Department of Biomedical Sciences, Program in Molecular, Cellular, and Integrative Neuroscience, Colorado State University, Fort Collins, Colorado, 80523, USA.
J Neurosci. 2012 Jan 25;32(4):1253-60. doi: 10.1523/JNEUROSCI.4652-11.2012.
Synaptotagmin is the major calcium sensor for fast synaptic transmission that requires the synchronous fusion of synaptic vesicles. Synaptotagmin contains two calcium-binding domains: C2A and C2B. Mutation of a positively charged residue (R233Q in rat) showed that Ca2+-dependent interactions between the C2A domain and membranes play a role in the electrostatic switch that initiates fusion. Surprisingly, aspartate-to-asparagine mutations in C2A that inhibit Ca2+ binding support efficient synaptic transmission, suggesting that Ca2+ binding by C2A is not required for triggering synchronous fusion. Based on a structural analysis, we generated a novel mutation of a single Ca2+-binding residue in C2A (D229E in Drosophila) that inhibited Ca2+ binding but maintained the negative charge of the pocket. This C2A aspartate-to-glutamate mutation resulted in ∼80% decrease in synchronous transmitter release and a decrease in the apparent Ca2+ affinity of release. Previous aspartate-to-asparagine mutations in C2A partially mimicked Ca2+ binding by decreasing the negative charge of the pocket. We now show that the major function of Ca2+ binding to C2A is to neutralize the negative charge of the pocket, thereby unleashing the fusion-stimulating activity of synaptotagmin. Our results demonstrate that Ca2+ binding by C2A is a critical component of the electrostatic switch that triggers synchronous fusion. Thus, Ca2+ binding by C2B is necessary and sufficient to regulate the precise timing required for coupling vesicle fusion to Ca2+ influx, but Ca2+ binding by both C2 domains is required to flip the electrostatic switch that triggers efficient synchronous synaptic transmission.
突触融合蛋白是快速突触传递的主要钙离子传感器，需要突触小泡的同步融合。突触融合蛋白包含两个钙离子结合域：C2A 和 C2B。带正电荷残基（大鼠中的 R233Q）的突变表明，C2A 域与膜之间的钙离子依赖性相互作用在引发融合的静电开关中起作用。令人惊讶的是，C2A 中抑制钙离子结合的天冬氨酸到天冬酰胺突变支持有效的突触传递，这表明 C2A 结合钙离子对于触发同步融合不是必需的。基于结构分析，我们在 C2A 中生成了一个单一钙离子结合残基的新型突变（果蝇中的 D229E），该突变抑制了钙离子结合，但保持了口袋的负电荷。这种 C2A 天冬氨酸到谷氨酸突变导致同步递质释放减少约 80%，并降低了释放的表观钙离子亲和力。以前在 C2A 中的天冬氨酸到天冬酰胺突变部分模拟了钙离子结合，通过降低口袋的负电荷来实现。我们现在表明，C2A 结合钙离子的主要功能是中和口袋的负电荷，从而释放突触融合蛋白的融合刺激活性。我们的结果表明，C2A 结合钙离子是触发同步融合的静电开关的关键组成部分。因此，C2B 结合钙离子对于调节与钙离子内流偶联的融合所需的精确时间是必要的，但两个 C 结构域的钙离子结合对于翻转触发有效同步突触传递的静电开关是必需的。
