Mozhayeva Marina G, Matos Maria F, Liu Xinran, Kavalali Ege T
Center for Basic Neuroscience, The University of Texas Southwestern Medical Center, Dallas, Texas 75390-9111, USA.
J Neurosci. 2004 Feb 18;24(7):1680-8. doi: 10.1523/JNEUROSCI.3801-03.2004.
Studies on the mechanisms that underlie the function of small central presynaptic terminals have been hampered by the inaccessibility of these synapses to soluble reagents. Here, we permeabilized hippocampal synapses in culture, manipulated their interior, and monitored the resulting changes in vesicle mobilization with the styryl dye FM2-10. Using this method, we found that 1 microm Ca2+ after incubation with GTP or GTP-gamma-S could mobilize approximately 90% of the total recycling pool, whereas 1 microm Ca2+ application after dialysis of permeabilized synapses with GDP-beta-S mobilized approximately 30% of the recycling vesicles, presumably corresponding to the readily releasable pool. In electron micrographs of permeabilized hippocampal synapses stimulated with 1 microm Ca2+, we could detect significant vesicle depletion after preincubation with GTP-gamma-S, whereas preincubation with GDP-beta-S left the total vesicle pool relatively intact. Taken together, in this system replenishment of the readily releasable pool by the reserve vesicles was strictly GTP dependent. In contrast, vesicle replenishment and release did not require ATP or N-ethylmaleimide-sensitive factor (NSF); however, this process involved formation of new soluble NSF-attachment protein receptor (SNARE) complexes as judged by its sensitivity to tetanus toxin. These results suggest that in hippocampal synapses, vesicle mobilization and replenishment of the readily releasable pool require GTP and Ca2+ but do not necessitate ATP-dependent priming and SNARE recycling.
对小型中枢突触前终末功能的机制研究一直受到这些突触难以接触到可溶性试剂的阻碍。在这里，我们使培养的海马突触通透化，对其内部进行操作，并用苯乙烯基染料FM2-10监测由此产生的囊泡动员变化。使用这种方法，我们发现与GTP或GTP-γ-S孵育后，1微摩尔Ca2+可动员约90%的总循环池，而在用GDP-β-S透析通透化突触后施加1微摩尔Ca2+可动员约30%的循环囊泡，推测对应于易释放池。在用1微摩尔Ca2+刺激的通透化海马突触的电子显微镜照片中，我们可以检测到与GTP-γ-S预孵育后有明显的囊泡耗竭，而与GDP-β-S预孵育使总囊泡池相对完整。综上所述，在这个系统中，储备囊泡对易释放池的补充严格依赖于GTP。相比之下，囊泡补充和释放不需要ATP或N-乙基马来酰亚胺敏感因子（NSF）；然而，从其对破伤风毒素的敏感性判断，这个过程涉及新的可溶性NSF附着蛋白受体（SNARE）复合物的形成。这些结果表明，在海马突触中，囊泡动员和易释放池的补充需要GTP和Ca2+，但不需要ATP依赖的启动和SNARE循环。
