WO2004013178A1

WO2004013178A1 - Fusion proteins between a fluorescent protein and an ionotropic receptor and uses thereof

Info

Publication number: WO2004013178A1
Application number: PCT/EP2003/008490
Authority: WO
Inventors: Régis GRAILHE; Lia Prado De Carvalho
Original assignee: Centre National de la Recherche Scientifique CNRS
Current assignee: Centre National de la Recherche Scientifique CNRS
Priority date: 2002-07-31
Filing date: 2003-07-31
Publication date: 2004-02-12
Anticipated expiration: 2005-01-31
Also published as: AU2003255339A1

Abstract

The present invention relates to a fusion protein between:- a subunit of an heteromeric neuronal nicotinic or serotoninergic ionotropic receptor, and, - a fluorescent protein chosen from fluorescent proteins obtained or derived from autofluorescent proteins, this protein being chosen in particular from: - green fluorescent protein (GFP), or - variants derived from GFP by addition, deletion or substitution of one or more amino acids, with the proviso that these variants conserve the fluorescence capability, - or fragments of GFP, or fragments of the above-mentioned variants, with the proviso that these fragments conserve the fluorescence capability, - or autofluorescent proteins that were isolated from other organisms than the Aequorea Victoria.

Description

FUSION PROTEINS BETWEEN A FLUORESCENT PROTEIN AND AN IONOTROPIC RECEPTOR AND USES THEREOF

The present invention relates to fusion proteins between a fluorescent protein and an ionotropic receptor, preferably a subunit of a nicotinic or serotoninergic ionotropic receptor. It also relates to the uses of these fusion proteins, in particular in a process for the detection and quantification of non-covalent interactions between such a fusion protein and a ligand said receptor.

The superfa ily of pentameric ligand-gated ion channels (LGIC) consists of the cationic nicotinic (nAChRs) and serotoninergic (5HT₃) receptors, as well as the anionic glycine, GABAA _C and GluCl receptors (Karlin & A abas, 1995; Le Novere & Changeux, 1995; Ortells & Lunt, 1995). These receptor channels mediate and modulate chemical interneuronal communication in the central (CNS) and peripheral nervous systems. To fulfil their physiological role, these proteins are efficiently exported to the cytoplasmic membrane and targeted to specific subcellular compartments. Several studies have enabled visualization of a few receptor channel tagged with GFP (Green Fluorescent Protein). Among these receptor channels, the first group belongs to the anionic receptor family. This is the case for the receptor GABA-A and Glycinergic type 1 (Kittler et al, 2000). These fluorescent anionic receptors were grafted with fluorescent proteins were found to be functional and were used to study their cellular compartmentalisation.

Cationic receptors which are members of the superfamily ligand-gated ion channels receptors, namely the serotoninergic 5HT and ,the cationic heteromeric nicotinic α_xβ_y receptors are major neurotransmitter receptors in the mammalian brain, and play a critical role in the mediation and the modulation of chemical interneuronal communications in the central and peripheral nervous systems. The serotoninergic 5- HT3 and nicotinic α_xβ_ynACh receptors are respectively organized as functional homopentamers and heteropentamers. Their transmembrane (TM) subunit topology is dictated by a large N-terminal extracellular domain, followed by four transmembrane segments, and a large cytoplasmic region which connects TM3 and TM4 (Fig. IB). High densities of 5HT₃A are found in the central nervous system (Barnes et al, 1990; Gehlert et al, 1991), whereas the α3β4nAChR is mainly distributed in the periphery, where it contributes to synaptic input in autonomic ganglia (Stollberg & Berg, 1987). However, it has been up to now difficult to characterize more precisely the intracellular trafficking and targeting of these transmembrane receptors or to functionally identify ligands and/or intracellular or extracellular partners of these receptors (Williams et al, 1998).

Effectively, several unsuccessful attempts have been done to characterize further the second group of receptor channels of the cationic receptor family, using the availability of the green fluorescent protein (GFP), initially cloned from the jellyfish Aequorea Victoria (Prasher et al, 1992; Lippincott-schwartz & Patterson, 2003), to enable visualization of the receptors channel-GFP fusions. Two ligand-gated cationic pentameric channels, were tagged with GFP protein (Genler et al, 2001; Palma et al, 2002). The GFP was tagged with the ε or γ subunits of nicotinic muscular receptor. When this one was grafted in the intracellular loop, the GFP fluorescent receptors were functional. However the grafted receptors were not functional when grafted in position N-terminal. When grafted with GFP in the C-terminal side of the nicotinic alpha ^'7, the receptors electrophysiological properties were altered by the presence of the GFP (Palma et al, 2002).

The aim of the present invention is to provide new fusion proteins between fluorescent protein variants and the N-terminus of receptor subunit, in particular the 5HT _A and 3,β4-nACh and α4,β2-nACh subunits without affecting the functionality and targeting thereof.

The present invention relates to a fusion protein between:

— a subunit of an heteromeric neuronal nicotinic or serotoninergic ionotropic receptor, and,

- a fluorescent protein chosen from fluorescent proteins obtained or derived from autofluorescent proteins, this protein being chosen in particular from:

- green fluorescent protein (GFP), or

- variants derived from GFP by addition, deletion or substitution of one or more amino acids, with the proviso that these variants conserve the fluorescence capability,

- or fragments of GFP, or fragments of the above-mentioned variants, with the proviso that these fragments conserve the fluorescence capability,

- or autofluorescent proteins that were isolated from other organisms than the Aequorea Victoria. It has been unexpectedly found by the present Inventors that the fluorescent protein (FP) variants such as the cyan fluorescent protein (CFP) and the yellow fluorescent protein (YFP), which are known to have distinct spectral properties, may be fused to the N-terminus of the ligand-gated cationic pentameric channels, in particular, the 5HT₃A and α3,β4-nACh and α4,β2-nACh subunits without affecting the functionality and targeting thereof. Actually, it has surprisingly been found that fusion at the N-terminal position did not impair the receptor-channel activity and enabled to follow the location or targeting of the receptors inside the cell and at the cell surface. Furthermore, it was found using antibodies directed against the fluorescent protein (FP) that the fluorescent receptors adopted a correct topological orientation across the plasma membrane, and was folded and assembled correctly. This was clearly unexpected in view of previous results that were obtained using similar cationic receptors of the superfamily of pentameric ligand-gated ion channels. The obtention of such functional fluorescent cationic receptors allow to follow the targeting and the regulation of the targeting by chemical or biological agents in different cells.

It has been also surprisingly found that the use of co-expression of functional recombinant subunits of the receptors, which have been grafted with two different fluorophores allow to follow in vivo the .motion of the subunits inside the receptors that had been activated by pharmacological compounds, thereby providing an in vivo dynamic screening of agonists or antagonists of the receptors.

It has been further surprisingly found that the use of these fluorescent receptors allow to dynamically characterize . interactions with fluorescent intracellular and/or extracellular partners.

An advantageous protein of the invention is characterized in that the fluorescent protein is chosen from:

- green fluorescent protein (GFP or EGFP),

- cyan fluorescent protein (CFP or ECFP),

- blue fluorescent protein (BFP or EBFP),

- yellow fluorescent protein (YFP or EYFP),

- GFPUV, or mutants thereof in which the codons are optimized for expression in human, bacterial or plant cells, or mutants thereof which have higher or lower excitation or emission wavelengths than those associated with the proteins defined above, or mutants that have different fluorescence lifetime properties.

The expression "optimized codons" indicates the replacement of codons of the wild-type protein with the host organism's preferred homologues thereof, without changing the code and thus without changing the protein sequence.

The green fluorescent protein (GFP) is decribed in Ward et al. (1980, Photochem. Photobiol. 31:611-615) and in Chalfie et al. (1995, Photochem. Photobiol. 62:651-656) and EGFP is decribed in Heim & Tsien (Current Biology, 1996, vol. No. 6, pp. 178- 182) and inMiyawaki et al (Nature 1997, vol-388, pp. 882-887).

The cyan fluorescent protein (CFP or ECFP) is described in Heim & Tsien (Current Biology, 1996, vol. No. 6, pp. 178-182) and in Miyawaki et al. (Nature 1997, vol. 388, pp. 882-887).

The yellow fluorescent protein (YFP or EYFP) is decribed in Cormack et al. (1995, Gene 173:33-38), in Heim, Cubitt and Tsien (1995, Nature), in Ehrig et al. (1995, FEBS Lett. 367:163-166) and in Miyawaki et al. (Nature 1997, vol. 388, pp. 882-887).

The GFPUV exhibiting the following mutations: F99S, M153T, V163A with excitation and emission wavelengths of 395 and 510, respectively, is described in Crameri et al. 1996 Nature Biotechnol. 14:315-319, or with the mutation T203I and the excitation and emission wavelengths of 400 and 512, respectively, is described in Ehrig et al. 1995 FEBS Lett. 367:163-166.

EGFP has the following mutations:

F64L S65T H231L

EYFP has the following mutations:

S65G V68L S72A T203Y

ECFP has the following mutations:

F64L S65T Y66W N146I

M153T V163A N212K

EBFP has the following mutations:

F64L S65T Y66H Y145F The main characteristics of the fluorescent proteins advantageously used in the process of the invention are given below:

All these fluorescent proteins are described in the international patent application PCT/FR98/01136.

The present invention also relates to a fusion protein such as defined above, characterized in that the fluorescent protein is bound to the N-terminal or C-terminal part of the receptor subunit via a peptide of formula Pro- Ala- Ala- Ala or an analogous derived sequence.

The present invention also relates to a fusion protein such as defined above, characterized in that it is preceded by a signal peptide such as the signal peptide of the subunit alphal glycine receptor from zebrafish (αZIL), said signal peptide being eventually bound via a linker such as the dipeptide AV or an analogous sequence to said fusion protein.

According to an advantageous embodiment, the fusion protein of the invention is characterized in that the subunit of the nicotinic receptors is chosen among the α and β subunits of the neuronal nitcotinic receptors family, particularly among the α3, 4, β2 and β4 subunits.

A preferred fusion protein of the invention is a fusion protein such as defined above, wherein the fluorescent protein is YFP and the subunit of a nicotinic or serotoninergic ionotropic receptor is the α3 nicotinic receptor subunit of the rat nicotinic acetylcholine receptor represented by SEQ ID NO : 1. This preferred fusion protein is called YFP-α3.

A preferred fusion protein of the invention is a fusion protein such as defined above, wherein the fluorescent protein is CFP and the subunit of a nicotinic or serotoninergic ionotropic receptor is the β4 nicotinic receptor subunit of the rat nicotinic acetylcholine receptor represented by SEQ ID NO : 2. This preferred fusion protein is called CFP-β4.

The present invention also relates to a fusion protein such as defined above, characterized in that the subunit of the serotoninergic receptor is the subunit 5-HT₃A.

A preferred fusion protein of the invention is a fusion protein such as defined above, wherein the fluorescent protein is YFP and the subunit of a nicotinic or serotoninergic ionotropic receptor is the mouse 5-HT₃A subunit represented by SEQ ID NO : 3.

The present also relates to a fusion protein such as defined above, characterized in that it is in the form of a pentamer^' of identical or different subunits of a nicotinic or serotoninergic ionotropic receptor, one at least of these subunits being bound to a fluorescent protein.

The present invention also relates to a fusion protein between α and β subunits of nicotinic receptors and a fluorescent protein such as defined above, characterized in that the pentamer comprises a mixture of α3 and β4 subunits, or of α4 and β2 subunits, in particular at the ratio of about 2 α subunits for 3 β subunits, or inversely in particular at the ratio of about 3 α subunits for 2 β subunits.

According to an advantageous embodiment, a fusion protein of the invention is characterized in that only the α subunits are bound to the fluorescent protein, or inversely only the β subunits are bound to the fluorescent protein.

The present invention also relates to a fusion protein such as defined above, characterized in that it comprises a mixture of subunits α3 and β4, wherein only the α3 subunits are bound to the fluorescent protein, which is preferably YFP, or only the j subunits β4 are bound to the fluorescent protein, which is preferably YFP.

The present invention also relates to a nucleotide sequence coding for a fusion protein such as defined above.

The present invention also relates to a vector, particularly a plasmid, containing a nucleotide sequence such as defined above.

The present invention also relates to a host cell chosen among any type of cells, and being in particular Human Embryonic Kidney Cells (HEK-293), transformed with a vector such as defined above.

The present invention also relates to a process for the preparation of a fusion protein such a defined above, characterized in that it comprises a step of culture of host cells such as defined previously transformed with a vector such as defined previously, and the recovering, eventually after a purification, of the fusion proteins such as produced by said cells.

The present invention also relates to a process such as defined above, for the preparation of fusion proteins between subunits of nicotinic receptors and a fluorescent protein in the form of a pentamer such as defined previously, characterized in that it comprises a step of mixing the different subunits of the nicotinic receptors, advantageously in equivalent proportions.

The present invention also relates to the use of fusion proteins such as defined above, for the implementation of a process of targeting in vitro of the nicotinic and/or serotoninergic receptors at the surface of the cells.

The present invention also relates to the use of fusion proteins such as defined above, for the implementation of a process of screening in vitro of ligands of the nicotinic and/or serotoninergic receptors or of inhibitors of such ligands.

The present invention also relates to the use of fusion proteins such as defined above, for the implementation of a process for the detection and quantification of non- covalent interactions between a fusion protein such as defined above and a ligand of the subunit of a nicotinic or serotoninergic ionotropic receptor comprised in said fusion protein, said ligand being labelled with a label consisting:

* either of a molecule which is capable of absorbing the light emitted by the fluorescent protein comprised in the fusion protein such as defined above,

* or of a fluorescent substance, this detection and quantification taking place by fluorescence energy transfer:

• between the above-mentioned fluorescent protein J comprised in the fusion protein or one of the variants such as defined above, or one of the fragments such as defined above, and the above-mentioned fluorescent substance, the fluorescent substance being such that either it is excitable at the emission wavelength of the above- mentioned fluorescent protein or of one of the above-mentioned variants, or of one of the above-mentioned fragments, or it emits at the excitation wavelength of the above- mentioned fluorescent protein, or of one of the above-mentioned variants, or of one of the above-mentioned fragments, or

• in between the above-mentioned fluorescent protein or one of its variants defined above, or one of the fragments defined above, and the above-mentioned molecule which is capable of absorbing the light emitted by the above-mentioned fluorescent protein.

The term "ligand" refers to any molecule which interacts non-covaleηtly and reversibly with another molecule.

The present invention also relates to the use of fusion proteins such as defined above, for the detection and quantification of non-covalent interactions between a fusion protein such as defined above and a ligand of the subunit of a nicotinic or serotoninergic ionotropic receptor, said receptor subunit being comprised in said fusion protein, said ligand being labelled

* either with a fluorescent substance, the labelling being carried out:

- either via a chemical route, the fluorescent substance then being a chemical compound,

- or via a recombinant route, the fluorescent substance then being a fluorescent peptide or protein which can be chosen in particular from the fluorescent proteins obtained or derived from autofluorescent proteins, this fluorescent substance being chosen in particular from:

• green fluorescent protein (GFP), or

• variants derived from GFP by addition, deletion or substitution of one or more amino acids, with the proviso that these variants conserve the fluorescence capability,

• or fragments of GFP, or fragments of the above-mentioned variants, with the proviso that these fragments conserve the fluorescence capability,

* or with a non-fluorescent substance belonging to the Acid Violet group [Acid Violet 5, CAS 10130-48-0; Acid Violet 1, CAS 4321-69-1; Acid Violet 17, CAS 4129- 84-4], the Acid Red group [Acid Red 1, CAS 3734-67-6; Acid Red 8, CAS 4787-93-3; Acid Red 37, CAS 6360-07-2; Acid Red 40, CAS 12167-45-2; Acid Red 106, CAS 6844-74-2; Acid Red 114, CAS 6459-94-5], alizarins, aluminon, azocarmine B [CAS 25360-72-9], basic fuschin [Basic Red 9, CAS 569-61-9], Bordeaux R [Acid Red 17, CAS 5858-33-3] and Carmine [CAS 1390-65-4].

The present invention also relates to a process for detecting and quantifying non- covalent interactions between a fusion protein such as defined above, and a ligand of the subunit of a nicotinic or serotoninergic ionotropic receptor said receptor subunit being comprised in said fusion protein, characterized in that: - cells or cell fragments are prepared containing a DNA sequence comprising the gene coding for a fluorescent protein comprised in the above-mentioned fusion protein, fused with the gene coding for a subunit of a nicotinic or serotoninergic ionotropic receptor comprised in the above-mentioned fusion protein, the fusion between the gene coding for the fluorescent protein and the gene coding for the above-mentioned. receptor being such that the properties of the above-mentioned receptor subunit are not modified by the presence of the above-mentioned fluorescent protein, namely:

• the interaction between the receptor subunit and the ligand is not modified,

• the response transduction function is not modified, the fluorescent protein being chosen from the fluorescent proteins obtained or derived from autofluorescent proteins, this protein being chosen in particular from:

• green fluorescent protein (GFP), or

- the above-mentioned cells or the above-mentioned cell fragments are placed in contact with a ligand for the above-mentioned receptor subunit, said ligand being labelled with a label consisting:

* either of a molecule capable of absorbing the light emitted by the fluorescent protein,

* or of a fluorescent substance, and either the fluorescent protein being the fluorescence energy donor and the label being the fluorescence energy acceptor, or the fluorescent protein being the fluorescence energy acceptor and the label being a fluorescent substance which is a fluorescence energy donor, and

- irradiation is carried out at a wavelength which makes it possible either to excite the fluorescent protein or to excite the fluorescent substance,

- it being possible for the above-mentioned steps of placing in contact and irradiation to be carried out either simultaneously or one after the other, or - the above-mentioned cells or the above-mentioned cell fragments are placed in contact with a ligand for the above-mentioned protein labelled with a label, the cells or the ligand having been irradiated before being placed in contact,

- either a reduction in the amplitude of the donor's emission and/or emission signal characteristic of the acceptor's emission is detected.

The present invention also relates to a process for detecting and quantifying non- covalent interactions between a fusion protein such as defined above, and a ligand of the subunit of a nicotinic or serotoninergic ionotropic receptor, said receptor subunit being comprised in said fusion protein, characterized in that:

- a fluorescent protein, comprised in the above-mentioned fusion protein, fused with a subunit of a nicotinic or serotoninergic ionotropic receptor, comprised in the above-mentioned fusion protein, the fusion between the fluorescent protein and the above-mentioned receptor subunit being such that the properties of the receptor subunit are not modified by the presence of the fluorescent protein, namely:

* the interaction between the receptor subunit and the ligand is not modified,

* the response transduction function is not modified, the fluorescent protein being chosen from the fluorescent proteins obtained or derived from autofluorescent proteins, this protein being chosen in particular from:

• green fluorescent protein (GFP), or

- the above-mentioned fluorescent protein fused with the receptor subunit is placed in contact with a ligand for the^* above-mentioned receptor subunit, this ligand being labelled with a label consisting:

* or of a fluorescent substance, and either the fluorescent protein being a fluorescence energy donor and the label being a fluorescence energy acceptor, or the fluorescent protein being a fluorescence energy acceptor and the label being a fluorescent substance which is a fluorescence energy donor, and

- it being possible for the above-mentioned steps of placing in contact and irradiation to be carried out either simultaneously or one after the other, or

- either a reduction in the amplitude of the donor's emission and/or an emission signal characteristic of the acceptor's emission is detected.

The present invention also relates to a process such as defined above, in which the fluorescent protein is CFP or YFP, and in which:

- either CFP or YFP is a fluorescence energy donor and the label is a fluorescence energy acceptor and is chosen from substances whose excitation spectrum overlaps the emission spectrum of CFP or YFP, and in particular, when the label is a fluorescent substance, it is chosen from: 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (Bodipy), eosin, erythrosin, tetramethylrhodamine, sulphorhodamine 101 sold by Molecular Probe under the name Texas Red, and derivatives thereof which, on the one hand, allow grafting, and, on the other hand, have an excitation spectrum which overlaps the emission spectrum of CFP or YFP, and, when the label is not a fluorescent substance, it is chosen from the Acid Violet group [Acid Violet 5, CAS 10130-48-0; Acid Violet 7, CAS 4321-69-1; Acid Violet 17, CAS 4129-84-4], the Acid Red group [Acid Red 1, CAS 3734- 67-6; Acid Red 8, CAS 4787-93-3; Acid Red 37, CAS 6360-07-2; Acid Red 40, CAS 12167-45-2; Acid Red 106, CAS 6844-74-2; Acid Red 114, CAS 6459-94- 5], alizarins, aluminon, azocarmine B [CAS 25360-72-9], basic fuschin [Basic Red 9, CAS 569-61-9], Bordeaux R [Acid Red 17, CAS 5858-33-3] and Carmine [CAS 1390-65-4],

- or the CFP or YFP is a fluorescence energy acceptor and the fluorescent substance is a fluorescence energy donor and is chosen from substances whose emission spectrum overlaps the excitation spectrum of CFP or YFP, and in particular from: coumarins, fluorescamine, 6-(N-methylanilino)naphthalene, (mansyl) and derivatives thereof which, on the one hand, allow grafting, and, on the other hand, have an excitation spectrum which overlaps the emission spectrum of CFP or YFP.

All these process are described with details in the international patent application PCTYFR98/01136. The present invention also relates to a method for identifying a ligand for a serotoninergic or nicotinic receptor comprising:

(i) providing a mixture of recombinant cells, each cell of which comprises.

(a) a first recombinant gene encoding a serotoninergic or nicotinic receptor protein, said receptor being labelled with a fluorescent protein such as defined above,

(b) a second recombinant gene encoding a polypeptide to be tested, liable to be a ligand of said receptor, said polypeptide being labelled with a label such as defined above, wherein said receptor is expressed on the cell membrane of said cell such that FRET signal is modulated by interaction between the fluorescent protein of the receptor and the label of the polypeptide to be tested; wherein said second recombinant gene is expressed and the polypeptide to be tested is transported to a location allowing interaction with the receptor expressed on the cell membrane, and wherein collectively the mixture of cells expresses a library of said polypeptides to be tested, said library capable of being expressed at a sufficient level such that modulation of the targeting of the receptor protein by a polypeptide to be tested within the library provides a detectable signal;

(ii) allowing cells to generate a detectable signal; and

(iii) identifying the polypeptide to be tested as a ligand for the serotoninergic or nicotinic receptor.

The present invention relates to functional fluorescent cationic receptors of the superfamily of pentameric ligand-gated ion channels, such as serotoninergic and nicotinic receptors, and the preparation thereof.

Various fluorescent proteins may be used and are well known in the art. One fluorescent protein which is commonly named green fluorescent protein (GFP), was initially cloned from the jellyfish Aequorea Victoria which folds as a fluorescent protein in the absence of additional reagent (Prasher et al, 1992). Derivatives of this protein were engineered in order to have distinct spectrum properties such as to emit in the blue (BFP), the cyan (CFP), the green (GFP) and the yellow (YFP) (Tsien, 1998). Other fluorescent proteins which were isolated from various organisms were shown to fold as a fluorescent protein in the absence of additional reagent and to be fluorescent in a specific range of the spectra such as the red for the Ds Red isolated from Discosoma striata or the HcRed isolated from the reef coral, Heteractis crispa. The structural conformation of all these fluorescent proteins is very similar, since the structure of these proteins is based on beta strand which forms a barrel. For most fluorescence imaging applications, the fluorescent label is a biologically inert participant that is used merely as a visible marker. Thanks to the barrel-like structures the chromophore is shielded from the external environment.

According to the present invention the fluorescent receptor channels belonging to the cationic receptor family are fully functional.

As set forth in SEQ ID NO : 1 the poly-amino acid sequence (amino acids 1-751) of the YFP-α3 nicotinic subunit receptor is composed from position 1 to 29 by the poly- amino acid sequence of the zebrafish alphal glycinergic receptor, from position 30 to 31 by a linker (AV), from position 32 to 268 by the fluorescent protein YFP, from position 269 to 277 by a linker (PMASPMPP), and from position 278 to 751 by the rat alpha3 nicotinic subunit amino sequence.

The CFP-β4 nicotinic subunit receptor (amino acids 1-752; SEQ ID NO : 2) of the present invention can be further characterized as having the same amino acid sequence as amino acids 1 to 31 and 269 to 277, as set forth in SEQ ID NO : 1 (for the YFP-α3 nicotinic subunit receptor) and from amino acids from position 32 to 268 by the fluorescent protein CFP, from position 269 to 277, and from position 278 to 752 by the rat beta4 nicotinic subunit amino sequence.

The YFP-5HT3A nicotinic subunit receptor (amino acids 1-740; SEQ ID NO : 3) of the present invention can be further characterized as having the same amino acid sequence as amino acids 1 to 268, as set forth in SEQ ID NO : 1, from position 269 to 272 by a linker (PAAA) and from position 278 to 740 by the mouse 5HT3A nicotinic subunit receptor.

Here, fluorescent protein (FP) variants such as the cyan fluorescent protein (CFP) and the yellow fluorescent protein (YFP), known to have distinct spectral properties, were fused to the N-terminus of the 5HT A and α3,β4-nACh subunits. Their electrophysiological properties were found to be similar to those of non-tagged receptors in whole-cell patch-clamp recordings performed in transiently transfected HEK-293. Fusion in position N-terminal contrary to the ε or γ subunits of nicotinic muscular receptor did not impair the receptor-channel activity. Preferred cationic pentameric ligand-gated ion channels include heteromeric nicotinic acetylcholine subunits receptors (AChR) 3, α4, α5, 6, β2, β3, β4 and serotoninergic subunits receptors 5-HT3A, 5-HT3B and 5-HT3C.

The present invention also provides for the use of the functional fluorescent cationic serotoninergic and nicotinic fluorescent receptors for the characterization of their targeting and regulation of such targeting within the cells in vivo by extracellular agents._. The present invention thus provides for a reliable and effective assay for screening and identifying pharmaceutically" effective compounds that specifically interact with and modulate the targeting of the cation channel serotoninergic and nicotinic receptors. The subject assay enables rapid screening of large numbers of polypeptides in a library in order to identify those polypeptides which inhibit or foster receptor targeting and bioactivity in the cells or in vivo. In general, the assay is characterized by the use of a library of recombinant cells, each cell of which include (i) the serotoninergic or nicotinic receptors whose targeting can be modulated by interaction with an exogenous polypeptide, and (ii) an expressible recombinant gene encoding said exogenous polypeptide to be tested from a polypeptide library. By the use of a gene library, the mixture of cells collectively expresses a population of test polypeptides.

Actually, these fluorescent functional receptors are useful to be used to screen for ligands that affect the cellular distribution thereof when expressed in cells.

The ability of particular constituents of the peptide library to modulate the targeting of the serotoninergic receptor can be scored, hi any event, a statistically significant change in the detection signal can be used to facilitate isolation of those cells from the mixture which contain a nucleic acid encoding a polypeptide to be tested which is an effector of the target receptor. By this method, polypeptides to be tested which induce or block receptors targeting in specific compartment in the cells can be identified, allowing the understanding of the mechanisms of such polypeptides. hi one embodiment of the present invention the reagent cells express the serotoninergic or nicotinic receptor endogenously. In yet other embodiments, the cells are engineered to express a heterologous target receptor protein. In either of these embodiments, it may be desirable to inactivate one or more endogenous genes of the host cells. For example, certain preferred embodiments in which a heterologous receptor is provided utilize host cells in which the gene for the homologous receptor has been inactivated. The present invention further provides for a dynamic assay for screening and identifying pharmacological compounds that specifically interact and modulate the activity of the serotoninergic or nicotinic receptor. The method uses the co-expression of functional recombinant subunits of the serotoninergic or nicotinic receptors grafted with two different fluorophores in order to follow by FRET the motion of the subunits inside the receptors which was activated by pharmacological compounds.

Fluorescence Resonance Energy Transfer (FRET) in Living Cells should enable to study ligand-receptor, and protein-protein interactions in living cells. This method involves two fluorophores defined as a pair: a donor fluorophore, the emission spectrum of which overlaps with the excitation spectrum of the other fluorophore, called acceptor fluorophore. When the two fluorophores are close enough, part of the energy absorbed by the donor is transferred to the acceptor. This transferred energy is then released as photons at the emission wavelength of the acceptor. The transferred energy amount is proportional to inverse 6th power of the distance between the two fluorophores. For example the method can imply the labelling of two proteins with a fluorophore pair (respectively with the donor and with the acceptor). When excited at the excitation wavelength of the donor fluorophore, if FRET occurs, light emission is observed at the emission wavelength of the acceptor. An alternative fluorescence imaging method is Fluorescence lifetime imaging (FLDvf). Here, the fluorescence decay time, the average time that the fluorophore (donor) resides in the excited state before emitting a fluorescent photon, is employed for imaging purposes. The fluorescence lifetime is independent of fluorescent concentration, excitation intensity, and fading due to photobleaching. Interestingly, FLIM may yield direct information about the local chemical environment and the state of the fluorescent molecules in the specimen. In particular the fluorescence lifetime of a donor position at proximity of an acceptor, in a FRET situation, is reduced. Indeed, the fluorescence lifetime of the donor can be easily related to the FRET efficiency, i conclusion both methods (measurement of the fluorescence intensity or the fluorescence lifetime) are sensitive to study inter- and intra-molecular interactions (detects distances variations between the fluorophore in the range of 50-100 angstroms).

Effectively, these receptors belong to the family of cationic pentameric ionotropic receptor specific for the acetylcholine and the serotonin, which are the natural neurotransmitters. These fluorescent functional receptors are useful to be used to screen for chemical compounds and proteins that non-covalently interact therewith. The detection of non-covalent interactions between the fluorescent receptor and one of its ligands labelled by a label consisting of either a molecule capable of absorbing the light emitted by the fluorescence protein, or a fluorescent substrate capable of emitting light with such wavelength capable of exciting the fluorescent protein, said detection talcing place by fluorescence energy transfer.

The fluorescent resonance energy transfer between subunits can be used to follow the conformation changes of the subunits in presence or absence of drugs. The subunits will be grafted with two different fluorescents protein adapted for the fluorescent resonance energy transfer measurement

The invention finally concerns, the use of these fluorescents proteins for screening by fluorescent resonance energy transfer the protein partners that are associated with these receptors in the central nervous system. The present invention thus provides for a dynamic assay for screening and identifying extracellular or intracellular partners by measuring the fluorescence signal and/or by FRET, and more particularly for the screening of protein partner genes of the cationic receptors, and the screening of new chemicals and drugs useful for example in the frame of the treatment of central nervous system diseases, and more particularly myasthenia gravis, Parkinson disease, epilepsy, nervous breakdown.

LEGENDS OF THE FIGURES.

Figures 1 and IB: Schematic representation of fluorescent subunits.

Figure 1A represents a schematic representation of the fusion proteins of the invention. The fluorescent protein (FP; dotted box) was inserted between a common leader peptide (LP) and the first amino acid of the mature rat α3 and β4 nicotinic receptor subunits (white box) and the mouse "5HT_3A receptor subunit (grey box). The numbered black boxes correspond to transmembrane domains.

Figure IB corresponds to a proposed transmembrane (TM) topology of FP-α3 tagged subunit (left), and a top view of FP-α3β4 pentameric organization (right).

Figures 2A-2F: Functional expression of FP-tagged α3β4-nAChR and 5HT_3AR.

Ach-evoked currents in HEK-293 cells expressing wild type and fluorescently tagged α3β4 and 5HT_3A receptors: α3β4 (Figure 2A), YFP-α3β4 (Figure 2B), α3CFP- β4 (Figure 2C), YFP-α3CFP-β4 (Figure 2D), and YFP-5HT_3A (Figure 2F). Horizontal bar indicate the time of ACh (acetylcholine) or 5HT applications, at concentrations indicated in the figures. Holding potential, -lOOmV. Current- voltage relation of the α3β4 FP-labeled channels; bold line corresponds to the wild type α3β4 receptor (Figure 2E).

Figures 3 -3D: Fluorescence signal from YFP-5HT_3AR, and α3CFP-β4 nAChR, in living HEK-293 cells.

Figures 3A and 3B show HEK-293 cells expressing the YFP-5HT_3A receptor. Figures 3C and 3D show HEK-293 cells expressing the α3CFP-β4 receptor. Figures 3A and 3C correspond to transmission images; Figures 3B and 3D to epifluorescence from the fluorescent receptors. Bar=10μm (Figures 3A-3D) Figures 4A-4C: Subcellular localization of YFP-5HT_3A receptor in HEK-293 cells and hippocampal neuron.

Figures 3 A and 3B correspond to the expression of YFP-5HT₃A receptor in HEK- 293 cell and Figure 3C corresponds to the expression of YFP-5HT₃A receptor in hippocampal single neuron. It should be noted that in HEK-293 a subcellular localization of the YFP-5HT₃A receptor in several micropodia. The YFP-5HT₃A receptor is localized in micropodia end points and spine-like formations of the hippocampal neurons. Scale: A, lOμm; B, 2μm; C, 30μm.

Figures 5 and 5B: Probing subunit-subunit interaction by measurement of lifetime values of fluorescent receptors.

Figure 5A corresponds to an illustration for the FRET-Fluorescence resonance energy transfer (arrows) between fluorescent proteins CFP (donor) to YFP (acceptor). Note that the co-expression of non-fluorescent subunit α should theoretically decrease the FRET efficiency between the fluorescents subunits CFP-β2 and YFP-β2 as illustrated by the number of arrows (schematic representation of the FRET).

Figure 2B corresponds to fluorescence lifetime images of CFP-β2 measured alone (2.6 ns ± 0.021). The fluorescence lifetime images of cells coexpressing CFP-β2 and YFP-β2 clearly display a decrease of the CFP-β2 lifetime. The existence of this low CFP-β2 lifetime (FLDVl data: 2.37 ns ± 0.031) suggests auto-association between fluorescent β2 subunit. The increase of CFP-β2 lifetime (FLTM data: 2.49 ns ± 0.029) in presence of non fluorescent 4 subunit demonstrate that the subunit is interacting with the β2 subunit. *, p<0.05; **, pO.OOl.

Example 1: DNA constructs and transfection of HEK-293 cells

The fluorescent-tagged nicotinic α3 and β4 and serotoninergic 5HT_3A subunits were generated using a strategy analogous to that previously reported for the zebrafish Glycine subunit (GlyRzl) (David-Watine et al, 1999). Rat α3, β4 nicotinic subunits and mouse 5HT_3A long splicing form sequences were amplified by polymerase chain reaction (PCR) from the codon encoding the first amino-acid to the stop codon. The resulting PCR products were digested with proper restriction enzymes and ligated into a pMT₃ based vector (Swick et al, 1992) containing, in frame, the peptide signal from the GlyRzl, a linker (AV), the enhanced cyan or yellow fluorescent proteins (CFP, YFP; Ozyme, France) and a linker. This linker was PAAA and PAAASPMPP for the 5-HT3 receptor and the xβy nicotinic receptors, respectively. All expression plasmid constructs were checked by restriction mapping and nucleotide sequencing. Cells were transiently transfected on 0.18 mm thick glass bottom dishes (MatTek, USA) pre-coated with 0.5 mg/ml poly(L-lysine) (Sigma). HEK-293 cell transfection of the constructions was carried out using the LipofectAMINE Reagent PLUS procedure (Gibco BRL) and visualized 2 to 7 days afterwards.

Example 2: Demonstration of the functionality of the fluorescent receptor by electrophysiology

To ascertain that the FP -tagged receptors designed here are fully functional, their electrophysiological properties were compared to those of non-tagged receptors in whole-cell patch-clamp experiments performed in transiently transfected HEK-293 cells. The co-expression of α3 and β4 nAChR subunits is rdquired to form functional hetero-pentameric channels (Duvoisin et al, 1989). In fifty percents of the cells transfected with wild type or fluorescent α3β4 nAChRs, ACh elicited inward currents at negative holding potentials (Fig. 2A). Responses were proportional to the concentration of ACh (100-300 μM range) and showed slow desensitization (Fig. 2A). Currents elicited by 300 μM ACh at a holding potential of -lOOmV varied from cell to cell (from 200 to 2000 pA), with a mean response of 710 ± 228pA (n=6). Current-voltage relationships displayed the strong rectification typical of nAChRs, with no outward current at positive potentials (Fig. 2E). Fusing a fluorescent protein to one subunit (YFP- 3β4, α3CFP-β4) or to both nAChR subunits (YFP- 3CFP-β4) still allowed the expression of functional receptors, hi cells transfected either with the FP-label fused to one subunit or with both FP-labeled subunits, ACh elicited currents displaying time course and current-voltage relationships typical of those obtained with their wild counterparts (Fig. 2B-E). The proportion of responding cells were also similar, however, the current amplitudes seemed to be smaller λvith FP-grafted receptors. In the case of cells transfected with a single FP- labeled subunit, the mean current evoked by 300 μM ACh was 335 ± 63 pA for α3CFP-β4 (n=10) and 175 ± 29 pA for YFP-α3β4 (n=6). When both subunits were tagged, the amplitude currents evoked by 300 μM ACh were 98 ± 53 pA (n=3).

Contrary to cells transfected with nAChR, almost 100% of the cells responded to serotonin, when transfected with either wild-type or YFP-tagged 5HT_3A subunits. In these cells, serotonin (10-100 μM) elicited robust rapidly desensitizing inward currents, with amplitude reaching several nA at -60 mV (Fig. 2F) whereas similar current- voltage relationships were observed (data not shown). The mean maximal current caused by a saturating concentration of serotonin (100 μM) in cells transfected with YFP-5HT₃A was 3125 ± 1709 pA (n=4). These currents were strongly inhibited by calcium or magnesium (2 mM) (data not shown and (Maricq et al, 1991; Eiselέ et al, 1993)) as is the case for cells transfected with untagged 5HT AR-

Our results thus show that the fusion of a FP to the N-terminus of either α3, or β4 nAChR subunits or to the serotonin 5HT₃A subunit generates receptors that can be expressed as fully functional ionic channels, which display the electrophysiological properties typical of native receptors. The weaker currents elicited by agonist and the j lower proportion of transfected cells expressing the α3β4 nAChR, compared to 5HT _AR appears characteristic of nicotinic receptors, regardless the presence or absence of the FP.

Example 3: Demonstration of the targeting of the fluorescent receptor by digital-imaging fluorescence microscopy

Epifluorescence microscopy: Fluorescence images were acquired using a Zeiss Axiovert fluorescent microscope that was fitted with a Sensicam charge-coupled device camera (12 bit) and controlled by MetaVue software package (Universal Imaging Corporation, USA). Filter cube specifications for the fluorescence channels were as follows (for excitation, emission, and main dichroic beam splitter, respectively): CFP (440 ± 21nm, 480 ± 30nm, 455nm); YFP (500 ± 25nm, 530nm LP, 525nm), (Chroma, USA).

Confocal microscopy: A Zeiss LSM510 laser scanning confocal microscope was used (Zeiss, Oberkochen, Germany). Detection and distinction between fluorescent signals were achieved by using appropriated conditions (for excitation, and emission, beam splitter, respectively): CFP ^' (Laser light at 458nm, BP 475-525nm, HFT 458/514nm), YFP (Laser light at 514nm, BP 530-600nm, HFT 458/514nm) and Texas Red / Rhodamine (Laser light at 543nm, LP 585nm, HFT 488/543nm). Additionally, to improve resolution and signal-to noise ratio, images were restored using the Huygens software 2.3.4a (Scientific Volume Imaging, Netherlands) and visualized using Imaris 3.1 software (Bitplane, Switzerland). Calculations were performed on a Silicon Graphics Octane workstation. Quantitative image processing and analysis of optical sections were performed with ImageJ software (http ://rsb info .nih. go v/ii ) .

Example 4: Demonstration of the targeting of the fluorescent receptors in rat hippocampal primary culture and transfection

Hippocampal neurons were prepared from 17 days-old rat embryos. Briefly, dissected hippocampi were mechanically and chemically dissociated in single-cell suspension with trypsin (0.25 mg/ml) (Brewer et al, 1993). The cells were centiifuged and suspended in Neurobasal medium (GibcoBRL) supplemented with Glutamax I (GibcoBRL), B27 (GibcoBRL), 100 U/ml penicillin, and 100 μg/ml streptomycin (DMEM medium; GibcoBRL). The dissociated cells were plated onto 0.18 mm thick glass bottom dishes pre-coated with 0.5 mg/ml poly(L-ornithine) (Sigma). After 5 days, primary culture neurons were transfected for four hours using LipofectAMINE Reagent PLUS method (Gibco BRL) and observed after 2 to 7 days. Every four days half of the culture medium was replaced with fresh Neurobasal supplemented medium. Tracking of transfected neurons was performed at low magnification, when fluorescent tagged receptors were co-expressed with a spectrally distinct fluorescent protein (example in Fig. 6: CFP). Example 5: Constructions of α3β4 nAChR and 5HT_3AR subunits tagged with fluorescent proteins

The cDNAs encoding for the CFP and YFP were introduced as described in Fig. 1. The corresponding fluorescently tagged subunits (FP -tagged) are termed YFP-α3, CFP-β4 and YFP-5HT_3A.

FP-tagged receptor subunits form functional channels

To ascertain that the FP-tagged subunits designed here assemble into fully functional receptor channels, their electrophysiological properties were compared to those of non-tagged receptors in .whole-cell patch-clamp recordings performed in transiently transfected HEK-293 cells. The co-expression of α3 and β4 nAChR subunits is required to form functional hetero-pentameric channels (Duvoisin et al, 1989). ha fifty percents of the cells transfected with wild-type α3β4 or fluorescent YFP-α3β4, α3CFP-β4 nAChRs, ACh elicited inward currents at a holding potential of 100 mV. Responses were proportional to the concentration of ACh (100-300 μM range) and showed slow desensitization with similar time course (Fig. 2A-C). Mean currents elicited by 300 μM ACh were 710 ± 228pA for α3β4 (n=6), 335 ± 63 pA for α3CFP-β4 (n=10) and 175 ± 29 pA for YFP-α3β4 (n=6). In all cases, current-voltage relationships displayed the strong inward rectification typical of nAChRs (Fig. 2D).

Contrary to cells transfected with nAChR, almost 100% of the cells transfected with either wild-type or YFP-tagged 5HT_3A subunits responded to serotonin, hi these cells, serotonin (10-100 μM) elicited robust rapidly desensitizing inward currents with an amplitude reaching several nA at -60 mV (Fig. 2E) and similar current-voltage relationships were observed (data not shown). The mean maximal current caused by a j saturating concentration of serotonin (100 μM) in cells transfected with YFP-5HT_3A was 3125 ± 1709 pA (n=4). These currents were strongly inhibited by calcium or magnesium (2 mM) (data not shown and (Maricq et al, 1991; Eisele et al, 1993)) as is the case in cells transfected with untagged 5HT_3AR

Our results thus show that the fusion of a FP to the N-terminus of either α3, or β4 nAChR subunits or to the serotonin 5HT₃A subunit generates receptors that can be expressed as fully functional ionic channels, which display the electrophysiological properties typical of native receptors. The weaker currents elicited by agonist and the lower proportion of transfected cells expressing the α3β4 nAChR, compared to 5HT_3AR appears characteristic of nicotinic receptors, regardless the presence or absence of the FP.

Example 6: Demonstration of differential expression of 5HT_3ARs and α3β4 nAChRs at the surface of HEK-293 cells

To determine whether the FP-tagged α3β4 nAChR and 5HT_3AR produce detectable fluorescence, HEK-293 cells were transiently transfected with various tagged subunits. The co-expression of YFP-α3 with wild-type β4 and the co-expression of CFP-β4 with wild-type α3 were investigated. 5HT₃A subunits are known to form functional homo-oligomers, thus the YFP-5HT_3A fusion was transfected alone (Maricq er a/., 1991).

Two to seven days after transfection, HEK-293 cells were visualized by epifluorescence (Fig. 3A) and confocal microscopy (Fig. 3G-L). Nicotinic and serotoninergic receptors displayed specific fluorescence distributions. For the YFP-α3β4 and α3CFP-β4 subunit co-expressions, the fluorescence was distributed within cytoplasmic compartments of the cell (Fig. 3D). Expression of YFP-5HT_3AR yielded fluorescence both in intracellular compartments and near or within the cytoplasmic membrane (see arrows in Fig. 3B). This difference was further revealed by transfecting the same cell with YFP-5HT₃A receptor together with 3CFP-β4 nAChR that clearly showed that the yellow fluorescence of YFP-5HT_3AR is co-localized with the cyan fluorescence of α3CFP-β4 in intracellular compartments, while only yellow fluorescence is observed in the cytoplasmic membrane.

One advantage of live imaging is that it enables visualization of the micro- extensions of the plasma membrane that are otherwise easily damaged by fixation procedures or mechanical force during specimen preparation. Indeed, more detailed analysis of images shows that the 5HT₃AR is specifically targeted to the tips of micropodia (Fig. 4B).

Example 7: Demonstration of the differential the intracellular targeting of 3β4 nAChRs and 5HT_3ARs in the endoplasmic reticulum

To further elucidate the distribution of receptors in cellular compartments, YFP- tagged subunits were co-transfected with the endoplasmic reticulum fluorescent marker ER-CFP. YFP-α3β4 expressed in HEK-293 cells along with ER-CFP protein marker revealed a strong co-localization of the two labels, indicating that the 3β4 receptor is mainly located in the ER. YFP-5HT_3AR was found in intracellular vesicles, and to a lesser extent in the ER. Time imaging sequence of the vesicles expressing the YFP-5HT₃AR follows linear trajectory consistent with a microtubule-mediated movement.

Example 8: Demonstration of the differential targeting of 5HT_3AR and α3β4 nAChR to the plasmalemma in hippocampal neurons

To determine the neuronal distribution of 5HT_3AR and α3β4 nAChR, primary cultures of rat hippocampal neurons were co-transfected with YFP-5HT_3A + α3CFP-β4 or with YFP-5HT_3A + CFP. Confocal microscopy showed that the YFP-5HT_3A and α3CFP-β4 receptors are located in somatic compartment co-localizing with the ER marker. Nevertheless, differences in receptor targeting were revealed by expressing α3CFP-β4 nAChRs and YFP-5HT_3ARS in the same cell. To account for differences in the level of protein expression and imaging parameters, the fluorescence signals of α3CFP-β4 receptors and YFP-5HT_3A receptors were normalized with respect to each other by matching the fluorescence in the cell body region. Two-channel, quantitative analysis of normalized fluorescent intensities indicated that unlike the α3CFP-β4 which remained in the cell body, the YFP-5HT_3A is highly localized in the dendrites.

Closer examination of hippocampal neurons transfected with the YFP-5HT_3A receptor revealed clear punctuate fluorescence in dendritic structures. More specifically, the chimeric receptor was targeted to the tips of the filipodia and spines (Figure 4C). To ascertain that 5HT_3AR seen in dendritic structures is facing t e extracellular milieu, we used a fluorescent ligand that specifically binds the extracellular domain of the 5HT₃AR. The antagonist GR-H (GR119566) is covalently attached to Rhodamine B isothiocyanate and is referred to hereafter as GR-rho (Schmid et al, 1998). Detailed analysis of GR-rho fluorescence revealed specific labeling at the cell body and the dendritic structures of the transfected hippocampal neurons (Fig. 6F-G). These results show that the 5HT _A receptors are efficiently expressed at the cell surface of neurites, and more specifically targeted to the tip of filopodias and spine-like structures. Example 9: Demonstration of subimit-subunit interaction by measurement of lifetime fluorescence for Fluorescent nicotinic receptor expressed in HEK-293 cells.

To study the association process between subunit belonging to the pentameric heteromeric nicotinic receptor family we have expressed α4β2 fluorescent nicotinic receptor in cells. The nicotinic β2 subunit was grafted in N-terminal with YFP or CFP fluorescent protein using the same strategy as developed as for the example 1. The expressing vectors encoding for the subunits' were transfected in HEK-293 cell lines using Lipofectamine method and tested after 48 hours.

Fluorescence Resonance Energy Transfer (FRET) in. Living Cells should enable to study protein-protein interactions in living cells. If the two fluorophores, the donor (CFP) and the acceptor (YFP) are close enough, part of the energy absorbed by the donor is transferred to the acceptor. In this respect, we have used fluorescence life time imaging techniques to probe the fluorescence lifetime of CFP-β2 fluorescent nicotinic subunit to measure the respective putative proximity of the YFP-β2 nicotinic subunit. When the donor is close (<100 A) of an acceptor, in a FRET situation, the lifetime is reduced. We used High Speed Lifetime Module (LIMO from Nikon) for performing fluorescence lifetime measurements in cell expressing fluorescently transfected receptors.

With such fluorescents subunits we were able to monitor protein-protein interaction by fluorescence resonance energy transfer (FLIM-TREF) between subunits belonging to the cationic super-family of pentameric ligand-gated ion channels.

Indeed, the CFP-β2 lifetime was decreased when the CFP-β2 nicotinic subunits were co-expressed with YFP-β2 subunits, and was increased¹⁾ in the presence of α4 non labelled nicotinic subunits (Figure 5).

REFERENCES

Barnes, J.M., Barnes, N.M., Champaneria, S., CostalL B. & Naylor, RJ. (1990)

Characterisation and autoradiographic localisation of 5-HT3 receptor recognition sites identified with [³H]-(S)-zacopride in the forebrain of the rat. Neuropharmacology, 29,

1037-1045. Brewer, G.J., Torricelli, J., Evege, E.K. & Price, P.J. (1993) Optimized survival of hippocampal neurons in B27 -supplemented Neurobasal, a new serum-free medium combination. J Neurosci Res, 35, 567-576. David- Watine, B., Shorte, S., Fucile, S., de Saint Jan, D., Korn, H. & Bregestovsld, P. (1999)

Functional integrity of green fluorescent protein conjugated glycine receptor chanels.

Neuropharmacology, 38, 785-792. Duvoisin, R., Deneris, E., Patrick, J. & Heinemann, S. (1989) The functional diversity of the neuronal nicotinic acetylcholine receptors is increased by a novel subunit, beta 4.

Neuron, 3, 487-496. Eisele, J.L., Bertrand, S., Galzi, J.L., Devillers-Thiery, A., Changeux, J.P. & Bertrand, D.

(1993) Chimeric nicotimc-serotoninergic receptor combines dictinct ligand binding and channel specificities. Nature, 366, 479-483. Gehlert, D.R., Gackenheimer, S.L., Wong, D.T. & Robertson, D.W. (1991) Localization of the 5-HT3 receptors in the rat brain using [3H]LY278584. Brain. Res., 553, 149-154. Genler, S;, Sander, A., Korngreen, A., Traina, G., Giese, G. & Witzermann, V. (2001)

Assembly and clustering of acetylcholine receptors containing GFP -tagged ε or γ subunits. Eur. J. Biochem, 268, 2209-2217. Karlin, A. & Akabas, M.H. (1995) Toward a structural basis for the .function of nicotinic acetylcholine receptors and their cousins. Neuron, 15, 1231-1244. Kittler, J.F., Wang, J., Connolly, C, N., Vicini, S., Smart, T., G. & Moss, S., J. (2000)

Analysis of GAB A- A receptor assembly in mammalian cell lines and hippocampal neurons using γ2 subunit Green fluorescent protein chimmeras. Mol and cell

Neuroscience, 16, 440-452. Le Novere, N. & Changeux, J.P. (1995) Molecular evolution of the nicotinic acetylcholine receptor: an example of multigene family in excitable cells. J.Mol Evol, 40, 155-172. Lippincott-schwartz, J. & Patterson, G.H. (2003) Development and use of fluorescent protein markers in living cells. Science, 300, 87-91. Maricq, AN., Peterson, A.S., Brake, A.J., Myers, R.M. & Julius, D. (1991) Primary structure and functional expression of the 5HT₃ receptor, a serotonin-gated channel. Science,

254, 432-437. Ortells, M.O. & Lunt, G.G. (1995) Evolutionary history of the ligand-gated ion-channel superfamily of receptors. Trends Neurosci, 18, 121-127. Palma, E., Mileo, A.M., Martinez-Torres, A., Eusebi, F. & Miledi, R. (2002) Some properties of the human neuronal α7 nicotinic acetylcholine receptors fused to the green fluorescent protein. Proc. Nail. Acad. Sci. USA, 99, 3950-3955. Prasher, D.C., Eckenrode, V.K., Ward, W.W., Prendergast, F.G. & Cormier, M.J. (1992)

Primary structure of the Aequorea ictoria green-fluorescent protein. Gene, 111, 229-

233. Schmid, E., Tairi, A.P., Hovius, R. & Vogel, H. (1998) Screening ligands for membrane protein receptors by total internal reflection fluorescence: the 5-HT3 serotonin receptor. Anal. Chem., 70, 1331-1338. Stollberg, J. & Berg, D.K. (1987) Neuronal acetylcholine receptors: fate of surface and internal pools in cell culture. J. Neurosci., 1, 1809-1815. Swick, A.G., Janicot, M., Cheneval-Kastelic, T., McLenithan, J.C. & Lane, M.D. (1992)

Promoter-cDNA-directed heterologous protein expression in Xenopus laevis oocytes.

Proc. Natl. Acad. Sci. USA., 89, 1812-1816. Tsien, R.Y. (1998) The green fluorescent protein. Annu. Rev. Biochem., 67, 509-544. Williams, B.M., Temburni, M.K., Levey, M.S., Bertrand, S., Bertrand, D. & Jacob, M.H.

(1998) The long internal loop of the alpha 3 subunit targets nAChRs to subdomains within individual synapses on neurons in vivo. Nat Neurosci, 7, 557-562.

Claims

1. A fusion protein between:

- a subunit of an heteromeric neuronal nicotinic or serotoninergic ionotropic receptor, and,

- green fluorescent protein (GFP), or

- or fragments of GFP, or fragments of the above-mentioned variants, with the proviso that these fragments conserve the fluorescence capability.

- or autofluorescent proteins that were isolated from other organisms than the Aequorea Victoria.

2. A fusion protein according to claim 1, characterized in that the fluorescent protein is chosen from:

- green fluorescent protein (GFP or EGFP),

- cyan fluorescent protein (CFP or ECFP),

- yellow fluorescent protein (YFP or EYFP),

- blue fluorescent protein (BFP or EBFP),

- GFPUV, or mutants thereof in which the codons are optimized for expression in human, bacterial or plant cells, or mutants thereof which have higher or lower excitation or emission wavelengths than those associated with the proteins defined above, or mutants that have different fluorescence lifetime properties,

3. A fusion protein according to claim 1 or 2, characterized in that the fluorescent prote is bound to the N-terminal or C-terminal part of the receptor subunit via a peptide of formula Pro-Ala- Ala- Ala or an analogous derived sequence.

4. A fusion protein according to any of claims 1 to 3, characterized in that it is preceded by a signal peptide such as the signal peptide of the subunit alphal glycine receptor from zebrafish (αZIL), said signal peptide being eventually bound via a linlcer such as the dipeptide AV or an analogous sequence to said fusion protein.

5. A fusion protein according to any of claims 1 to 4, characterized in that the subunit of the nicotinic receptors is chosen among the α and β subunits of the neuronal nicotinic receptors family, particularly among the α3, α4, β2 and β4 subunits.

6. A fusion protein according to claim 5, wherein the fluorescent protein is YFP and the subunit of a nicotinic or serotoninergic ionotropic receptor is the α3 nicotinic receptor subunit of the rat nicotinic acetylcholine receptor (YFP-α3) represented by SEQ ID NO : 1.

7. A fusion protein according to claim 5, wherein the fluorescent protein is CFP and the subunit of a nicotinic or serotoninergic ionotropic receptor is the β4 nicotinic receptor subunit of the rat nicotinic acetylcholine receptor (CFP-β4) represented by SEQ LD NO : 2.

8. A fusion protein according to any of claims 1 to 4, characterized in that the subunit of the serotoninergic receptor is the subunit 5-HT _A.

9. A fusion protein according to claim 8, wherein the fluorescent protein is YFP and the subunit of a nicotinic or serotoninergic ionotropic receptor is the mouse 5- HT_3A subunit represented by SEQ LD NO : 3.

10. A fusion protein according to any of claims 1 to 9, characterized in that it is in the form of a pentamer of identical or different subunits of a nicotinic or serotoninergic ionotropic receptor, one at least of these subunits being bound to a fluorescent protein.

11. A fusion protein between α and β subunits of nicotinic receptors and a fluorescent protein such as defined in claim 10, characterized in that the pentamer comprises a mixture of α3 and β4 subunits, or of α4 and β2 subunits, in particular at the ratio of about 2 α subunits for 3 β subunits, or inversely in particular at the ratio of about 3 α subunits for 2 β subunits.

12. A fusion protein according to claim 11, characterized in that only the α subunits are bound to the fluorescent protein, or inversely only the β subunits are bound to the fluorescent protein.

13. A fusion protein according to any of claims 10 to 12, characterized in that it comprises a mixture of subunits α3 and β4, wherein only the α3 subunits are bound to the fluorescent protein, which is preferably YFP, or only the subunits β4 are bound to the fluorescent protein, which is preferably YFP.

14. A nucleotide sequence coding for a fusion protein according to any of claims 1 to 9.

15. A vector, particularly a plasmid, containing a nucleotide sequence according to claim 14.

16. A host cell chosen among any type of cells, and being in particular Human Embryonic Kidney Cells (HEK-293), transformed with a vector according to claim 15.

17. A process for the preparation of a fusion protein according to any of claims 1 to 13, characterized in that it comprises a step of culture 'of host cells according to claim 16 transformed with a vector according to claim 15, and the recovering, eventually after a purification, of the fusion proteins such as produced by said cells.

18. A process according to claim 17 for the preparation of fusion proteins between subunits of nicotinic receptors and a fluorescent protein in the form of a pentamer according to any of claims 11 to 13, characterized in that it comprises a step of mixing the different subunits of the nicotinic receptors, advantageously in equivalent proportions.

19. The use of fusion proteins according to any of claims 1 to 13, for the implementation of:

- a process of targeting in vitro of the nicotinic and/or serotoninergic receptors at the surface of the cells,

- a process of screening in vitro of ligands of the nicotinic and/or serotoninergic receptors or of inhibitors of such ligands,

- a process for the detection and quantification of non-covalent interactions between a fusion protein according to any of claims 1 to 13 and a ligand of the subunit of a nicotinic or serotoninergic ionotropic receptor, said receptor subunit being comprised in said fusion protein, said ligand being labelled with a label consisting:

* either of a molecule which is capable of absorbing the light emitted by the fluorescent protein comprised in the fusion protein such as defined in any of claims 1 to 13,

• between the above-mentioned fluorescent protein comprised in the fusion protein or one of the variants such as defined in claim 1, or one of the fragments such as defined in claim 1, and the above-mentioned fluorescent substance, the fluorescent substance being such that either it is excitable at the emission wavelength of the above- mentioned fluorescent protein or of one of the above-mentioned variants, or of one of the above-mentioned fragments, or it emits at the excitation wavelength of the above- mentioned fluorescent protein, or of one of the above-mentioned variants, or of one of the above-mentioned fragments, or

20. The use of fusion proteins according to any of claims 1 to 13, for the detection and quantification of non-covalent interactions between a fusion protein according to any of claims 1 to 13 and a ligand of the subunit of a nicotinic or serotoninergic ionotropic receptor, said receptor subunit being comprised in said fusion protein, said ligand being labelled * either with a fluorescent substance, the labelling being carried out:

• green fluorescent protein (GFP), or

* or with a non-fluorescent substance belonging to the Acid Violet group [Acid Violet 5, CAS 10130-48-0; Acid Violet 7, CAS 4321-69-1; Acid Violet 17, CAS 4129- 84-4], the Acid Red group [Acid Red 1, CAS 3734-67-6; Acid Red 8, CAS 4787-93-3; Acid Red 37, CAS 6360-07-2; Acid Red 40, CAS 12167-45-2; Acid Red 106, CAS 6844-74-2; Acid Red 114, CAS 6459-94-5], alizarins, aluminon, azocarmine B [CAS 25360-72-9], basic fuschin [Basic Red 9, CAS 569-61-9], Bordeaux R [Acid Red 17, CAS 5858-33-3] and Carmine [CAS 1390-65-4].

21. A process for detecting and quantifying non-covalent interactions between a fusion protein according to any of claims 1 to 13, and a ligand of the subunit of a nicotinic or serotoninergic ionotropic receptor said receptor snbunit being comprised in said fusion protein, characterized in that:

- cells or cell fragments are prepared containing a DNA sequence comprising the gene coding for a fluorescent protein comprised in the above-mentioned fusion protein, fused with the gene coding for a subunit of a nicotinic or serotoninergic ionotropic receptor comprised in the above-mentioned fusion protein, the fusion between the gene coding for the fluorescent protein and the gene coding for the above-mentioned receptor being such that the properties of the above-mentioned receptor subunit are not modified by the presence of the above-mentioned fluorescent protein, namely: • the interaction between the receptor subunit and the ligand is not modified,

• green fluorescent protein (GFP), or

- the above-mentioned cells or the above-mentioned cell fragments are placed in contact with a ligand for the above-mentioned protein labelled with a label, the cells or the ligand having been irradiated before being placed in contact,

22. A process for detecting and quantifying non-covalent interactions between a fusion protein according to any of claims 1 to 13, and a ligand of the subunit of a nicotinic or serotoninergic ionotropic receptor, said receptor subunit being comprised in said fusion protein, characterized in that:

- a fluorescent protein, comprised in the above-mentioned fusion protein, fused with a subunit of a nicotinic or serotoninergic ionotropic receptor, comprised in the above-mentioned fusion protein, the fusion between the fluorescent protein .and the above-mentioned receptor subunit being such that the properties of the receptor subunit are not modified by the presence of the fluorescent protein, namely:

* the interaction between the receptor subunit and the ligand is not modified,

• green fluorescent protein (GFP), or

- the above-mentioned fluorescent protein fused with the receptor subunit is placed in contact with a ligand for the above-mentioned receptor subunit, this ligand being labelled with a label consisting:

- it being possible for the above-mentioned steps of placing in contact and irradiation to be carried out either simultaneously or one after the other, or - either a reduction in the amplitude of the donor's emission and/or an emission signal characteristic of the acceptor's emission is detected.

23. A process according to claim 22, in which the fluorescent protein is CFP or YFP, and in which:

~ either CFP or YFP is a fluorescence energy donor and the label is a fluorescence energy acceptor and is chosen from substances whose excitation spectrum overlaps the emission spectrum of CFP or YFP, and in particular, when the label is a fluorescent substance, it is chosen from: 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (Bodipy), eosin, erythrosin, tetramethyhhodamine, sulphorhodamine 101 sold by Molecular Probe under the name Texas Red, and derivatives thereof which, on the one hand, allow grafting, and, on the other hand, have an excitation spectrum which overlaps the emission spectrum of CFP or YFP, and, when the label is not a fluorescent substance, it is chosen from the Acid Violet group [Acid Violet 5, CAS 10130-48-0; Acid Violet 7, CAS 4321-69-1; Acid Violet 17, CAS 4129-84-4], the Acid Red group [Acid Red 1, CAS 3734- 67-6; Acid Red 8, CAS 4787-93-3; Acid Red 37, CAS 6360-07-2; Acid Red 40, CAS 12167-45-2; Acid Red 106, CAS 6844-74-2; Acid Red 114, CAS 6459-94- 5], alizarins, aluminon, azocarmine B [CAS 25360-72-9], basic fuschin [Basic Red 9, CAS 569-61-9], Bordeaux R [Acid Red 17, CAS 5858-33-3] and Carmine [CAS 1390-65-4],

~ or the CFP or YFP is a fluorescence energy acceptor and the fluorescent substance is a fluorescence energy donor and is chosen from substances whose emission spectrum overlaps the excitation spectrum of CFP or YFP> and in particular from: coumarins, fluorescamine, 6-(N-methylanilino)naphthalene, (mansyl) and derivatives thereof which, on the one hand, allow grafting, and, on the other hand, have an excitation spectrum which overlaps the emission spectrum of CFP or YFP.

24. A method for identifying a ligand for a serotoninergic or nicotinic receptor comprising:

(i) providing a mixture of recombinant cells, each cell of which comprises

(a) a first recombinant gene encoding a serotoninergic or nicotinic receptor protein, said receptor being labelled with a fluorescent protein such as defined in claim 1,

(b) a second recombinant gene encoding a polypeptide to be tested, liable to be a ligand of said receptor, said polypeptide being labelled with a label such as defined in claim 19, wherein said receptor is expressed on the cell membrane of said cell such that FRET signal is modulated by interaction between the fluorescent protein of the receptor and the label of the polypeptide to be tested; wherein said second recombinant gene is expressed and the polypeptide to be tested is transported to a location allowing interaction with the receptor expressed on the cell membrane, and wherein collectively the mixture of cells expresses a library of said polypeptides to be tested, said library capable of being expressed at a sufficient level such that modulation of the targeting of the receptor protein by a polypeptide to be tested within the library provides a detectable signal;

(ii) allowing cells to generate a detectable signal; and