Institut Pasteur, Université Paris Cité, Inserm U06, Institut de l'Audition, Paris, France; Bordeaux Neurocampus, Université de Bordeaux, Bordeaux 33076, France.
Institut Pasteur, Université Paris Cité, Inserm U06, Institut de l'Audition, Paris, France.
Prog Neurobiol. 2024 Sep;240:102658. doi: 10.1016/j.pneurobio.2024.102658. Epub 2024 Aug 3.
Our understanding of how otoferlin, the major calcium sensor in inner hair cells (IHCs) synaptic transmission, contributes to the overall dynamics of synaptic vesicle (SV) trafficking remains limited. To address this question, we generated a knock-in mouse model expressing an otoferlin-GFP protein, where GFP was fused to its C-terminal transmembrane domain. Similar to the wild type protein, the GFP-tagged otoferlin showed normal expression and was associated with IHC SV. Surprisingly, while the heterozygote Otof mice exhibited a normal hearing function, homozygote Otof mice were profoundly deaf attributed to severe reduction in SV exocytosis. Fluorescence recovery after photobleaching revealed a markedly increased mobile fraction of the otof-GFP-associated SV in Otof IHCs. Correspondingly, 3D-electron tomographic of the ribbon synapses indicated a reduced density of SV attached to the ribbon active zone. Collectively, these results indicate that otoferlin requires a free intravesicular C-terminal end for normal SV docking and fusion.
我们对于内耳毛细胞（IHC）突触传递中主要的钙离子感受器—— otoferlin 如何影响突触小泡（SV）运输的整体动力学的理解仍然有限。为了解决这个问题，我们构建了一个表达 otoferlin-GFP 蛋白的 knock-in 小鼠模型，其中 GFP 融合到其 C 端跨膜结构域。与野生型蛋白相似，GFP 标记的 otoferlin 表现出正常的表达，并与 IHC SV 相关。令人惊讶的是，虽然杂合子 Otof 小鼠表现出正常的听力功能，但纯合子 Otof 小鼠却严重失聪，这归因于 SV 胞吐作用的严重减少。光漂白后荧光恢复显示 otof-GFP 相关 SV 在 Otof IHC 中的可动分数显著增加。相应地， ribbonsynapses 的 3D 电子断层扫描表明，与 ribbon active zone 相连的 SV 密度降低。总的来说，这些结果表明 otoferlin 需要游离的腔内 C 端来实现正常的 SV docking 和融合。
