Ueki Misuzu, Maki Takahisa, Iwamoto Masayuki
Department of Molecular Neuroscience, Faculty of Medical Sciences, University of Fukui, Fukui 910-1193, Japan; Life Science Innovation Center, University of Fukui, Fukui 910-8507, Japan.
Department of Molecular Neuroscience, Faculty of Medical Sciences, University of Fukui, Fukui 910-1193, Japan; Life Science Innovation Center, University of Fukui, Fukui 910-8507, Japan.
Biochim Biophys Acta Biomembr. 2025 Jun;1867(5-6):184425. doi: 10.1016/j.bbamem.2025.184425. Epub 2025 May 21.
Cell membranes regulate water flow to maintain homeostasis, cell volume, and osmotic balance. Aquaporins (AQPs) enable selective water transport, making precise permeability measurements essential for understanding their function. The current methods have limitations, including high resource demands and poor control over membrane properties like bilayer tension. In this study, the droplet interface bilayer (DIB) system was used to measure aquaporin water channel activity. Unlike conventional water permeability assays, this method uniquely quantifies lipid bilayer tension by determining droplet internal pressure. This pressure-determined DIB (PDIB) method was used to investigate the water permeability of a lipid bilayer reconstituted with Escherichia coli aquaporin Z (AqpZ). Water permeability increased in an AqpZ concentration-dependent manner at bilayer tensions of 2.2-3.0 mN/m and was inhibited by mercury (IC, 340 μM). Fluorescence microscopy was performed to visualize and quantify AqpZ molecules, thereby allowing us to derive an approximate estimate of the unitary water permeability. Although this study established the PDIB method and demonstrated its applicability to AqpZ, this technique may also facilitate future investigations on the effects of lipid bilayer tension on aquaporin function and the fundamental mechanisms of water transport across biological membranes.
细胞膜调节水的流动以维持体内平衡、细胞体积和渗透平衡。水通道蛋白（AQPs）实现选择性水运输，因此精确的渗透率测量对于理解其功能至关重要。当前的方法存在局限性，包括资源需求高以及对双层张力等膜特性的控制不佳。在本研究中，液滴界面双层（DIB）系统被用于测量水通道蛋白水通道活性。与传统的水渗透率测定方法不同，该方法通过确定液滴内部压力独特地量化脂质双层张力。这种基于压力测定的DIB（PDIB）方法被用于研究用大肠杆菌水通道蛋白Z（AqpZ）重构的脂质双层的水渗透率。在2.2 - 3.0 mN/m的双层张力下，水渗透率以AqpZ浓度依赖的方式增加，并受到汞的抑制（IC，340 μM）。进行荧光显微镜检查以可视化和量化AqpZ分子，从而使我们能够得出单一水渗透率的近似估计值。尽管本研究建立了PDIB方法并证明了其对AqpZ的适用性，但该技术也可能有助于未来对脂质双层张力对水通道蛋白功能的影响以及水跨生物膜运输的基本机制的研究。
