Department of Biology and Center for Molecular Investigation of Neurological Disorders, Penn State University, University Park, PA 16802, United States.
Mol Cell Neurosci. 2013 Sep;56:244-54. doi: 10.1016/j.mcn.2013.06.002. Epub 2013 Jun 11.
The COMPLEXIN (CPX) proteins play a critical role in synaptic vesicle fusion and neurotransmitter release. Previous studies demonstrated that CPX functions in both activation of evoked neurotransmitter release and inhibition/clamping of spontaneous synaptic vesicle fusion. Here we report a new cpx mutant in Drosophila melanogaster, cpx(1257), revealing spatially defined and separable pools of CPX which make distinct contributions to the activation and clamping functions. In cpx(1257), lack of only the last C-terminal amino acid of CPX is predicted to disrupt prenylation and membrane targeting of CPX. Immunocytochemical analysis established localization of wild-type CPX to active zone (AZ) regions containing neurotransmitter release sites as well as broader presynaptic membrane compartments including synaptic vesicles. Parallel biochemical studies confirmed CPX membrane association and demonstrated robust binding interactions of CPX with all three SNAREs. This is in contrast to the cpx(1257) mutant, in which AZ localization of CPX persists but general membrane localization and, surprisingly, the bulk of CPX-SNARE protein interactions are abolished. Furthermore, electrophysiological analysis of neuromuscular synapses revealed interesting differences between cpx(1257) and a cpx null mutant. The cpx null exhibited a marked decrease in the EPSC amplitude, slowed EPSC rise and decay times and an increased mEPSC frequency with respect to wild-type. In contrast, cpx(1257) exhibited a wild-type EPSC with an increased mEPSC frequency and thus a selective failure to clamp spontaneous release. These results indicate that spatially distinct and separable interactions of CPX with presynaptic membranes and SNARE proteins mediate separable activation and clamping functions of CPX in neurotransmitter release.
CPX 蛋白在突触囊泡融合和神经递质释放中起着关键作用。之前的研究表明，CPX 在诱发神经递质释放的激活和自发突触囊泡融合的抑制/钳制中都发挥作用。在这里，我们报告了果蝇中的一个新的 cpx 突变体 cpx(1257)，揭示了 CPX 的空间上定义和可分离的池，它们对激活和钳制功能有不同的贡献。在 cpx(1257)中，CPX 的最后一个 C 末端氨基酸的缺失预计会破坏 CPX 的异戊二烯化和膜靶向。免疫细胞化学分析确定野生型 CPX 定位于含有神经递质释放位点的活性区（AZ）以及包括突触囊泡在内的更广泛的突触前膜区。平行的生化研究证实了 CPX 与膜的结合，并证实 CPX 与所有三种 SNARE 都有很强的结合相互作用。这与 cpx(1257)突变体形成对比，在 cpx(1257)突变体中，CPX 的 AZ 定位仍然存在，但普遍的膜定位，以及令人惊讶的是，CPX-SNARE 蛋白相互作用的大部分都被消除了。此外，对肌神经突触的电生理分析揭示了 cpx(1257)与 cpx 缺失突变体之间的有趣差异。cpx 缺失突变体表现出 EPSC 幅度明显减小、EPSC 上升和下降时间变慢以及 mEPSC 频率增加，与野生型相比。相比之下，cpx(1257)表现出与野生型相似的 EPSC，但 mEPSC 频率增加，因此选择性地不能钳制自发释放。这些结果表明，CPX 与突触前膜和 SNARE 蛋白的空间上不同和可分离的相互作用介导了 CPX 在神经递质释放中的可分离激活和钳制功能。
