Goodyear Richard J, Gale Jonathan E, Ranatunga Kishani M, Kros Corné J, Richardson Guy P
School of Life Sciences, University of Sussex, Brighton BN1 9QG, United Kingdom.
J Neurosci. 2008 Oct 1;28(40):9939-52. doi: 10.1523/JNEUROSCI.1124-08.2008.
The aminophospholipid phosphatidylserine (PS) is normally restricted to the inner leaflet of the plasma membrane. During certain cellular processes, including apoptosis, PS translocates to the outer leaflet and can be labeled with externally applied annexin V, a calcium-dependent PS-binding protein. In mouse cochlear cultures, annexin V labeling reveals that the aminoglycoside antibiotic neomycin induces rapid PS externalization, specifically on the apical surface of hair cells. PS externalization is observed within approximately 75 s of neomycin perfusion, first on the hair bundle and then on membrane blebs forming around the apical surface. Whole-cell capacitance also increases significantly within minutes of neomycin application, indicating that blebbing is accompanied by membrane addition to the hair cell surface. PS externalization and membrane blebbing can, nonetheless, occur independently. Pretreating hair cells with calcium chelators, a procedure that blocks mechanotransduction, or overexpressing a phosphatidylinositol 4,5-biphosphate (PIP2)-binding pleckstrin homology domain, can reduce neomycin-induced PS externalization, suggesting that neomycin enters hair cells via transduction channels, clusters PIP2, and thereby activates lipid scrambling. The effects of short-term neomycin treatment are reversible. After neomycin washout, PS is no longer detected on the apical surface, apical membrane blebs disappear, and surface-bound annexin V is internalized, distributing throughout the supranuclear cytoplasm of the hair cell. Hair cells can therefore repair, and recover from, neomycin-induced surface damage. Hair cells lacking myosin VI, a minus-end directed actin-based motor implicated in endocytosis, can also recover from brief neomycin treatment. Internalized annexin V, however, remains below the apical surface, thereby pinpointing a critical role for myosin VI in the transport of endocytosed material away from the periphery of the hair cell.
氨基磷脂磷脂酰丝氨酸（PS）通常局限于质膜的内小叶。在某些细胞过程中，包括细胞凋亡，PS会转移至外小叶，并且可以用外部施加的膜联蛋白V进行标记，膜联蛋白V是一种钙依赖性的PS结合蛋白。在小鼠耳蜗培养物中，膜联蛋白V标记显示氨基糖苷类抗生素新霉素会诱导PS快速外化，特别是在毛细胞的顶端表面。在新霉素灌注约75秒内可观察到PS外化，首先出现在毛束上，然后出现在围绕顶端表面形成的膜泡上。在应用新霉素几分钟内，全细胞电容也显著增加，这表明膜泡形成伴随着毛细胞表面的膜增加。然而，PS外化和膜泡形成可以独立发生。用钙螯合剂预处理毛细胞（该过程会阻断机械转导）或过表达磷脂酰肌醇4,5 -二磷酸（PIP2）结合的普列克底物蛋白同源结构域，可以减少新霉素诱导的PS外化，这表明新霉素通过转导通道进入毛细胞，聚集PIP2，从而激活脂质翻转。短期新霉素处理的效果是可逆的。新霉素洗脱后，顶端表面不再检测到PS，顶端膜泡消失，表面结合的膜联蛋白V被内化，分布在毛细胞的核上细胞质中。因此，毛细胞可以修复并从新霉素诱导的表面损伤中恢复。缺乏肌球蛋白VI（一种参与内吞作用的基于肌动蛋白的负端定向马达）的毛细胞也可以从短暂的新霉素处理中恢复。然而，内化的膜联蛋白V仍留在顶端表面下方，从而确定了肌球蛋白VI在将内吞物质从毛细胞周边转运走的过程中的关键作用。
