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Summary Ligand-gated ion channels (LGICs) are natural biosensors generating electrical signals in response to the binding of specific ligands. Creating de novo LGICs for biosensing applications is technically challenging. We have previously designed modified LGICs by linking G protein-coupled receptors (GPCRs) to the Kir6.2 channel. In this article, we extrapolate these design concepts to other channels with different structures and oligomeric states, namely a tetrameric viral Kcv channel and the dimeric mouse TREK-1 channel. After precise engineering of the linker regions, the two ion channels were successfully regulated by a GPCR fused to their N-terminal domain. Two-electrode voltage-clamp recordings showed that Kcv and mTREK-1 fusions were inhibited and activated by GPCR agonists, respectively, and antagonists abolished both effects. Thus, dissimilar ion channels can be allosterically regulated through their N-terminal domains, suggesting that this is a generalizable approach for ion channel engineering., Graphical abstract, Highlights • GPCR regulation of diverse potassium channels through N-terminal fusions • Fusion to the viral PBCV-1 Kcv channel requires a specific linker and receptor • Tetrameric Kcv and dimeric mTREK-1 channels fusions respond to GPCR ligands • The engineered fusions broaden the properties of the channels' electrical signals, Motivation For biosensing applications, electrical signals are attractive since they can be recorded by micro- or nano-electronic systems for developing miniaturized detection devices in biomedical or environmental fields. One of the main limiting steps is the design of sensing elements that specifically recognize ligands such as biomarkers or chemical compounds. Ligand-gated ion channels are natural biosensors that specifically recognize ligands and generate an electrical signal. They have naturally evolved to be finely tuned by regulatory domains or proteins resulting in an appropriate electrical signal. The objective of this work is to leverage these natural biosensors by engineering diverse artificial ion channels that have the desired properties for biosensing or basic research applications. The main challenge is to design de novo allosteric regulations between ion channels and physiologically unrelated membrane proteins. Previously, different G protein-coupled receptors were successfully coupled to a specific ion channel, Kir6.2. In this work, we demonstrate that other ion channels (viral and murine) with distinct oligomerization properties can also be fused to GPCRs via their N-terminal domains. These results open avenues for diversifying engineered ligand-gated ion channels., Ligand-gated ion channels are natural biosensors that generate highly sensitive and fast electrical signals. García-Fernández et al. broaden the regulatory properties of electrical signal output by fusing different potassium channels to GPCRs. Functional fusion to tetrameric and dimeric channels demonstrates the versatility of this approach.

Ion channel-coupled receptors (ICCRs) are original man-made ligand-gated ion channels created by fusion of G protein-coupled receptors (GPCRs) to the inward-rectifier potassium channel Kir6.2. GPCR conformational changes induced by ligand binding are transduced into electrical current by the ion channel. This functional coupling is closely related to the length of the linker region formed by the GPCR C-terminus (C-ter) and Kir6.2 N-terminus (N-ter). Manipulating the GPCR C-ter length allows to finely tune the channel regulation, both in amplitude and sign (opening or closing Kir6.2). In this work, we demonstrate that the primary sequence of the channel N-terminal domain is an additional parameter for the functional coupling with GPCRs. As for all Kir channels, a cluster of basic residues is present in the N-terminal domain of Kir6.2 and is composed of 5 arginines which are proximal to the GPCR C-ter in the fusion proteins. Using a functional mapping approach, we demonstrate the role of specific arginines (R27 and R32) for the function of ICCRs, indicating that the position and not the cluster of positively-charged arginines is critical for the channel regulation by the GPCR. Following observations provided by molecular dynamics simulation, we explore the hypothesis of interaction of these arginines with acidic residues, and using site-directed mutagenesis, we identified aspartate D307 and glutamate E308 residues as critical for the function of ICCRs. These results demonstrate the critical role of the N-terminal and C-terminal charged residues of Kir6.2 for its allosteric regulation by the fused GPCR.

International audience; G-protein-coupled receptors (GPCR) are the most widely used system of communication used by cells. They sense external signals and translate them into intracellular signals. The information is carried mechanically across the cell membrane, without perturbing its integrity. Agonist binding on the extracellular side causes a change in receptor conformation which propagates to the intracellular side and causes release of activated G-proteins, the first messengers of a variety of signaling cascades.Permitting access to powerful electrophysiological techniques, ion channels can be employed to monitor precisely the most proximal steps of GPCR signaling, receptor conformational changes, and G-protein release. The former is achieved by physical attachment of a potassium channel to the GPCR to create an Ion-Channel Coupled Receptor (ICCR). The latter is based on the use of G-protein-regulated potassium channels (GIRK). We describe here how these two systems may be used in the Xenopus oocyte heterologous system with a robotic system for increased throughput.

Ion channel-coupled receptors (ICCR) are artificial proteins built from a G protein-coupled receptor and an ion channel. Their use as molecular biosensors is promising in diagnosis and high-throughput drug screening. The concept of ICCR was initially validated with the combination of the muscarinic receptor M2 with the inwardly rectifying potassium channel Kir6.2. A long protein engineering phase has led to the biochemical characterization of the M2-Kir6.2 construct. However, its molecular mechanism remains to be elucidated. In particular, it is important to determine how the activation of M2 by its agonist acetylcholine triggers the modulation of the Kir6.2 channel via the M2-Kir6.2 linkage. In the present study, we have developed and validated a computational approach to rebuild models of the M2-Kir6.2 chimera from the molecular structure of M2 and Kir6.2. The protocol was first validated on the known protein complexes of the μ-opioid Receptor, the CXCR4 receptor and the Kv1.2 potassium channel. When applied to M2-Kir6.2, our protocol produced two possible models corresponding to two different orientations of M2. Both models highlights the role of the M2 helices I and VIII in the interaction with Kir6.2, as well as the role of the Kir6.2 N-terminus in the channel opening. Those two hypotheses will be explored in a future experimental study of the M2-Kir6.2 construct.

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).

International audience; Structural studies of G protein-coupled receptors (GPCRs) extensively use the insertion of globular soluble protein domains to facilitate their crystallization. However, when inserted in the third intracellular loop (i3 loop), the soluble protein domain disrupts their coupling to G proteins and impedes the GPCRs functional characterization by standard G protein-based assays. Therefore, activity tests of crystallization-optimized GPCRs are essentially limited to their ligand binding properties using radioligand binding assays. Functional characterization of additional thermostabilizing mutations requires the insertion of similar mutations in the wild-type receptor to allow G protein-activation tests. We demonstrate that ion channel-coupled receptor technology is a complementary approach for a comprehensive functional characterization of crystallization-optimized GPCRs and potentially of any engineered GPCR. Ligand-induced conformational changes of the GPCRs are translated into electrical signal and detected by simple current recordings, even though binding of G proteins is sterically blocked by the added soluble protein domain.

International audience; Chemokines are chemotactic cytokines comprised of 70-100 amino acids. The chemokines CXCL12 and CCL5 are the endogenous ligands of the CXCR4 and CCR5 G protein-coupled receptors that are also HIV co-receptors. Biochemical, structural and functional studies of receptors are ligand-consuming and the cost of commercial chemokines hinders their use in such studies. Here, we describe methods for the expression, refolding, purification, and functional characterization of CXCL12 and CCL5 constructs incorporating C-terminal epitope tags. The model tags used were hexahistidines and Strep-Tag for affinity purification, and the double lanthanoid binding tag for fluorescence imaging and crystal structure resolution. The ability of modified and purified chemokines to bind and activate CXCR4 and CCR5 receptors was tested in Xenopus oocytes expressing the receptors, together with a Kir3 G-protein activated K(+) channel that served as a reporter of receptor activation. Results demonstrate that tags greatly influence the biochemical properties of the recombinant chemokines. Besides, despite the absence of any evidence for CXCL12 or CCL5 C-terminus involvement in receptor binding and activation, we demonstrated unpredictable effects of tag insertion on the ligand apparent affinity and efficacy or on the ligand dissociation. These tagged chemokines should constitute useful tools for the selective purification of properly-folded chemokines receptors and the study of their native quaternary structures.

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.

Ion Channel-Coupled Receptors (ICCRs) are artificial receptor-channel fusion proteins designed to couple ligand binding to channel gating. We previously validated the ICCR concept with various G protein-coupled receptors (GPCRs) fused with the inward rectifying potassium channel Kir6.2. Here we characterize a novel ICCR, consisting of the light activated GPCR, opsin/rhodopsin, fused with Kir6.2. To validate our two-electrode voltage clamp (TEVC) assay for activation of the GPCR, we first co-expressed the apoprotein opsin and the G protein-activated potassium channel Kir3.1(F137S) (Kir3.1*) in Xenopus oocytes. Opsin can be converted to rhodopsin by incubation with 11-cis retinal and activated by light-induced retinal cis→trans isomerization. Alternatively opsin can be activated by incubation of oocytes with all-trans-retinal. We found that illumination of 11-cis-retinal-incubated oocytes co-expressing opsin and Kir3.1* caused an immediate and long-lasting channel opening. In the absence of 11-cis retinal, all-trans-retinal also opened the channel persistently, although with slower kinetics. We then used the oocyte/TEVC system to test fusion proteins between opsin/rhodopsin and Kir6.2. We demonstrate that a construct with a C-terminally truncated rhodopsin responds to light stimulus independent of G protein. By extending the concept of ICCRs to the light-activatable GPCR rhodopsin we broaden the potential applications of this set of tools.

Ion Channel-Coupled Receptors (ICCRs) are artificial proteins comprised of a G protein-coupled receptor and a fused ion channel, engineered to couple channel gating to ligand binding. These novel biological objects have potential use in drug screening and functional characterization, in addition to providing new tools in the synthetic biology repertoire as synthetic K(+)-selective ligand-gated channels. The ICCR concept was previously validated with fusion proteins between the K(+) channel Kir6.2 and muscarinic M(2) or dopaminergic D(2) receptors. Here, we extend the concept to the distinct, longer β(2)-adrenergic receptor which, unlike M(2) and D(2) receptors, displayed barely detectable surface expression in our Xenopus oocyte expression system and did not couple to Kir6.2 when unmodified. Here, we show that a Kir6.2-binding protein, the N-terminal transmembrane domain of the sulfonylurea receptor, can greatly increase plasma membrane expression of β(2) constructs. We then demonstrate how engineering of both receptor and channel can produce β(2)-Kir6.2 ICCRs. Specifically, removal of 62-72 residues from the cytoplasmic C-terminus of the receptor was required to enable coupling, suggesting that ligand-dependent conformational changes do not efficiently propagate to the distal C-terminus. Characterization of the β(2) ICCRs demonstrated that full and partial agonists had the same coupling efficacy, that an inverse agonist had no effect and that the stabilizing mutation E122 W reduced agonist-induced coupling efficacy without affecting affinity. Because the ICCRs are expected to report motions of the receptor C-terminus, these results provide novel insights into the conformational dynamics of the β(2) receptor.

Nanoscale electrical biosensors are promising tools for diagnostics and high-throughput screening systems. The electrical signal allows label-free assays with a high signal-to-noise ratio and fast real-time measurements. The challenge in developing such biosensors lies in functionally connecting a molecule detector to an electrical switch. Advances in this field have relied on synthetic ion-conducting pores1 and modified ion channels2,3,4 that are not yet suitable for biomolecule screening. Here we report the design and characterization of a novel bioelectric-sensing platform engineered by coupling an ion channel, which serves as the electrical probe, to G-protein-coupled receptors (GPCRs), a family of receptors that detect molecules outside the cell. These ion-channel-coupled receptors may potentially detect a wide range of ligands recognized by natural or altered GPCRs5,6, which are known to be major pharmaceutical targets7. This could form a unique platform for label-free drug screening8. The challenge in developing electrical biosensors lies in connecting a molecule detector to an electrical switch. Attaching ion channels to certain cell receptors forms a detector–switch pair that converts chemical information into a measurable electrical signal, creating a platform suitable for screening drugs and other molecules.

Inspired by the natural example of the K-ATP channel in which an ion channel (Kir6.2) is regulated by an associated, unrelated membrane protein, the ABC protein SUR, we have engineered Ion-Channel Coupled Receptors (ICCRs) by physical coupling of G-protein coupled receptors (GPCRs) with Kir6.2.A first ICCR was constructed using the muscarinic M2 receptor and Kir6.2. Our strategy consisted of creating various fusion proteins by linking the receptor C-terminus to the channel N-terminus and progressively removing residues from either termini until functional coupling was achieved, i.e., agonist binding to the receptor modified channel activity. The fusion proteins were heterologously expressed in Xenopus oocytes and characterized with the two-electrode voltage-clamp and patch-clamp techniques.Successful coupling was achieved with limited deletions of the channel N-terminal with an optimum of 25 residues. The optimal construct was reversibly upregulated by the M2 agonist, acetylcholine. To further establish proof-of-concept, a second ICCR was obtained by using the dopaminergic D2 receptor. This ICCR was also regulated by D2 agonists and antagonists although, unexpectedly, the D2 ICCR responses were the opposite of those of the M2 ICCR, i.e., agonists caused channel downregulation.We observed that, i) agonist modulation of Kir6.2 was concentration-dependent and saturatable, ii) agonist effects were abolished by receptor antagonists, iii) the GPCRs within the fusion remained functional as verified by their capacity to activate coexpressed G-protein-activated Kir3 channels. iv) receptor-mediated responses were independent of G-protein activation because they persisted in the presence of pertussis toxin, and v) ICCRs remained functional in cell-free, outside-out patch conditions.ICCRs could be useful tools for the study of GPCR activation and K+ channel gating and could also serve as biosensors for drug screening and diagnostics.Ref :Moreau et al, Nature Nanotechnology. 2008, in press.

It is possible to achieve functional coupling between a receptor and an ion channel by covalent linkage so that ligand binding to the receptor modifies channel gating. This was demonstrated with the inward rectifier potassium channel Kir6.2 (the pore subunit of the KATP channel) and the muscarinic M2 and dopaminergic D2 G-protein coupled receptors (GPCRs) [Moreau et al., 2008, Nature Nanotech].To extend this concept of Ion-Channel Coupled Receptor (ICCR), we designed new contructs by engineering fusion between Kir6.2 and 3 GPCRs: the s2 adrenergic, cannabinoid 1 (CB1) and dopaminergic D3 receptors. The receptor C-ter and channel N-ter extremities were pared to promote efficient coupling as in M2 and D2 ICCRs and joined covalently. The fusions were heterologously expressed in Xenopus oocytes and characterized by the two-electrode voltage clamp technique. Construct names ‘G-Kxx-yy’ indicate the GPCR name (G), the residues clipped off from the GPCR C-ter (xx) and from the Kir6.2 N-ter (yy).A D3-based ICCR, D3-K0-25, behaved like the D2-based ICCR, showing channel inhibition upon dopamine application. Two s2-based ICCRs were successfully constructed, s2-K62-25 and s2-K73-25. Only when co-expressed with TMD0 (the Kir6.2-anchoring domain of SUR, the regulatory subunit of the KATP channel) to augment surface expression, these two ICCRs were reversibly activated by the agonist isoproterenol and inhibited by the antagonist alprenolol. Similarly, a CB1-based ICCR, CB1-K0-25, was activated by the agonist W102, an effect that was enhanced by the presence of TMD0.Thus, the ICCR concept is readily applicable to class A GPCRs. Besides their obvious interest in drug screening, the new ICCRs should be valuable tools to investigate the intermolecular events involved in the modulation of Kir6.2 gating and the nature of the GPCR conformational changes evoked by their ligands.

Ligand-gated ion channels combine within a single polypeptide chain a binding site for a specific signalling molecule and a selective ion-conducting pore, allosterically linked so that ligand binding controls pore opening, thus allowing fast chemical-to-electrical signal transduction as required in synapses.Ion Channel-Coupled Receptors (ICCRs) are artificial proteins comprised of a G protein-coupled receptor fused to an ion channel, engineered to create a physical coupling between ligand binding and channel gating. The ICCR concept was previously validated with fusion proteins between the K+ channel Kir6.2 and either muscarinic M2, or dopaminergic D2 unmodified receptors (Moreau et al., Nature Nanotech. 3:620-625, 2008). Here, we extend the concept to the β2-adrenergic and the opsin receptors.These receptors raised new challenges as they are significantly different from M2 and D2 receptors in terms of structure (poor sequence homology, much longer cytoplasmic C-terminal), function (coupled to distinct G-proteins, distinct effectors), and surface expression (extremely low in some cases in our Xenopus oocyte heterologous expression system). We show here how surface expression can be enhanced by co-expression of an accessory Kir6.2-binding protein, the first transmembrane domain of the sulfonylurea receptor SUR, and how receptor-channel coupling can be achieved by modifications of the receptors that preserve their ligand-binding properties. The resulting β2 and opsin ICCRs constitute blueprints for innovative tools to study the molecular mechanics of receptors and channels and could find applications not only in diagnostics and drug screening, but also in synthetic biology as adrenergic-gated and light-gated K+ channels.

Ion Channel-Coupled Receptors (ICCRs) are artificial ion channels created by the fusion of a Gprotein-coupled receptor to a Kir6.2 channel. In this concept, the channel acts a direct reporter ofthe conformational changes of the GPCRs, allowing the detection by simple current recordingsof agonists and antagonists binding in concentration-dependent manner.The signal being directly correlated to the receptor activity, independently of G protein signallingpathways, we exploited this advantage to extend the field of applications of ICCRs during thisthesis. We developed 4 applications: 1) the functional characterization of the optimized GPCRsfor crystallization by insertion of the T4 phage lysozyme domain in the ICL3 loop; 2) thedetection of a cholesterol-dependence of the GPCRs; 3) the detection of the so-called "biasedligands" to simplify their screening; and 4) the functional mapping of the Kir6.2 channel gatesunder control of membrane proteins interaction with the N-terminus domain., Les Récepteurs Couplés à un Canal Ionique (ICCRs) sont des canaux ioniques artificielscréés par fusion d'un Récepteur Couplé aux Protéines G (RCPG) au canal ionique Kir6.2. Dansce concept, le canal agit comme un rapporteur direct des changements conformationnels desRCPGs permettant de détecter par simple mesure de courant, la fixation d'agonistes etd'antagonistes proportionnellement à leur concentration.Le signal induit étant directement corrélé à l'activité du récepteur, indépendamment desvoies de signalisation des protéines G, nous avons exploité cet avantage pour étendre le champd'applications des ICCRs au cours de cette thèse. Nous avons développé quatre applications quisont: 1) la caractérisation fonctionnelle des RCPG optimisés pour la cristallisation par insertionde domaine du lysozyme du phage T4 dans la boucle ICL3; 2) la détection de la dépendance desRCPGs au cholestérol; 3) la détection de ligands dits "biaisés" pour faciliter leur criblage; et 4) lacartographie fonctionnelle des portes du canal Kir6.2 régulées par des protéines membranairesinteragissant par le domaine N-terminal.