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      Calcein Fluorescence Quenching to Measure Plasma Membrane Water Flux in Live Mammalian Cells

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          Summary

          Aquaporins (AQPs) are membrane channel proteins that facilitate the movement of water down osmotic gradients across biological membranes. This protocol allows measurements of AQP-mediated water transport across the plasma membrane of live mammalian cells. Calcein is a fluorescent dye that is quenched in a concentration-dependent manner. Therefore, on short timescales, its concentration-dependent fluorescence can be used as a probe of cell volume, and therefore a probe of water transport into or out of cells.

          For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020) and Kitchen and Conner (2015). For the underlying methodology development, please refer to Fenton et al. (2010) and Solenov et al. (2004).

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          Highlights

          • Calcein fluorescence quenching can be used to monitor cell volume

          • This can be used to quantify plasma membrane water permeability

          • The protocol is suitable for all adherent cell types

          Abstract

          Aquaporins (AQPs) are membrane channel proteins that facilitate the movement of water down osmotic gradients across biological membranes. This protocol allows measurements of AQP-mediated water transport across the plasma membrane of live mammalian cells. Calcein is a fluorescent dye that is quenched in a concentration-dependent manner. Therefore, on short timescales, its concentration-dependent fluorescence can be used as a probe of cell volume, and therefore a probe of water transport into or out of cells.

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          Most cited references9

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

          Summary 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.
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            Sevenfold-reduced osmotic water permeability in primary astrocyte cultures from AQP-4-deficient mice, measured by a fluorescence quenching method.

            A calcein fluorescence quenching method was applied to measure osmotic water permeability in highly differentiated primary cultures of brain astrocytes from wild-type and aquaporin-4 (AQP-4)-deficient mice. Cells grown on coverglasses were loaded with calcein for measurement of volume changes after osmotic challenge. Hypotonic shock producing twofold cell swelling resulted in a reversible approximately 12% increase in calcein fluorescence, which was independent of cytosolic calcein concentration at levels well below where calcein self-quenching occurs. Calcein fluorescence was quenched in <200 ms in response to addition of cytosol in vitro, indicating that the fluorescence signal arises from changes in cytosol concentration. In astrocytes from wild-type CD1 mice, calcein fluorescence increased reversibly in response to hypotonic challenge with a half-time of 0.92 +/- 0.05 s at 23 degrees C, corresponding to an osmotic water permeability (Pf) of approximately 0.05 cm/s. Pf was reduced 7.1-fold in astrocytes from AQP-4-deficient mice. Temperature dependence studies indicated an increased Arrhenius activation energy for water transport in AQP-4-deficient astrocytes (11.3 +/- 0.5 vs. 5.5 +/- 0.4 kcal/mol). Our studies establish a calcein quenching method for measurement of cell membrane water permeability and indicate that AQP-4 provides the principal route for water transport in astrocytes.
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              Molecular characterization of a broad selectivity neutral solute channel.

              In all living cells, coordination of solute and water movement across cell membranes is of critical importance for osmotic balance. The current concept is that these processes are of distinct biophysical nature. Here we report the expression cloning of a liver cDNA encoding a unique promiscuous solute channel (AQP9) that confers high permeability for both solutes and water. AQP9 mediates passage of a wide variety of non-charged solutes including carbamides, polyols, purines, and pyrimidines in a phloretin- and mercury-sensitive manner, whereas amino acids, cyclic sugars, Na+, K+, Cl-, and deprotonated monocarboxylates are excluded. The properties of AQP9 define a new evolutionary branch of the major intrinsic protein family of aquaporin proteins and describe a previously unknown mechanism by which a large variety of solutes and water can pass through a single pore, enabling rapid cellular uptake or exit of metabolites with minimal osmotic perturbation.
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                Author and article information

                Contributors
                Journal
                STAR Protoc
                STAR Protoc
                STAR Protocols
                Elsevier
                2666-1667
                02 November 2020
                18 December 2020
                02 November 2020
                : 1
                : 3
                : 100157
                Affiliations
                [1 ]School of Life & Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, UK
                [2 ]Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA
                [3 ]Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA 02115, USA
                [4 ]Department of Pharmacology, College of Pharmacy, University of Mosul, Mosul 41002, Iraq
                [5 ]Department of Experimental Medical Science, Lund University, PO Box 118, 221 00 Lund, Sweden
                [6 ]Institute of Clinical Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
                Author notes
                []Corresponding author p.kitchen1@ 123456aston.ac.uk
                [∗∗ ]Corresponding author r.m.bill@ 123456aston.ac.uk
                [7]

                These authors contributed equally

                [8]

                Technical Contact

                [9]

                Lead Contact

                Article
                S2666-1667(20)30144-1 100157
                10.1016/j.xpro.2020.100157
                7757292
                33377051
                5c86809c-1b88-449f-b44f-11f3a009ea00
                © 2020 The Author(s)

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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