Abstract
Cholesterol dynamically regulates P2X7 receptor function in both physiological and pathological conditions. Studies suggest that cholesterol suppresses P2X7 receptor activity through direct binding or through indirect effects on the biophysical properties of the membrane. Notably, the palmitoylated C-terminus seems to counteract the action of cholesterol to make it less inhibitory. However, the mechanism underlying cholesterol-dependent regulation of P2X7 receptor remains unclear. Here we describe detailed methods that facilitate the quantification of P2X7 channel activity while controlling the amount of cholesterol in the system. We will first describe the use of methyl-β-cyclodextrin (MCD), a cyclic oligosaccharide consisting of seven glucose monomers, to decrease or increase plasma membrane cholesterol levels. We will then describe protocols for the reconstitution of purified P2X7 in proteoliposomes of defined lipid composition. These methods can be combined with commonly used techniques such as dye-uptake assays or electrophysiology. We also describe a fluorescence assay to measure cholesterol-binding to P2X7. These approaches are complementary to cryo-EM or molecular dynamics simulations, which are also powerful tools for investigating cholesterol-P2X7 interactions. An improved understanding of the mechanisms of action of cholesterol on P2X7 may contribute to elucidate the roles of this receptor in ageing, inflammation, and cancer, whose progression correlates with the level of cholesterol.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Zakany F, Kovacs T, Panyi G, Varga Z (2020) Direct and indirect cholesterol effects on membrane proteins with special focus on potassium channels. Biochim Biophys Acta Mol Cell Biol Lipids 1865(8):158706. https://doi.org/10.1016/j.bbalip.2020.158706
Murrell-Lagnado RD (2017) Regulation of P2X purinergic receptor signaling by cholesterol. Curr Top Membr 80:211–232. https://doi.org/10.1016/bs.ctm.2017.05.004
Gonnord P, Delarasse C, Auger R, Benihoud K, Prigent M, Cuif MH, Lamaze C, Kanellopoulos JM (2009) Palmitoylation of the P2X7 receptor, an ATP-gated channel, controls its expression and association with lipid rafts. FASEB J 23(3):795–805. https://doi.org/10.1096/fj.08-114637
Barth K, Weinhold K, Guenther A, Young MT, Schnittler H, Kasper M (2007) Caveolin-1 influences P2X7 receptor expression and localization in mouse lung alveolar epithelial cells. FEBS J 274(12):3021–3033. https://doi.org/10.1111/j.1742-4658.2007.05830.x
Gangadharan V, Nohe A, Caplan J, Czymmek K, Duncan RL (2015) Caveolin-1 regulates P2X7 receptor signaling in osteoblasts. Am J Physiol Cell Physiol 308(1):C41–C50. https://doi.org/10.1152/ajpcell.00037.2014
Garcia-Marcos M, Perez-Andres E, Tandel S, Fontanils U, Kumps A, Kabre E, Gomez-Munoz A, Marino A, Dehaye JP, Pochet S (2006) Coupling of two pools of P2X7 receptors to distinct intracellular signaling pathways in rat submandibular gland. J Lipid Res 47(4):705–714. https://doi.org/10.1194/jlr.M500408-JLR200
Robinson LE, Shridar M, Smith P, Murrell-Lagnado RD (2014) Plasma membrane cholesterol as a regulator of human and rodent P2X7 receptor activation and sensitization. J Biol Chem 289(46):31983–31994. https://doi.org/10.1074/jbc.M114.574699
Karasawa A, Michalski K, Mikhelzon P, Kawate T (2017) The P2X7 receptor forms a dye-permeable pore independent of its intracellular domain but dependent on membrane lipid composition. Elife 6:e31186. https://doi.org/10.7554/eLife.31186
McCarthy AE, Yoshioka C, Mansoor SE (2019) Full-length P2X7 structures reveal how Palmitoylation prevents channel desensitization. Cell 179(3):659–670e613. https://doi.org/10.1016/j.cell.2019.09.017
Casares D, Escriba PV, Rossello CA (2019) Membrane lipid composition: effect on membrane and organelle structure, function and compartmentalization and therapeutic avenues. Int J Mol Sci 20(9):2167. https://doi.org/10.3390/ijms20092167
Kopp R, Krautloher A, Ramirez-Fernandez A, Nicke A (2019) P2X7 interactions and signaling—making head or tail of it. Front Mol Neurosci 12:183. https://doi.org/10.3389/fnmol.2019.00183
Tall AR, Yvan-Charvet L (2015) Cholesterol, inflammation and innate immunity. Nat Rev Immunol 15(2):104–116. https://doi.org/10.1038/nri3793
Barbera N, Ayee MAA, Akpa BS, Levitan I (2018) Molecular dynamics simulations of Kir2.2 interactions with an ensemble of cholesterol molecules. Biophys J 115(7):1264–1280. https://doi.org/10.1016/j.bpj.2018.07.041
Barbera N, Levitan I (2019) Chiral specificity of cholesterol orientation within cholesterol binding sites in inwardly rectifying K(+) channels. Adv Exp Med Biol 1115:77–95. https://doi.org/10.1007/978-3-030-04278-3_4
Grouleff J, Irudayam SJ, Skeby KK, Schiott B (2015) The influence of cholesterol on membrane protein structure, function, and dynamics studied by molecular dynamics simulations. Biochim Biophys Acta 1848(9):1783–1795. https://doi.org/10.1016/j.bbamem.2015.03.029
Karasawa A, Kawate T (2017) Expression and purification of a mammalian P2X7 receptor from Sf9 insect cells. Bio Protoc 7(17):e2544. https://doi.org/10.21769/BioProtoc.2544
Mahammad S, Parmryd I (2015) Cholesterol depletion using methyl-beta-cyclodextrin. Methods Mol Biol 1232:91–102. https://doi.org/10.1007/978-1-4939-1752-5_8
Pottosin II, Valencia-Cruz G, Bonales-Alatorre E, Shabala SN, Dobrovinskaya OR (2007) Methyl-beta-cyclodextrin reversibly alters the gating of lipid rafts-associated Kv1.3 channels in Jurkat T lymphocytes. Pflugers Arch 454(2):235–244. https://doi.org/10.1007/s00424-007-0208-4
Acknowledgments
T.K. was supported by the National Institutes of Health (GM114379).
R.D.M-L was supported by the Biotechnology and Biological Sciences Research Council (BB/F001320/1).
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Murrell-Lagnado, R.D., Kawate, T. (2022). Methods for Studying Cholesterol-Dependent Regulation of P2X7 Receptors. In: Nicke, A. (eds) The P2X7 Receptor. Methods in Molecular Biology, vol 2510. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2384-8_13
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2384-8_13
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2383-1
Online ISBN: 978-1-0716-2384-8
eBook Packages: Springer Protocols