Jean Revilloud

Coupling ion channels to receptors for biomolecule sensing.

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 pores and modified ion channels 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 GPCRs, which are known to be major pharmaceutical targets. This could form a unique platform for label-free drug screening.

Coassembly of Different Sulfonylurea Receptor Subtypes Extends the Phenotypic Diversity of ATP-sensitive Potassium (KATP) Channels

KATP channels are metabolic sensors and targets of potassium channel openers (KCO; e.g., diazoxide and pinacidil). They comprise four sulfonylurea receptors (SUR) and four potassium channel subunits (Kir6) and are critical in regulating insulin secretion. Different SUR subtypes (SUR1, SUR2A, SUR2B) largely determine the metabolic sensitivities and the pharmacological profiles of KATP channels. SUR1- but not SUR2-containing channels are highly sensitive to metabolic inhibition and diazoxide, whereas SUR2 channels are sensitive to pinacidil. It is generally believed that SUR1 and SUR2 are incompatible in channel coassembly. We used triple tandems, T1 and T2, each containing one SUR (SUR1 or SUR2A) and two Kir6.2Δ26 (last 26 residues are deleted) to examine the coassembly of different SUR. When T1 or T2 was expressed in Xenopus laevis oocytes, small whole-cell currents were activated by metabolic inhibition (induced by azide) plus a KCO (diazoxide for T1, pinacidil for T2). When coexpressed with any SUR subtype, the activated-currents were increased by 2- to 13-fold, indicating that different SUR can coassemble. Consistent with this, heteromeric SUR1+SUR2A channels were sensitive to azide, diazoxide, and pinacidil, and their single-channel burst duration was 2-fold longer than that of the T1 channels. Furthermore, SUR2A was coprecipitated with SUR1. Using whole-cell recording and immunostaining, heteromeric channels could also be detected when T1 and SUR2A were coexpressed in mammalian cells. Finally, the response of the SUR1+SUR2A channels to azide was found to be intermediate to those of the homomeric channels. Therefore, different SUR subtypes can coassemble into KATP channels with distinct metabolic sensitivities and pharmacological profiles.

Three C-terminal residues from the sulphonylurea receptor contribute to the functional coupling between the KATP channel subunits SUR2A and Kir6.2

Cardiac ATP‐sensitive potassium (KATP) channels are metabolic sensors formed by the association of the inward rectifier potassium channel Kir6.2 and the sulphonylurea receptor SUR2A. SUR2A adjusts channel gating as a function of intracellular ATP and ADP and is the target of pharmaceutical openers and blockers which, respectively, up‐ and down‐regulate Kir6.2. In an effort to understand how effector binding to SUR2A translates into Kir6.2 gating modulation, we examined the role of a 65‐residue SUR2A fragment linking transmembrane domain TMD2 and nucleotide‐binding domain NBD2 that has been shown to interact with Kir6.2. This fragment of SUR2A was replaced by the equivalent residues of its close homologue, the multidrug resistance protein MRP1. The chimeric construct was expressed in Xenopus oocytes and characterized using the patch‐clamp technique. We found that activation by MgADP and synthetic openers was greatly attenuated although apparent affinities were unchanged. Further chimeragenetic and mutagenetic studies showed that mutation of three residues, E1305, I1310 and L1313 (rat numbering), was sufficient to confer this defective phenotype. The same mutations had no effects on channel block by the sulphonylurea glibenclamide or by ATP, suggesting a role for these residues in activatory – but not inhibitory – transduction processes. These results indicate that, within the KATP channel complex, the proximal C‐terminal of SUR2A is a critical link between ligand binding to SUR2A and Kir6.2 up‐regulation.

β2-Adrenergic Ion-Channel Coupled Receptors as Conformational Motion Detectors

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 M2 or dopaminergic D2 receptors. Here, we extend the concept to the distinct, longer β2-adrenergic receptor which, unlike M2 and D2 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 β2 constructs. We then demonstrate how engineering of both receptor and channel can produce β2-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 β2 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 β2 receptor.

Remodelling of the SUR–Kir6.2 interface of the KATP channel upon ATP binding revealed by the conformational blocker rhodamine 123

ATP‐sensitive K+ channels (KATP channels) are metabolic sensors formed by association of a K+ channel, Kir6, and an ATP‐binding cassette (ABC) protein, SUR, which allosterically regulates channel gating in response to nucleotides and pharmaceutical openers and blockers. How nucleotide binding to SUR translates into modulation of Kir6 gating remains largely unknown. To address this issue, we have used a novel conformational KATP channel inhibitor, rhodamine 123 (Rho123) which targets the Kir6 subunit in a SUR‐dependent manner. Rho123 blocked SUR‐less Kir6.2 channels with an affinity of ∼1 μm, regardless of the presence of nucleotides, but it had no effect on channels formed by the association of Kir6.2 and the N‐terminal transmembrane domain TMD0 of SUR. Rho123 blocked SUR + Kir6.2 channels with the same affinity as Kir6.2 but this effect was antagonized by ATP. Protection from Rho123 block by ATP was due to direct binding of ATP to SUR and did not entail hydrolysis because it was not mimicked by AMP, did not require Mg2+ and was reduced by mutations in the nucleotide‐binding domains of SUR. These results suggest that Rho123 binds at the TMD0–Kir6.2 interface and that binding of ATP to SUR triggers a change in the structure of the contact zone between Kir6.2 and domain TMD0 of SUR that causes masking of the Rho123 site on Kir6.2.

C-terminal engineering of CXCL12 and CCL5 chemokines: functional characterization by electrophysiological recordings.

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.

Tuning the allosteric regulation of artificial muscarinic and dopaminergic ligand-gated potassium channels by protein engineering of G protein-coupled receptors.

Ligand-gated ion channels enable intercellular transmission of action potential through synapses by transducing biochemical messengers into electrical signal. We designed artificial ligand-gated ion channels by coupling G protein-coupled receptors to the Kir6.2 potassium channel. These artificial channels called ion channel-coupled receptors offer complementary properties to natural channels by extending the repertoire of ligands to those recognized by the fused receptors, by generating more sustained signals and by conferring potassium selectivity. The first artificial channels based on the muscarinic M2 and the dopaminergic D2L receptors were opened and closed by acetylcholine and dopamine, respectively. We find here that this opposite regulation of the gating is linked to the length of the receptor C-termini, and that C-terminus engineering can precisely control the extent and direction of ligand gating. These findings establish the design rules to produce customized ligand-gated channels for synthetic biology applications.

Functional Mapping of the N-terminal Arginine Cluster and C-terminal Acidic Residues of Kir6.2 channel Fused to a G Protein-Coupled Receptor

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 Kir6.2N-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.

Artificial Ligand-Gated Channels Engineered by Assembly of Potassium Channels and G-Protein Coupled Receptors

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.