Abstract
Assays based on bioluminescence resonance energy transfer (BRET) provide a sensitive and simple method to study protein–protein interactions in live cells. Here we describe a protocol using BRET technique to investigate potential interactions between P2X subunits. This approach combined with bimolecular fluorescence complementation (BiFC) can also be employed to determine the stoichiometry of heteromeric P2X receptors.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Wampler JE, Hori K, Lee JW, Cormier MJ (1971) Structured bioluminescence. Two emitters during both the in vitro and the in vivo bioluminescence of the sea pansy, Renilla. Biochemistry 10:2903–2909
Ward WW, Cormier MJ (1979) An energy transfer protein in coelenterate bioluminescence. Characterization of the Renilla green-fluorescent protein. J Biol Chem 254:781–788
Young MT, Fisher JA, Fountain SJ, Ford RC, North RA, Khakh BS (2008) Molecular shape, architecture, and size of P2X4 receptors determined using fluorescence resonance energy transfer and electron microscopy. J Biol Chem 283:26241–26251. https://doi.org/10.1074/jbc.M804458200
Fisher JA, Girdler G, Khakh BS (2004) Time-resolved measurement of state-specific P2X2 ion channel cytosolic gating motions. J Neurosci 24:10475–10487. https://doi.org/10.1523/JNEUROSCI.3250-04.2004
Khakh BS, Fisher JA, Nashmi R, Bowser DN, Lester HA (2005) An angstrom scale interaction between plasma membrane ATP-gated P2X2 and alpha4beta2 nicotinic channels measured with fluorescence resonance energy transfer and total internal reflection fluorescence microscopy. J Neurosci 25:6911–6920. https://doi.org/10.1523/JNEUROSCI.0561-05.2005
Limapichat W, Dougherty DA, Lester HA (2014) Subtype-specific mechanisms for functional interaction between alpha6beta4∗ nicotinic acetylcholine receptors and P2X receptors. Mol Pharmacol 86:263–274. https://doi.org/10.1124/mol.114.093179
Shrivastava AN, Triller A, Sieghart W, Sarto-Jackson I (2011) Regulation of GABA(A) receptor dynamics by interaction with purinergic P2X(2) receptors. J Biol Chem 286:14455–14468. https://doi.org/10.1074/jbc.M110.165282
Richler E, Chaumont S, Shigetomi E, Sagasti A, Khakh BS (2008) Tracking transmitter-gated P2X cation channel activation in vitro and in vivo. Nat Methods 5:87–93. https://doi.org/10.1038/nmeth1144
Koshimizu TA, Kretschmannova K, He ML, Ueno S, Tanoue A, Yanagihara N, Stojilkovic SS, Tsujimoto G (2006) Carboxyl-terminal splicing enhances physical interactions between the cytoplasmic tails of purinergic P2X receptors. Mol Pharmacol 69:1588–1598. https://doi.org/10.1124/mol.105.019802
Compan V, Ulmann L, Stelmashenko O, Chemin J, Chaumont S, Rassendren F (2012) P2X2 and P2X5 subunits define a new heteromeric receptor with P2X7-like properties. J Neurosci 32:4284–4296. https://doi.org/10.1523/JNEUROSCI.6332-11.2012
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Compan, V., Rassendren, F. (2020). Multimeric Purinoceptor Detection by Bioluminescence Resonance Energy Transfer. In: Pelegrín, P. (eds) Purinergic Signaling. Methods in Molecular Biology, vol 2041. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9717-6_11
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9717-6_11
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9716-9
Online ISBN: 978-1-4939-9717-6
eBook Packages: Springer Protocols