In Vitro Assay to Evaluate Cation Transport of Ionophores

Overview

Journal Bio Protoc

Specialty Biology

Date 2022 Dec 19

PMID 36532682

Authors

Huriye D Uzun

Melissa Vazquez-Hernandez

Julia E Bandow

Thomas Gunther Pomorski

Affiliations

Soon will be listed here.

Abstract

Ion homeostasis is a fundamental regulator of cellular processes and depends upon lipid membranes, which function as ion permeability barriers. Ionophores facilitate ion transport across cell membranes and offer a way to manipulate cellular ion composition. Here, we describe a calcein quenching assay based on large unilamellar vesicles that we used to evaluate divalent cation transport of the ionophore 4-Br-A23187. This assay can be used to study metal transport by ionophores and membrane proteins, under well-defined conditions. Proteomics (2022), DOI: 10.1002/pmic.202200061 Graphical abstract.

References

Senges C, Warmuth H, Vazquez-Hernandez M, Uzun H, Sagurna L, Dietze P . Effects of 4-Br-A23187 on Bacillus subtilis cells and unilamellar vesicles reveal it to be a potent copper ionophore. Proteomics. 2022; 22(17):e2200061. PMC: 10140759. DOI: 10.1002/pmic.202200061. View

Kunding A, Mortensen M, Christensen S, Stamou D . A fluorescence-based technique to construct size distributions from single-object measurements: application to the extrusion of lipid vesicles. Biophys J. 2008; 95(3):1176-88. PMC: 2479610. DOI: 10.1529/biophysj.108.128819. View

Erdahl W, Chapman C, Taylor R, Pfeiffer D . Ca2+ transport properties of ionophores A23187, ionomycin, and 4-BrA23187 in a well defined model system. Biophys J. 1994; 66(5):1678-93. PMC: 1275887. DOI: 10.1016/S0006-3495(94)80959-2. View

Maherani B, Arab-Tehrany E, Kheirolomoom A, Geny D, Linder M . Calcein release behavior from liposomal bilayer; influence of physicochemical/mechanical/structural properties of lipids. Biochimie. 2013; 95(11):2018-33. DOI: 10.1016/j.biochi.2013.07.006. View

Bae W, Yoon T, Jeong C . Direct evaluation of self-quenching behavior of fluorophores at high concentrations using an evanescent field. PLoS One. 2021; 16(2):e0247326. PMC: 7895399. DOI: 10.1371/journal.pone.0247326. View

OLSON F, Hunt C, Szoka F, Vail W, Papahadjopoulos D . Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes. Biochim Biophys Acta. 1979; 557(1):9-23. DOI: 10.1016/0005-2736(79)90085-3. View

Su Z, Ran X, Leitch J, Schwan A, Faragher R, Lipkowski J . How Valinomycin Ionophores Enter and Transport K across Model Lipid Bilayer Membranes. Langmuir. 2019; 35(51):16935-16943. DOI: 10.1021/acs.langmuir.9b03064. View

Picard V, Govoni G, Jabado N, Gros P . Nramp 2 (DCT1/DMT1) expressed at the plasma membrane transports iron and other divalent cations into a calcein-accessible cytoplasmic pool. J Biol Chem. 2000; 275(46):35738-45. DOI: 10.1074/jbc.M005387200. View

Mayer L, Hope M, Cullis P . Vesicles of variable sizes produced by a rapid extrusion procedure. Biochim Biophys Acta. 1986; 858(1):161-8. DOI: 10.1016/0005-2736(86)90302-0. View

10.

Erdahl W, Chapman C, Taylor R, Pfeiffer D . Effects of pH conditions on Ca2+ transport catalyzed by ionophores A23187, 4-BrA23187, and ionomycin suggest problems with common applications of these compounds in biological systems. Biophys J. 1995; 69(6):2350-63. PMC: 1236472. DOI: 10.1016/S0006-3495(95)80104-9. View

11.

Erdahl W, Chapman C, Wang E, Taylor R, Pfeiffer D . Ionophore 4-BrA23187 transports Zn2+ and Mn2+ with high selectivity over Ca2+. Biochemistry. 1996; 35(43):13817-25. DOI: 10.1021/bi961391q. View

12.

Dutta S, Watson B, Mattoo S, Rochet J . Calcein Release Assay to Measure Membrane Permeabilization by Recombinant Alpha-Synuclein. Bio Protoc. 2020; 10(14). PMC: 7500579. DOI: 10.21769/bioprotoc.3690. View

13.

Billesbolle C, Azumaya C, Kretsch R, Powers A, Gonen S, Schneider S . Structure of hepcidin-bound ferroportin reveals iron homeostatic mechanisms. Nature. 2020; 586(7831):807-811. PMC: 7906036. DOI: 10.1038/s41586-020-2668-z. View