Thomas D M, Elferink L A
Wayne State University, Department of Biological Sciences, Detroit, Michigan 48202, USA.
J Neurosci. 1998 May 15;18(10):3511-20. doi: 10.1523/JNEUROSCI.18-10-03511.1998.
Synaptotagmin 1 is proposed to function as a low affinity calcium sensor for calcium-triggered exocytosis from neural and neuroendocrine cells. Because of the calcium-binding properties of the C2A domain of synaptotagmin 1, calcium-dependent interactions through this domain may modulate neurotransmitter release. We addressed this question by using alanine-scanning mutagenesis to generate a series of mutations within the C2A domain of synaptotagmin 1. The effects of these mutations on synaptotagmin 1 C2A function were analyzed for (1) calcium-dependent phospholipid binding, (2) calcium-dependent binding to syntaxin 1A, a plasma membrane protein critical for vesicle docking or fusion, and (3) calcium-regulated secretion after microinjection into neuroendocrine pheochromocytoma (PC12) cells. Our analyses reveal that a polylysine motif at residues 189-192 confers an inhibitory effect on secretion by recombinant synaptotagmin C2A fragments. The synaptotagmin 1 C2A polylysine motif functions independently of calcium-mediated interactions with phospholipids and syntaxin 1A. Furthermore, alpha-latrotoxin reverses the inhibitory effect of injected recombinant C2A fragments, suggesting that they perturb the cellular calcium-sensing machinery by interfering with synaptotagmin 1 activity in vivo. Our results indicate that novel calcium-independent interactions mediated through the C2A polylysine motif of synaptotagmin 1 function to modulate neurotransmitter release.
突触结合蛋白1被认为是一种低亲和力钙传感器，用于神经和神经内分泌细胞中钙触发的胞吐作用。由于突触结合蛋白1的C2A结构域具有钙结合特性，通过该结构域的钙依赖性相互作用可能会调节神经递质的释放。我们通过丙氨酸扫描诱变在突触结合蛋白1的C2A结构域内产生一系列突变来解决这个问题。分析了这些突变对突触结合蛋白1 C2A功能的影响，包括：（1）钙依赖性磷脂结合；（2）与 syntaxin 1A（一种对囊泡对接或融合至关重要的质膜蛋白）的钙依赖性结合；（3）显微注射到神经内分泌嗜铬细胞瘤（PC12）细胞后钙调节的分泌。我们的分析表明，189 - 192位残基处的多聚赖氨酸基序对重组突触结合蛋白C2A片段的分泌具有抑制作用。突触结合蛋白1 C2A多聚赖氨酸基序的功能独立于与磷脂和syntaxin 1A的钙介导相互作用。此外，α- 银环蛇毒素可逆转注射的重组C2A片段的抑制作用，这表明它们通过在体内干扰突触结合蛋白1的活性来扰乱细胞钙传感机制。我们的结果表明，通过突触结合蛋白1的C2A多聚赖氨酸基序介导的新型非钙依赖性相互作用可调节神经递质的释放。
