支持了这样的假设，即C2A和C2B钙离子结合口袋末端的碱性残基介导的突触结合蛋白与突触前膜之间的相互作用，对于体内突触传递过程中钙离子内流与囊泡融合的偶联至关重要。我们关于突触结合蛋白在将钙离子结合与囊泡融合偶联中直接作用的模型，将这一发现与多项体外和体内研究的结果结合了起来。
