Ion Channel Reporter for Monitoring the Activity of Engineered GPCRs

CHAPTER TWENTY

Ion Channel Reporter for Monitoring the Activity of Engineered GPCRs Christophe J. Moreau*,†,{,1, Katarzyna Niescierowicz*,†,{, Lydia N. Caro},2, Jean Revilloud*,†,{, Michel Vivaudou*,†,{,1 *Institut de Biologie Structurale (IBS), University of Grenoble Alpes, Grenoble, France † CNRS, IBS, LabEx ICST, Grenoble, France { CEA, IBS, Grenoble, France } Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada 1 Corresponding authors: e-mail address: [email protected]; [email protected]

Contents 1. Introduction 1.1 G protein-coupled receptors 1.2 GPCR crystallization 1.3 ICCR history and principle 2. ICCR Design 2.1 Genetic engineering of the GPCR–Kir6.2 fusion 2.2 Linker optimization for functional coupling 2.3 T4L engineering and site-directed mutagenesis 3. Functional Characterization 3.1 Xenopus oocytes preparation 3.2 mRNA microinjection 3.3 TEVC recordings 4. Current Advantages and Limitations of the ICCR Technology 4.1 Advantages and limitations 4.2 Troubleshooting Acknowledgments References

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Abstract 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 2

Present address: University of Toronto, Toronto, ON M5S 1A8, Canada.

Methods in Enzymology, Volume 556 ISSN 0076-6879 http://dx.doi.org/10.1016/bs.mie.2014.12.017

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2015 Elsevier Inc. All rights reserved.

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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). Twoelectrode 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).

1. INTRODUCTION The ion channel-coupled receptor (ICCR) technology was initially published in 2008 (Moreau, Dupuis, Revilloud, Arumugam, & Vivaudou, 2008) and is based on the fusion of G protein-coupled receptors (GPCRs) to an ion channel. The ion channel acts as a proximal reporter of the receptor function independently of G protein activation. Among the possible application of this GPCR functional assay, the article published in 2014 (Niescierowicz, Caro, Cherezov, Vivaudou, & Moreau, 2014) describes the functional characterization of GPCRs optimized for their crystallization by attaching a fusion domain at the intracellular side and unable to interact with their cognate G proteins. This chapter aims to provide exhaustive details on the design, the characterization, the advantages, and the limitations of this method.

1.1 G protein-coupled receptors GPCRs represent the largest family of membrane proteins in the human genome (Fredriksson, Lagerstr€ om, Lundin, & Schi€ oth, 2003), which are involved in transmission of circulating extracellular messages (neurotransmitters, hormones, signaling lipids, peptides, ions, odor molecules, or photons) to intracellular effectors leading to appropriate cell responses or environment sensing. Activation of G proteins by the ligand-bound GPCR

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results in GDP–GTP exchange and separation of the Gα subunit from the Gβγ subunits. Both separated parts of G proteins bind to different effectors leading to alteration of cytoplasmic concentration of second messengers, such as Ca2+, cyclic AMP, inositol triphosphate, diacylglycerol, or leading to activation of G protein-activated channels (Kir3 channels).

1.2 GPCR crystallization While structural information on GPCRs is essential for understanding the ligand binding specificity and the mechanisms of receptor activation, these proteins are notoriously difficult to crystallize, mainly due to their intrinsic flexibility. The first GPCR structure, that of the bovine visual rhodopsin in the dark state, was determined in 2000 (Palczewski et al., 2000). Rhodopsin, however, is quite different from other receptors, because it is activated by light rather than by a diffusible ligand and in the dark it is stabilized in a fully inactive state by a covalently bound chromophore retinal. Additionally, rhodopsin is expressed at a high level in the eye’s retina and can be extracted and purified from natural sources, while other receptors have to be heterologously expressed for crystallization trials. It took another 7 years until the structure of the first human GPCR (β2-adrenergic receptor) bound to a diffusible ligand was published in 2007 (Cherezov et al., 2007; Rasmussen et al., 2007; Rosenbaum et al., 2007), beginning the era of GPCR structural biology. The main technological advancements that enabled structural studies of GPCRs include (i) development of methods for heterologous expression in insect cells, (ii) discovery of a new method of membrane protein crystallization in lipidic cubic phase (Landau & Rosenbusch, 1996) and subsequent development of tools, instruments, and protocols for automation of crystallization and crystal detection, (iii) development of receptor stabilization technologies, including antibodies toward the intracellular side of GPCRs in Fab form (Rasmussen et al., 2007) and insertion of the T4 phage lysozyme domain (T4L) in the third intracellular (i3) loop (Cherezov et al., 2007; Rosenbaum et al., 2007), and (iv) development of microcrystallography. This toolbox for GPCR crystallization has rapidly expanded with (i) development of an alanine scanning mutagenesis approach enabling receptor stabilization in different functional states Serrano-Vega, Magnani, Shibata, & Tate, 2008; Warne et al., 2008), (ii) identification of other exogenous soluble fusion domains such as the thermostabilized apocytochrome b562RIL (BRIL) (Chun et al., 2012; Liu et al., 2012) and the rubredoxin (Tan et al.,

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2013), (iii) introduction of smaller camelid antibodies called nanobodies (Rasmussen, DeVree, et al., 2011a), and (iv) insertion of soluble domains in the N-terminus (Fenalti et al., 2014; Rasmussen, Choi, et al., 2011; Siu et al., 2013; Wang et al., 2013; Wu et al., 2014) and in the i2 loop (Hollenstein et al., 2013). Alone or in combination, these methods led to the structural determination of 25 other GPCRs from four different classes over the past 7 years. Among all receptor engineering technologies, insertion of soluble domains in intracellular loops frequently combined with thermostabilizing mutations of the receptors has so far provided the highest number of crystallized receptors. While engineering GPCRs facilitates their crystallization, it could also affect their function. Standard GPCR functional assays are based on G protein activation or β-arrestin recruitment. These assays are used to characterize optimized GPCRs for crystallization that keep the G protein binding site intact, such as T4L addition at the GPCR N-terminus [(T4L) GPCR], stabilizing antibodies, and thermostabilizing mutants. However, the steric hindrance engendered by the insertion of soluble domains in the i3 loop [GPCR(T4L)] obstructs the access of G proteins or β-arrestins to their binding site and, consequently, makes the standard G protein or β-arrestins assays obsolete. β-Arrestin-based assays are also impeded by truncation of the GPCR C-termini. Functional characterization of GPCR(T4L) is therefore limited. The method of choice consists of comparing binding constants between wildtype (wt) and engineered receptors with radioligand binding or competition assays. Results give invaluable information on possible alterations of the ligand binding site. But to assess potential impacts on subsequent conformational changes of the activated receptors, additional methods were developed such as fluorescence spectroscopy of a bimane probe attached at the cytoplasmic end of the helix VI in the β2-adrenoreceptor (Rosenbaum et al., 2007), luminescence resonance energy transfer between the cytoplasmic end of the helix VI and the C-terminus of the arginine-vasopressin type 2 receptor (Rahmeh et al., 2012), or FRET between intra and intersubunits of different GPCRs (Hlavackova et al., 2012; Monnier et al., 2011). In 2008, we developed a complementary approach based on an electrical signal generated by an ion channel fused to the GPCRs C-termini. This method, designated ICCR, is suitable for the functional characterization of GPCR(T4L) as it does not rely on G protein activation and requires C-terminal truncation of long C-terminal domains. The following sections explain the ICCR technology in more details.

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1.3 ICCR history and principle 1.3.1 Origin of the concept ICCRs are artificial ligand-gated ion channels created by fusion of GPCRs C-termini to an ion channel, which creates an original sensor able to generate an electrical signal that correlates with receptor activity. This concept was inspired by a natural channel, the ATP-sensitive potassium channel in which the homotetrameric potassium channel subunit Kir6.x is allosterically regulated by four sulfonylurea receptor (SUR) subunits. Binding of ligands on SUR regulates the Kir6.x gating by long-range propagation of conformational changes from the SUR ligand binding sites to the channel gates. Therefore, Kir6.x channels naturally possess all molecular determinants to be regulated by another membrane protein, a characteristic that led to the ICCR concept (Fig. 1).

1.3.2 Brief introduction to the methods As GPCRs do not interact spontaneously with Kir6 channels, they were fused to the Kir6.2 channel subunit by genetic engineering as described in details in Section 2.1. The simplest fusion strategy consists of linking the cytoplasmic GPCR C-terminus to the cytoplasmic channel N-terminus as schematized in Fig. 2. GPCR cDNAs are fused to the Kir6.2 cDNA by two-step PCRs. The vector we used is derived from the pGEMHE vector designed for overexpression of proteins in Xenopus oocytes. The ICCRs are heterologously expressed in Xenopus oocytes, which are commonly used in ion channel studies for the reasons that they are large cells (1 mm in diameter), easy to handle (single-cell manipulation, microinjection of nucleic acids), easy to maintain (salt buffers with antibiotics, incubator at 19°C in standard laboratories) (Cens & Charnet, 2014), and having a plasma membrane homologous to that of mammalian cells in terms of lipid composition (cholesterol (Hill et al., 2005), phosphatidylinositol 4,5-bisphosphate (PIP2),. . .). Protein expression is obtained by microinjection of mRNA that is synthesized by in vitro transcription. Optimal expression of ICCRs at the plasma membrane is obtained after 2 days of incubation at 19 °C of the injected oocytes. The functional characterization of the fusion proteins was performed with the two-electrode voltage-clamp (TEVC) technique. This technique basically consists of impaling a Xenopus oocyte with two electrodes for clamping the membrane voltage and recording ion currents during

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Figure 1 Ion channel-coupled receptor (ICCR) principle. (A) Simplified schematic of the natural KATP channels composed of the Kir6.2 pore subunit and the regulatory sulfonylurea receptor subunit. The real molecular architecture is four Kir6.2 subunits surrounded by four SUR subunits. Binding of potassium channel openers (KCOs) on SUR triggers conformational changes of SUR that are transduced to Kir6.2, resulting in opening of the channel, flow of potassium ions, and generation of an electrical signal. (B) ICCRs were created with the same principle by genetic fusion of G protein-coupled receptors (GPCRs) to Kir6.2. Binding of ligands on GPCRs generates an electrical signal that is easily detectable with standard electrophysiological techniques. The real architecture of ICCRs is four Kir6.2 surrounded by four GPCRs.

applications of ligands on the whole oocyte. TEVC offers the advantages of an absence of membrane protein purification step, real-time observation of ion channel activity, application of ligands on the extracellular side of the plasma membrane, and low-cost buffers. 1.3.3 ICCR surface expression is independent of the Kir6.2 endoplasmic reticulum retention signal A recurrent difficulty with modified membrane proteins is the low level of expression at the plasma membrane because these proteins do not pass through the cell’s quality control machinery. The first tested fusion of

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Figure 2 Methods used for the design and the functional characterization of ICCRs. Genetic fusion of GPCRs to Kir6.2 is obtained by two-step PCR, resulting in precise insertion of the GPCR cDNA in 50 of the Kir6.2 cDNA. The plasmid used (pGEMHE) is dedicated to protein overexpression in Xenopus laevis oocytes. mRNA is obtained by in vitro transcription before microinjection in Xenopus oocytes. After 48 h at 19 °C, ICCRs are functionally characterized by the two-electrode voltage-clamp (TEVC) technique that provides real-time recordings of whole-cell currents. In our conditions (50 mV, high external K+) and by convention, the current is negative. The channel is partially open (red (gray in the print version) trace) in basal state and can be activated by GPCR ligands (blue (black in the print version) trace) or blocked by potassium channel blockers such as Ba2+ (gray trace).

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full-length M2 and Kir6.2 proteins (noted M2-K) revealed a correct expression of this artificial channel into the plasma membrane of Xenopus oocytes. The surface expression was estimated by the current amplitude generated by Kir6.2 in the basal state. Because each open channel produces a unitary current of the same amplitude, whole-cell currents are the sum of unitary currents from all simultaneously open channels. Surprisingly, the surface expression of M2-K was not affected by the presence or absence of a well-known endoplasmic reticulum (ER) retention signal in Kir6.2 that controls the stoichiometry of the natural SUR/Kir6.x octameric complex (Tucker, Gribble, Zhao, Trapp, & Ashcroft, 1997). This indicates that the fused M2 receptor is able to mask the Kir6.2 ER retention signal either directly or indirectly via interacting proteins. 1.3.4 Experimental proof of concept of ICCRs To test the feasibility of the ICCR concept, the human muscarinic M2 receptor was chosen as an archetypal GPCR. Direct fusion of the M2 receptor to Kir6.2 resulted in nonfunctional ICCR, since application of the endogenous M2 agonist acetylcholine (ACh) did not trigger the expected change in current (Moreau et al., 2008). To rule out the hypothesis that the receptor activity could be affected by its fusion to the channel, we used a G protein-activated potassium channel (Kir3.x) as a reporter of G protein activation by the M2 receptor. The M2 receptor is preferentially coupled to Gi/o proteins, which activate Kir3.x channels through interaction with Gβγ subunits. Coexpression of M2-K and Kir3.x channels in Xenopus oocytes and activation of Kir3.x upon ACh application demonstrated that the fused receptor was functional (Fig. 3). The Kir6.2 activity was also controlled, and the fused channel did not show any differences in sensitivity to its physiological ligand (ATP) compared to the unfused channel. Since both proteins were functional in the fusion construct, the absence of signal in the presence of ACh meant a lack of communication between the receptor and the channel. This critical problem has been solved by shortening the linker region (Fig. 4). The nomenclature used to name the engineered ICCR is: GPCR-K-x-y; where x is the number of residues deleted at the GPCR C-terminus and y is the number of residues deleted at the Kir6.2 N-terminus. For instance, an ICCR created by truncations of the last 10 residues of M2 and the first 20 residues from Kir6.2 is written M2-K-10-20. Functional characterization of all M2-K ICCRs with shortened linkers revealed that the signal amplitude increased with increasing deletions of the

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Figure 3 G protein activation assay based on Kir3.x channels. (A) Schematics of the oocyte plasma membrane containing a muscarinic ICCR insensitive to the GPCR ligand (left panel) and the same ICCR coexpressed with the Kir3.4* channel (right panel). Kir3.4* stands for the Kir3.4S143T mutant that forms homomeric channels. Activation of endogenous Gi/o proteins by the fused GPCR is detected by activation of the Kir3 channel. (B) TEVC traces showing the Kir6.2 current generated by the ACh-insensitive ICCR (left panel) and the current predominantly generated by Kir3.4* channels. Activation of Kir3 channels by ACh at 5 μM demonstrates the ability of the fused GPCR to activate Gi/o proteins upon ligand binding. Ba2+ is at 3 mM in all recordings. Adapted from Moreau et al. (2008).

channel N-terminus. An optimum signal was observed with 25 residues deleted in Ki6.2 (M2-K-0-25). An optimum and a limit were observed with 25 residues deleted in Ki6.2 (M2-K-0-25). Accordingly, truncation of the first 25 residues of Kir6.2 is a prerequisite for the design of new functional ICCRs based on other GPCRs. At the date of publication of this article, four additional GPCRs have been reported to be functionally fused to Kir6.2: the human long D2L dopaminergic receptor (Moreau et al., 2008), the bovine opsin receptor (Caro, Moreau, Estrada-Mondrago´n, Ernst, & Vivaudou, 2012), the human β2-adrenergic receptor (Caro, Moreau, Revilloud, & Vivaudou, 2011), and the human oxytocin (OXT) receptor (Niescierowicz et al., 2014). All these receptors were directly fused to Kir6.2 deleted with the first 25 residues, but only D2L (and M2) produced a functional ICCR with intact receptor C-terminus. The opsin, β2, and OXT receptors possess much

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Figure 4 Engineering of ICCRs for functional coupling of the M2 GPCR to Kir6.2. (A) Schematic of the M2-K ICCR omitting front and rear subunits for clarity. The C-terminus of the M2 muscarinic receptor is fused to the N-terminus of Kir6.2. H8 represents the last α-helix of the GPCR, parallel to the membrane plane. β1 is the first secondary structure observed in crystallographic structures of Kir channels and forms a small β-sheet. (B) Zoom in the fusion region showing all deletions performed in the M2 C-terminus or in the Kir6.2N-terminus. The nomenclature is M2-K-x-y, x being the number of truncated residues in M2 and y the ones in Kir6.2. (C) Bar chart showing the percentage of current change induced by 5 μM of acetylcholine. The optimal deletion providing the highest current amplitude corresponds to the deletion of 25 residues in Kir6.2 (M2-K-0-25). Adapted from Moreau et al. (2008).

Figure 5 C-terminal engineering of GPCR for functional coupling. Alignment of all published GPCRs that created functional ICCR after truncation of their C-terminus. Markers define the position of the D2L-like and the M2-like C-termini.

longer C-terminal domains than M2 and D2L receptors (Fig. 5) and, consequently, extend the fusion region that hinders the coupling with Kir6.2. Functional coupling was obtained by shortening the GPCR C-terminal domains down to a size matching that of the C-terminal domain of M2 or D2L. This strategy was successful and is now used routinely for the

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creation of new ICCRs. Therefore, any new GPCRs fused to ΔN25Kir6.2 are created in three forms: (1) full-length receptor, (2) M2-like C-ter deletion, and (3) D2L-like C-ter deletion. Localization of the GPCR C-terminal deletions is simplified by the presence of upstream residues conserved in most GPCRs, like the cysteine residue at the end of helix VIII serving as a putative palmitoylation site. Recent attempts to couple GPCRs lacking that cysteine are still unsuccessful probably due to the variability in the helix VIII length and the difficulty to accurately predict the end of this helix in the primary sequence. In the case of the β2 and the opsin receptors, surface expression of the ICCRs required coexpression of the first transmembrane domain (TMD0) of SUR that spontaneously interacts with Kir6.2 (Chan, Zhang, & Logothetis, 2003) and acts as a chaperone in ICCRs. TMD0 is now routinely used for surface-expression-deficient-ICCRs to boost their expression into the Xenopus oocyte plasma membrane. Unexpectedly, we have observed that the sign of the signal can be inverted depending on the fused GPCR. Thus, binding of agonists on M2 and β2 receptors resulted in an increase of the current amplitude, while binding of agonists on D2L and light-activation of rhodopsin partially closed the channel. When Kir6.2 is neither fully open nor fully closed, ICCRs can detect both activation and inhibition of the channel. The level of activity of Kir6.2 is controlled by the intracellular concentration of ATP that inhibits the channel. The ATP sensitivity of ICCR-embedded Kir6.2 is similar to that of the unfused channel (IC50  100 μM) (Moreau et al., 2008; Tucker et al., 1997), resulting in partially active Kir6.2 channels in Xenopus oocytes. 1.3.5 Pharmacological properties of ICCRs ICCRs detect agonists with the same specificity as the fused GPCR. The halfmaximal effective concentration (EC50) is determined by concentration– effect curves obtained by successive applications of increasing concentrations of ligands over the same oocyte. The duration of the applications is adjusted from a few seconds to a few minutes, depending on the on-rate of ligand binding. While real-time recordings are performed on single cells, statistics are calculated from data obtained on different oocytes from different batches. ICCRs detect neutral antagonists. Antagonist effects are studied by recording the response to a known agonist in the absence and in the presence of the investigated compound. Antagonists abolish the agonist effect leading to signal amplitude returning back to the basal current level (Fig. 6).

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Figure 6 Antagonist sensitivity of ICCRs. TEVC traces showing the antagonism of atropine (left panel) and sulpiride (right panel) on M2 and D2L ICCRs, respectively. The agonists acetylcholine (ACh) and quinpirole and the antagonist sulpiride are at 5 μM, while atropine is at 1 μM. Adapted from Moreau et al. (2008).

Other classes of ligands have been tested such as the partial agonist salbutamol for the β2-adrenoreceptor (Caro et al., 2011), but the maximal response was similar to the one obtained with the full agonist isoproterenol. This demonstrates a lack of discrimination between partial and full β2adrenoreceptor agonists of the ICCR assay. Inverse agonists, such as oxybutinin on the M2 receptor and ICI118,551 on the β2-adrenoreceptor, were also tested but they did not produce any detectable signal with the ICCR technology. 1.3.6 Assessment of G protein activation with Kir3.x channels To test if the fused GPCRs are still able to activate heterotrimeric G proteins, we used a reporter of G protein activation: the G proteinactivated potassium channels (Kir3.x) (Picciocchi et al., 2014). Agonistinduced activation of Gi/o protein-coupled receptors triggers the release of the Gβγ subunits, which activate the Kir3.x channels (Fig. 3). When coexpressed with ICCRs, Kir3.x clearly reports the Gi/o protein activation by a large increase of the potassium current amplitude. In this configuration, the measured current is the sum of the currents generated by the ICCRembedded Kir6.2 and the Kir3.x channels. This does not interfere with the measurements because the Kir3.x channels are robustly expressed and produce a larger signal than Kir6.2. Gi/o proteins are endogenously expressed in Xenopus oocytes, and experiments with the opsin receptor demonstrated that they can also replace the Gt proteins (transducin). Moreover, we extended this Kir3 assay to Gs protein-coupled receptors, such as the β2-adrenoreceptor, by coexpressing and overexpressing the Gαs subunits in Xenopus oocytes (Lim, Dascal, Labarca, Davidson, & Lester, 1995), and

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in this condition, we observed activation of Kir3.x channels by β2-adrenergic agonists. Gq proteins are also endogenously expressed in Xenopus oocytes. Their activation triggers the activation of the endogenous phospholipase C, resulting in depletion of PIP2 and transient elevation of cytosolic Ca2+ concentration. PIP2 being a mandatory phospholipid for opening Kir6.2 and Kir3.x channels, activation of Gq protein-coupled receptors hinders the ICCR and Kir3.x assays by closing the two channels. This problem was encountered with the wt OXT receptor which is coupled to Gq and Gi/o proteins: no current could be observed for the wt OXTR-K construct. Testing a specific Gq protein inhibitor would validate this interpretation and extend the ICCR and Kir3.x assays to Gq protein-coupled receptors. A new Gq protein inhibitor is now commercially available (UBO-QIC, University of Bonn, Germany), but it has not been tested yet on ICCRs. As Kir6.2 and Kir3.x channels are both inward-rectifying potassium channels, recording of Kir6.2- or Kir3.x-generated currents is performed in the same experimental conditions, including buffers composition and imposed membrane potential. We also simplified the Kir3.x assay with single point mutants of Kir3.x subunits. The Kir3 family is composed of four human isoforms from Kir3.1 to Kir3.4. In physiological conditions, Kir3.x channels are mainly heterotetramers composed, for instance, of the Kir3.1/Kir3.4 subunits in the heart or Kir3.1/Kir3.2 in the brain. Mutations of Kir3.1 in F137S (Kir3.1*) or Kir3.4 in S143T (Kir3.4*) create Kir3 channels that are able to form homotetramers (Vivaudou et al., 1997). Thus, coinjection of only one Kir3.x*-coding RNA with ICCR-coding RNA is sufficient to perform the G protein activation assay. 1.3.7 Extrapolation of the ICCR concept to GPCR(T4L) The principle of the ICCR technology is based on the detection of GPCR conformational changes through the regulation of the fused Kir6.2. This process is independent of G protein actions, even though G proteins still interact with the GPCRs in ICCR as demonstrated by the Kir3.x assay. Indeed, recording the activity of ICCRs in excised pieces of plasma membrane (outside-out patch-clamp) or in the presence of toxins-inactivating Gi/o proteins (pertussis toxins), demonstrated that ICCRs are activated independently of G protein actions. Based on this observation, we envisioned that ICCR could serve as functional assay for engineered GPCRs that are unable to interact with

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G proteins. One well-known example of such G protein-“uncoupled” receptors are the GPCRs optimized for crystallization by replacement of the i3 loop by the T4L domain. The T4L domain prevents the interaction with G proteins and impedes the use of standard G protein-based assays. Since ICCRs acts as reporters of agonists- and antagonists-induced conformational changes, this method would be a complementary assay for detecting altered-states of ligand-bound GPCR(T4L) without protein purification steps. Three different GPCR(T4L) were used to validate this concept: the M2(T4L), β2(T4L), and the OXT(T4L) receptors. M2(T4L) and β2(T4L) were created by insertion of the T4L domain in functional ICCRs, while the OXTR(T4L) was fused entirely to the channel as it was already optimized for crystallization attempts (T4L insertion in the i3 loop, codon optimization for insect cell expression, and truncation of the last 42 residues). Optimized OXTR(T4L) is well-suited to the ICCR technology as it lacks the long C-terminal domain of the wild-type, which is a prerequisite for the functional coupling to Kir6.2. Unexpectedly, the presence of the T4L domain in the i3 loop abolished the surface expression of the 3 ICCR(T4L), but deletion of the Kir6.2 ER retention signal restored it. Simple deletion of the i3 loop (GPCRΔi3) did not induce the same consequences, suggesting that insertion of the T4L domain could disrupt an interaction with a GPCR partner, possibly an escort protein (Achour, Labbe´-Jullie´, Scott, & Marullo, 2008), that masks the Kir6.2 retention signal in the ER. Consequently, surface expression of ICCR(T4L) requires the truncation of the last 36 residues of Kir6.2 to remove the ER retention signal. In the case of the β2(T4L) ICCR, coexpression of the TMD0 was also required for sufficient surface expression as it was the case for the wt β2 ICCR. Functional characterization of GPCR(T4L)-K revealed that ICCRs are indeed able to detect agonists- and antagonists-induced conformational changes of GPCR(T4L) as observed for wt GPCR-K (Fig. 7). Concentration–effect curves indicate an unchanged EC50 between GPCR(T4L) and wt GPCR which is in agreement with the unchanged Kd measured by radioligand binding and competition assays. The only difference observed between GPCR-K and GPCR(T4L)-K was a lower maximal effect for the T4L constructs, suggesting that the engineered receptors are in partial-active state. Intermediate active states of agonist-bound receptors were also observed in crystallographic structures of optimized GPCRs

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Figure 7 Functional characterization of ICCR(T4L). (A) TEVC traces showing the inhibition by ACh of an engineered M2 ICCR (left panel) and the same response of the M2(T4L) ICCR (right panel). M2(T4L) is engineered by replacement of the M2 third intracellular loop with the phage T4 lysozyme domain. As demonstrated by the effect of atropine that blocks the ACh-induced activation, the ICCR(T4L) is also sensitive to antagonists. ACh is at 5 μM and atropine at 1 μM. KΔ means truncation of the last 36 residues of Kir6.2 in order to remove an endosplasmic reticulum retention signal. (B) Concentration–effect curves of carbachol (CCh), a synthetic muscarinic agonist, of the M2, M2(T4L), and M2(Δi3) ICCRs. M2(Δi3) corresponds to the M2 receptor deleted of its third intracellular loop. Adapted from Niescierowicz et al. (2014).

(Venkatakrishnan et al., 2013) and by NMR studies (Kim et al., 2013; Nygaard et al., 2013), the fully active state being reached in the presence of G proteins or stabilizing nanobodies. Surprisingly, the M2 ICCR lacking the i3 loop (M2Δi3-K) displayed the same partial agonist phenotype while it was still able to interact with G proteins, indicating that the i3 loop could be involved in the stabilization of the receptor in the fully active state. Comparison of the concentration–effect curves of wt OXTR-K and OXTR(T4L)-K was not possible due to the lack of recorded current of the wt ICCR. Postulating that this absence of current is due to the Gq protein activity, we expect to overcome this obstacle with the recently commercially available Gq protein inhibitor: UBO-QIC (University of Bonn).

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2. ICCR DESIGN 2.1 Genetic engineering of the GPCR–Kir6.2 fusion 2.1.1 Materials • cDNA of GPCRs • cDNA of Kir6.2 in pGEMHE vector. Ideally the gene deleted of its first 25 codons • Quikchange Lightning PCR kit (Ambion) • Geneclean kit (Qiagen) • PCR machine (Eppendorf ) • DNA electrophorese set up • Low melting agarose • TAE buffer • RNAse-free space and equipments (centrifuge, micropipettes, tips, tubes) • RNAse-free milli-Q water • mMessage mMachine T7 transcription kit (Life Technologies) 2.1.2 Genetic engineering The fusion of the GPCR to the Kir6.2 channel is performed by linking the 30 end of the receptor cDNA to the 50 end of the Kir6.2 cDNA. As the size of the fusion region being critical for the function of ICCRs, insertion of restriction sites for enzymatic subcloning is not practical. Consequently, we used a two-step PCR protocol for subcloning (Fig. 8). The first step consists of amplifying the receptor gene with additional flanking regions that will recognize the Kir6.2 pGEMHE template at the exact fusion position. The forward and reverse oligonucleotide primers are 50 nucleotides long and their 30 moiety hybridizes to the receptor gene extremities while their 50 moieties will hybridize the pGEMHE and the Kir6.2 sequence in the second PCR. • Typical PCR conditions are described in Table 1, but they could require optimizations of the hybridization temperature and template quantity: • Ten microliters of 6  DNA loading buffer are added to the total PCR product and loaded into a 0.8% (w/w) low-melting agarose gel. • Migration in standard tris–acetate–EDTA buffer at 100 V approximately 40 min for a 6-cm-long gel. • The band corresponding to the size of the GPCR gene is extracted from the gel and purified according to the standard protocol of the Geneclean kit.

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Figure 8 Genetic engineering of ICCRs by two-step PCR. The GPCR cDNA is amplified in a first PCR with hybrid primers containing in 50 the sequences of the PCR2 template that overlap the site of insertion. pX means any plasmid. The PCR1 product is purified from an agarose gel and used as “megaprimers” for the second PCR. In PCR2, the template is Kir6.2 cDNA inserted in the pGEMHE vector. The final product corresponds to the ICCR cDNA inserted in the pGEMHE vector.

The PCR product of the first PCR is used as “megaprimers” for a second PCR with the Kir6.2 cDNA already inserted in the pGEMHE vector (Liman, Tytgat, & Hess, 1992). This vector contains untranslated regions of the Xenopus laevis globin that favor overexpression of the recombinant proteins (Fig. 9).

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Table 1 PCR1 conditions Reaction mix

PCR program

Solution

Volume (μl)

Cycle

Temperature (°C)

Time

dH2O

QS 50

1

95

2 min

10  buffer (kit)

5

40

95

30 s

GPCR cDNA (100 ng/μl)

1

60

30 s

Pimer F (20 μM)

1.25

68

30 s/kb

Pimer R (20 μM)

1.25

68

30 s/kb

dNTPs (kit)

1

6

1

Quiksol (kit)

1.5

Pfu Turbo polymerase (kit)

1

1

Figure 9 The pGEMHE vector. This vector contains 50 and 30 Xenopus globin UTRs that enhance protein expression in Xenopus oocytes. Inserts are under control of the T7 promoter. The linearization site located downstream of the polyA-polyC sequence contains unique restriction sites different from the multicloning site (MCS). Apr stands for ampicillin-resistance coding sequence.

•

• •

Typical conditions for the second PCR are described in Table 2: Tip: Heating the “megaprimers” at 95 °C for 2 min, mixing, and cooling down the sample rapidly on ice could increase the efficiency of the reaction. Carefully digest at 37 °C the PCR product with 1 μl of DpnI provided in the kit. Transform by thermic-shock (42 °C 45 s, ice 2 min) 45 μl of the ultracompetent XL10-Gold Escherichia coli bacteria provided in the kit with 2–5 μl of the DpnI-treated PCR2 product.

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Table 2 PCR2 conditions Reaction mix

PCR program

Solution

Volume/ quantity

Temperature Cycle (°C) Time

dH2O

QS 50 μl

1

95

2 min

10  buffer (kit)

5 μl

30

95

30 s

Kir6.2 pGEMHE (100 ng/μl)

1 μl

58

1 min

Purified PCR1 product: “Megaprimers” (ideally >50 ng/μl)

500 ng

68

30 s/kb

dNTPs (kit)

1 μl

68

30 s/kb

Quiksol (ki)

1.5 μl

6

1

Pfu Turbo polymerase (kit)

1 μl

•

• •

•

1

Add 250 μl of S.O.C. medium (Hanahan, 1983) and incubate 1 h at 37 °C. Spread the whole volume on an LB-agar plate containing ampicillin and incubate overnight at 37 °C. Tip: In absence of colonies, repeat the PCR2 and decrease the temperature of hybridization. Pick few colonies for overnight culture at 37 °C in 2–5 ml of LB medium with ampicillin. Purify the plasmid with standard Qiagen Miniprep protocol and analyze the restriction profile to identify positive clones containing the GPCR– Kir6.2 fusion. From positive clones, purify the plasmid with the cleaner Qiagen Midiprep kit and sequence the full coding sequence for control of random mutations. Tip: A simpler but more expensive alternative consists in ordering the desired GPCR-K-0-25 synthetic gene subcloned in the pGEMHE vector. Delivery periods are generally within 3 weeks.

2.1.3 mRNA synthesis Microinjections of nucleic acids in Xenopus oocytes are possible under DNA or RNA forms. Injection of DNA avoids the delicate manipulation of RNA but it requires more expertise for its injection in the nucleus. We prefer to inject mRNA in the cytosol and the pGEMHE vector has been specifically designed for in vitro synthesis of mRNA. The kit we routinely used is the

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mMessage mMachine T7 (Life Technologies), the ICCR coding sequence being under the control of the T7 promoter. • All operations are performed in RNAse-free conditions. • Linearize overnight 10 μg of the Midiprep-purified DNA using the unique restriction sites inserted in 30 of the gene and after the poly-A sequence. • Purify the DNA using standard phenol/chloroform protocol and control the presence of a single band at the expected size by agarose gel electrophoresis. • Prepare the transcription reaction mix according to the protocol from the kit and incubate 2 h at 37 °C. • RNA is purified with the standard phenol/chloroform protocol, and RNA pellets are resuspended in 20 μl of RNAse-free water. • The quality of RNA is estimated by nondenaturating agarose gel electrophoresis and quantified by UV spectrophotometry. • An intermediate 3  stock is prepared at 0.26 μg/μl for a fusion protein of 800 residues-long (Kir6.2 ¼ 390 residues). • Ready-to-inject mRNA stock is prepared by adding 1 μl of the 3  intermediate stock in 2 μl of RNAse-free water. The total volume of 3 μl is enough for the injection of 50 oocytes. All RNA stocks are stored at 80 °C. • mRNA coding for Kir3.x channels and TMD0 (F195 from SUR1, Chan et al., 2003) are prepared from cDNA inserted in pGEMHE, and concentrations of the mRNA 3 intermediate stock are 0.15 and 0.06 μg/μl, respectively. • Coinjections of Kir3.x or TMD0 mRNA with ICCR are made by mixing the following volumes of 3  mRNA stock: 1 μl ICCR + 1 μl Kir3.x or TMD0 + 1 μl RNAse-free water.

2.2 Linker optimization for functional coupling Once the GPCR is fused to Kir6.2 deleted of its first 25 residues (GPCR-K0-25), the functional coupling often requires deletions of the GPCR C-terminus to mimick the M2 and the D2L C-terminus length. Precise deletions are obtained by one-step PCR using symmetrical primers overlapping both sides of the deletion region (Fig. 10). The same PCR kit (Quikchange Lightning; Agilent) and the same conditions as the previous PCR1 are used. The constructs follow the same protocol from DpnI digestion to Midiprep DNA purification as previously described.

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Figure 10 Deletions by PCR. Symmetric primers overlapping the region to delete are used in classical mutagenic PCR to adapt the size of the fusion region of ICCRs.

2.3 T4L engineering and site-directed mutagenesis The ICCR(T4L) are created either by insertion of the T4L domain or by direct fusion of the GPCR(T4L) to ΔN25Kir6.2 using in both cases the two-step PCR protocol. In the first case of T4L insertion, the T4L sequence is amplified from a template containing the T4L domain and supplemented with flanking sequences recognizing both sides of the site of insertion. During the second PCR, the i3 loop is simultaneously deleted when the “megaprimers” containing the T4L sequence is inserted. The insertion of the T4L domain in place of the i3 loop abolishes surface expression of the ICCRs, unless the Kir6.2 ER retention signal is removed. This is easily obtained by truncation of the last 36 residues (Tucker et al., 1997) of the channel with a stop codon inserted by PCR. wt Kir6.2 C-terDEDHSLLEALTLASARGPLRKRSVPMAKAK PKFSISPDSLS Kir6.2ΔC36 DEDHS1 RKR is the ER retention signal (Zerangue, Schwappach, Jan, & Jan, 1999). While only insertion of the T4L domain was tested, the ICCR can theoretically be extrapolated to other soluble domains such as the cytochrome BRIL domain also inserted in place of the i3 loop of some crystallized GPCRs (Chun et al., 2012). Frequently, additional thermostabilizing mutations are inserted in crystallized GPCR(T4L) or GPCR(BRIL). The functional impacts of such mutations require the characterization by standard G protein-based assays of GPCRs with intact i3 loop. The ICCR could simplify this step by 1

Stop codon.

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performing the functional characterization of these mutants directly in the ICCR(T4L/BRIL). Mutations are inserted in the ICCR(T4L/BRIL) by PCR, or the full-length mutated GPCR(T4L/BRIL) is directly fused to ΔN25Kir6.2.

3. FUNCTIONAL CHARACTERIZATION 3.1 Xenopus oocytes preparation 3.1.1 Material • Mature X. laevis females (Xenopus Express company), in governmentapproved animal facilities, protocols, and personnel training • Thin forceps • Collagenase from Clostridium histolyticum type IA (Sigma-Aldrich #C9891)) • Glass Pasteur pipette adjusted to the size of oocytes • 60-mm Plastic Petri dishes • Orbital or rotary agitator • Incubator at 19 °C (Thermostat cabinet; Aqualytic) • Stereo binocular microscope (Leica #MZ6) 3.1.2 Protocol • X. laevis oocytes are surgically removed using a standard protocol (Nakagawa & Touhara, 2013). Tip: Several service companies deliver ready-to-inject oocytes (Ecocyte Bioscience, Nasco, Xenoocyte). By experience, nonisolated oocytes are less fragile and stored longer. Freshly extracted oocytes are attached in “grape form” to conjonctive tissues. They are enzymatically individualized with collagenase. This step is critical for the quality of oocytes: overdigestion weakens the oocytes while underdigestion impedes the microinjection step. Following parameters are given for 5 ml of oocytes in 15-ml tube. Variation of incubation time has been observed from a lot of collagenase to another. • Mechanically open the oocyte-containing lobes with thin forceps (or a scalpel). • Incubate them in a small Petri dish or 15 ml tube containing 2 mg/ml of collagenase in Ca2+-free buffer A (Barth’s modified buffer). Buffer A: NaCl 88 mM, KCl 1 mM, NaHCO3 2.4 mM, HEPES 16 mM, MgSO4 0.82 mM, pH 7.4. • Agitate under gentle rotation for 2 h at 19 °C.

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•

• • • • •

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Control the completeness of the digestion: majority of isolated oocytes without apparent blood vessels on their surface. If oocytes are underdigested, incubate them longer with controls every 15 min. Carefully wash the oocytes in 50-ml tubes with buffer A: 5  5 min. Rinse with 50 ml buffer B. Buffer B: Buffer A + Ca(NO3)2 0.3 mM, CaCl2 0.41 mM, pH 7.4. Manually select healthy stage V–VI oocytes, which are the biggest ones with two hemispheres of dark and white colors. Incubate them overnight at 19 °C in Buffer B + Penicillin 100 U/ml, streptomycin 100 μg/ml, gentamycin 100 μg/ml. The day after, remove the few dead oocytes before microinjection.

3.2 mRNA microinjection 3.2.1 Material • NanoJect II (Drummond Instrument) • Pipette puller P97 (Sutter Instrument) • Capillaries 3.500 Drummond #3-000-203-G/X (Drummond) • 96-Well plates with conical bottom, Nunc (VWR # 732–0812) • 60-mm plastic Petri dish with dark background and bottom nylon mesh (0.8 mm grid). • Glass Pasteur pipette adjusted to the size of oocytes. • Microforge MF-83 (Narishige) • Light mineral oil (Sigma-Aldrich #M5904) • Hypodermic needle (Popper) • Luer-lock 20 ml syringe. • Stereo binocular microscope • Mini centrifuge 3.2.2 Protocol • Pull the pipettes with the P97 puller. Parameters (may vary with filament type and position): Heat ¼ 460, Pull ¼ 50, Vel ¼ 20, Time ¼ 30, P ¼ 500. Ramp ¼ 462 • Precisely break the pipettes using the microforge: seven divisions with the 33 lens. • Using the syringe with the hypodermic needle, fill one pipette with the mineral oil. • Fit the pipette to NanoJect (with piston pushed out three-fourth of the way).

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Fast spin the 1  mRNA solution to pellet potential impurities that could clog the pipette. • Fill the pipette with the mRNA solution. Three microliter for 50 oocytes. • Place the oocytes in the Petri dish lined with the grid to immobilize them • Inject oocytes one by one with 50 nl of mRNA solution. • Store the injected oocytes individually in wells of a 96-well plate filled with 180 μl of Buffer B + antibiotics. Label the lid with the injected construct. • Change the pipette and repeat the injection for different mRNAs. • Incubate the oocytes at 19 °C for >48 h without agitation. Alternatives: (1) The MultiChannel Systems company sells the RoboInject automate, which collects mRNA and injects automatically oocytes distributed manually in 96-well plates. Rapidity and simplicity are the advantages of this robot, while the higher required volume (6–10 μl), the absence of needle change (wash in RNAse-free water), and the lower success rate compared to manual injections are its weaknesses. (2) Some companies like Ecocyte Bioscience also offer mRNA and even cDNA microinjection of Xenopus oocytes. We did not try this type of service. •

3.3 TEVC recordings 3.3.1 Material Manual setup • Computer • Faraday cage • Digidata 1440A (Axon Instruments) • Amplifier Geneclamp500B (Axon Instruments) • Stereo binocular microscope • Light source with optic fiber (Hund Wetzlar # FLQ150) • Solution exchanger ValveLink 8.2 (Automate Scientific) • Vacuum or peristaltic Pump • Antivibration table • pClamp10 software • “High-K+” buffer: CaCl2 1.8 mM, HEPES 5 mM, KCl 91 mM, MgCl2 10 mM, pH 7.4. • Pipette puller P97 (Sutter Instrument) • Capillaries KimbleChase #34502-99 0.8-1.1  100 mm Robot setup • Hi-Clamp automate • Hi-Clamp magnetic microstirrers

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• • • • • •

Computer 96-Well plates with conical bottom Nunc (VWR # 732–0812) “High-K+” buffer ND96 buffer: NaCl 91 mM, KCl 2 mM, CaCl2 1.8 mM, MgCl2 1 mM, HEPES 5 mM, pH 7.4 Pipette puller P97 (Sutter Instrument) Thin wall capillaries with filament ref: TW150F-4 (WPI)

3.3.2 Protocol for manual TEVC • Pull the pipettes in the P97 puller. Parameters (may vary with the filament position): Heat ¼ 462, Pull ¼, Vel ¼ 28, Time ¼ 60. Ramp ¼ 462 • Fill the pipettes with 3 M KCl, remove the potential bubbles by gentle flicking, and install them in the pipette holder. • Prepare the solutions with the High-K+ buffer, including the ligands and a 3 mM BaCl2 solution (K+ channel blocker) • Fill the solution reservoir in the following order: (1) High-K+ buffer (control) • • • • •

•

• •

(2) Ligand

(3) BaCl2

Install the oocyte chamber under the binocular, and set the perfusion head, ground electrodes, and the pump system for solution waste. Place an oocyte in the oocyte chamber Position the pipettes extremity in the flowing high-K+ buffer Control the pipette resistance (1 MΩ) and reset to 0 the junction potential Impale the oocyte and start the pClamp protocol. Parameters: Episodic stimulation, 0 mV 300 ms, 50 mV 500 ms, 0 mV 500 ms, +50 mV 500 ms, holding potential 0 mV. Total ¼ 5 s ¼ 1 sweep repeated in continuous loop. Surface expression of ICCR is observed with current >0.2 μA and lower current amplitude at +50 mV compare to the one at 50 mV. This asymmetry of current amplitude is called rectification and is a signature of Kir channels due to intracellular Mg2+ and polyamines that partially blocks the outward K+ flow. Start the recording on the pClamp software. When the signal is stable, change solutions in the following order for testing one ligand concentration: (2) Ligand (3) High-K+ buffer (4) BaCl2. (1) High-K+ buffer Note the time or number of sweeps for each solution change.

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Analyze data with the ClampFit software and/or home-made excel macros (MV). Current amplitude is normalized to the current amplitude in the first application of high-K+ buffer, and the baseline is adjusted to the remaining current amplitude in BaCl2.

3.3.3 Protocol for automated TEVC • Pull the pipettes in the P97 puller. Parameters (may vary with the filament position): Heat ¼ 460, Pull ¼ 50, Vel ¼ 20, Time ¼ 50. Ramp ¼ 462 • Prime the pump system with the ND96 buffer in pump 1 and the high-K+ buffer in pump 2. • Fill the pipettes with 3 M KCl, remove the potential bubbles by gentle flicking • Install them on the Hi-Clamp pipette holder, insert the Ag/AgCl electrodes, and cover the KCl solution with light mineral oil to avoid evaporation. • Place the holder in the robot. • Calibrate the lower position of pipettes in the oocyte basket using the Hi-Clamp software. • Place the 96-well plate containing the oocytes on the right side of the robot table, and place the 96-well plate containing the buffers and compounds on the left side. • Program your experiment with the Hi-clamp software icons to set: • oocytes position • names of injected constructs • position of compounds • names of compounds • concentration of compounds • incubation time (10–20 s in most cases) • membrane potential (50 mV) • The sequence of tested solutions is similar to the manual setup (1) High-K+ buffer (2) Ligand (3) High-K+ buffer (4) BaCl2. or for concentration-effect curves with ascending order:

•

(3) Ligand . . . (x) BaCl2. (1) High-K+ (2) Ligand buffer concentration1 concentration2 Start the program. The robot automatically takes the oocytes, impales them, records, changes solutions, and puts back the oocytes in wells.

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•

Data are processed, visualized, and analyzed with in-house Visual basic software and Microsoft Excel (available upon request from MV). The provided Hi-Clamp DataMining and DataMerger software is geared toward ligand-gated channels and difficult to interface with Microsoft Excel. Tip: As control, perform concentration–effect curves of ligands on Kir6.2ΔC36 alone. We discovered effect of some GPCR ligands on Kir6.2 at the highest concentrations.

4. CURRENT ADVANTAGES AND LIMITATIONS OF THE ICCR TECHNOLOGY 4.1 Advantages and limitations See Table 3. Table 3 Advantages and limitations of the ICCR assay Advantages Limitations

G protein-independent assay

Currently, semi-empiric design of the fusion-based on functional ICCRs

Suited to i3 (or i2) loop-modified GPCRs with truncated C-terminus

Low surface expression of M2-K ICCR reported by an external group in a mammalian cell line

No protein purification

Current success with Class-A GPCRs but not yet with class-B GPCRs

Rapidity of the measurements

No expression with two tested Gq protein-coupled receptors

Real-time monitoring of the signal

No detection of the tested inverse agonists

Single-cell experiments

Rare effects of ligands on Kir6.2 alone (mainly at high concentrations)

Sequential applications of ligands Detect agonists and antagonists in concentration-dependent manner Low-cost reagents Low to no internalization in Xenopus oocytes Endogenous Gi/o proteins Optional G protein activation assay with Kir3.x channels

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4.2 Troubleshooting See Table 4. Table 4 Troubleshooting Problem Remedy

Difficult PCR

Change primers Sequence templates

No surface expression

Remove the Kir6.2 ER retention signal (ΔC36) Coexpress the TMD0 domain Prepare fresh mRNA stocks

No functional coupling Verify deletion of the first 25 residues from Kir6.2 Shorten C-terminus of the GPCR as in M2 and D2 Coexpress TMD0 domain Test different ligands (agonists and antagonists at different concentrations: concentration–effect curves) Ligand effect on the channel alone

Perform concentration–effect curves on the ICCR and the channel alone. Subtract channel effect or adjust the concentration to observe the maximal effect on the ICCR without effect on the channel

ACKNOWLEDGMENTS We warmly thank Dr. Vadim Cherezov for corrections and improvements of the manuscript, for the OXTR and OXTR(T4L) genes, and for OXT stocks. We are grateful to D. Rosenbaum and B. Kobilka for the β2AR(T4L) construct, S. Seino for mouse Kir6.2, and K. Chan for the TMD0(SUR1)-F195 construct. This work was supported by grants from the Agence Nationale de la Recherche (ICCR project, Grant ANR-09-PIRI-0010 and VenomPicoScreen project, Grant ANR-11RPIB-022-04) to M.V., a studentship from the Region Rhone-Alpes to K.N., and a studentship from the French Ministry of Research to L.N.C. The Grenoble laboratory is a member of the French National Laboratory of Excellence (Ion Channel Science and Therapeutics) supported by a network grant from ANR.

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