College of Health and Life Sciences, Aston University, Birmingham, UK.
School of Biosciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford, UK.
J Neurochem. 2024 Feb;168(2):100-114. doi: 10.1111/jnc.16029. Epub 2023 Dec 16.
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.
水通道蛋白-4（AQP4）广泛表达于中枢神经系统的神经胶质细胞中，并促进多种损伤后的脑水肿，如创伤或中风。缺乏特异性和治疗性的 AQP4 抑制剂突出表明需要探索控制神经胶质细胞膜水通透性的替代途径。哺乳动物细胞中 AQP4 的细胞表面丰度会根据氧水平和渗透压的变化而迅速波动，这表明囊泡运输在其从细胞表面的转位中起作用。然而，AQP4 运输的分子机制尚未完全阐明。在这项工作中，早期和再循环内体被研究为 AQP4 快速转位的可能候选物，同时还研究了细胞骨架动力学的变化。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。当暴露于低渗条件下时，AQP4-eGFP 从细胞内囊泡快速转位到细胞表面。与 AQP4-eGFP 共表达的 mCherry-Rab5 和 -Rab11 的显性负形式可阻止低渗诱导的 AQP4-eGFP 运输，并分别导致在细胞表面或细胞内囊泡处的聚集。内吞抑制剂的使用表明 AQP4 的内化是依赖于胞质动力蛋白的。细胞骨架动力学修饰药物也影响 AQP4 从细胞表面的转位。AQP4 运输机制在表达高水平内源性 AQP4 的原代人星形胶质细胞中得到了验证。这些结果突出了早期和再循环内体以及细胞骨架动力学在 AQP4 对低渗和缺氧应激的转位中的作用，并表明在生理条件下 AQP4 在细胞内囊泡和细胞表面之间进行持续循环。
