Multimeric Purinoceptor Detection by Bioluminescence Resonance Energy Transfer
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.
Key wordsBioluminescence BRET P2X BiFC Stoichiometry Heteromer
- 3.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.M804458200CrossRefPubMedPubMedCentralGoogle Scholar
- 5.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.0561-05.2005CrossRefPubMedPubMedCentralGoogle Scholar
- 9.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.105.019802CrossRefPubMedGoogle Scholar