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Aquaporin water channels (AQPs) are membrane proteins that maintain cellular water homeostasis. The interactions between human AQPs and other proteins play crucial roles in AQP regulation by both gating and trafficking. Here, we describe a protocol for characterizing the interaction between a human AQP and a soluble interaction partner using microscale thermophoresis (MST). MST has the advantage of low sample consumption and high detergent compatibility enabling AQP protein-protein interaction investigation with a high level of control of components and environment. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020) and Roche et al. (2017).

Aquaporins (AQPs) are membrane-bound water channels that play crucial roles in maintaining the water homeostasis of the human body. Here, we present a protocol for high-yield recombinant expression of human AQPs in the methylotropic yeast Pichia pastoris and subsequent AQP purification. The protocol typically yields 1–5 mg AQP per g of yeast cell at >95% purity and is compatible with any membrane protein cloned into Pichia pastoris, although expression levels may vary. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020) and Frick et al. (2014).

Aquaporins (AQPs) are water channels embedded in the cell membrane that are critical in maintaining water homeostasis. We describe a protocol for determining the water permeation capacity of AQPs reconstituted into proteoliposomes. Using a stopped-flow setup, AQP embedded in proteoliposomes are exposed to an osmogenic gradient that triggers water flux. The consequent effects on proteoliposome size can be tracked using the fluorescence of an internalized fluorophore. This enables controlled characterization of water flux by AQPs. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020). [Abstract copyright: © 2022 The Authors.]