PDF(Pubmed)
Abstract：
The aquaporin-4 (AQP4) water channel is abundantly expressed in the glial cells of the central nervous system and facilitates brain swelling following diverse insults, such as traumatic injury or stroke. Lack of specific and therapeutic AQP4 inhibitors highlights the need to explore alternative routes to control the water permeability of glial cell membranes. The cell surface abundance of AQP4 in mammalian cells fluctuates rapidly in response to changes in oxygen levels and tonicity, suggesting a role for vesicular trafficking in its translocation to and from the cell surface. However, the molecular mechanisms of AQP4 trafficking are not fully elucidated. In this work, early and recycling endosomes were investigated as likely candidates of rapid AQP4 translocation together with changes in cytoskeletal dynamics. In transiently transfected HEK293 cells a significant amount of AQP-eGFP colocalised with mCherry-Rab5-positive early endosomes and mCherry-Rab11-positive recycling endosomes. When exposed to hypotonic conditions, AQP4-eGFP rapidly translocated from intracellular vesicles to the cell surface. Co-expression of dominant negative forms of the mCherry-Rab5 and -Rab11 with AQP4-eGFP prevented hypotonicity-induced AQP4-eGFP trafficking and led to concentration at the cell surface or intracellular vesicles respectively. Use of endocytosis inhibiting drugs indicated that AQP4 internalisation was dynamin-dependent. Cytoskeleton dynamics-modifying drugs also affected AQP4 translocation to and from the cell surface. AQP4 trafficking mechanisms were validated in primary human astrocytes, which express high levels of endogenous AQP4. The results highlight the role of early and recycling endosomes and cytoskeletal dynamics in AQP4 translocation in response to hypotonic and hypoxic stress and suggest continuous cycling of AQP4 between intracellular vesicles and the cell surface under physiological conditions.
摘要：
水通道蛋白4（AQP4）水通道在中枢神经系统的神经胶质细胞中大量表达，并促进各种损伤后的脑肿胀，如外伤或中风。缺乏特异性和治疗性AQP4抑制剂凸显了探索控制神经胶质细胞膜水渗透性的替代途径的必要性。哺乳动物细胞中AQP4的细胞表面丰度随着氧水平和张力的变化而迅速波动，提示囊泡运输在其进出细胞表面的易位中的作用。然而,AQP4转运的分子机制尚未完全阐明.在这项工作中,研究了早期和再循环内体作为AQP4快速易位和细胞骨架动力学变化的可能候选者.在瞬时转染的HEK293细胞中，大量的AQP-eGFP与mCherry-Rab5阳性早期内体和mCherry-Rab11阳性再循环内体共定位。当暴露于低渗条件时，AQP4-eGFP从细胞内囊泡快速转运到细胞表面。mCherry-Rab5和-Rab11的显性阴性形式与AQP4-eGFP的共表达可防止低渗性诱导的AQP4-eGFP运输，并分别导致细胞表面或细胞内囊泡的浓度。内吞抑制药物的使用表明AQP4内化是动态蛋白依赖性的。细胞骨架动力学修饰药物也会影响AQP4在细胞表面的易位。AQP4转运机制在原代人星形胶质细胞中得到验证，表达高水平的内源性AQP4。结果强调了早期和再循环内体和细胞骨架动力学在AQP4易位中对低渗和低氧应激的反应中的作用，并表明在生理条件下AQP4在细胞内囊泡和细胞表面之间的连续循环。
