Assessing water permeability of aquaporins in a proteoliposome-based stopped-flow setup

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.

High-yield overproduction and purification of human aquaporins from Pichia pastoris

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)

Targeting Aquaporin-4 Subcellular Localization to Treat Central Nervous System Edema

Swelling of the brain or spinal cord (CNS edema) affects millions of people every year. All potential pharmacological interventions have failed in clinical trials, meaning that symptom management is the only treatment option. The water channel protein aquaporin-4 (AQP4) is expressed in astrocytes and mediates water flux across the blood-brain and blood-spinal cord barriers. Here we show that AQP4 cell-surface abundance increases in response to hypoxia-induced cell swelling in a calmodulin-dependent manner. Calmodulin directly binds the AQP4 carboxyl terminus, causing a specific conformational change and driving AQP4 cell-surface localization. Inhibition of calmodulin in a rat spinal cord injury model with the licensed drug trifluoperazine inhibited AQP4 localization to the blood-spinal cord barrier, ablated CNS edema, and led to accelerated functional recovery compared with untreated animals. We propose that targeting the mechanism of calmodulin-mediated cell-surface localization of AQP4 is a viable strategy for development of CNS edema therapies

The rapid “teabag” method for high-end purification of membrane proteins

Overproduction and purification of membrane proteins are generally challenging and time-consuming procedures due to low expression levels, misfolding, and low stability once extracted from the membrane. Reducing processing steps and shortening the timespan for purification represent attractive approaches to overcome some of these challenges. We have therefore compared a fast “teabag” purification method with conventional purification for five different membrane proteins (MraY, AQP10, ClC-1, PAR2 and KCC2). Notably, this new approach reduces the purification time significantly, and the quality of the purified membrane proteins is equal to or exceeds conventional methods as assessed by size exclusion chromatography, SDS-PAGE and downstream applications such as ITC, crystallization and cryo-EM. Furthermore, the method is scalable, applicable to a range of affinity resins and allows for parallelization. Consequently, the technique has the potential to substantially simplify purification efforts of membrane proteins in basic and applied sciences

Characterization of human aquaporin protein-protein interactions using microscale thermophoresis (MST)

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)

Human adipose glycerol flux is regulated by a pH gate in AQP10

Obesity is a major threat to global health and metabolically associated with glycerol homeostasis. Here we demonstrate that in human adipocytes, the decreased pH observed during lipolysis (fat burning) correlates with increased glycerol release and stimulation of aquaglyceroporin AQP10. The crystal structure of human AQP10 determined at 2.3 Å resolution unveils the molecular basis for pH modulation-an exceptionally wide selectivity (ar/R) filter and a unique cytoplasmic gate. Structural and functional (in vitro and in vivo) analyses disclose a glycerol-specific pH-dependence and pinpoint pore-lining His80 as the pH-sensor. Molecular dynamics simulations indicate how gate opening is achieved. These findings unravel a unique type of aquaporin regulation important for controlling body fat mass. Thus, targeting the cytoplasmic gate to induce constitutive glycerol secretion may offer an attractive option for treating obesity and related complications

Structure of the human ClC-1 chloride channel

ClC-1 protein channels facilitate rapid passage of chloride ions across cellular membranes, thereby orchestrating skeletal muscle excitability. Malfunction of ClC-1 is associated with myotonia congenita, a disease impairing muscle relaxation. Here, we present the cryo-electron microscopy (cryo-EM) structure of human ClC-1, uncovering an architecture reminiscent of that of bovine ClC-K and CLC transporters. The chloride conducting pathway exhibits distinct features, including a central glutamate residue ("fast gate") known to confer voltage-dependence (a mechanistic feature not present in ClC-K), linked to a somewhat rearranged central tyrosine and a narrower aperture of the pore toward the extracellular vestibule. These characteristics agree with the lower chloride flux of ClC-1 compared with ClC-K and enable us to propose a model for chloride passage in voltage-dependent CLC channels. Comparison of structures derived from protein studied in different experimental conditions supports the notion that pH and adenine nucleotides regulate ClC-1 through interactions between the so-called cystathionine-β-synthase (CBS) domains and the intracellular vestibule ("slow gating"). The structure also provides a framework for analysis of mutations causing myotonia congenita and reveals a striking correlation between mutated residues and the phenotypic effect on voltage gating, opening avenues for rational design of therapies against ClC-1-related diseases