Department of Ophthalmology & Visual Sciences, Truhlsen Eye Institute, University of Nebraska Medical Center, Omaha, NE.
Department of Pharmacology & Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE.
J Gen Physiol. 2018 Apr 2;150(4):591-611. doi: 10.1085/jgp.201711919. Epub 2018 Mar 19.
Endocytosis is an essential process at sites of synaptic release. Not only are synaptic vesicles recycled by endocytosis, but the removal of proteins and lipids by endocytosis is needed to restore release site function at active zones after vesicle fusion. Synaptic exocytosis from vertebrate photoreceptors involves synaptic ribbons that serve to cluster vesicles near the presynaptic membrane. In this study, we hypothesize that this clustering increases the likelihood that exocytosis at one ribbon release site may disrupt release at an adjacent site and therefore that endocytosis may be particularly important for restoring release site competence at photoreceptor ribbon synapses. To test this, we combined optical and electrophysiological techniques in salamander rods. Pharmacological inhibition of dynamin-dependent endocytosis rapidly inhibits release from synaptic ribbons and slows recovery of ribbon-mediated release from paired pulse synaptic depression. Inhibiting endocytosis impairs the ability of second-order horizontal cells to follow rod light responses at frequencies as low as 2 Hz. Inhibition of endocytosis also increases lateral membrane mobility of individual Ca channels, showing that it changes release site structure. Visualization of single synaptic vesicles by total internal reflection fluorescence microscopy reveals that inhibition of endocytosis reduces the likelihood of fusion among vesicles docked near ribbons and increases the likelihood that they will retreat from the membrane without fusion. Vesicle advance toward the membrane is also reduced, but the number of membrane-associated vesicles is not. Endocytosis therefore appears to be more important for restoring later steps in vesicle fusion than for restoring docking. Unlike conventional synapses in which endocytic restoration of release sites is evident only at high frequencies, endocytosis is needed to maintain release from rod ribbon synapses even at modest frequencies.
内吞作用是突触释放部位的一个基本过程。突触囊泡不仅通过内吞作用回收，而且在囊泡融合后，需要通过内吞作用去除蛋白质和脂质，以恢复活性区的释放部位功能。脊椎动物光感受器的突触胞吐作用涉及到突触带，它有助于将囊泡聚集在突触前膜附近。在这项研究中，我们假设这种聚类增加了一个带释放位点的胞吐作用可能破坏相邻位点释放的可能性，因此内吞作用对于恢复光感受器带突触的释放部位能力可能特别重要。为了验证这一点，我们在蝾螈棒状细胞中结合了光学和电生理学技术。依赖动力蛋白的内吞作用的药理学抑制迅速抑制来自突触带的释放，并减缓来自成对脉冲突触抑制的带介导释放的恢复。抑制内吞作用会损害第二级水平细胞跟随棒状光反应的能力，频率低至 2 Hz。内吞作用的抑制也会增加单个 Ca 通道的侧膜流动性，表明它改变了释放部位的结构。通过全内反射荧光显微镜对单个突触囊泡的可视化显示，内吞作用的抑制降低了停靠在带附近的囊泡融合的可能性，并增加了它们在不融合的情况下从膜上退缩的可能性。囊泡向膜的推进也减少了，但膜相关囊泡的数量没有增加。因此，内吞作用对于恢复囊泡融合的后期步骤似乎比恢复停靠更为重要。与传统突触不同，传统突触中只有在高频率时才会出现内吞作用恢复释放部位的现象，而在棒状带突触中，即使在适度的频率下，内吞作用也需要维持释放。
