Evaluation of aquaporin Z water permeability in bilayers using droplet interface systems with internal-pressure–defined membrane tension

Abstract

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 (IC₅₀, 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.