Functional Assessment of Crystallization-Optimized G Protein-Coupled Receptors using Ion Channel-Coupled Receptors

Ion Channel-Coupled Receptors (ICCRs) are artificial ligand-gated ion channels created by genetic fusion of G protein-coupled receptors (GPCRs) to a K+ inward rectifier channel (Kir6.2) such that the channel is a direct reporter of the receptor conformational changes. This concept has been validated with 4 prototypical GPCRs: the M2 muscarinic, the D2L dopaminergic, the β2 adrenergic and the opsin receptors (Moreau et al., Nature Nanotech 3:620 2008, Caro et al. PLoS ONE 6:e18226 2011, Caro et al. PLoS ONE 7:e43766 2012). Voltage-clamp recordings showed that ICCRs detect the agonist- and antagonist-bound states of the receptor via direct physical coupling. This GPCR-channel communication proceeds without any involvement of G proteins and the electrical signal amplitude is correlated with the ligand concentration.The intrinsic instability of the GPCRs has proved a challenge to crystallographic studies. A successful approach, introduced in 2007 by Cherezov et al (Science. 318:1258) and subsequently applied to obtain 12 GPCR structures, consists in the insertion of the T4 phage lysozyme domain in the 3rd intracellular loop of the receptors. However, this modification abolishes G protein binding and prohibits related functional assays. Current characterization of crystallization-optimized GPCR(T4L) is performed by radiolabeled ligand assays or localized FRET techniques. Requiring no biochemical steps, ICCRs are an alternative tool to functionally characterize modified GPCRs that are unable to bind or activate G proteins and not amenable to most GPCR functional assays. We demonstrate here the validity of this tool with 3 different GPCRs (M2-muscarinic, β2-adrenergic and oxytocin receptors). The final application of this study would be the integration of this technology in the current crystallographic platforms dedicated to GPCR structure determination or for structure-function studies independent of G protein interaction.