Novel ion-conductive channel fusion subunit and method of using the same

Abstract

The present invention provides a channel fusion subunit comprising an ion-conducting channel subunit bound to a probe, comprising: the probe comprises an ion sensor bound therebetween to at least one bioluminescent donor molecule and at least one fluorescent acceptor molecule; the probe is bound to the N-terminus, C-terminus, or within an intracellular or extracellular loop of the channel subunit via either the bioluminescent donor molecule or the fluorescent acceptor molecule; The bioluminescent donor molecule and the fluorescent acceptor molecule are selected so that the emission spectrum of the bioluminescent donor molecule overlaps with the absorption spectrum of the fluorescent acceptor molecule, thereby enabling non-radiative energy transfer between the bioluminescent energy donor and the fluorescent acceptor molecule by non-radiative dipole-dipole coupling. Concerning channel fusion subunits.

Description

Technical Field

[0001] [1] The present invention generally relates to novel ion-conductive channel fusion subunits and / or novel ion-conductive channel fusions, nucleic acids encoding the channel fusions and channel subunit fusions, amino acid sequences comprising the channel fusions and channel subunit fusions, recombinant cells and expression vectors, and their use for high-throughput screening (HTS) methods, particularly for screening inhibitors or activators of ion-conductive channels.

Background Art

[0002] [2] The life of higher organisms, including humans, requires the rapid and efficient transmission of electrical and chemical signals that synchronize countless diverse cellular processes. The interior of animal and plant cells is electrically negative relative to the outside. The magnitude of this potential difference is generally 5 - 90 mV, and most of this potential is generated across the cell membrane. The membrane potential difference of a given cell is set by the balance between the activities of ion transporters and channels that create and maintain the electrochemical gradient, and the activities of ion channels, passive diffusion, and other factors that allow ions to flow across the plasma membrane. Electrical signals are extremely important for cell function and are mainly mediated by ion channels or ion-conductive channels, which are special groups of proteins that are permeable to charged atoms (ions) present in the body across the membranes of all living cells. In a simple sense, ion-conductive channels can be regarded as the biological equivalent of transistors because they respond to stimuli by controlling the passage of electric current across the cell membrane by switching between an "on" (open) state and an "off" (closed) state. By doing so, ion-conductive channels control the electrical state of the cell and enable basic processes such as the beating of the heart, muscle contraction, and information processing in the nervous system. Since ion-conductive channels allow the movement of ions across the cell membrane, they are fully responsible for the entire area of electrical signal transmission in biological systems. Furthermore, ion channels affect the intracellular calcium concentration directly by allowing the flow of calcium or indirectly by modulating the membrane potential; this, in turn, controls various functions including energy production, synaptic transmission, muscle contraction, hormone secretion, and gene transcription.

[0003] [3] Specifically, an ion-conductive channel can be defined as a pore-forming protein that enables a specific type of ion to pass through the channel by its electrochemical gradient and controls a slight voltage difference across the plasma membrane of a cell. These ion-conductive channels are present in the cell membranes of all living cells. Ion-conductive channels can be classified into a chloride ion channel family, a potassium ion channel family, a sodium ion channel family, a calcium ion channel family, a proton channel family, and a universal ion channel family according to the type of ion.

[0004] [4] Considering the central and important role of ion-conductive channels in many physiological processes, they represent a target class with great potential for intervention in a wide range of disease states. In fact, the successful introduction of a number of ion-conductive channel drug classes (e.g., calcium antagonists, sodium channel blockers, gamma-aminobutyric acid or GABA receptor enhancers, and sulfonylureas) into the market has firmly established ion-conductive channels as effective drug targets. However, most commercially available ion-conductive channel drugs discovered in the "pre-molecular era", i.e., first-generation ion channel drugs, are relatively non-selective, often exhibit dose-limiting side effects, and are rarely 100% effective. Therefore, there is clearly a need for improved ion-conductive channel drugs that target ion-conductive channels with higher selectivity, fewer side effects, and improved efficacy, i.e., a new generation of ion channel drugs. Several diseases are associated with ion-conductive channel function. For example, diseases resulting from ion-conductive channel dysfunction in the central nervous system include anxiety, depression, epilepsy, insomnia, memory impairment, and chronic pain. Other diseases resulting from ion-conductive channel dysfunction include cardiac arrhythmia and type II diabetes.

[0005] [5] The initial methods developed to measure the activity of ion-conductive channels were based on the recording of living cells. Overexpression of the ion-conductive channel of interest was often required in such approaches, which changed the physiological state of the cells and led to unpredictable results regarding channel activity. This often resulted in toxicity to the cells and their apoptosis. In these methods, the activity of ion channels was mainly measured by patch clamp or fluorescence.

[0006] [6] Patch clamp systems involve electrophysiology-based measurements. The electrode is immersed inside the cell, and the reference electrode is outside the solution containing the cell. When ions pass through the channel, a current is generated across the membrane and measured by the electrode. If the channel is non-functional or blocked by a compound, no current is recorded. Fluorescence-based systems use ion-specific (or pH-dependent) fluorescent molecules. However, such ion-specific fluorescent molecules do not exist for all ions. To overcome the drawbacks of cell-based methods, cell-free methods still based on patch clamp or fluorescence measurements have been developed. In these cell-free methods, ion-conductive channels are likely expressed, purified, and reconstituted in model membranes, such as droplet interface bilayers (DIBs), in a microfluidic platform.

[0007] [7] To discover new targets for treatment and new pharmaceutical compounds that target ion channels, it is useful to be able to screen chemical libraries of a large number of compounds by automated high-throughput assays. However, the above cell-based methods or cell-free methods have the limitation of not facilitating high-throughput screening (HTS). In fact, most current HTS methods typically involve compounds in flow, in microfluidic devices or millifluidic devices. Thus, this precludes the use of the patch-clamp method due to the use of electrodes. The main limitation of the patch-clamp method is its low throughput. Typically, a highly trained operator can test fewer than 10 compounds per day using the patch-clamp method. Furthermore, this technique is not easily automated and yields complex results that require extensive analysis. The use of fluorescence-based methods is also not straightforward as fluorescence smears are observed under flow of fluorescent compounds that essentially generate noise and limit the readout information.

[0008] [8] Fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) technologies have been developed for drug discovery and HTS methods to monitor protein-protein interactions.

[0009] [9]International Publication No. WO2016 / 131832 describes a first-generation intramolecular BRET probe that includes a transient receptor potential (TRP) channel subunit fused to a bioluminescent donor molecule and a fluorescent acceptor molecule at either the C-terminus or N-terminus of the channel subunit. Such BRET probes enabled the monitoring of transient receptor potential (TRP) channel conformational changes and TRP channel activation in living cells. However, these first-generation BRET probes are only efficient in measuring changes in the conformation of ion channels, and since changes in the conformation of ion channels are not necessarily related to actual activation or inhibition, they do not necessarily show the actual activation or inhibition of ion-conductive channels in a reliable manner.

Summary of the Invention

Problems to be Solved by the Invention

[0010]

[10] Therefore, one problem in increasing the drug discovery rate was to provide a method that enables the measurement of the passage of ions through channels embedded in the membrane in the HTS method. Accordingly, the present application relates to a novel BRET probe that enables the reliable measurement of the activation or inhibition of ion-conductive channels by measuring the actual passage of ions using BRET technology, and to methods of using these new probes for drug screening and mapping signal transduction pathways.

Means for Solving the Problems

[0011]

[11] The present invention relates to a nucleic acid encoding a novel fusion protein, and a novel fusion protein comprising an ion-conductive channel fusion or a novel ion-conductive channel fusion subunit. These fusions comprise an ion sensor having at least one bioluminescent donor molecule and at least one fluorescent acceptor molecule bound or sandwiched therebetween, with an ion-conductive channel or a subunit thereof bound to the C-terminus, N-terminus, or within an intracellular or extracellular loop of a probe, and the ion sensor is configured to undergo a conformational change in the presence of ions transported by the channel subunit.

[0012]

[12] The present invention also relates to an expression vector comprising a nucleotide sequence encoding a novel ion-conductive channel fusion or a novel ion-conductive channel fusion subunit, a recombinant cell comprising such an expression vector, a method for producing an ion channel subunit, and a fusion protein and a fusion subunit obtainable by said method.

[13] The present invention further relates to a method for screening for candidate compounds that can modulate ion flux through an ion-conductive channel by measuring fluctuations in bioluminescence resonance energy transfer, and thus activate and / or inhibit said channel. BRIEF DESCRIPTION OF THE DRAWINGS

[0013]

Figure 1

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Mode for Carrying Out the Invention

[0014]

[27] The techniques of recombinant proteins, cell culture, and molecular cloning utilized in the present invention are standard procedures well-known to those skilled in the art. Such techniques are described and explained throughout the literature of sources such as J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0015]

[28] The term "bioluminescent donor molecule" refers to any molecule capable of generating luminescence after either an action on a suitable substrate or excitation of itself by an external source.

[29] The term "fluorescent acceptor molecule" refers to any compound that can receive the energy released as a result of the activity of a bioluminescent donor molecule and re-emit it as light energy.

[30] Bioluminescent resonance energy transfer (BRET) refers to a proximity assay based on the non-radiative transfer of energy between a bioluminescent donor molecule and any suitable fluorescent acceptor molecule. Non-radiative transfer means the transfer of energy from an energy donor to an energy acceptor without the emission of light by the donor.

[0016]

[31] The term "spatial position" refers to the three-dimensional arrangement of a bioluminescent donor molecule relative to an acceptor molecule that changes as a result of the binding of a compound to a voltage-dependent ion channel or as a result of a change in a specific physical parameter.

[32] The term "dipole orientation" refers to the direction in three-dimensional space of the dipole moment associated with either the donor molecule and / or the acceptor molecule with respect to their orientation in three-dimensional space. The dipole moment is the result of the fluctuation of charges over the entire molecule. The resulting resonance energy transfer is an electromagnetic phenomenon in which quantum energy non-radiatively transfers from an excited bioluminescent donor to a nearby fluorescent acceptor molecule.

[0017]

[33] The term "resonance energy transfer" refers to the fact that the energy transfer is due to intermolecular dipole-dipole coupling, i.e., the process does not involve the emission and reabsorption of photons. The donor typically emits light at a shorter wavelength that overlaps with the absorption spectrum of the acceptor molecule.

[34] The term "protein-protein interaction" or "binding" refers to the specific complementary recognition and association of two proteins with a dissociation constant Kd preferably of 10 -5 M, more preferably 10 -7 M, most preferably 10 -9 M or less.

[0018]

[35] A modulator, activator or inhibitor is any agent that can alter the functional activity of an intracellular ion channel and can represent a channel opener (functional agonist), a channel blocker (functional antagonist).

[36] As used herein, the terms "fusion channel unit" or "fusion channel" generally refer to a polypeptide or protein channel or channel subunit composed of two or more amino acid sequences, where each amino acid sequence encodes a protein or a part thereof, and the two or more amino acid sequences are not found to be naturally linked, and the two or more amino acid sequences are physically linked by peptide bonds.

[0019]

[37] As used herein, the term "domain" refers to a region of a protein that is at least 2 amino acids less than the full protein and retains at least the biological activity of the full protein. A domain can range in size from 10 to 1000 amino acids, such as 50 to 60 amino acids, 100 to 400 amino acids or 200 to 300 amino acids. A domain is a functional unit of a full protein and refers to a functional unit having the biological activity of the full protein. For example, a domain of a useful protein according to the present invention can refer to a region of the protein that binds to a second protein.

[0020]

[38] The term "more sensitive" refers to a greater change in the resonance energy transfer ratio between a bioluminescent donor molecule and a fluorescent acceptor molecule.

[39] The term "contacting" refers to the addition of candidate molecules, a series of candidate molecules, and / or samples that can activate or inhibit an ion-conductive channel and thus can be screened by HTS methods using a new generation of BRET probes.

[0021]

[40] "Substantially purified" or "purified" means that the ion-conductive channel or channel subunit is separated from one or more lipids, nucleic acids, other polypeptides, or other contaminating molecules that are associated in its native state. A substantially purified polypeptide preferably contains less than 60% of other components that are naturally associated, more preferably less than 75%, and even more preferably less than 90%.

[0022]

[41] As used herein, a "cell membrane" preparation refers to a preparation of the cell lipid membrane and is different from a cell homogenate in that at least a part (i.e., at least 10%, preferably more) of the non-membrane-bound cell components has been removed from the homogenate. "Membrane-bound" refers to a polypeptide that is either incorporated into the lipid membrane or physically associated with a component incorporated into the lipid membrane.

[0023]

[42] The terms "polypeptide" and "protein" are generally used interchangeably and refer to a single polypeptide chain that may or may not be modified by the addition of non-amino acid groups. It is understood that such polypeptide chains can associate with other polypeptides or proteins or other molecules, such as cofactors. The terms "protein" and "polypeptide" as used herein also include variants, mutants, biologically active fragments, modified forms, analogs and / or derivatives of the polypeptides described herein.

[0024]

[43] The percent identity of the polypeptide is determined by GAP analysis (GCG program) with a gap insertion penalty of 5 and a gap extension penalty of 0.3. The query sequence is at least 25 amino acids in length, and GAP analysis aligns the two sequences over a region of at least 25 amino acids. More preferably, the query sequence is at least 50 amino acids in length, and GAP analysis aligns the two sequences over a region of at least 50 amino acids. More preferably, the query sequence is at least 100 amino acids in length, and GAP analysis aligns the two sequences over a region of at least 100 amino acids. Even more preferably, the query sequence is at least 250 amino acids in length, and GAP analysis aligns the two sequences over a region of at least 250 amino acids. Even more preferably, GAP analysis aligns the two sequences over their entire lengths.

[0025]

[44] As used herein, "biologically active fragment" is a part of a polypeptide as described herein that maintains the defined activity of the full-length polypeptide. For example, a biologically active fragment of a voltage-dependent ion subunit should be able to bind to a target compound and consequently cause a conformational change. A biologically active fragment can be of any size as long as it maintains the defined activity. Preferably, the biologically active fragment is at least 150 amino acids in length, more preferably at least 250 amino acids in length.

[0026]

[45] As used herein, "biologically active variant" refers to a molecule that differs from a naturally occurring molecule and / or a defined molecule by one or more amino acids, but maintains the defined activity as defined above for biologically active fragments. Biologically active variants typically have at least 50%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 97%, and even more preferably at least 99% identity to the naturally occurring molecule and / or the defined molecule.

[0027]

[46] For a defined polypeptide or polynucleotide, it is understood that numerical values of percent identity higher than those provided above encompass preferred embodiments. Thus, where applicable in light of the minimum percent identity values, a polypeptide or polynucleotide preferably comprises an amino acid sequence that is at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant specified amino acid sequence.

[0028]

[47] "Isolated polynucleotide" (including DNA, RNA, or combinations thereof, single-stranded or double-stranded, in sense or antisense orientation, or a combination of both) means a polynucleotide that is at least partially separated from the polynucleotide sequences that are associated or linked in its native state. Preferably, the isolated polynucleotide contains no less than 60%, preferably no less than 75%, and most preferably no less than 90% of the other components with which they are naturally associated.

[48] As used herein, the terms "determine", "measure", "evaluate", and "assay" are used interchangeably and include both quantitative and qualitative determinations.

[0029]

[49] As used herein, "recombinant vector" refers to an isolated genetic factor used to introduce a heterologous nucleic acid into a cell for either its expression or replication. The selection and use of such vehicles are well within the skill of the art. Recombinant expression vectors include vectors capable of expressing nucleic acids operably linked to regulatory sequences, such as a promoter region capable of effecting the expression of the nucleic acid. Thus, an expression vector refers to a recombinant DNA construct or a recombinant RNA construct, such as a plasmid, phage, recombinant virus, or other vector that, when introduced into a suitable host cell, results in the expression of the cloned nucleic acid. Suitable expression vectors are well known to those skilled in the art and include those that can replicate episomally or be integrated into the host cell genome, including those that are replicable in eukaryotic cells and / or prokaryotic cells.

[0030]

[50] The term "ion-conductive channel" shall refer to an ion-permeable pore in the lipid membrane of all cells, which is known to open and close in response to stimuli and gate the flow of specific small ions. In fact, the lipid bilayer of all cells forms a barrier that is almost impermeable to the flow of ions and water. What exists within the membrane is a family of proteins called ion channels or ion-conductive channels, which provide a selective pathway for ion flux. Thus, such ion-conductive channels are ion-selective channels, for example, sodium-, potassium-, calcium-, chloride-selective or non-selective channels.

[0031]

[51] Ion flux through ion-conductive channels contributes to cell depolarization, makes the membrane potential more positive, and generates cytoplasmic regulatory signal transduction. For example, the controlled conductance generated by ion channels is necessary for intercellular signal transduction and nerve excitability. The group of ion channels that open during depolarization of excitable cells is classified as voltage-gated / voltage-dependent and is responsible for electrical activity in nerve, muscle, and heart tissues. For example, in neurons, the ion current flowing through voltage-gated sodium ion (Na + ) channels is responsible for rapid spike-like action potentials. During the action potential, most Na + channels open for a very short time. These short openings result in transient Na + currents. However, a subset of voltage-gated Na + channels do not close rapidly and remain open for a relatively long time. Thus, these channels generate persistent Na + currents. The balance between transient Na + currents and persistent Na + currents is important for maintaining normal physiological functions and electrical signal transduction throughout the nervous system.

[0032]

[52] Over the past 50 years, an increasing number of diseases have been shown to result from dysregulation of ion-conductive channels. This class of diseases is called channelopathies. Abnormal persistent sodium currents can contribute to the onset or progression of many channelopathies because normal function is disrupted when neurons inappropriately signal. For example, abnormal persistent sodium currents are thought to induce deleterious phenomena including, for example, neuropathy, neurodegenerative diseases, movement disorders, cardiac arrhythmias, epileptic seizures, neuronal death, behavioral disorders, and dementia. For example, in the case of neuropathy subsumed in epilepsy, there may be short electrical "storms" arising from neurons that are inherently unstable due to genetic defects as in various types of genetic epilepsy, or from neurons destabilized by metabolic abnormalities such as hypoglycemia or alcohol. In other cases, such abnormal discharges may originate from local regions of the brain, as in patients with epilepsy caused by head injury or brain tumor. In the case of ischemic injury such as cerebral ischemia and myocardial ischemia, electrical activity arising from neurons with increased persistent sodium channel expression or activity may be prolonged. Such abnormal electrical activity can cause or contribute to neuronal death, which can lead to debilitating injury or death of the individual. Abnormal electrical activity can also contribute to neurodegenerative disorders including, but not limited to, Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, and multiple sclerosis. Thus, abnormal persistent sodium currents can contribute to the onset or progression of pathological conditions by disrupting the normal transmembrane gradient for sodium, causing reverse operation of the sodium-calcium exchanger, and causing intracellular calcium influx that damages axons. Thus, selective reduction of the expression or activity of sodium channels capable of mediating persistent currents, as compared to any reduction of normal voltage-gated (transient) sodium currents, may be useful for treating channelopathies associated with increased persistent sodium currents.

[0033]

[53] Therefore, there is a need for a new screening method that can be used to identify any persistent ion channel modulators, inhibitors, or activators useful for treating channelopathies. The present invention meets this need by providing high-throughput screening for identifying ion channel modulators, activators, or inhibitors.

[0034]

[54] One technique for evaluating protein-protein interactions is based on fluorescence resonance energy transfer (FRET). FRET is a proximity assay based on the non-radiative transfer of energy, i.e., the transfer of energy that occurs without the emission of photons and results in a dipole-dipole interaction between an energy donor and an energy acceptor. In FRET, an excited-state fluorophore donor transfers its energy to a ground-state fluorescent acceptor molecule, which is thereby activated. Following activation, the acceptor returns to the ground state by emitting photons at its own wavelength. The energy transfer efficiency depends on the distance between the donor and acceptor, the degree of spectral overlap, and the relative orientation of the acceptor dipole and the donor dipole.

[0035]

[55] Experiments have been conducted in the past on neuronal voltage-gated N-type (Cav2.2) calcium using a FRET-based calcium biosensor named TN-XL (see Tay et al., (Nature Communications, Vol, 3; 778, 2012)). The authors used a specific approach of fusing TN-XL (an optical, genetically encoded calcium ion indicator) to the carboxy terminus of the major subunit of the CaV2.2 Ca 2+ channel, which was targeted to this environment, and when Ca 2+It was investigated whether it could respond. These experiments could not provide a reliable and useful means for measuring the activity of ion-conductive channels. In fact, to measure TN-XL responsiveness, TIRF imaging had to be used to measure single-cell CFP and FRET fluorescence signals. Here, the ratio of these signals provides the conventional sensor readout information for Ca 2+ . Furthermore, to improve the TN-XL signal-to-noise ratio in the surface membrane, TIRF microscopy was utilized to selectively irradiate only the CaV2.2 / TN-XL channels near the surface, thereby attenuating the background signal from the incompletely targeted intracellular channels. Additionally, to facilitate the rise of intracellular Ca 2+ , additional steps such as raising the extracellular Ca 2+ to 10 mM and internal Ca 2+ buffering (1 mM EGTA) were employed. A further limitation of the study was related to the incomplete targeting of active CaV2.2 / TN-XL channels to the surface membrane in the TIRF volume, which required approximate correction of the static background fluorescence. Potential errors in such corrections result in a crude approximate estimate of calcium spikes, and thus such techniques are not at all suitable for HTS.

[0036]

[56] Thus, this study revealed the complexities and difficulties encountered when using FRET-based calcium biosensors, which require the complex integration of TIRF / patch-clamp electrophysiology and CaV2.2 / TN-XL fusion, instead of a direct and simple method based on the fusion construct alone.

[0037]

[57] An alternative to FRET technology is BRET technology. The BRET method is similar to the FRET method where the donor is a bioluminescent molecule. Thus, an external excitation source is not required, thereby avoiding the disadvantages of photo-bleaching and autofluorescence of cells / tissues. Typically, the bioluminescent donor used may be luciferase, and the acceptor may be any suitable fluorophore similar to that of FRET. The use of luciferase avoids the need for illumination as the addition of the substrate initiates bioluminescence emission and thus results in resonance energy transfer between the bioluminescent donor and the fluorescent acceptor. The transfer efficiency of both FRET and BRET also depends on the degree of spectral overlap or partial spectral overlap between the donor and acceptor, the relative orientation between the emission dipole of the donor and the absorption dipole of the acceptor, and the distance between the donor and acceptor, and further depends on the efficacy of ion sensors strategically placed between the donor and acceptor in the novel fusion construct. Thus, it is expected that those skilled in the art will not be able to utilize similar BRET-based ion biosensors to provide similar results and thus for drug discovery or HTS methods.

[0038]

[58] In contrast to previous teachings and experiments using FRET, the Applicant has surprisingly shown for the first time that it is possible to construct and use BRET-based ion sensors to accurately and reliably evaluate ion fluxes and thus the actual activation or inhibition of ion-conductive channels. The Applicant has shown that the new BRET probes can be used in screening systems that target ion-conductive channels with excellent efficiency, particularly in HTS methods that do not require further analysis, such as patch clamp experiments, etc.

[0039]

[59] Accordingly, the present invention relates to a novel ion-conductive channel protein subunit fusion comprising an ion-conductive channel subunit bound to a probe, the probe comprising an ion sensor in which at least one bioluminescent donor molecule and at least one fluorescent acceptor molecule are bound or "sandwiched" therebetween. The probe may be bound to either the bioluminescent donor molecule or the fluorescent acceptor molecule at its C-terminus, its N-terminus, or within an intracellular or extracellular loop, to the channel subunit, optionally via a linker. The ion sensor is configured to undergo a conformational change in the presence of an ion transported by the channel subunit. The bioluminescent donor molecule and the fluorescent acceptor molecule are selected such that the emission spectrum of the bioluminescent donor molecule overlaps with the absorption spectrum of the acceptor molecule, enabling non-radiative energy transfer between the bioluminescent energy donor and the fluorescent acceptor via non-radiative dipole-dipole coupling. Thus, the novel channel protein fusion subunit may be used as a novel BRET probe for detecting activation, inhibition, or modulation of the channel by measuring the actual passage of ions through the ion-conductive channel.

[0040]

[60] The present invention also relates to a novel ion-conductive channel fusion obtained from the assembly of the fusion subunits as described above. Thus, such a novel ion-conductive channel protein fusion is bound to a probe that itself contains an ion sensor sandwiched between at least one bioluminescent donor molecule and at least one fluorescent acceptor molecule, at the C-terminus, N-terminus, or within either an intracellular or extracellular loop of the subunit. The ion sensor is configured to undergo a conformational change in the presence of ions transported by the channel fusion. Preferably, all subunits of the ion-conductive channel protein fusion can be channel subunit fusions. As described above, the bioluminescent donor molecule and the fluorescent acceptor molecule are selected such that the emission spectrum of the bioluminescent donor molecule overlaps with the absorption spectrum of the acceptor molecule, enabling non-radiative energy transfer between the bioluminescent energy donor and the fluorescent acceptor via non-radiative dipole-dipole coupling. Thus, the novel channel protein fusion may be used as a novel BRET probe for detecting activation, inhibition, or modulation of the channel by measuring the actual passage of ions through the ion-conductive channel.

[0041]

[61] The present invention also relates to a nucleotide sequence encoding a channel fusion subunit comprising a subunit of an ion-conductive channel bound to a probe, the probe itself containing an ion sensor bound therebetween to at least a bioluminescent donor molecule and at least a fluorescent acceptor molecule. As described above, the ion sensor is configured to undergo a conformational change in the presence of ions transported by the channel subunit, and the probe is bound to the N-terminus or C-terminus of the channel subunit, or within an intracellular or extracellular loop, via either a bioluminescent donor molecule or a protein fluorescent acceptor molecule. The nucleotide sequence or construct according to the invention may further comprise a linker sequence between the nucleotide sequence encoding the ion-conductive channel subunit and the nucleotide sequence encoding the probe, if desired.

[0042]

[62] Since most ion channels have such a common three-dimensional structure, all known ion-conductive channels are within the scope of the present invention. Typically, these ion-conductive channels have at least three functional domains: a first domain corresponding to an ion-conductive pore, i.e., a selectivity filter that distinguishes between ions that pass through and those that do not; a second domain corresponding to a gate, i.e., a part of the channel that can open and close the conductive pore; and a third domain corresponding to a detector of stimuli that responds to potential changes or chemical signals. Thus, the detector of stimuli typically couples to the channel gate to control the probability that they open and close the pore.

[0043]

[63] Channels are formed by subunits that each pass through the plasma membrane at least twice. Various subunits assemble to form the channel. Whether the channel is formed by a homomeric assembly of the same subunits, or by a heteromeric combination of alpha subunits and other subunits, either heteromeric or homomeric, depends on the channel type. At the center of the assembled channel is a pore, a narrow opening that allows ions, e.g., Na + , K + , Ca 2+ , and / or Cl - to flow across the membrane. The pore is responsible for the permeability to the selected ions.

[0044]

[64] Thus, ion-conductive channels are composed of several subunits organized in the plasma membrane to create a pore through which cations or anions can move. Ion-conductive channels may be classified by gating, i.e., that which opens and closes the channel, and may particularly include voltage-dependent ion channels or ligand-dependent ion channels. Voltage-gated ion channels activate / inactivate depending on the potential gradient across the plasma membrane, while ligand-gated ion channels activate / inactivate depending on the binding of a ligand to the channel.

[0045]

[65] Voltage-gated ion channels are membrane proteins that conduct ions rapidly and are controlled by the voltage across the membrane. Therefore, these channel proteins are selective for specific ions, such as calcium ions, potassium ions, sodium ions, or chloride ions. The most studied and well-known are the voltage-gated Na + , K + , and Ca 2+ channels. They generally have a common tetrameric structural arrangement formed by four identical or non-identical subunits, with a central pore and highly conserved pore-lining residues for channels with similar charge selectivity; a common gating strategy using charged membrane voltage sensors; and share auxiliary subunits that regulate channel protein trafficking and function. For example, channels composed of four non-identical subunits include the K + channel, or channels composed of four identical domains include the Na + and Ca 2+ channels. Each of the subunits or domains has six transmembrane segments and a pore loop. The fifth and sixth transmembrane segments (S5 and S6) and the pore loop are responsible for ion conduction. Among these, prominent ones are the voltage-gated Na + , K + , and Ca 2+ channels that underlie the action potentials and rapid calcium signaling in electrically excitable cells. The rapid voltage-dependent opening of the Na + channel explains the 0 / 1 (all-or-nothing) excitation that starts at a sharp threshold by depolarizing the stimulus, and the regenerative spread of excitation when the action potential propagates along the axon or muscle fiber.

[0046]

[66] Some hold a voltage sensor domain with a small positive charge, while others have at most a weak voltage dependence. Among these are cyclic nucleotide-gated (CNG) channels and non-selective cation channels of the TRP family. CNG channels are useful for vertebrate phototransduction and olfaction. The TRP family includes diverse forms that are useful in invertebrate phototransduction and in sensory receptors that detect heat, cold, capsaicin, mustard, ginger, and possibly touch, pressure, and movement. Another family of ion channels, inward rectifier K + channels, do not have a voltage sensor domain but have a pore-forming domain with the same sequence identity and ion selectivity as voltage-gated K + channels.

[67] Voltage-dependent ion channels have a track record as drug discovery targets, and many ion channel modulators are currently in clinical use for the treatment of pain, epilepsy, hypertension, and other disease states. They include the molecular basis for essential physiological functions, including body fluid secretion, electrolyte balance, and bioenergy and membrane excitability.

[0047]

[68] Voltage-dependent ion channels are known to exist in several different conformational states called gating states. Voltage-gated ion channels can be considered to exist in one of three gating states: closed (no ion permeation), open (ion flux occurs), and inactivated (no ion permeation; the channel cannot open in response to depolarization), although it should be noted that some channels do not exhibit an inactivated state. The transitions between gating states are voltage-dependent, and at any given time, an equilibrium exists between these gating states, and the proportion of channels in each state depends on the cell membrane potential. Many voltage-dependent ion channel modulators have been shown to preferentially bind to specific gating states. For example, the voltage-gated sodium channel blocker lamotrigine is thought to bind to the open and inactivated states of the brain sodium channel protein. Preferential binding to a specific gating state can occur by an increase in the channel affinity for the ion channel modulator or simply by improved access of the drug to its binding site on the channel.

[0048]

[69] A complete description of the voltage-gated ion channels within the scope of the present invention is presented in the following table.

[0049]

Table 1-1

Table 1-2

[0050]

Table 2-1

Table 2-2

[0051]

Table 3-1

Table 3-2

Table 3-3

Table 3-4

[0052]

Table 4

[0053]

Table 5

[0054]

Table 6

[0055]

Table 7

[0056]

Table 8-1

Table 8-2

Table 8-3

Table 8-4

Table 8-5

[0057]

Table 9-1

Table 9-2

[0058]

Table 10-1

Table 10-2

[0059]

Table 11

[0060]

[70] Generally, ligand-gated ion channels (LICs, LGICs), also known as ion-channel receptors, are integral membrane proteins containing pores that allow the flow of selected ions, such as Na + , K + , Ca 2+ and / or Cl - to pass through the membrane in response to the binding of a chemical messenger, i.e., a ligand.

[0061]

[71] Therefore, these channels are extracellular and contain a fourth domain that includes a ligand-binding domain. Such ligands can be, for example, neurotransmitters. When the presynaptic neuron is excited, neurotransmitters are released from the vesicles into the synaptic cleft. The neurotransmitter then binds to a receptor located on the postsynaptic neuron, causing a conformational change in the ion channel and, upon activation or inhibition, leading to the flow of ions across the cell membrane. This results in either depolarization for excitatory receptor responses or hyperpolarization for inhibitory responses. The function of these channels located postsynaptically is to directly and very rapidly convert the chemical signal of the neurotransmitter released presynaptically into a postsynaptic electrical signal.

[0062]

[72] These ligand-gated ion channels are subdivided with respect to the type of ion they conduct, e.g., anionic or cationic, and further into families defined by endogenous ligands. They include cation-selective receptors such as the acetylcholine receptor, 5-HT3 receptor, ionotropic glutamate receptor, and P2X receptor, and A anion-selective receptors such as the GABA receptor, glycine receptor, and zinc-activated channel. With respect to their conformation, the nicotinic acetylcholine receptor, 5-HT3 receptor, GABAA (γ-aminobutyric acid A) receptor, glycine receptor, and further zinc-activated channels are pentameric structures and are often called Cys-loop receptors due to the presence of a defined loop of residues formed by a disulfide bond between two cysteine residues in the N-terminal extracellular domain of their constituent subunits. The ionotropic glutamate receptor and P2X receptor are tetrameric and trimeric structures, respectively. The binding sites in the extracellular N-terminal ligand-binding domain confer receptor specificity on them for specific endogenous ligands including acetylcholine (AcCh), serotonin, glycine, glutamate, and γ-aminobutyric acid (GABA), etc.

[0063]

[73] The 5-HT3 receptor, or 5-hydroxytryptamine (serotonin) receptor, exists as a pentamer of four TM subunits that form an endogenous cation selectivity. 5-HT 3A (https: / / www.uniprot.org / uniprot / P46098), 5-HT 3B (https: / / www.uniprot.org / uniprot / P46098), 5-HT 3C (https: / / www.uniprot.org / uniprot / Q8WXA8), 5-HT 3D (https: / / www.uniprot.org / uniprot / Q70Z44), 5-HT 3E (https: / / www.uniprot.org / uniprot / A5X5Y0), five human 5-HT3 receptor subunits have been cloned, and the homo-oligomeric assembly of 5-HT3A as well as the hetero-oligomeric assembly of 5-HT3A and 5-HT3B subunits have been fully characterized (5-HT3A and 5-HT3AB receptors). The 5-HT3C subunit as well as the 5-HT3D and 5-HT3E subunits, similar to the 5-HT3B subunit, do not form functional homomers, but when combined with the 5-HT3A subunit, they affect not the pharmacological profile but the functional expression. Serotonin receptors are a group of G protein-coupled receptors and ligand-gated ion channels found in the central and peripheral nervous systems that mediate both excitatory and inhibitory neurotransmission. They are activated by the neurotransmitter serotonin, which acts as their natural ligand, and modulate the release of many neurotransmitters, including glutamate, GABA, dopamine, epinephrine / norepinephrine, and acetylcholine, as well as many hormones, such as oxytocin, prolactin, vasopressin, cortisol, corticotropin, and substance P. These receptors affect various biological and neurological processes, such as aggression, anxiety, appetite, cognition, learning, memory, mood, nausea, and sleep.

[0064]

[74] The zinc-activated ion channel (ZAC), also known as ZAC1, L2mLICZ, LICZ1 (https: / / www.uniprot.org / uniprot / Q401N2). ZAC forms a cation-permeable ligand-gated ion channel of the cys-loop family. This channel is most likely to exist as a homo-pentamer of four transmembrane subunits that form an endogenous cation-selective channel that is equipermeable to Na + , K + and Cs + , but impermeable to Ca 2+ and Mg 2+ . ZAC is expressed in the prostate, thyroid, trachea, lung, brain (adult and fetus), spinal cord, skeletal muscle, heart, placenta, pancreas, liver, kidney and stomach. The endogenous ligand of ZAC is thought to be Zn 2+ , but ZAC has also been found to activate spontaneously.

[0065]

[75] GABA A(Gamma-aminobutyric acid A) receptors or channels are ion-channel receptors. They are pentamers of four transmembrane subunits, including six alpha subunits: alpha1-alpha6 subunits (https: / / www.uniprot.org / uniprot / P14867, P47869, P34903, P48169, P31644, Q16445), three beta subunits: beta1-beta3 subunits (https: / / www.uniprot.org / uniprot / P14867, P47870, P28472), three gamma subunits: gamma1-gamma3 subunits (https: / / www.uniprot.org / uniprot / Q8N1C3, P18507, Q99928), one delta (δ) subunit (https: / / www.uniprot.org / uniprot / O14764), one epsilon (ε) subunit (https: / / www.uniprot.org / uniprot / P78334), one pi (π) subunit (https: / / www.uniprot.org / uniprot / O00591), one theta (θ) subunit (https: / / www.uniprot.org / uniprot / Q9UN88), and three rho subunits: rho1-rho3 receptor subunits (https: / / www.uniprot.org / uniprot / P24046, P28476, A8MPY1). Many GABA A receptor subtypes contain alpha-, beta-, and gamma-subunits with a stoichiometry of 2α.2β.1γ. However, in the CNS, the α1β2γ2 hetero-oligomer constitutes the largest population of GABA A receptors in the CNS, followed by the α2β3γ2 and α3β3γ2 isoforms. Currently, eleven native GABA A receptors, namely, α1β2γ2, α1βγ2, α3βγ2, α4βγ2, α4β2δ, α4β3δ, α5βγ2, α6βγ2, α6β2δ, α6β3δ, and ρ, have been classified as finally identified. The endogenous ligand is gamma-aminobutyric acid (GABA), which is the main inhibitory neurotransmitter in the central nervous system. When activated, GABAA The receptor selectively conducts chloride (Cl - ) through its pore. Several inhibitors have been identified, including muscimol, gaboxadol, bicuculline, benzodiazepine, non-benzodiazepine, alprazolam, diazepam, and picrotoxin.

[0066]

[76] The glycine receptor, also known as GlyR or GLRi, is a receptor for the amino acid neurotransmitter glycine. GlyR is an ion channel-type receptor that exerts its effect through chloride current. The receptor is expressed either as a homo-pentamer of α subunits or as a complex of 2α and 3β subunits. The amino acid sequences of glycine receptor subunits α1-α3 and β are provided at https: / / www.uniprot.org / uniprot / P23415, P23416, O75311, and P48167, respectively. It is one of the most widely distributed inhibitory receptors in the central nervous system and plays an important role in mediating inhibitory neurotransmission in various physiological processes, particularly in the spinal cord and brainstem. The receptor can be activated by a series of simple amino acids including glycine, β-alanine, and taurine, and can be selectively blocked by the high-affinity competitive antagonist strychnine. Caffeine is a competitive antagonist of GlyR.

[0067]

[77] Ion channel-type glutamate receptors bind to the neurotransmitter glutamate. They form tetramers with each subunit consisting of an extracellular amino-terminal domain (ATD) involved in subunit assembly, an extracellular ligand-binding domain (LBD) that binds glutamate, and a transmembrane domain (TMD) that forms the ion channel. Each subunit of the tetramer has a binding site for glutamate formed by two LBD sections that form a clam shell-like shape. Only two of these sites in the tetramer need to be occupied to open the ion channel. Ion channel-type glutamate receptors include members of the NMDA (N-methyl-D-aspartic acid), AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), and kainic acid receptor classes.

[0068]

[78] NMDA receptors assemble as heteromers containing GluN1 (https: / / www.uniprot.org / uniprot / Q05586), GluN2A (https: / / www.uniprot.org / uniprot / Q12879), GluN2B (https: / / www.uniprot.org / uniprot / Q13224), GluN2C, GluN2D (https: / / www.uniprot.org / uniprot / Q12879), GluN3A (https: / / www.uniprot.org / uniprot / Q8TCU5), and GluN3B (https: / / www.uniprot.org / uniprot / O60391) subunits. AMPA receptors assemble as homomers or heteromers containing GluA1, GluA2, GluA3, and GluA4 subunits. Kainate receptors may be expressed as homomers of the GluK1 subunit (https: / / www.uniprot.org / uniprot / P39086), GluK2 subunit (https: / / www.uniprot.org / uniprot / Q13002), or GluK3 subunit (https: / / www.uniprot.org / uniprot / Q13003). The GluK1-3 subunits can also assemble into heterotetramers, such as GluK1 / K2.

[0069]

[79] P2X receptors are ATP-gated channels that open in response to binding of extracellular nucleotide ATP. They form trimers with two transmembrane domains having intracellular C-terminal and N-terminal ends, and gate Na + , K + and Ca 2+ . Native P2X receptors can exist as either homotrimers or heterotrimers containing P2X1-7 subunits (https: / / www.uniprot.org / uniprot / P51575, Q9UBL9, P56373, Q99571, Q93086, O15547, Q99572).

[0070]

[80] The nicotinic acetylcholine receptor (nAChR) is a member of the Cys-loop family consisting of a pentamer of protein subunits (typically, ααβγδ). The subunits can be of class alpha (α1-α10) called ACHRA (https: / / www.uniprot.org / uniprot / P02708), class beta (β1-β4) called ACHRB (https: / / www.uniprot.org / uniprot / P11230), class gamma (γ) or ACHRG (https: / / www.uniprot.org / uniprot / P07510), class delta (δ) or ACHRD (https: / / www.uniprot.org / uniprot / Q07001), class epsilon (ε) or ACHRE (https: / / www.uniprot.org / uniprot / Q04844). The binding site for acetylcholine is on the interface of each alpha subunit. When acetylcholine binds, the conformation of the receptor changes, the pore that was about 3 angstroms wide expands to about 8 angstroms, allowing ions to pass through. This pore allows + Na ions to flow into the cell according to the electrochemical gradient. When a sufficient number of channels open at once, the + inward flow of positive charge carried by Na ions depolarizes the postsynaptic membrane sufficiently to generate an action potential. nAChRs respond to the neurotransmitter acetylcholine and drugs such as the agonist nicotine. They are found in the central and peripheral nervous systems, muscle, and many other tissues of many organisms. At the neuromuscular junction, they are the major receptors in muscle for motor nerve-muscle transmission that controls muscle contraction. In the peripheral nervous system, they transmit the output signal from presynaptic cells to postsynaptic cells within the sympathetic and parasympathetic nervous systems, and they are the receptors found on skeletal muscle that receive the released acetylcholine and transmit the signal for muscle contraction.

[0071]

[81] Thus, according to the present invention, the ion-conductive channel fusion subunit is a subunit of an ion-conductive channel of either a ligand-gated ion channel or a voltage-gated ion channel as described above, which is bound or coupled at the C-terminus or N-terminus to a probe comprising an ion sensor sandwiched between at least one bioluminescent donor molecule and at least one fluorescent acceptor molecule. Alternatively, the ion sensor sandwiched between the bioluminescent donor and fluorescent acceptor molecules may also be inserted within an intracellular loop or an extracellular loop of the ion-conductive channel subunit. When the subunits of the ion-conductive channel are expressed in cells, they can thus reassemble to form an active ion-conductive channel fusion protein that can be used for the HTS method according to the present invention. Such channel fusion proteins can be formed by the same type of subunits and are homopolymer channels (homo-dimers, homo-tetramers, etc.) or can be formed by different types of subunits and are heteropolymer channels (hetero-dimers, hetero-tetramers, etc.).

[0072]

[82] The bioluminescent donor molecule and the fluorescent acceptor molecule are selected such that the emission spectrum of the bioluminescent donor molecule overlaps with the absorption spectrum of the acceptor molecule, so that the light energy delivered by the bioluminescent donor molecule is at a wavelength capable of exciting the acceptor molecule, thus enabling non-radiative energy transfer between the bioluminescent energy donor and the fluorescent acceptor via non-radiative dipole-dipole coupling.

[0073]

[83] The bioluminescent donor refers to any moiety capable of converting chemical energy into light energy by acting on a suitable substrate. For example, it may refer to an enzyme that converts a substrate into an activated product. Here, the activated product then releases energy upon relaxation. The activated product (produced by the activity of the bioluminescent protein on the substrate) is the source of the luminescence produced by the bioluminescent protein, and this luminescence travels to the acceptor molecule.

[0074]

[84] Several bioluminescent donor molecules can be used in the present invention. Luminescence systems are known and have been isolated from many luminescent organisms including bacteria, protozoa, coelenterates, mollusks, fish, squid, flies, fungi, worms, crustaceans, and beetles, particularly fireflies of the genus Pyrophorus, and fireflies of the genera Photinus, Photuris, and Luciola. Additional organisms that exhibit bioluminescence are listed in International Publications WO00 / 024878 and WO99 / 049019.

[0075]

[85] One well-characterized example is a class of proteins known as luciferases. Luciferase proteins catalyze an energy-generating chemical reaction in which a specific biochemical, luciferin (a naturally occurring substrate), is oxidized by an enzyme with luciferase activity. Both prokaryotes and eukaryotes, including bacteria, algae, fungi, insects, fish, and other marine forms of species, can emit light energy in this manner, each having specific luciferase activity and luciferin, which are chemically different from those of other organisms. The luciferin / luciferase system is very diverse in form, chemistry, and function. For example, there is luciferase activity that facilitates continuous chemiluminescence, as shown by some bacteria and fungi, and luciferase activity that is adapted to facilitate sporadic or stimulus-induced luminescence, as in the case of dinoflagellates. As a phenomenon involving the conversion of chemical energy to light energy, bioluminescence is not limited to living organisms and does not require the presence of organisms. It is simply a type of chemiluminescent reaction that requires luciferase activity derived from a biological catalyst at a certain stage. Therefore, the preservation or construction of the essential activity and chemical substances is sufficient to obtain a means of causing the bioluminescence phenomenon.

[0076]

[86] Such bioluminescent donor molecules can be, for example, luciferases well-known in the art. Luciferases are enzymes or oxygenases that catalyze a luminescence reaction, such as a bioluminescence reaction.

[87] All known types of luciferase, whether naturally occurring or mutated, are encompassed within the scope of the present invention.

[0077]

[88] Examples of bioluminescent proteins having luciferase activity can be found in U.S. Patent Nos. 5,229,285, 5,219,737, 5,843,746, 5,196,524, and 5,670,356. Two of the most widely used luciferases are as follows: (i) Renilla luciferase (from R. reniformis), a 35 kDa protein that uses coelenterazine as a substrate and emits light at 480 nm; and (ii) Firefly luciferase (from Photinus pyralis), a 61 kDa protein that uses luciferin as a substrate and emits light at 560 nm.

[0078]

[89] The bioluminescent donor molecule may be a luciferase selected from Renilla luciferase, Firefly luciferase, Coelenterate luciferase, North American glow worm luciferase, click beetle luciferase, railroad worm luciferase, Gaussia luciferase, Aequorin, Arachnocampa luciferase, luciferases derived from copepods such as Gaussia princeps, Pleuromamma abdominalis, Metridia pacifica, Metridia curticauda, Metridia asymmetrica, Metridia okhotensis, Metridia longa, Lucicutia ovaliformis, Heterorhabdus tanneri, Pleuromamma scutullata, or biologically active variants or fragments thereof.

[90] A preferred selection of the molecular weight range of luciferase for use in accordance with the present invention can be in the range of 15 to 70 kDa, or 15 to 60 kDa, or 15 to 50 kDa, or 15 to 40 kDa, or 15 to 30 kDa.

[0079]

[91] Among preferred luciferases, GLuc, NanoLuc (NLuc), MLuc7, HtLuc, LoLuc, PaLuc1, PaLuc2, MpLuc1, McLuc1, MaLuc1, MoLuc1, MoLuc2, MLuc39, PsLucl, LocLuc1-3, HtLuc2 Renilla, TurboLuc16 (Tluc) or homologs or orthologs thereof or mutants or functional derivatives thereof can be mentioned. In various embodiments, a mutant or functional derivative of luciferase retains at least 100%, 90%, 80%, 70%, 60% or 50% of the bioluminescence activity from which the mutant or functional derivative is derived. As described by Takenaka et al. (Mol Biol Evol 29(6): 1669-1681, 2012), the firefly luciferase contains two domains, each of which contains conserved sequences across various luciferases. In some embodiments, any luciferase containing the consensus sequence C-x(3)-C-L-x(2)-L-x(4)-C-x(8)-P-x-R-C (SEQ ID NO: 2437) in each of domains 1 and 2 described by Takenaka et al. may be used in the methods described herein.

[0080]

[92] The most preferred luciferase is a protein derived from the 19 kDa subunit of luciferase extracted from the deep-sea shrimp Oplophorus gracilirostris, as described by S. Inouye et al. (FEBS Letters, 481 (2000) 19-25), and the nanoluciferase in which the second subunit is a 35 kDa protein can be mentioned. This nanoluciferase is sold by Promega under the trade name NanoLuc®, and as described by Hall M. et al. (ACS Chem. Biol. 2012, 7, 1848-1857) and International Publication WO2012 / 061530, it utilizes novel substrates (furimazine or bisdeoxycoelenterazine and 6h-f-coelenterazine) to produce high-intensity glow-type luminescence.

[0081]

[93] An alternative non-luciferase bioluminescent molecule can be any enzyme that can act on a suitable substrate to generate a luminescence signal. Specific examples of such enzymes include β-galactosidase, lactamase, horseradish peroxidase, alkaline phosphatase, β-glucuronidase, or β-glucosidase. Synthetic luminescent substrates for these enzymes are well-known in the art and are commercially available from companies such as Tropix Inc. (Bedford, MA, USA).

[0082]

[94] As an example, several bioluminescent donor molecules are listed in Table 12 below.

Table 12

[0083]

[95] The selection of the substrate for the bioluminescent donor molecule can affect the wavelength and intensity of the light produced by the bioluminescent protein. A widely known substrate is coelenterazine, which is present in cnidarians, chaetognaths, ctenophores, decapod shrimps, mysids, radiolarians, and some fish taxa. For example, for Renilla luciferase, coelenterazine analogs / derivatives that produce luminescence between 418 and 512 nm are available. A coelenterazine analog / derivative (400A, DeepBlueC) has been described to emit light at 400 nm by Renilla luciferase (WO01 / 46691). Other examples of coelenterazine analogs / derivatives include EnduRen and ViviRen.

[0084]

[96] Luciferin is a bioluminescent pigment found in organisms capable of bioluminescence and is oxidized in the presence of the enzyme luciferase to produce oxyluciferin and energy in the form of light. Luciferin, or 2-(6-hydroxybenzothiazol-2-yl)-2-thiazoline-4-carboxylic acid, was first isolated from the firefly Photinus pyralis. Since then, various forms of luciferin have been discovered and studied from various organisms, mainly from the ocean, such as fish and squid, but many have also been identified in, for example, worms, beetles, and various other insects.

[0085]

[97] There are at least five common types of luciferin, each of which is chemically distinct and is catalyzed by chemically and structurally distinct luciferases that use a wide range of different cofactors. The first is firefly luciferin, the substrate of firefly luciferase, which requires ATP for catalysis (EC 1.13.12.7). The second is bacterial luciferin, found in some squid and fish, which consists of a long-chain aldehyde and reduced riboflavin phosphate. Bacterial luciferase is FMNH-dependent. The third is dinoflagellate luciferin, a tetrapyrrole-based chlorophyll derivative found in dinoflagellates (marine plankton), the organisms responsible for nocturnal ocean bioluminescence. Dinoflagellate luciferase catalyzes the oxidation of dinoflagellate luciferin and consists of three identical catalytic active domains. The fourth is imidazolopyrazine vargulin, which is found in certain ostracods and deep-sea fish, such as Porichthys. Finally, there is coelenterazine (imidazolepyrazine), the emitter of the protein aequorin, which is found in radiolarians, ctenophores, cnidarians, squid, copepods, chaetognaths, fish, and shrimp.

[0086]

[98] Some acceptor molecules, either proteinaceous or non-proteinaceous, can be used in the novel BRET probes within the molecule according to the present invention.

[0087]

[99] Acceptor proteins include green fluorescent protein (GFP), variants of green fluorescent protein (e.g., GFP10), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, mAmetrine, LSS-mOrange, LSS-mKate, Emerald, Topaz, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), HcRed, t-HcRed, DsRed, DsRed2, mRFPl, Porphyrin, Renilla GFP, Monster GFP, paGFP, Kaede protein, phycobiliprotein, TagCFP, mTagCFP2, Czurite, ECFP2, mKalamal, Sirius, Sapphire, T-Sapphire, ECFP, Cerulean, SCFP3C, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP, mTFPl, EGFP, Emerald, Superfolder GFP, monomeric Czami Green, TagGFP2, mUKG, mWasabi, Clover, Citrine, Venus, SYFP2, TagYFP, monomeric Kusabira-Orange, ιηΚΟκ, mK02, mOrange, mOrange2, mRaspberry, mCherry, mStrawberry, mScarlet, mTangerine, tdTomato, TagRFP, TagRFP-T, mCpple, mRuby, mRuby2, mPlum, HcRed-Tandem, mKate2, mNeptune, NirFP, TagRFP657, IFP1.4 and iRFP or biologically active variants or fragments of any one of the foregoing.

[0088]

[0100] The most frequently used bioluminescent or fluorophores are the green fluorescent protein from Aequorea victoria, as well as a number of other variants (GFP) obtained, for example, by mutagenesis and chimeric protein technology. GFP is classified based on the specific components of their chromophores, and each class has different excitation and emission wavelengths: Class 1, the wild-type mixture of neutral phenol and anionic phenolate; Class 2, phenolate anion; Class 3, neutral phenol; Class 4, phenolate anion with a stacked electron system; Class 5, indole; Class 6, imidazole; and Class 7, phenyl.

[0089]

[0101] A preferred fluorescent molecule or fluorescent acceptor molecule may be mNeonGreen, which is a monomeric yellow-green fluorescent protein derived from a tetrameric fluorescent protein from the lancelet Branchiostoma lanceolatum and is evaluated as the "brightest monomeric green or yellow fluorescent protein". The previously developed mNeonGreen monomeric protein is referred to herein as wild-type mNeonGreen. mNeonGreen variants with improved photostability and fluorescence compared to the wild-type protein are also included herein. For example, see the mNeonGreen variants of wild-type mNeonGreen of SEQ ID NO: 65 reported in International Publication No. WO2019 / 055498, which contain one or more mutations selected from V52H, D53E, N65Y, E69M or E69Q, A110Q, S131I or S131C, T139R, S143D, K152H, A158P, T164Q, S166K, A169G, W172P, S175E, K177M, T178L, T188D or T188E, K190T, T194K, G196K or G196E, N197D, S203C, T204Q, T208H, N218D, and Y226F.

[0090]

[0102] Other representative acceptor molecules can include, but are not limited to, the following: sgGFP, sgBFP, BFP blue-shifted GFP (Y66H), cyan GFP, DsRed, monomeric RFP, EBFP, ECFP, GFP (S65T), GFP red-shifted (rsGFP), non-UV excited (wtGFP), UV excited (wtGFP), GFPuv, HcRed, rsGFP, sapphire GFP, sgBFP (trademark), sgBFP (trademark) (super glow BFP), sgGFP (trademark), sgGFP (trademark) (super glow GFP), yellow GFP, semiconductor nanoparticles (e.g., Raman nanoparticles), 1,5IAEDANS; 1,8-ANS; 4-methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-carboxyfluorescein (5-FAM); 5-carboxynaphthofluorescein; 5-carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5-carboxyfluorescein); 5-HAT (hydroxytryptamine); 5-hydroxytryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 5-TAMRA (5-carboxytetramethylrhodamine); 6-carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-amino-4-methylcoumarin; 7-aminoactinomycin D (7-AAD); 7-hydroxy-4-methylcoumarin; 9-amino-6-chloro-2-methoxyacridine; ABQ; acid fuchsin; ACMA (9-amino-6-chloro-2-methoxyacridine); acridine orange; acridine red; acridine yellow; acriflavin; Acriflavin Feulgen SITSA; aequorin (luminescent protein); AFP (autofluorescent protein; Quantum Biotechnologies); Alexa Fluor 350 (trademark); Alexa Fluor 430 (trademark); Alexa Fluor 488 (trademark); Alexa Fluor 532 (trademark); Alexa Fluor 546 (trademark); Alexa Fluor 568 (trademark); Alexa Fluor 594 (trademark); Alexa Fluor 633 (trademark); Alexa Fluor 647 (trademark); Alexa Fluor 660 (trademark); Alexa Fluor 680 (trademark);Alizarin Complexone; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; AMCA (Aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin; Aminomethylcoumarin (AMCA); Aniline Blue; Anthracyl Stearate; APC (Allophycocyanin); APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7GLL; Atabrine; ATTO-TAG (trademark) CBQCA; ATTO-TAG (trademark) FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyl Oxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulfate; β-Lactamase; Bimane; Bisbenzamide; Bisbenzimide (Hoechst); Bis-BTC; Blancophor FFG; Blancophor SV; BOBO (trademark)-1; BOBO (trademark)-3; Bodipy 492 / 515; Bodipy 493 / 503; Bodipy 500 / 510; Bodipy 505 / 515; Bodipy 530 / 550; Bodipy 542 / 563; Bodipy 558 / 568; Bodipy 564 / 570; Bodipy 576 / 589; Bodipy 581 / 591; Bodipy 630 / 650-X; Bodipy 650 / 665-X; Bodipy 665 / 676; Bodipy F1; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO (trademark)-1; BO-PRO (trademark)-3; Brilliant Sulfoflavin FF; BTC; BTC-5N; Calcein; Calcein Blue; Calcium Crimson (trademark); Calcium Green; Calcium Green-1 Ca; 2+ Dye; Calcium Green-2 Ca 2+ ; Calcium Green-5N Ca 2+ ; Calcium Green-C18 Ca2+; Calcium Orange; CalcoFluor White; Carboxy-X-Rhodamine (5-ROX); Cascade Blue (trademark); Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; Chlorophyll; Chromomycin A; Chromomycin A; CL-NERF; CMFDA; Coumarin phalloidin; C-Phycoerythrin; CPM Methylcoumarin; CTC; CTC Formazan; Cy2 (trademark); Cy3.18; Cy3.5 (trademark); Cy3 (trademark); Cy5.18; Cy5.5 (Trademark); Cy5 (Trademark); Cy7 (Trademark); Circular AMP Fluorosensor (FiCRhR); Dabsyl; Dansyl; Dansylamine; Dansylcadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl Fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3'DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydrorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (Not comparable); DiA (4-Di-16-ASP); Dichlorodihydrofluorescein Diacetate (DCFH); DiD - Lipophilic Tracer; DiD (DilC18(5)); DIDS; Dihydrorhodamine 123 (DHR); Dil (DilC18(3)); Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DilC18(7)); DM-NERF (High pH); DNP; Dopamine; DTAF; DY-630-NHS; DY-635-NHS; ELF97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide; Ethidium Homodimer 1 (EthD-1); EuKrysin; EukoLight; Europium(III) Chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FIF (Formaldehyde-Induced Fluorescence); FITC; Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43 (Trademark); FM 4-46; Fura Red (Trademark) (High pH); Fura Red (Trademark) / Fluo-3; Fura-2; Fura-2 / BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer (CCF2); Gloxalic Acid; Granular blue;. Hematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine; Indo-1, high calcium; Indo-1, low calcium; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO-JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine rhodamine; Lissamine rhodamine B; Calcein / ethidium homodimer; LOLO-1; LO-PRO-1; Lucifer yellow; Lysotracker blue; Lysotracker blue white; Lysotracker green; Lysotracker red; Lysotracker yellow; LysoSensor blue; LysoSensor green; LysoSensor yellow / blue; Mag green; Magdala red (Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-Indo-1; Magnesium green; Magnesium orange; Malachite green; Marina Blue; Maxilon brilliant flavin 10 GFF; Maxilon brilliant flavin 8 GFF; Merocyanine; Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitomycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (methyl green pyronin stilbene); NBD; NBD amine; Nile red; Nitrobenzoxadiazole; Norepinephrine; Nuclear fast red; Nuclear yellow; Nylosan Brilliant lavin E8G; Oregon Green; Oregon Green 488-X; Oregon Green (trademark); Oregon Green (trademark) 488; Oregon Green (trademark) 500; Oregon Green (trademark) 514; Pacific Blue;Pararosaniline(Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-Texas Red [Red 613]; Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontchrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primuline; Procion Yellow; Propidium Iodide (PI); PyMPO; Pyrene; Pyronin; Pyronin B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Red 613 [PE-TexasRed]; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5GLD; Rhodamine 6G; Rhodamine B; Rhodamine B200; Rhodamine B Extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Falicidin; Rhodamine Faloidin; Rhodamine Red; Rhodamine WT; Rose Bengal; R-Phycoocyanin; R-Phycoerythrin (PE); S65A; S65C; S65L; S65T; SBFI; Serotonin; Serbron Brilliant Red 2B; Serbron Brilliant Red 4G; Serbron Brilliant Red B; Serbron Orange; Serbron Yellow L; SITS; SITS (Primuline); SITS (Stilbene Isothiosulfonate); SNAFL Calcein; SNAFL-1; SNAFL-2; SNARF Calcein; SNARF1; Sodium Green; SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ (6-Methoxy-N-(3-Sulfopropyl) Quinolinium); Stilbene; Sulforhodamine B can C; Sulforhodamine Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO 24;SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange; Tetracycline; Tetramethylrhodamine (TRITC); Texas Red (trademark); Texas Red X (trademark) conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiazole Orange; Tinopol CBS (Calcofluor White); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC Tetramethylrhodamine Isothiocyanate; True Blue; TrueRed; Ultralite; Uranine B; Uvitex SFC; WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; YO-PRO-1; YO-PRO-3; YOYO-1; YOYO-3, Sybr Green, Thiazole Orange (mutual chelate dye), or a combination thereof.;

[0091]

[0103] Alternatively, the acceptor molecule may be a fluorescent nanocrystal. Nanocrystals have several advantages as fluorescent labels compared to organic molecules, including resistance to photodegradation, improved brightness, non-toxicity, and a size-dependent narrow emission spectrum that allows for simultaneous monitoring of several processes. Furthermore, the absorption spectrum of the nanocrystals is continuous above the first peak, allowing all sizes and thus all colors to be excited with a single excitation wavelength.

[0092]

[0104] The acceptor molecule may also be a fluorescent microsphere. These are typically made from a polymer and contain a fluorescent molecule (e.g., fluorescein, GFP or YFP) incorporated into the polymer matrix, which can be conjugated to various reagents. The fluorescent microsphere can be labeled internally or on the surface. Internal labeling typically produces very bright and stable particles with a narrow fluorescence emission spectrum. In internal labeling, the surface groups remain available for conjugating a ligand (e.g., a protein) to the surface of the bead. Internally labeled beads are widely used in imaging applications because they exhibit higher resistance to photobleaching.

[0093]

[0105] Multiple combinations of bioluminescent donor molecules and fluorescent acceptor molecules can be used to increase the sensitivity of detection of inhibitor candidates or activator candidates being tested. The criteria to consider when determining a suitable pair for RET are the relative emission / fluorescence spectra of the acceptor molecule compared to the emission / fluorescence spectra of the bioluminescent donor molecule. The emission spectrum of the bioluminescent protein overlaps with the absorption spectrum of the acceptor molecule, such that the light energy from the bioluminescent protein emission can excite the acceptor molecule and is of a wavelength that promotes fluorescence of the acceptor molecule when the two molecules are in appropriate proximity and orientation to each other. Good spectral properties, such as brightness, photostability may also be considered.

[0094]

[0106] The original BRET system uses Renilla luciferase as the donor, EYFP (or Topaz) as the acceptor, and a coelenterazine derivative as the substrate. When these components are combined in a BRET assay, maximum light is generated in the range of 475 - 485 nm for the bioluminescent protein and 525 - 535 nm for the acceptor molecule, resulting in a spectral resolution of 40 - 60 nm. Renilla luciferase generates a broad emission peak that substantially overlaps with GFP emission, which in turn contributes to a decrease in the signal-to-noise of the system. Various coelenterazine derivatives are known in the art, including coel400a, which generates light at various wavelengths (different from those generated by wild-type coelenterazine) as a result of Renilla luciferase activity. One of ordinary skill in the art will understand that, since the emission peak of the donor has changed, it is necessary to select an acceptor molecule that absorbs light at that wavelength, thereby enabling efficient energy transfer. Spectral overlap between the donor emission and the acceptor light absorption peak is, inter alia, one of the conditions for efficient energy transfer. Class 3 and 1 GFP are known to absorb light at 400 nm and re-emit between 505 - 511 nm. This results in a wavelength difference of approximately 111 nm between the donor emission and the acceptor emission.

[0095]

[0107] Preferred selections of bioluminescent donor and fluorescent acceptor molecules suitable for use in the intramolecular BRET probes according to the present invention include the combination of NanoLuc and mNeonGreen.

[0108] As will be understood by those skilled in the art, the bioluminescent donor molecule and the fluorescent acceptor molecule are inserted such that they activate an ion sensor and / or an ion-conductive channel subunit as a result of a spatial change in the position and / or dipole orientation of the bioluminescent donor molecule relative to the fluorescent acceptor molecule. The spatial arrangement of the ion channel, the ion sensor probe, the bioluminescent donor molecule, and the fluorescent acceptor molecule is such that the resulting fusion channel protein is functional. Energy transfer mainly depends on: (i) the overlap between the emission spectrum and the excitation spectrum of each of the donor molecule and the acceptor molecule, and (ii) proximity of about 100 angstroms (Å) between the donor molecule and the acceptor molecule. The donor molecule in BRET generates light via chemiluminescence and is thus suitable for imaging small animals. Furthermore, the BRET system does not use an external light excitation source and potentially provides higher sensitivity for live subjects due to a low signal-to-noise ratio.

[0096]

[0109] The ion sensor used in the probe according to the present invention is a sensor that accepts calcium, potassium, sodium, chloride ions, or any sensor that accepts ion intensity. It represents a sensing domain or a sensitive domain that is responsive or sensitive to the presence and level of signal ions, such as calcium, potassium, sodium, chloride ions, etc., or can detect / perceive them. The sensor or the sensing domain then typically changes conformation in response to the binding of signal ions or molecules or changes in intracellular conditions.

[0110] Therefore, the intramolecular BRET probe according to the present invention is useful for monitoring conformational changes and ion channel activation of ion channel structures in living cells.

[0097]

[0111] According to the present invention, the ion sensor may be selected from a calcium-binding protein, such as a troponin C calcium-binding domain or a calmodulin calcium-binding protein, a potassium-binding protein (KBP), a sodium-binding protein, such as the NhAs-1 protein, or a chloride-binding protein.

[0112] Calcium sensing proteins or calcium-binding proteins are well known in the art and they may be any protein that acts in a second messenger system. Examples thereof include calmodulin, calnexin, calreticulin and gelsolin.

[0098]

[0113] Calmodulin (CaM) (abbreviation for calcium-modulating protein) is a multifunctional intermediate calcium-binding messenger protein expressed in all eukaryotic cells. This is an intracellular target of the second messenger Ca 2+ and binding of Ca 2+ is necessary for the activation of calmodulin. Once bound to Ca 2+ , calmodulin acts as part of the calcium signaling pathway by modifying its interaction with various target proteins, such as kinases or phosphatases.

[0114] Calnexin (CNX) is an endogenous protein of 67 kDa in the endoplasmic reticulum (appearing variously as a band of 90 kDa, 80 kDa, or 75 kDa in Western blotting depending on the source of the antibody). This consists of a large (50 kDa) N-terminal calcium-binding luminal domain, a single transmembrane helix and a short (90 residue) acidic cytoplasmic tail.

[0099]

[0115] Calreticulin, also known as calregulin, CRP55, CaBP3, calsequestrin-like protein, and endoplasmic reticulum resident protein 60 (ERp60), is a protein encoded by the CALR gene in humans. Calreticulin is Ca 2+It is a multifunctional soluble protein that binds to ions and inactivates them. Ca 2+ has low affinity but binds with high capacity and may be released during signaling. Calreticulin is located in a storage compartment associated with the endoplasmic reticulum (ER) and is considered an ER resident protein.

[0116] Gelsolin is an actin-binding protein that is an important regulator of the assembly and disassembly of actin filaments. Gelsolin is one of the most potent members of the actin-cutting gelsolin / superfamily as it cuts with nearly 100% efficiency.

[0100]

[0117] Potassium sensors or potassium-binding proteins are also well known in the art and can include any potassium sensor protein that binds to positively charged potassium ions and can generate a detectable signal when the positively charged potassium ions bind to the potassium sensor. Binding of K+ can, for example, induce a conformational shift in the potassium sensor, which can then enable the signaling domain to generate a detectable signal. Preferably, binding of K+ to the potassium sensor can induce a conformational shift in the peptide, which can then enable the signaling domain to generate a detectable signal. In a preferred embodiment, the potassium sensor comprises the amino acid sequence of a KBP as described by Ashraf et al. (Structure 2016, May 3; 24(5) 741-9). The K+-binding protein (KBP) is also known as YgaU and is characterized as a soluble 16 kDa cytoplasmic protein from Escherichia coli. This is a highly specific K+-binding protein that is required for normal growth in the presence of high levels of external K. Potassium ions bind exclusively to the BON domain, which undergoes a conformational change upon binding. The KBP further comprises a LysM domain that can interact with the BON domain.

[0101]

[0118] Sodium ion-binding proteins or sodium sensors may include, for example, the NhaS gene product, NhaS, which is a protein characterized by binding to and sequestering sodium ions (Na + +). Functional fragments of sodium ion-binding proteins that include NhaS are also included within the scope, and such fragments are characterized by their ability to bind sodium ions. See, for example, U.S. Patent No. 5,346,815A.

[0102]

[0119] Chloride-binding proteins or chloride sensors can include, for example, OEC, Chameleon, and other chloride-requiring enzymes. The polypeptide structure of OEC has been extensively described, among others, by Coleman W.J. (Photosynth Res (1990) 23: 1. https: / / doi.org / 10.1007 / BF00030059). Chloride biosensors, such as YFP-based chloride biosensor Chameleon, consist of two fluorescent proteins, CFP and Topaz, linked by a flexible polypeptide linker of 24 amino acids, as described by Arosio et al. (Front Cell Neurosci. 2014;8:258. 2014 Aug 29. doi:10.3389 / fncel.2014.00258) and Watts et al. (April 10, 2012, https: / / doi.org / 10.1371 / journal.pone.0035373). In some cases, the acceptor shows chloride sensitivity, and thus, in such cases, the chloride sensor may be the acceptor itself. In this regard, the inventors may cite YFP-H148Q, a genetically modified YFP photostable fluorescent protein that also shows enhanced chloride sensitivity as described by Zhong S et al. (2014 Jun 5;9(6):e99095. doi: 10.1371 / journal.pone.0099095). Thus, such modified YFP may be used as a fluorescent acceptor molecule and as an ionic chloride sensor for studying the activation or inhibition of ion-conductive channels by chloride ion flux.

[0103]

[0120] Accordingly, the present invention provides a useful tool for exploring the passage of ions and thus the actual activation or inhibition of ion-conductive channels as described above. As a result, by multiplexing different BRET-biosensors of such ion-conductive channels, novel BRET probes within the molecule thus offer the possibility of reliably and rapidly testing various candidates.

[0104]

[0121] High-throughput screening of ion channel function requires sensitive and simple assays and equipment that report ion channel activity in living cells. Accordingly, the present invention provides a nucleic acid or polynucleotide comprising a nucleotide sequence encoding a channel fusion subunit comprising an ion-conductive channel subunit bound to a probe, wherein the probe comprises an ion sensor bound therebetween to at least one bioluminescent donor molecule and at least one protein fluorescent acceptor molecule, wherein the ion sensor is configured to undergo a conformational change in the presence of ions transported by the channel subunit, wherein the probe is bound via either the bioluminescent donor molecule or the protein fluorescent acceptor molecule to the N-terminus, C-terminus, intracellular or extracellular loop of the channel subunit, wherein the bioluminescent donor molecule and the protein fluorescent acceptor molecule are selected such that the emission spectrum of the bioluminescent donor molecule overlaps the absorption spectrum of the fluorescent acceptor molecule and the light energy delivered by the bioluminescent donor molecule is at a wavelength capable of exciting the fluorescent acceptor molecule, relating to a nucleic acid or polynucleotide.

[0105]

[0122] The nucleic acid or polynucleotide of the present invention may be inserted into several commercially available expression vectors. Non-limiting examples include prokaryotic plasmid vectors such as the pUC series, pBluescript (Stratagene), pET series expression vectors (Novagen) or pCRTOPO (Invitrogen), and vectors suitable for expression in mammalian cells such as pREP (Invitrogen), pcDNA3 (Invitrogen), pCEP4 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pIZD35, pLXIN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (Edge Biosystems), pTriEx-Hygro (Novagen) and pCINeo (Promega). Examples of plasmid vectors suitable for Pichia pastoris include, for example, plasmids pAO815, pPIC9K and pPIC3.5K (all Invitrogen). The nucleic acid or polynucleotide of the present invention mentioned above may also be inserted into a vector such that a translational fusion with another polynucleotide is generated. The other polynucleotide may encode a protein that can, for example, increase solubility and / or facilitate purification of the fusion protein. Non-limiting examples include pET32, pET41, pET43. The vector may also contain one or more additional expressible polynucleotides encoding chaperones that facilitate correct protein folding. Suitable bacterial expression hosts include, for example, strains derived from BL21 (e.g., BL21(DE3), BL21(DE3)PlysS, BL21(DE3)RIL, BL21(DE3)PRARE) or Rosetta®.In a preferred embodiment, the expression vector according to the present invention is a DNA or RNA vector capable of transforming a eukaryotic host cell and resulting in stable or transient expression of the TRP channel fusion subunit, and the vector is a plasmid, a virus, such as an adenovirus, an adeno-associated virus (AAV), a lentivirus, Epstein-Barr, herpes simplex, papilloma, polyoma, retro, SV40, vaccinia, any retroviral vector, an influenza virus vector, and other non-viral vectors including naked DNA, or a liposome.

[0106]

[0123] Transcription regulatory factors (part of the expression cassette) that ensure expression in prokaryotic or eukaryotic cells are well known to those skilled in the art. These factors include regulatory sequences that ensure the initiation of transcription (e.g., translation initiation codon, promoter, enhancer, and / or insulator), an internal ribosome entry site (IRES) within the sequence, and, optionally, a polyA signal that ensures the termination of transcription and the stabilization of the transcript. Further regulatory elements may include transcription enhancers and translation enhancers, and / or a naturally associated promoter region or a heterologous promoter region. Preferably, the polynucleotide of the present invention is operably linked to such expression control sequences to enable expression in prokaryotic or eukaryotic cells. The vector may further include a nucleotide sequence encoding a secretion signal as a further regulatory element. Such sequences are well known to those skilled in the art. Further, depending on the expression system used, a leader sequence capable of directing the expressed polypeptide to a cellular compartment may be added to the coding sequence of the polynucleotide of the present invention. Such leader sequences are well known in the art.

[0107]

[0124] Possible examples of regulatory elements that ensure the start of transcription include the cytomegalovirus (CMV) promoter, SV40-promoter, RSV-promoter (Rous sarcoma virus), lacZ promoter, human elongation factor 1α-promoter, CMV enhancer, CaM-kinase promoter, Autographa californica nuclear polyhedrosis virus (AcMNPV) polyhedron promoter or SV40-enhancer. For expression in prokaryotes, numerous promoters are described, including, for example, the tac-lac-promoter, lacUV5 or trp promoter. Examples of further regulatory elements in prokaryotic and eukaryotic cells include transcription termination signals downstream of the polynucleotide, such as the SV40-polyA site or tk-polyA site or the SV40, lacZ and AcMNPV polyadenylation signals.

[0108]

[0125] The present invention further provides a recombinant host cell containing an expression vector for the expression of an ion-conductive channel fusion unit as described above, wherein the vector contains a polynucleotide comprising a nucleic acid comprising a nucleotide sequence encoding a channel fusion subunit comprising a subunit of an ion-conductive channel bound to a probe, wherein the probe comprises an ion sensor having at least one bioluminescent donor molecule and at least one protein fluorescent acceptor molecule bound therebetween, wherein the ion sensor is configured to undergo a conformational change in the presence of ions transported by the channel subunit, wherein the probe is bound via either the bioluminescent donor molecule or the protein fluorescent acceptor molecule to the N-terminus, C-terminus or within an intracellular or extracellular loop of the channel subunit, The bioluminescent donor molecule and the protein fluorescent acceptor molecule are selected such that the emission spectrum of the bioluminescent donor molecule overlaps with the absorption spectrum of the fluorescent acceptor molecule, and the light energy delivered by the bioluminescent donor molecule is at a wavelength capable of exciting the fluorescent acceptor molecule. Relates to recombinant host cells.

[0109]

[0126] Accordingly, the recombinant cells according to the present invention are genetically engineered to contain a polynucleotide construct of an ion channel fusion subunit as described above. The cell or host can be any prokaryotic or eukaryotic cell, and preferably can be a stable cell line. Suitable eukaryotic hosts can be mammalian cells, amphibian cells, fish cells, insect cells, fungal cells or plant cells. The eukaryotic cell can be an insect cell, for example, Spodoptera frugiperda cell, a yeast cell, for example, Saccharomyces cerevisiae or Pichia pastoris cell, a fungal cell, for example, Aspergillus cell or a vertebrate cell. Suitable prokaryotes can be E. coli (e.g., E. coli strains HB101, DH5a, XL1Blue, Y1090 and JM101), Salmonella typhimurium, Serratia marcescens, Burkholderia glumae, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas stutzeri, Streptomyces lividans, Lactococcus lactis, Mycobacterium smegmatis or Bacillus subtilis.

[0110]

[0127] Accordingly, the recombinant host cell according to the present invention contains an expression vector, the channel fusion subunit is expressed, and can co-assemble with other homomeric or heteromeric channel subunits in vitro and in vivo to form a functional fusion channel. The present invention embodies a manufacturing process of the channel fusion subunit as described above, including culturing the recombinant cell according to the present invention and expressing the channel fusion subunit.

[0111]

[0128] The present invention also relates to cell-free compositions and assays comprising at least an ion channel subunit combined with, coupled or bound to an ion sensor nucleotide sequence disposed between a nucleotide sequence encoding a bioluminescent donor molecule and a nucleotide sequence encoding at least one fluorescent acceptor molecule. The cell-free composition comprises an ion channel fusion unit or a functional fusion channel as described above, and the fusion subunit is embedded in a lipid bilayer, preferably the bilayer of a liposome. Such a lipid bilayer typically comprises two amphiphilic layers having an inner common hydrophobic bilayer and two hydrophilic surfaces. The lipid bilayer can be naturally occurring or artificial. Such a lipid bilayer can be a cell membrane or biomembrane derived from, for example, the cell membrane of a mammal or yeast into which a voltage-dependent ion channel fusion subunit has been inserted.

[0112]

[0129] Methods for preparing cell-free compositions from cells are well known in the art and involve obtaining recombinant cells as described above and disrupting the cell membranes. These methods generally include repeated cycles of freezing and thawing, grinding, treatment of cells with ultrasound in an ultrasonic processor, homogenization, and use of a French press, addition of surfactants and / or enzymes, glass bead lysis, differential centrifugation, and several density gradient procedures using various gradient media. These techniques are described in detail, inter alia, in "Current Protocols in Protein Science"; John E. Caligan; Ben M. Dunn; Hidde L. Ploegh; David W. Speicher; Paul T. Wingfield; Wiley and Sons). Combinations of these methods are usually employed to isolate or prepare cell membrane extracts. Generally, cells are lysed either by mechanical means or using surfactants, and the membrane fraction is isolated via differential centrifugation. Liposomes containing recombinant cells according to the present invention can be produced by disrupting the phospholipid membrane of cells that express proteins in water, for example, by sonication. The phospholipids reassemble into liposome spheres containing an aqueous core. At low shear rates, multilamellar liposomes with multiple layers are produced. Continuous high-shear sonication forms smaller unilamellar liposomes that are more suitable for the application of the present invention.

[0113]

[0130] The present invention further provides a method for evaluating whether a test compound functions as a ligand for an ion-conductive channel, comprising: (i) providing a cell comprising a nucleotide sequence encoding an ion-conductive channel subunit bound to a probe, wherein the probe comprises an ion sensor having at least one bioluminescent donor molecule and at least one fluorescent acceptor molecule bound therebetween, the probe is bound via either the bioluminescent donor molecule or the fluorescent acceptor molecule to the N-terminus, or the C-terminus, or within an intracellular or extracellular loop of the channel subunit, The bioluminescent donor molecule and the fluorescent acceptor molecule are selected such that the emission spectrum of the bioluminescent donor molecule overlaps with the absorption spectrum of the fluorescent acceptor molecule, enabling non-radiative energy transfer by non-radiative dipole-dipole coupling between the bioluminescent energy donor and the fluorescent acceptor. (ii) contacting the cell with a test compound, and (iii) determining the resulting reaction or interaction and output A method is provided that includes the steps of.

[0114]

[0131] A method for screening a compound candidate in real time enables measuring ion flux and thus activation or inhibition of an ion-conductive channel, and includes contacting the candidate with a recombinant host cell or cell-free composition according to the present invention, providing a substrate for the bioluminescent donor molecule, and (iii) measuring fluctuations in the BRET signal. Such a method enables screening for agonists or antagonists of ion-conductive channels in real time.

[0132] In a further aspect, the present invention provides a kit for screening agonist or inhibitor compound candidates of an ion-conductive channel, comprising a nucleic acid or polynucleotide of the present invention, a vector of the present invention, a recombinant cell or host cell of the present invention, or a cell-free composition or composition of the present invention, and / or a biosensor of the present invention.

[0115]

[0133] Throughout this application, various references are cited, and the disclosures of these publications are hereby incorporated by reference in their entirety to more fully describe the state of the art to which the present invention pertains.

Examples

[0116] Example 1: Construction of a second-generation intramolecular probe A novel intramolecular BRET probe as shown in FIG. 1 was constructed using an ion-binding protein sandwiched between YFP and Luc and fused to the intracellular termini of several ion-conductive channels to measure ion flux in the microenvironment of the voltage-gated ion channel pore.

[0117] Example 1.1: Construction of potassium-binding BRET probe A K+-sensitive, genetically encoded bioluminescence resonance energy transfer (BRET)-based probe was constructed by cloning the cDNA encoding the amino acid sequence of KBP such that it was fused, as described by Ashraf et al. (2016) (Structure 2016, May 3; 24(5) 741-9), without a stop codon, to the cDNA encoding nanoluciferase at the 5'-terminus and to the cDNA encoding mNeon green fluorescent protein at the 3'-terminus to obtain a NanoLuc-KBP-mNeonGreen BRET probe, or fused to the cDNA encoding mNeonGreen at the 5'-terminus and to the cDNA encoding nanoluciferase protein at the 3'-terminus to obtain a NanoLuc-KBP-mNeonGreen BRET probe. The peptide spacer between the N-terminal acceptor or donor protein is not critical for the operability of the sensor. Thus, the RMQDA peptide spacer may be added, if necessary, between the N-terminal acceptor or donor protein and the KBP protein, and the PLAEL peptide spacer may be added between the KBP protein and the C-terminal donor or acceptor protein.

[0118] Example 1.2: Construction of ion intensity-sensitive BRET probe An ion intensity-sensitive, genetically encoded bioluminescence resonance energy transfer (BRET)-based probe was constructed by cloning a cDNA encoding an ion intensity sensor sequence, designated SOFI herein, as described by Liu B. et al. (2017) (ACS Chem. Biol. 2017, 12, 2510-2514), fusing it to a cDNA encoding nanoluciferase at the 5'-end and to a cDNA encoding mNeon green fluorescent protein at the 3'-end to obtain a NanoLuc-KBP-mNeonGreen BRET probe, or fusing it to a cDNA encoding mNeonGreen at the 5'-end and to a cDNA encoding nanoluciferase protein at the 3'-end to obtain a NanoLuc-SOFI-mNeonGreen BRET probe. The peptide spacer between the N-terminal acceptor or donor protein is not critical for the operability of the sensor. Thus, an RMQDA peptide spacer may be added between the N-terminal acceptor or donor protein and the calflux protein as needed, and a PLAEL peptide spacer is added between the calflux protein and the C-terminal donor or acceptor protein.

[0119] Example 1.3: Construction of a calcium-binding BRET probe Ca 2+A gene-encoded bioluminescence resonance energy transfer (BRET)-based probe for chloride sensitivity was constructed by cloning a cDNA encoding the amino acid sequence of calflux, fusing it without a stop codon to a cDNA encoding nanoluciferase at the 5'-end and to a cDNA encoding mNeon green fluorescent protein at the 3'-end to obtain a NanoLuc-calflux-mNeonGreen BRET probe, or fusing it to a cDNA encoding mNeonGreen at the 5'-end and to a cDNA encoding nanoluciferase protein at the 3'-end to obtain a NanoLuc-calflux-mNeonGreen BRET probe. The peptide spacer between the N-terminal acceptor or donor proteins is not important for the operability of the sensor. Therefore, an RMQDA peptide spacer may be added between the N-terminal acceptor or donor protein and the calflux protein as needed, and a PLAEL peptide spacer may be added between the calflux protein and the C-terminal donor or acceptor protein.

[0120] Example 1.4: Construction of a Chloride-Binding BRET Probe A chloride-sensitive, gene-encoded bioluminescence resonance energy transfer (BRET)-based probe was constructed by cloning a cDNA encoding the amino acid sequence of YFP or a YFP variant and fusing it to a cDNA encoding nanoluciferase at its 3'-end, and introducing point mutations conferring chloride sensitivity into the YFP construct (e.g., mutations H148Q and I152L as described by Galietta et al., FEBS letters, 2001, Jun 22;499(3):220-4. DOI:10.1016 / s0014-5793(01)02561-3).

[0121] Example 2: Expression vector carrying a sensor-based BRET probe Expression of this protein in the target mammalian cells is enabled by transfection of an expression vector containing cDNA encoding a fusion protein comprising the channel and ion sensor of interest, using BRET technology. Transient or stable expression of the vector in the targeted mammalian cells enabled direct measurement of the activity of the targeted channel by the BRET probe according to the invention. This protein enabled the release of a signal (light) during the addition of a luciferase substrate, such as Celenterazine H. Luciferase catalyzed the oxidation of the substrate. This biochemical reaction results in luminescence at a wavelength characteristic of the luciferase / substrate pair. When an acceptor having an excitation spectrum that matches the emission spectrum of the luciferase / substrate pair is in close proximity in the appropriate orientation, non-radiative transfer of energy (BRET signal) occurs between the donor and the acceptor. This energy transfer is characterized by the fluorescence of the acceptor at its emission wavelength (Figure 2). Typically, for energy to be transferred between a donor and a compatible acceptor, the two moieties must be within about 10 nm of each other, which is a distance compatible with interacting protein-protein.

[0122] Example 3: Calcium-sensitive BRET probe targeting the TRPV1 channel Intramolecular BRET probe: hTRPV1-nLuc-Calflux-YFP was prepared, where the DNA sequence encoding the human protein TRPV1 is fused at the C-terminus to a calcium sensor, and the sensor is sandwiched between nanoluciferase and YFP or mNeonGreen (Calflux probe, Yang J. et al. Nature Communication, 2016, 27;7:13268. doi: 10.1038 / ncomms13268.). The applicant has shown that the basal BRET signal of the probe hTRPV1-nLuc-Calflux-YFP (or mNeonGreen) is modulated in a dose-dependent manner in addition to the activation of cells by capsaicin (CAPS), which is a major agonist of TRPV1 (Figure 4A).

[0123] This activation was absolutely specific to the passage of ions across TRPV1 for the following reasons. - AMG517, an antagonist of TRPV1, shifted the dose-response to CAPS to the right and showed competitive inhibition as expected (Figure 4A). - The nLuc-Calflux-YFP probe did not respond to CAPS stimulation in the absence of co-expressed TRPV1 ion channels when expressed alone without fusion to TRPV1 (Figure 4A).

[0124] - The hTRPV1-nLuc-Calflux D13A / D51A-YFP probe containing double mutations that render the Calflux probe insensitive to calcium did not respond to CAPS stimulation. This control was important to show that the conformational change induced by CAPS on the TRPV1 channel was not involved in the observed increase in the BRET signal (Figure 4A). - Non-specific diffusion of calcium into cells by using ionomycin (a calcium ionophore) did not cause a dose-dependent increase in the BRET signal when measured with the free nLuc-Calflux-mNeonGreen probe, but only caused a slight increase in the signal obtained with the TRPV-nLuc-Calflux-mNeonGreen probe (Figure 4B).

[0125] These data clearly demonstrated that the BRET probe described herein targeting the TRPV1 channel specifically reflected the passage of ions across the TRPV1 channel and thus reflected the actual activity of TRPV1. Analysis of this dose-response curve showed that CAPS induced TRPV1 activation with an EC50 of 328 ± 33 nM, demonstrating the sensitivity of the BRET probe to CAPS with pharmacological properties similar to those found in scientific literature using patch clamp.

[0126] Example 4: Sensor-based BRET Probe Targeting Canal KCa2.3 A novel BRET probe (SK3-nLuc-KBP-mNeonGreen, see Figure 5A) was constructed to measure the activity of potassium channel SK3 (KCa2.3) by coupling the cDNA of the SK3 channel to a potassium sensor (protein KBP, Ashrafet KU et al., 2016, Structure 24, 741-749) sandwiched between luciferase and mNeonGreen. SK channels are a family of potassium channels activated by an increase in cytoplasmic calcium concentration. The opening of the SK3 channel induces a small outward flow of potassium ions diffusing through the open pore according to the electrochemical gradient of potassium ions. After adding ionomycin at different concentrations to induce an increase in the cytoplasmic sol of calcium and then activating the SK3 channel, the BRET signal measured in HEK293T cells expressing the SK3-nLuc-KBP-mNeonGreen probe decreased, indicating an efflux of potassium to the extracellular space (Figure 5B). The BRET sensor is based on the published KBP (Bischof H et al., 2017, Nat Commun. 8(1):1422), but this sensor was not coupled to the channel to specifically measure the activity of the channel as presented in the present invention.

[0127] Example 5: Sensor-based BRET Probe Targeting Receptor P2X2 The inventors constructed an rP2X2-nLuc-Calflux-YFP probe, where the DNA sequence encoding the rat protein P2X2 is fused at the C-terminus to a calcium sensor sandwiched between nanoluciferase and YFP or mNeonGreen (Calflux probe, Yang J. et al., Nature Communication, 2016, 27;7:13268. doi: 10.1038 / ncomms13268.). The inventors were able to show that the basal BRET signal of rP2X2-nLuc-Calflux-YFP (or mNeonGreen) was modulated in a dose-dependent manner after cell activation with ATP, the natural agonist of P2X2 (Figure 6A). This activation is specific for the passage of ions through P2X2 for the following reasons.

[0128] When the 1 / nLuc-Calflux-YFP probe was expressed alone without fusion to P2X2, the BRET probe did not respond to ATP stimulation (Figure 6B). When the 2 / nLuc-Calflux-YFP probe was fused to the TRPV1 channel, this probe did not respond to ATP stimulation (Figure 6C), but did respond to stimulation with capsaicin (CAPS), an agonist of TRPV1. Analysis of the dose-response curve showed that ATP activated the P2X2-NLuc-CalFlux-YFP probe with an EC50 of 888 ± 274 nM, indicating that the ATP potency to activate the P2X2 BRET probe is similar to the ATP potency to activate native P2X2 as seen in scientific literature using prior art.

[0129] Example 6: Sensor-based BRET probe targeting the NMDA receptor The inventors constructed a novel BRET probe that enables the measurement of NMDA receptor activity by coupling the cDNA of the NR1 subunit to the BRET sensor nLuc-Calflux-YFP (Figure 7). The NMDA receptor is a heterotetramer containing two NR1 subunits and two NR2 or NR3 subunits. The separately expressed subunits NR1, NR2, or NR3 cannot form a functional receptor.

[0130] The inventors were able to show that the BRET signal generated when the BRET probe NR1-nLuc-Calflux-YFP was expressed alone was stable and not modulated by increasing doses of NMDA (Figure 7A). However, co-expression of this probe with the subunit NR2A (Figure 7B) or NR2B (Figure 7C) resulted in the generation of a functional receptor, and an increase in signal was observed in HEK293T cells co-expressing NR1-nLuc-Calflux-YFP and the NR2A subunit after stimulation of the cells with NMDA.

[0131] Analysis of the dose-response curves showed that NMDA activated the NR1-nLuc-Calflux-YFP / NR2A and NR1-nLuc-Calflux-YFP / NR2B ion channels with EC50 values of 79.7 μM + / - 17.2 μM and 12.3 + / - 7.32 μM, respectively. These data are in complete agreement with the scientific literature and thus show that the heteromeric receptor containing the NR2A subunit is less sensitive to NMDA than the receptor containing the NR2B subunit.

[0132] Example 7: Sensor-based BRET probe targeting the calcium-permeable cation channel TRPM8 Intramolecular BRET probe: hTRPM8-nLuc-Calflux-YFP was prepared, where the DNA sequence encoding the human protein TRPM8 is fused at the C-terminus to a calflux-based calcium BRET sensor, and the sensor is sandwiched between nanoluciferase and YFP or mNeonGreen.

[0133] Example 8: Sensor-based BRET probe targeting the calcium-permeable cation channel TRPV4 Intramolecular BRET probe: hTRPV4-nLuc-Calflux-YFP was prepared, where the DNA sequence encoding the human protein TRPV4 is fused at the C-terminus to a calflux-based calcium BRET sensor.

[0134] Example 9: Sensor-based BRET probe targeting the sodium channel Nav1.7 Intramolecular BRET probe: Nav1.7-nLuc-SOFI-YFP was prepared, where the DNA sequence encoding the human protein TRPV4 is fused at the C-terminus to a SOFI-based ion intensity BRET sensor.

[0135] Example 10: Sensor-based BRET probe targeting the potassium channel Kv7.1 Intramolecular BRET probe: Kv7.1-nLuc-KBP-YFP was prepared, where the DNA sequence encoding the human protein TRPV4 is fused at the C-terminus to a KBP-based potassium BRET sensor.

[0136] Example 11: Sensor-based BRET probe targeting one chloride channel LRRC8A Intramolecular BRET probe: LRRC8A-nLuc-YFP H148Q;I152L was prepared, where the DNA sequence encoding the human protein LRRC8A is fused at the C-terminus to a chloride-based BRET sensor (YFP H148Q;I152L) containing NanoLuc and a chloride-sensitive YFP mutant.

[0137] Example 12: Evaluation of calcium ion flow through TRPV1 using a calcium sensor-based BRET probe targeting the TRPV1 ion channel As described above in Example 3, the intramolecular BRET biosensor probe hTRPV1-nLuc-Calflux-mNeonGreen was prepared, where the DNA sequence encoding the human protein TRPV1 is fused to the calcium sensor at the C-terminus, and the sensor is sandwiched between nanoluciferase and mNeonGreen (Calflux probe, Yang J. et al., Nature Communication, 2016, 27;7:13268. doi: 10.1038 / ncomms13268.).

[0138] HEK293T cells expressing the mNeonGreen-Calflux-nLuc-hTRPV1 BRET biosensor were treated for BRET analysis. The basal BRET measured at rest increased dramatically in a primary kinetics after exposure to the prototype TRPV1 agonist capsaicin (CAPS), but only slightly and transiently increased after exposure to ATP, which is known to induce calcium release from intracellular stores via activation of the endogenous P2Y receptor (Figure 8A). These results indicate that the mNeonGreen-Calflux-nLuc-hTRPV1 BRET biosensor can specifically probe the calcium entering the cell via TRPV1 and is only slightly activated by the calcium increase in the cell. To exclude that the measured CAPS-induced BRET increase is not related to a conformational change in the quaternary structure of the TRPV1 ion channel, the inventors used the hTRPV1-nLuc-Calflux D / A-sYFP2 BRET probe containing the calcium-insensitive mutant Calflux reporters (D13A and D51A). In cells expressing this mutant BRET probe, no significant BRET increase could be detected after CAPS activation (Figure 8B), so the inventors concluded that the Calflux-based BRET biosensor fused to TRPV1 effectively responds to the fluctuations in the amount of calcium ions in the nanoscale environment inside the TRPV1 pore into the cytoplasm. Next, the inventors used patch-clamp or calcium flux measurements on cells expressing endogenous or overexpressed TRPV1 to demonstrate that the agonist-induced BRET increase is dose-dependent and that the half-maximal response (Figure 8C) is consistent with what has been reported in the literature. The pharmacological selectivity of the BRET change promoted by the ligand was further demonstrated by the competitive nature of the effect, as the well-known TRPV1 competitive antagonist AMG517 inhibited the CAPS-induced BRET increase, as shown by a shift to a higher value of the CAPS potency (Figure 8C).The inventors also examined whether the mNeonGreen-Calflux-nLuc-hTRPV1 BRET biosensor could distinguish between known TRPV1 agonists and antagonists that were expected to exhibit different potencies. In these experiments, HEK293T cells transiently expressing the mNeonGreen-Calflux-nLuc-hTRPV1 BRET biosensor were challenged with increasing amounts of four TRPV1 agonists (Figure 8D) or increasing amounts of various TRPV1 antagonists (Figure 8E). For each test compound, the inventors were able to measure dose-dependent BRET modulation that correlated with the known ability of the test ligand to activate TRPV1 or inhibit CAPS-induced TRPV1 activation. Importantly, RN1734, a known specific antagonist of TRPV4, was unable to inhibit CAPS-activated TRPV1. Overall, these data strongly suggest that agonist-promoted BRET changes in both probe conformations correspond to activation of the TRPV1 channel in living cells.

[0139] Example 13: Evaluation of calcium ion flux through TRPV4, TRPM8, and PIEZO1 using calcium sensor-based BRET probes targeting these ion channels As described above in Examples 7 and 8, the intramolecular BRET biosensor probes hTRPM8-nLuc-Calflux-mGreen and hTRPV4-nLuc-Calflux-mGreen were prepared, where the DNA sequence encoding the human protein TRPM8 is fused at the C-terminus to a calflux-based calcium BRET sensor, and the sensor is sandwiched between nanoluciferase and mNeonGreen.

[0140] To exemplify and validate the concept of calcium sensor-based BRET probes fused to other ion channels, the inventors first evaluated whether the activities of two other TRP ion channels, TRPV4 and TRPM8, could also be measured using such BRET probes. As shown in FIGS. 9A and 9B, the BRET signals measured for HEK293T cells transiently expressing mNeonGreen-Calflux-nLuc-hTRPV4 and mNeonGreen-Calflux-nLuc-hTRPM8 BRET probes increased dose-dependently after the addition of two specific agonists of TRPV4 and TRPM8, GSK1016790A and WS12, respectively, to the cell culture medium. The measured half-maximal responses were consistent with those reported in the literature using patch-clamp or calcium flux measurements for cells transiently expressing TRPV4 or TRPM8. The pharmacological selectivity of the BRET changes promoted by the ligands was further demonstrated by the competitive nature of the effect, as both of the two well-known competitive antagonists of TRPV4 and TRPM8, HC060747 and M8B, shifted the corresponding agonist potencies to higher values with a rightward shift. Overall, these data strongly suggest that agonist-promoted BRET changes in TRPV4 and TRPM8 BRET probes correspond to the activation of these two ion channels in living cells.

[0141] Next, according to the results obtained using TRPV1, TRPV4, and TRPM8, the inventors constructed an mNeonGreen-Calflux-nLuc-PIEZO1 BRET probe that should be useful for evaluating calcium currents through the PIEZO1 channel when the N-terminal tip of PIEZO1 is located in the cytoplasm as suggested by others. As shown in Fig. 9C, the BRET signal measured in HEK293T cells transiently expressing the mNeonG-Calflux-nLuc-PIEZO1 BRET probe increased in a dose-dependent manner after adding increasing amounts of YODA1, a known agonist of PIEZO1, to the cell culture medium. This demonstrates the functionality of the inventors' calcium sensor-based BRET probe targeting PIEZO1.

[0142] Example 14: Evaluation of potassium and sodium ion currents through non-TRP ion channels using calcium sensor-based BRET probes targeting TREK1, Kir6.1, and NaV1.8 channels To further demonstrate that ion sensor-based BRET probes may be useful for probing other types of ion channels, the inventors constructed mNeon-KBP-nLuc-TREK1 and mNeon-KBP-nLuc-KiR6.1 (where the KBP potassium sensor was sandwiched between mNeonGreen and NanoLuc and fused to the N-terminus of both the TREK1 and KiR6.1 potassium channels), as well as the mNeon-SOFI-nLuc-NaV1.8 BRET probe (where the ion intensity sensor was sandwiched between mNeonGreen and nanoLuc and fused to the N-terminus of the tetrodotoxin (TTX)-resistant voltage-gated sodium channel NaV1.8). The TREK1 BRET probe was activated using the chemical activator BL1249. The Kir6.1 BRET probe was activated with KCl. It is known that a strong outward potassium current occurs in HEK293T cells after depolarization. Thus, extracellular potassium ions are expected to re-enter the cell through heterologously expressed inwardly rectifying ion channels, such as Kir6.1, thereby increasing the local concentration of potassium in the intracellular vicinity of the Kir ion channel. NaV1.8 was activated in the presence of deltamethrin, a pyrethroid toxin known to prolong the open state of tetrodotoxin-resistant sodium channels. Repeated activation of TREK1 with increasing amounts of BL1249 dose-dependently increased the basal BRET measured using the mNeon-KBP-nLuc-TREK1 BRET probe, an effect that could not be detected using mock activation conditions (Figure 10A). The increase in the BRET signal indicates an increase in the potassium concentration in the cytoplasmic microenvironment of the TREK1 pore after repeated activation of TREK1 by BL1241. Surprisingly, nonetheless, this observation can be explained considering that TREK1 induces hyperpolarization of the cell membrane by leaking potassium extracellularly, thus lowering the intracellular potassium concentration to equilibrate with the extracellular potassium concentration. Under those conditions, potassium has been shown to flow inward through the TREK1 open pore, which is consistent with the inventors' observations.As expected, mock activation with DMSO alone was unable to induce activation of the TREK1 BRET probe (Figure 10A). The inventors were further able to evaluate that KCl depolarization could induce an increase in the BRET signal measured for HEK293T cells expressing mNeon-KBP-nLuc-KiR6.1 (Figure 10B), which also agreed with the known inward rectifying activity of the Kir6.1 ion channel. Finally, deltamethrin successfully induced a decrease in the BRET signal measured for HEK293T cells expressing mNeon-SOFI-nLuc-NaV1.8 (Figure 10C), which indicates an increase in ionic strength in the cytoplasmic side of the NaV1.8 pore microenvironment, consistent with the idea that sodium is flowing inward following its opening through the NaV1.8 ion channel pore. Overall, these results demonstrate that the intramolecular BRET biosensor can probe conformational changes that occur during the gating of ion channels belonging to various ion channel families, not just the TRP-ion channel family.

[0143] Example 15: Sensor-based BRET probes targeting all P2X receptors The inventors constructed human and mouse P2X-NeonGreen-Calflux-nLuc probes in which the DNA sequences encoding seven human or mouse P2X subunits were individually fused at the C-terminus to a calflux calcium sensor sandwiched between nanoluciferase and mNeonGreen (left panel of Figure 11). The inventors were able to show that the basal BRET signals of the hP2X2, hP2X4, hP2X5, hP2X7 and mP2X4-NeonGreen-Calflux-nLuc probes were modulated in a dose-dependent manner after cell activation with ATP (or Bz-ATP for hP2X7, right panel of Figure 11).

[0144] Analysis of the dose-response curves showed that ATP or BzATP activated the P2X-NeonGreen-Calflux-nLuc probe with an EC50 (1.63 + / - 0.18, 0.49 + / - 0.06, 0.48 + / - 0.07, 49.00 + / - 11.51 μM for hP2X2,4,5,7 and mP2X4, respectively) similar to the ATP or BzATP potency that activates native P2X as found in the scientific literature and measured using prior art.

[0145] Example 16: Sensor-based BRET Probes Targeting the AMPA (GluA1) Receptor The inventors constructed a second-generation BRET probe that enables the measurement of GluA1 receptor activity by coupling the cDNA of subunit GluA1 to the BRET sensor nLuc-Calflux-YFP (Figure 12, left panel). To block desensitization of the AMPA receptor by glutamate, the inventors introduced the L497Y point mutation as previously described (Stern-Bach Y, Russo S, Neuman M, and Rosenmund C (1998) A point mutation in the glutamate binding site blocks desensitization of AMPA receptors. Neuron 21:907-918).

[0146] The inventors were able to show that the BRET signal generated when expressing the BRET probe GluA1-L497Y-nLuc-Calflux-YFP alone was stable and modulated by increasing doses of glutamate (Figure 12, right panel). Analysis of the dose-response curve showed that glutamate activated the biosensor with an EC50 of 8.98 + / - 1.019 μM. These data are consistent with scientific literature reporting the affinity of the homomeric GluA1-L497Y receptor for glutamate obtained by patch-clamp recordings in HEK293 cells.

[0147] Example 17: Sensor-based BRET probes targeting the Cys-loop receptor family The inventors constructed second-generation BRET probes that enable the measurement of the activities of cationic and anionic Cys-loop receptors. For cationic receptors, the inventors inserted the cDNA of the sensors NLuc-CalFlux-sYFP2 (NL-CF-YFP) or NeonGreen-Calflux-nLuc (NG-CF-NL) into the second intracellular loop of the 5-HT3A receptor or the alpha-4 and beta-2 nicotinic receptors, respectively (Figure 13A). The inventors were able to show that the BRET signals generated when expressing the BRET probe 5-HT3A-NL-CF-YFP (Figure 13C) or a combination of alpha-4 and beta-2 nicotinic subunits fused to NG-CF-NL (Figure 13B) were stable and modulated by their respective agonist stimulations.

[0148] The inventors also developed a BRET probe that enables the sensing of anion flux by fusing the cDNA of a chloride-sensitive variant of YFP (YFP Chlore or YPetCl) to the cDNA of nLuc (Figure 13D). When expressed in HEK cells, this probe can detect the anion influx generated by VRAC channel opening after a hypotonic challenge (Figure 13F). Based on this probe, the inventors fabricated a biosensor that enables the measurement of the activity of anionic GABA receptors. The inventors inserted the cDNA of the YPetCl sensor into the second intracellular loop of the GABARho1 subunit (Figure 13E). The inventors were able to show that the BRET signals generated when expressing the BRET probe with or without combination with the untagged GABARho1 subunit were stable and modulated by GABA stimulation (Figure 13G).

Claims

**Claim 1** A channel fusion subunit comprising an ion-conductive channel subunit coupled to a probe, wherein the probe comprises an ion sensor coupled therebetween to at least one bioluminescent donor molecule and at least one fluorescent acceptor molecule, wherein the probe is coupled via either the bioluminescent donor molecule or the fluorescent acceptor molecule to the N-terminus, C-terminus, intracellular or extracellular loop of the channel subunit, wherein the bioluminescent donor molecule and the fluorescent acceptor molecule are selected such that the emission spectrum of the bioluminescent donor molecule overlaps the absorption spectrum of the fluorescent acceptor molecule, enabling non-radiative energy transfer by non-radiative dipole-dipole coupling between the bioluminescent energy donor and the fluorescent acceptor, Channel fusion subunit. **Claim 2** The channel fusion subunit according to claim 1, wherein the ion-conductive channel subunit is a voltage-dependent ion channel or a ligand-dependent ion channel. **Claim 3** The channel fusion subunit according to claim 1 or 2, wherein the ion sensor is a sensor for calcium ions, potassium ions, sodium ions, chloride ions or ion strength. **Claim 4** The channel fusion subunit according to any one of claims 1 to 3, wherein the ion sensor is selected from a calcium-binding protein, a troponin C calcium-sensing domain or calmodulin calcium-binding protein, a potassium-binding protein (KBP), a sodium-binding protein, the NhAs-1 protein, or a chloride-binding protein. **Claim 5** The channel fusion subunit according to any one of claims 1 to 4, further comprising a linker between the ion-conductive channel subunit and the probe. **Claim 6** The channel fusion subunit according to any one of claims 1 to 5, wherein the bioluminescent donor molecule is selected from Renilla luciferase, Firefly luciferase, Coelenterate luciferase, North American glow worm luciferase, click beetle luciferase, railroad worm luciferase, Gaussia luciferase, Aequorin, Arachnocampa luciferase, NanoLuciferase derived from the deep-sea shrimp Oplophorus gracilirostris, GLuc, NanoLuc (NLuc), MLuc7, HtLuc, LoLuc, PaLuc1, PaLuc2, MpLucl, McLucl, MaLuc1, MoLuc1, MoLuc2, MLuc39, PsLucl, LocLucl-3, HtLuc2 Renilla, TurboLucl6 (TLuc), or a homolog or ortholog thereof.

7. The channel fusion subunit according to any one of claims 1 to 5, wherein the bioluminescent donor molecule is a non-luciferase bioluminescent protein selected from β-galactosidase, lactamase, horseradish peroxidase, alkaline phosphatase, β-glucuronidase or β-glucosidase.

8. The fluorescent acceptor molecule is a non-luciferase bioluminescent protein selected from green fluorescent protein (GFP), a variant of green fluorescent protein (GFP10), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, mAmetrine, LSS-mOrange, LSS-mKate, Emerald, Topaz, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), HcRed, t-HcRed, DsRed, DsRed2, mRFPl, Porphyrin, Renilla GFP, Monster GFP, paGFP, Kaede protein, mNeonGreen of Branchiostoma lanceolatum or phycobiliprotein, TagCFP, mTagCFP2, Czurite, ECFP2, mKalamal, Sirius, Sapphire, T-Sapphire, ECFP, Cerulean, SCFP3C, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP, mTFPl, EGFP, Emerald, Superfolder GFP, monomeric Czami Green, TagGFP2, mUKG, mWasabi, Clover, Citrine, Venus, SYFP2, TagYFP, monomeric Kusabira-Orange, ιηΚΟκ, mK02, mOrange, mOrange2, mRaspberry, mCherry, mStrawberry, mScarlet, mTangerine, tdTomato, TagRFP, TagRFP-T, mCpple, mRuby, mRuby2, mPlum, HcRed-Tandem, mKate2, mNeptune, NirFP, TagRFP657, IFP1.4 or iRFP, the channel fusion subunit according to any one of claims 1 to 7.

9. A recombinant cell expressing the channel fusion subunit according to any one of claims 1 to 8.

10. A method for screening a candidate compound capable of modulating the ion flux through a channel, comprising: contacting the candidate compound with the recombinant cell according to claim 9; providing a substrate for the bioluminescent donor molecule; and measuring the variation in bioluminescence resonance energy transfer. A method comprising the steps of:

11. A nucleic acid comprising a nucleotide sequence encoding a channel fusion subunit comprising an ion-conductive channel subunit bound to a probe, wherein the probe comprises an ion sensor bound therebetween to at least one bioluminescent donor molecule and at least one protein fluorescent acceptor molecule, wherein the ion sensor is configured to undergo a conformational change in the presence of an ion transported by the channel subunit, wherein the probe is bound via either the bioluminescent donor molecule or the protein fluorescent acceptor molecule to the N-terminus, C-terminus, or within an intracellular or extracellular loop of the channel subunit, wherein the bioluminescent donor molecule and the protein fluorescent acceptor molecule are selected such that the emission spectrum of the bioluminescent donor molecule overlaps the absorption spectrum of the fluorescent acceptor molecule and the light energy delivered by the bioluminescent donor molecule is at a wavelength capable of exciting the fluorescent acceptor molecule. A nucleic acid.

12. The nucleic acid according to claim 11, wherein the ion-conductive channel is a voltage-dependent ion channel or a ligand-dependent ion channel, and the ion sensor is a sensor for calcium ions, potassium ions, sodium ions, chloride ions, or a sensor for ion strength.

13. The nucleic acid according to claim 11 or 12, further comprising a linker sequence between the nucleotide sequence encoding the ion-conductive channel subunit and the nucleotide sequence encoding the probe.

14. The nucleic acid according to any one of claims 11 to 13, wherein the ion sensor is selected from a troponin C calcium-sensing domain or a calmodulin calcium-binding protein, a potassium-binding protein (KBP), a sodium-binding protein, an NhAs-1 protein, or a chloride-binding protein.

15. The bioluminescent donor molecule is luciferase selected from Renilla luciferase, Firefly luciferase, Coelenterate luciferase, North American glow worm luciferase, click beetle luciferase, railroad worm luciferase, Gaussia luciferase, Aequorin, Arachnocampa luciferase, NanoLuciferase derived from the deep-sea shrimp Oplophorus gracilirostris, GLuc, NanoLuc (NLuc), MLuc7, HtLuc, LoLuc, PaLuc1, PaLuc2, MpLucl, McLucl, MaLuc1, MoLuc1, MoLuc2, MLuc39, PsLucl, LocLucl-3, HtLuc2 Renilla, TurboLucl6 (TLuc), or a homolog or ortholog thereof, or the bioluminescent donor molecule is a non-luciferase bioluminescent protein selected from β-galactosidase, lactamase, horseradish peroxidase, alkaline phosphatase, β-glucuronidase or β-glucosidase, the nucleic acid according to any one of claims 11 to 14.

16. The nucleic acid according to any one of claims 11 to 15, wherein the fluorescent acceptor molecule is selected from green fluorescent protein (GFP), a variant of green fluorescent protein (GFP10), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, mAmetrine, LSS-mOrange, LSS-mKate, Emerald, Topaz, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), HcRed, t-HcRed, DsRed, DsRed2, mRFPl, Posilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein, mNeonGreen of Branchiostoma lanceolatum or phycobiliprotein, TagCFP, mTagCFP2, Czurite, ECFP2, mKalamal, Sirius, Sapphire, T-Sapphire, ECFP, Cerulean, SCFP3C, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP, mTFPl, EGFP, Emerald, Superfolder GFP, monomeric Czami Green, TagGFP2, mUKG, mWasabi, Clover, Citrine, Venus, SYFP2, TagYFP, monomeric Kusabira-Orange, ιηΚΟκ, mK02, mOrange, mOrange2, mRaspberry, mCherry, mStrawberry, mScarlet, mTangerine, tdTomato, TagRFP, TagRFP-T, mCpple, mRuby, mRuby2, mPlum, HcRed-Tandem, mKate2, mNeptune, NirFP, TagRFP657, IFP1.4 or iRFP, a non-luciferase bioluminescent protein.

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