Ion channel reporter for monitoring the activity of engineered GPCRs

• Published in: Methods in enzymology Vol. 556; p. 425
• Main Authors: Moreau, Christophe J, Niescierowicz, Katarzyna, Caro, Lydia N, Revilloud, Jean, Vivaudou, Michel
• Format: Journal Article
• Language: English
• Published: United States 2015
• Subjects: Amino Acid Sequence; Animals; Bacteriophage T4 - chemistry; Bacteriophage T4 - metabolism; Electrodes; Humans; Ion Channels; Models, Molecular; Molecular Sequence Data; Muramidase - chemistry; Muramidase - metabolism; Oocytes - metabolism; Patch-Clamp Techniques - instrumentation; Patch-Clamp Techniques - methods; Potassium Channels, Inwardly Rectifying - chemistry; Potassium Channels, Inwardly Rectifying - genetics; Potassium Channels, Inwardly Rectifying - metabolism; Protein Engineering - methods; Protein Structure, Tertiary; Receptors, G-Protein-Coupled - chemistry; Receptors, G-Protein-Coupled - genetics; Receptors, G-Protein-Coupled - metabolism; Viral Proteins - chemistry; Viral Proteins - metabolism; Xenopus laevis - genetics; G protein-coupled receptor; Xenopus oocytes; Ion channel-coupled receptors; Crystallization; GPCR; TEVC; Ion channels; ICCR; Structure; T4L domain
• ISSN: 1557-7988
• DOI: 10.1016/bs.mie.2014.12.017

Ion channel-coupled receptor (ICCR) is a recent technology based on the fusion of G protein-coupled receptors (GPCRs) to an ion channel. Binding of ligands on the GPCR triggers conformational changes of the receptor that are mechanically transmitted to the ion channel gates, generating an electrical signal easily detectable with conventional electrophysiological techniques. ICCRs are heterologously expressed in Xenopus oocytes and offers several advantages such as: (i) real-time recordings on single cells, (ii) standard laboratory environment and inexpensive media for Xenopus oocytes maintenance, (iii) absence of protein purification steps, (iv) sensitivity to agonists and antagonists in concentration-dependent manner, (v) compatibility with a Gi/o protein activation assay based on Kir3.x channels, and (vi) ability to detect receptor activation independently of intracellular effectors. This last characteristic of ICCRs led to the development of a functional assay for G protein-"uncoupled" receptors such as GPCRs optimized for crystallization by alteration of their third intracellular (i3) loop. One of the most widely used approaches consists in replacing the i3 loop with the T4 phage lysozyme (T4L) domain that obstructs the access of G proteins to their binding site. We recently demonstrated that the ICCR technology can functionally characterize GPCRs(T4L). Two-electrode voltage-clamp (TEVC) recordings revealed that apparent affinities and sensitivities to ligands are not affected by T4L insertion, while ICCRs(T4L) displayed a partial agonist phenotype upon binding of full agonists, suggesting that ICCRs could detect intermediate-active states. This chapter aims to provide exhaustive details from molecular biology steps to electrophysiological recordings for the design and the characterization of ICCRs and ICCRs(T4L).