Patent Publication Number: US-2023160909-A1

Title: Novel ion conducting channel fusion subunits and methods of use thereof

Description:
FIELD OF THE INVENTION 
     The present invention relates generally to novel ion conducting channel fusion subunits and/or novel ion conducting channel fusions, nucleic acids encoding such channel fusions and channel subunit fusions, amino acid sequences, recombinant cells and expression vectors comprising such channel fusions and channel subunit fusions, as well as method of use thereof particularly for high throughput screening (HTS) methods for screening inhibitors or activators of the ion conducting channels. 
     BACKGROUND OF THE INVENTION 
     Life of higher organisms including humans requires quick and efficient transmission of electric and chemical signals that synchronize myriad diverse cellular processes. The interior of animal and plant cells is electrically negative with respect to the exterior. The magnitude of this potential difference is generally between 5 and 90 mV, with most of the potential being developed across the cell membrane. The transmembrane potential of a given cell is set by the balance of the activities of ion transporters and channels which 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 vital for cellular functions and are mediated primarily by ion channels or ion conducting channels, a specialized group of proteins which span the membranes of all living cells and which are permeable to electrically charged atoms (ions) present in the body. In a simple sense, ion conducting channels can be considered the biological equivalent of transistors as they respond to stimuli by switching between “on” (open) and “off” (closed) states, thereby controlling the passage of electric current across the cell membrane. In doing so, ion conducting channels control the electrical state of cells and allow such fundamental processes as the beating of the heart, contraction of muscles and information processing in the nervous system. Since ion conducting channels allow the movement of ions across cell membranes, they are completely responsible for the entire range of electrical signaling within the living system. In addition, ion channels either directly, by allowing calcium flux, or indirectly, by modulating membrane potential, affect the intracellular calcium concentration; this in turn regulates a variety of functions, including energy production, synaptic transmission, muscle contraction, hormone secretion and gene transcription. 
     Specifically, ion conducting channels can be defined as pore-forming proteins that allow certain specific types of ions to pass through the channel by their electrochemical gradient to control slight voltage difference between plasma membranes of cells. These ion conducting channels are present in cell membrane of all living cells. Ion conducting channels can be divided into chloride ion channel family, potassium ion channel family, sodium ion channel family, calcium ion channel family, proton channel family and universal ion channel family in accordance with types of ions. 
     Given the central and pivotal role of ion conducting channels in many physiological processes, they represent a target class with enormous potential for intervention in a wide range of disease states. Indeed, the successful introduction of numerous ion conducting channel drug classes (e.g., calcium antagonists, sodium channel blockers, γ-aminobutyric acid or GABA receptor potentiators and sulphonylureas) onto the market has firmly established ion conducting channels as valid drug targets. However, most marketed ion conducting channel drugs that were discovered in the pre-molecular era, i.e., first-generation ion channel drugs, are relatively nonselective, often exhibit dose-limiting side effects and are rarely 100% effective. Thus, there remains a clear need for improved ion conducting channel drugs, i.e., new-generation ion channel drugs, that are more selective and that target ion conducting channels with fewer side effects and improved efficacy. Several diseases have been associated with ion conducting channel functionality. For instance, diseases resulting from ion conducting channel dysfunctions in the central nervous system include anxiety, depression, epilepsy, insomnia, memory problems and chronic pain. Other diseases resulting from ion conducting channel dysfunctions include cardiac arrhythmia, and type II diabetes. 
     The early methods developed to measure the activity of ion conducting channels were based on live cell recordings. Overexpression of the ion conducting channel of interest was often required for such approaches, which changes the cellular physiological state with unpredictable consequences on channel activity. This leads in many instances to toxicity for cell and their apoptosis. In these methods, the activity of the ion channels was measured mainly by patch-clamp or fluorescence. 
     The patch-clamp systems involve an electrophysiology-based measurement. An electrode is dipped into the cell and a reference electrode is outside in the solution containing the cell. When ions go through the channels, a current is generated across the membrane and is measured by the electrodes. If the channels are non-functional or blocked by compounds, no current will be recorded. The fluorescence-based systems use ion-specific (or pH-dependent) fluorescent molecules. However, such ion-specific fluorescent molecules do not exist yet for all ions. In order to overcome the drawbacks of the cell-based methods, cell-free methods have been developed still based on patch-clamp or fluorescence measures. In these cell-free methods, ion conducting channels are expressed, purified and reconstituted in model membranes, such as droplet interface bilayer (DIB), possibly in a microfluidic platform. 
     In order to discover new targets for therapy and new pharmaceutical compounds targeting ion channels, it is useful to be able to screen chemical libraries of numerous compounds by automated, high-throughput assays. However, the above cell-based or cell-free methods have limitations that do not facilitate a high-throughput screening (HTS). Indeed, most current HTS methods involve compounds under flow, typically in microfluidic or millifluidic devices. Thus, this precludes the use of patch-clamp methods due to the use of electrodes. A major limitation of the patch clamp technique is its low throughput. Typically, a single, highly trained operator can test fewer than ten compounds per day using the patch clamp technique. Furthermore, the technique is not easily amenable to automation, and produces complex results that require extensive analysis. The use of fluorescence-based methods is also not convenient since a fluorescent smear is observed under flow of a fluorescent compound which basically generates noise and limits the readout. 
     Fluorescence resonance energy transfer (FRET) and Bioluminescence resonance energy transfer (BRET) techniques have been developed to monitor protein-protein interactions, for drug discovery and HTS methods. 
     International publication No. WO2016/131832 describes first generation of intramolecular BRET probes comprising a transient receptor potential (TRP) channel subunit fused to a bioluminescent donor molecule and a fluorescent acceptor molecule either on the C-terminal or N-terminal extremity of the channel subunit. Such BRET probes allowed monitoring transient receptor potential (TRP) channel structural conformational changes and TRP channel activation in living cells. However, while these first generation of BRET probes are only efficient in measuring the change of conformation of ion channels, they do not always show in a reliable fashion actual activation or inhibition of the ion conducting channels since the change of conformation of the ion channels is not necessarily connected to actual activations or inhibitions. 
     One challenge to increase the discovery rate of drugs was thus to provide a method allowing measuring the passage of ions through membrane-imbedded channels in HTS methods. The present application is thus directed to novel BRET probes allowing to reliably measure activation or inhibition of ion conducting channels by measuring the actual passages of ions using BRET techniques and methods of use of these new probes for drug screening and mapping of signal transduction pathways. 
     SUMMARY OF THE INVENTION 
     The present invention relates to nucleic acids encoding novel fusion proteins as well as the novel fusion proteins comprising ion conducting channel fusions or novel ion conducting channel fusion subunits. These fusions comprise an ion conducting channel, or a subunit thereof bound in C-terminal, N-terminal, or within an intracellular or extracellular loop, to a probe which comprising an ion sensor bound in between or sandwiched between at least one bioluminescent donor molecule and at least one fluorescent acceptor molecule, and wherein the ion sensor is configured to undergo a conformational change in the presence of an ion transported by the channel subunit. 
     The present invention also relates to expression vectors comprising nucleotide sequences encoding the novel ion conducting channel fusions or novel ion conducting channel fusion subunits, recombinant cells comprising such expression vectors, process for the production of ion channel subunit, as well as to the fusion proteins and fusion subunits obtainable by said process. 
     This invention further relates to a method for screening a candidate compound capable of modulating the ion flux through the ion conducting channel, and thus activating and/or inhibiting said channel by with measuring a variation of bioluminescent resonance energy transfer. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG.  1   : is a schematic representation of a novel intramolecular BRET probe using a putative 6-transmembrane domain Calcium channel as an example. The sensor sandwiched by Luc and YFP in both orientations is fused to the C-terminus of the channel. The fusion of the ion BRET biosensor to the N-terminus of the channel will also be constructed. 
         FIG.  2   : is a schematic representation of a novel BRET probe allowing to measure the TRP channel activity (6 transmembrane domains) by measuring the amount of ions diffusing under the channel during its opening via the BRET probe sensitive to ions which is fused in C-terminal to the channel of interest. During the passage of the ions across the pore of the opened channel, the ions diffuse towards the cytoplasm and are caught at the exit of the pore by the BRET probe which is fused to the channel of interest. The BRET probe will undergo a conformation change which will be reflected on the measure of the BRET signal. The groups Luc and YFP may be placed on either side of the ion sensor and/or may be fused in N-terminal of the channel of interest. 
         FIG.  3   : is a schematic representation of a BRET probe of 2 nd  generation allowing to measure the activity of P2X channel (2 transmembrane domains) via the detection of calcium ions passing through the channel during its opening and which are bound to the BRET probe fused to the C-terminal tail of the P2X receptor. During the passage of the calcium ions across the pore of the opened channel, the ions diffusing towards the cytoplasm are caught at the exit of the pore by the BRET probe which is fused to the channel of interest. This BRET probe will undergo a conformational change caused by the binding of the ions, which will be reflected by an increase of the BRET signal. 
         FIG.  4   : Dose-response to Capsaïcine (CAPS) and lonomycine (IONO) as measured in HEK293T cells transitory expressing TRPV1-nLuc-calflux-YFP or TRPV1-nLuc-calflux-mNeonGreen probe, the TRPV1-nLuc-calflux D13A/D51A-YFP (TRPV1-nLuc-CalfluxD&gt;A-YFP) probe or the nLuc-Calflux-mNeonGreen probe alone without (A) or with (B) TRPV1. 
         FIG.  5   : is a schematic representation of the SK3-nLuc-KBP-mNeonGreen probe (A) and the measure of BRET signal as obtained during the activation of the channel in presence of lonomycine (B). 
         FIG.  6   : shows the dose-response of ATP as measured in HEK293T cells transiently expressing P2X2-nLuc-Calflux-YFP probe (A) or nLuc-Calflux-YFP probe (B) or TRPV1-nLuc-Calflux-YFP probe(C). Cells are stimulated with increasing doses of ATP 5 min after the addition of coelenterazine H. At the end of the reading, cells are stimulated with capsaïcine (CAPS), an agonist of TRPV1. These data allowed us to generate dose response and to determine the EC50 of the P2X2-nLuc-Calflux-YFP probe for ATP (D). 
         FIG.  7   : Dose-response for NMDA as measured in HEK293T cells transiently expressing the NR1-nLuc-Calflux-YFP probe alone (A) or coexpressed with NR2A (B) or NR2B (C) subunits. Cells are stimulated with increasing doses of NMDA 4 min after the addition of coelenterazine H. With these data, we generated dose-response and determine the EC50 for NMDA of heteromeric receptors comprising NR2A or NR2B subunits (D). 
         FIG.  8   : shows the characterization of the mNeon-Calflux-nLuc-hTRPV1 BRET probe. A) Kinetic measurement of the effect of 10 μM CAPS on HEK293T cells expressing the mNeon-Calflux-nLuc-hTRPV1 BRET probe. CAPS was injected at 30s. Data represent one out of five independent experiments. B) Dose-response curves of CAPS-induced BRET changes measured in HEK293T cells expressing the hTRPV1-nLuc-Calflux-sYFP2 or the hTRPV1-nLuc-CalfluxD/A-sYFP2 BRET probes. C) Dose-response curves of CAPS-induced BRET changes measured in HEK293T cells expressing the mNeon-Calflux-nLuc-hTRPV1 BRET probe in absence or in presence of 10 μM of the TRPV1 antagonist AMG517. D) Dose-response curves of the BRET changes measured in HEK293T cells expressing the mNeon-Calflux-nLuc-hTRPV1 BRET probe in presence of increasing quantities of various TRPV1 agonists. E) Dose-response curves of the BRET changes measured in HEK293T cells expressing the mNeon-Calflux-nLuc-hTRPV1 BRET probe in presence of increasing quantities of various TRPV1 antagonists. Data represent the average of at least three independent experiments. 
         FIG.  9   : shows (A) a dose response curve of the TRPV4 agonist GSK1016790A to trigger activation of the mNeon-Calflux-nLuc-hTRPV4 BRET probe transiently expressed in HEK293T cells that were pre-treated or not with 10 μM of the TRPV4 antagonist HC067047. B) Dose response curve of the TRPM8 agonist WS12 to trigger activation of the mNeon-Calflux-nLuc-hTRPM8 BRET probe transiently expressed in HEK293T cells that were pre-treated or not with 10 μM of the TRPM8 antagonist M8B hydrochloride. C) Dose response curve of the PIEZO1 agonist YODA1 to trigger activation of the mNeon-Calflux-nLuc-PIEZO1 BRET probe transiently expressed in HEK293T cells. Data represent the average of at least three independent experiments. 
         FIG.  10   : shows (A) kinetic measurement of the effect of increasing quantities of the TREK1 agonist BL1249 or saline buffer alone to activate the mNeon-Calflux-nLuc-hTRPV1 BRET probe transiently expressed in HEK293T cells. B) Kinetic measurement of the effect of KCl injection on the BRET measured from HEK293T cells transiently expressing mNeon-Calflux-nLuc-KiR6.1 BRET probe. C) Kinetic measurement of the effect of Deltamethrine injection on the BRET measured from HEK293T cells transiently expressing mNeon-SOFI-nLuc-NaV1.8 BRET probe. 
         FIG.  11   : shows dose-response curves of ATP or BzATP-induced BRET changes measured in HEK293T cells expressing human (h) or mouse (m) P2X-mNeonGreen-CalFlux-NanoLuc BRET probes (NG-CF-NL). Data for hP2X1, hPX3 and hP2X6 subunit are not represented as these subunits desensitize too fast or do not form functional homomeric receptors. Values were normalized to the maximal efficacy measured individually for each probe and represent the mean of three independent experiments. 
         FIG.  12   : shows dose-response curves of Glutamate-induced BRET changes measured in HEK293T cells expressing rat GluA1-NLuc-CalFlux-YFP BRET probes (NL-CF-YFP). Values were normalized to the maximal efficacy and represent the mean of three independent experiments. 
         FIG.  13   : Biosensors allowing to track Cys-loop receptor family activity. Insertion of NG-CF-NL or NL-CF-YFP probes in the second intracellular loop of cationic receptors (A) allows to track activity of nicotinic (B) or serotonin receptors (C). A BRET probe called YPetCI was also developed to sense intracellular anionic flux (D and F). When inserted in the second intracellular loop of GABARho1 subunit this probe allows to track activity of the GABA receptor (G). 
     
    
    
     DETAILED DESCRIPTION 
     Recombinant protein, cell culture, and molecular cloning techniques 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 in 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 &amp; Sons (including all updates until present). 
     The terms “bioluminescent donor molecule” refer to any molecule able to generate luminescence following either action on a suitable substrate, or its own excitation by an external source. 
     The terms “fluorescent acceptor molecule” refer to any compound which can accept energy emitted as a result of the activity of a bioluminescent donor molecule and re-emit it as light energy. 
     Bioluminescent resonance energy transfer (BRET) refers a proximity assay based on the non-radiative transfer of energy between the bioluminescent donor molecule and any suitable fluorescent acceptor molecule. Non-radiative transfer means transfer of energy from an energy donor to an energy acceptor without the emission of light by the donor. 
     The terms “spatial location” refer to the three-dimensional positioning of the bioluminescent donor molecule relative to the acceptor molecule which changes as a result of the compound binding to the voltage-dependent ion channel or of the change in specific physical parameters. 
     The terms “dipole orientation” refer to the direction in three-dimensional space of the dipole moment associated either with the donor molecule and/or the acceptor molecule relative to their orientation in three-dimensional space. The dipole moment is a consequence of a variation in electrical charge over a molecule. The resulting resonance energy transfer is an electromagnetic phenomenon in which quantum energy is transferred non-radiatively from an excited bioluminescent donor to a fluorescent acceptor molecule within close proximity. 
     The terms “resonance energy transfer” refer to the fact that energy transfer is by means of intermolecular dipole-dipole coupling, that is, the process does not involve emission and reabsorption of photons. The donor typically emits at shorter wavelengths that overlap with the absorption spectrum of the acceptor molecule. 
     The terms “protein-protein interaction” or “binding” refer to the specific complementary recognition and association of two proteins with a dissociation constant, Kd of preferably 10 −5 M, more preferably 10 −7 M, most preferably 10 −9 M or less. 
     Modulators, activators or inhibitors are any agents capable of altering the functional activity of ion channel within a cell and may represent a channel opener (functional agonist), a channel blocker (functional antagonist). 
     As used herein, the terms “fusion channel unit(s)” or “fusion channel(s)” generally refer to a polypeptide or protein channel or channel subunit that is comprised of two or more amino acid sequences, wherein each amino acid sequence encodes a protein or a portion thereof, wherein the two or more amino acid sequences are not found linked in nature, and wherein the two or more amino acid sequences are physically linked by a peptide bond. 
     As used herein, the term “domain” refers to a region of a protein that is at least 2 amino acids less than the whole protein and which retains at least the biological activity of the whole protein. A domain may range in size from 10-1000 amino acids in length, e.g. 50-60 amino acids, 100-400 amino acids or 200-300 amino acids. Domain refers to a functional unit of a complete protein having a biological activity of the complete protein. For example, the domain of a protein useful according to the invention may refer to a region of a protein that binds to a second protein. 
     The terms “more sensitive” refer to a greater change in resonance energy transfer ratio between the bioluminescent donor molecule and the fluorescent acceptor molecule. 
     The term “contacting” refers to the addition of a candidate molecule, a series of candidate molecules, and/or samples capable of activating or inhibiting the ion conducting channel and which may thus be screened by HTS methods using the new-generation BRET probe. 
     By “substantially purified” or “purified” is meant an ion conducting channel or channel subunit which has been separated from one or more lipids, nucleic acids, other polypeptides, or other contaminating molecules with which it is associated in its native state. It is preferred that the substantially purified polypeptide is at least 60% free, more preferably at least 75% free, and more preferably at least 90% free from other components with which it is naturally associated. 
     As used herein, “cell membrane” preparation refers to a preparation of cellular lipid membranes and is distinct from a cellular homogenate, in that at least a portion (i.e., at least 10%, and preferably more) of non-membrane-associated cellular constituents have been removed from the homogenate. “Membrane associated” refers to a polypeptide that is either integrated into a lipid membrane or is physically associated with a component that is integrated into a lipid membrane. 
     The terms “polypeptide” and “protein” are generally used interchangeably and refer to a single polypeptide chain which may or may not be modified by addition of non-amino acid groups. It would be understood that such polypeptide chains may associate with other polypeptides or proteins or other molecules such as co-factors. The terms “proteins” and “polypeptides” as used herein also include variants, mutants, biologically active fragments, modifications, analogous and/or derivatives of the polypeptides described herein. 
     The % identity of a polypeptide is determined by GAP analysis (GCG program) with a gap creation penalty of 5, and a gap extension penalty of 0.3. The query sequence is at least 25 amino acids in length, and the 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 the 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 the 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 the GAP analysis aligns the two sequences over a region of at least 250 amino acids. Even more preferably, the GAP analysis aligns the two sequences over their entire length. 
     As used herein, a “biologically active fragment” is a portion of a polypeptide as described herein, which maintains a defined activity of the full-length polypeptide. For example, a biologically active fragment of a voltage-dependent ion subunit must be capable of binding the target compound, resulting in a conformational change. Biologically active fragments can be any size as long as they maintain the defined activity. Preferably, biologically active fragments are at least 150, more preferably at least 250 amino acids in length. 
     As used herein, a “biologically active variant” is a molecule which differs from a naturally occurring and/or defined molecule by one or more amino acids but maintains a defined activity, such as defined above for biologically active fragments. Biologically active variants are typically 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% identical to the naturally occurring and/or defined molecule. 
     With regard to a defined polypeptide or polynucleotide, it will be appreciated that % identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that the polypeptide or polynucleotide comprises an amino acid sequence which 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 nominated amino acid sequences. 
     By an “isolated polynucleotide”, including DNA, RNA, or a combination of these, single or double stranded, in the sense or antisense orientation or a combination of both, we mean a polynucleotide which is at least partially separated from the polynucleotide sequences with which it is associated or linked in its native state. Preferably, the isolated polynucleotide is at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated. 
     As used herein, the terms “determining,” “measuring,” “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations. 
     As used herein, “recombinant vector” refers to a discrete genetic element that is used to introduce heterologous nucleic acid into cells for either expression or replication thereof. Selection and use of such vehicles are well within the skill of the artisan. A recombinant expression vector includes vectors capable of expressing nucleic acids that are operatively linked to regulatory sequences, such as promoter regions that are capable of effecting expression of such nucleic acids. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned nucleic acid. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells including those that remain episomal or those, which integrate into the host cell genome. 
     The terms “ion conducting channel” are intended to refer to ion-permeable pores in the lipid membranes of all cells, which are known to open and close in response to stimuli, gating the flow of specific small ions. Indeed, the lipid bilayer membrane of all cells forms a barrier that is largely impermeable to the flux of ions and water. Residing within the membrane is a family of proteins called ion channels or ion conducting channels, which provide selective pathways for ion flux. Such ion conducting channels are thus ion-selective channels, for example sodium-, potassium, calcium-, chloride-selective or non-selective channels. 
     Flux of ions through ion conducting channels contributes to depolarization of cells causing the membrane potential to become more positive and generating cytoplasmic regulatory signaling. For example, regulated conductance produced by ion channels are required for intercellular signaling and neuronal excitability. We may cite a group of ion channels that open upon depolarization of excitable cells are classified as voltage-gated and are responsible for electrical activity in nerve, muscle and cardiac tissue. For example, in neurons, ion currents flowing through voltage-gated sodium ion (Na + ) channels are responsible for rapid spike-like action potentials. During action potentials the majority of Na +  channels open very briefly. These brief openings result in transient Na +  currents. However, a subset of voltage-gated Na +  channels does not close rapidly but remain open for relatively long intervals. These channels therefore generate sustained or persistent Na +  currents. The balance between transient and persistent Na +  current is crucial for maintaining normal physiological function and electrical signaling throughout the entire nervous system. 
     Over the past 5 decades, an increasing number of diseases have been shown to result from the dysregulation of ion conducting channels. This class of disease has been termed channelopathies. Aberrant persistent sodium current can contribute to the development or progression of many channelopathies because normal function is disrupted when neurons discharge signals inappropriately. For example, abnormal persistent sodium current is thought to induce deleterious phenomena, including, e.g., neuropathies, neurodegenerative diseases, movement disorders, cardiac arrhythmia, epileptic seizure, neuronal cell death, behavioral disorders and dementia etc. . . . . For example, in the case of the neuropathies embraced by epilepsy, there can be a brief electrical “storm” arising from neurons that are inherently unstable because of a genetic defect as in various types of inherited epilepsy, or from neurons made unstable by metabolic abnormalities such as low blood glucose, or alcohol. In other cases, the abnormal discharge can come from a localized area of the brain, such as in patients with epilepsy caused by head injury or brain tumor. In the case of ischemic injuries, such as, e.g., cerebral ischemia and myocardial ischemia, there can be prolonged electrical activity arising from neurons in which persistent sodium channel expression or activity is increased. Such aberrant electrical activity can cause or contribute to neuronal death, which can lead to debilitating injury or death of an individual. Aberrant electrical activity also can contribute to neurodegenerative disorders such as, without limitation, Parkinson&#39;s disease, Alzheimer&#39;s disease, Huntington&#39;s disease, amyotrophic lateral sclerosis and multiple sclerosis. Thus, aberrant persistent sodium current can contribute to development or progression of pathological conditions by collapsing the normal cell transmembrane gradient for sodium, leading to reverse operation of the sodium-calcium exchanger, and resulting in an influx of intracellular calcium, which injures the axon. Therefore, selective reduction in the expression or activity of sodium channels capable of mediating persistent current relative to any reduction in normal voltage-gated (transient) sodium current can be useful for treating channelopathies associated with increased persistent sodium current. 
     Thus, there exists a need for new screening methods that can be used to identify persistent ion channel modulators either inhibitors or activators useful for treating channelopathies. The present invention satisfies this need by providing high-throughput screens for identifying ion channel modulators, activators or inhibitors. 
     One technique for assessing protein-protein interaction is based on fluorescence resonance energy transfer (FRET). FRET is a proximity assay based on the non-radiative transfer of energy i.e. a transfer of energy which occurs without emission of photons, and which results in a dipole-dipole interaction between a donor of energy and an acceptor of energy. In FRET, one excited state fluorophore donor transfers its energy to a ground state fluorescent acceptor molecule that become activated. Following activation, the acceptor returns at basal stage by emitting a photon at its own wavelength. Energy transfer efficiency is dependent on the distance between the donor and acceptor, the extent of the spectral overlap and the relative orientation of the acceptor and donor dipoles. 
     Experiments on the neuronal voltage-gated N-type (Cav2.2) calcium using a FRET-based calcium biosensor named TN-XL have been conducted in the past (See Tay et al., (Nature Communications, Vol, 3; 778, 2012). The authors explored whether troponin-based TN-XL, an optical, genetically encoded calcium ion indicator, could respond to Ca 2+  when targeted to this environment using a specific approach of fusing TN-XL to the carboxy terminus of the principle subunit of CaV2.2 Ca 2+  channels. These experiments failed to provide any reliable and useful means for measuring the activity of an ion conducting channel. Indeed, in order to gauge TN-XL responsiveness, authors had to measure using TIRF imaging, the single-cell CFP and FRET fluorescence signals where the ratio of these signals furnishes the customary sensor readout of Ca 2+ . In addition, in order to improve the TN-XL signal-to-noise ratio at the surface membrane, the authors had to utilize TIRF microscopy to selectively illuminate only those CaV2.2/TN-XL channels near the surface, thereby attenuating the background signal from imperfectly targeted intracellular channels. Further in order to facilitate elevation of intracellular Ca 2+ , additional steps like internal Ca 2+  buffering (1 mM EGTA) with elevated 10 mM extracellular Ca 2+  was employed. Further limitation of the study concerned the incomplete targeting of active CaV2.2/TN-XL channels to the surface membrane in the TIRF volume, necessitating approximate correction of static background fluorescence. The potential error in such correction rendered the calcium spike determination as a coarse approximate estimate, accordingly such a technique is not at all suitable for HTS. 
     Accordingly, this study clearly revealed the complexity and difficulties encountered when using the FRET-based calcium biosensor, requiring a complex integration of TIRF/patch-clamp electrophysiology and CaV2.2/TN-XL fusion, instead of a straightforward and simple method based on the fusion construct alone. 
     Alternative to FRET technique is the BRET technique. BRET method is similar to that of FRET wherein the donor is a bioluminescent molecule. No external source of excitation is thus required, thereby avoiding inconveniences of photobleaching and autofluorescence of the 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 a luciferase avoids the need for illumination as the addition of a substrate initiates bioluminescent emission and thus resulting to resonance energy transfer between a bioluminescent donor and a fluorescent acceptor. Both FRET and BRET transfer efficiency also depends on the degree of the 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 by the efficacy of the ion sensor placed strategically between the donor and acceptor in the novel fusion construct. It would thus have been expected to one skilled person in the art that similar BRET-based ion biosensor would have provided similar results and thus could not be exploitable for drug discovery or HTS method. 
     In contrast with previous teaching and experimentations with FRET, the present applicant surprisingly showed for the first time that it was possible to construct and use BRET-based ion sensor in order to assess flux of ions and thus actual activation or inhibition of ion conducting channels in an accurate and reliable manner. The applicant showed that the new BRET probe may be used in a screening system that targets ion conducting channels with superior efficiency and particularly for HTS method without any requirements of further analysis such as for patch-clamp experiments, etc. . . . . 
     The present invention thus relates to novel ion conducting channel protein subunit fusions comprising an ion conducting channel subunit bound to a probe, which probe comprises an ion sensor bound in between or “sandwiched” between at least one bioluminescent donor molecule and at least one fluorescent acceptor molecule. The probe may be bound to the channel subunit, optionally via a linker, at its C-terminal, its N-terminal end, or within an intracellular or extracellular loop, either the bioluminescent donor molecule or the fluorescent acceptor molecule. 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 fluorescent acceptor molecule are selected so that the emission spectrum of the bioluminescent donor molecule overlaps with the absorbance spectrum of the acceptor molecule, making it possible the non-radiative energy transfer between the bioluminescent energy donor and the fluorescent acceptor through nonradiative dipole-dipole coupling. The novel channel protein fusion subunits may thus be used as novel BRET probes for detecting activation, inhibition or modulation of ion conducting channels by measuring the actual passages of ions through said channels. 
     The present invention also relates to novel ion conducting channel fusions which result from the assembling of the fusion subunits as described above. Therefore, such novel ion conducting channel protein fusion may comprise one or more of its subunits bound either in C-terminal, in N-terminal, or within an intracellular or extracellular loop of the subunit to a probe which itself comprises an ion sensor being sandwiched between at least one bioluminescent donor molecule and at least one fluorescent acceptor molecule. The ion sensor is configured to undergo a conformational change in the presence of an ion transported by the channel fusion. Preferably, all subunits of the ion conducting channel protein fusion may be channel subunit fusions. As indicated above, the bioluminescent donor molecule and fluorescent acceptor molecule are selected so that the emission spectrum of the bioluminescent donor molecule overlaps with the absorbance spectrum of the acceptor molecule, making it possible the non-radiative energy transfer between the bioluminescent energy donor and the fluorescent acceptor through nonradiative dipole-dipole coupling. The novel channel protein fusions may thus be used as novel BRET probes for detecting activation, inhibition or modulation of ion conducting channels by measuring the actual passages of ions through said channels. 
     The present invention also relates to a nucleotide sequence encoding a channel fusion subunit comprising a subunit of an ion conducting channel bound to a probe which itself probe comprises an ion sensor bound in between to at least a bioluminescent donor molecule and to at least a protein fluorescent acceptor molecule. As described above, the ion sensor is configured to undergo a conformational change in the presence of an ion transported by the channel subunit, and the probe is bound to the N-terminal or the C-terminal, or within an intracellular or extracellular loop of said channel subunit via either the bioluminescent donor molecule or the protein fluorescent acceptor molecule. The nucleotide sequence or construct according to the present invention may optionally further comprise a linker sequence between the nucleotide sequence encoding the ion conducting channel subunit and the nucleotide sequence encoding the probe. 
     All the known ion conducting channels are within the scope of the present invention as most ion-channels have such common three-dimensional structure. Typically, these ion conducting channels at least have three functional domains: a first domain corresponding to the ion conducting pore, i.e., a selectivity filter that distinguishes among the ions that do go through and the ions that do not; a second domain corresponding to the gate, i.e., a part of the channel that can open and close the conducting pore; and a third domain corresponding to the detector of stimuli that responds to electrical potential changes or chemical signals. The detectors of stimuli thus typically couple to the channel gates to control the probability that they open or close the pores. 
     The channel is formed by subunits that each cross the plasma membrane at least twice. Various subunits are assembled together to form a channel. Whether the channel is formed by homomeric assembly of the same subunits or the heteromeric combination of an alpha subunit and other subunits heteromeric or homomeric subunits depends on the channel type. In the center of the assembled channel is the pore, a narrow opening that allows ions such as Na + , K + , Ca 2+ , and/or Cl −  to flow across the membrane. The pore is responsible for the permeability to the selected ion(s). 
     Ion conducting channel are thus made up of several subunits which are organized in the plasma membrane to create a pore through which cations or anions can move. Ion conducting channels may be classified by gating, i.e. what opens and closes the channels, and may include in particular voltage-dependent ion channels or ligand-dependent ion channel. Voltage-gated ion channels activate/inactivate depending on the voltage gradient across the plasma membrane, while ligand-gated ion channels activate/inactivate depending on binding of ligands to the channel. 
     Voltage-dependent ion channels are membrane proteins that conduct ions at high rates regulated by the voltage across the membrane. These channel proteins are thus selective for particular ions, such as for example, calcium, potassium, sodium, or chloride ions. Most studied and well-known are voltage-gated Na + , K + , and Ca 2+  channels. They generally share several common tetrameric structural configuration formed by four homologous or heterologous subunits with a central pore with highly conserved pore-lining residues for channels with similar charge selectivity; common gating strategy using a charged membrane voltage sensor; and auxiliary subunits that regulate channel protein trafficking and function. For example, channels are made up of four heterologous subunits include K +  channels, or channels that are made of four homologous domains include 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. Prominent among these are the voltage-gated Na + , K + , and Ca 2+  channels that underlie the action potentials and the rapid calcium signaling of electrically excitable cells. Fast voltage-dependent opening of Na +  channels accounts for all-or-nothing excitation initiated with a sharp threshold by depolarizing stimuli and for the regenerative spread of excitation as an action potential propagates along an axon or muscle fiber. 
     Some are at best weakly voltage dependent, although they retain voltage-sensor domains with a few positive charges. Included among these are the cyclic nucleotide gated (CNG) and the TRP families of non-selective cation channels. The CNG channels serve vertebrate phototransduction and olfaction. The TRP family includes diverse forms serving in phototransduction of invertebrates and in sensory receptors detecting hot, cold, chili peppers, mustard, ginger, and possibly touch, pressure, and motion. Another family of ion channels, the inwardly rectifying K +  channels, has no voltage-sensor domain but has a pore-forming domain with much sequence identity and ion selectivity identical to that of voltage-gated K +  channels. 
     Voltage-dependent ion channels are a proven target for drug discovery, and many ion channel modulators are currently in clinical use for the treatment of pain, epilepsy, hypertension and other disease states. They comprise the molecular basis for essential physiological functions including fluid secretion, electrolyte balance, and bioenergetics and membrane excitability. 
     Voltage-dependent ion channels are known to exist in several different conformational states, referred to as gating states. A voltage-gated ion channel can be viewed as residing in one of 3 gating states-closed (no ion permeation), open (ion flux occurs) and inactivated (no ion permeation; channel cannot be opened by depolarization), although it should be noted that some channels do not exhibit an inactivated state. Transition between gating states is voltage-dependent, and at any given time, equilibrium exists between these gating states, with the proportion of channels residing in each state depending upon the cellular membrane potential. Many voltage-dependent ion channel modulators have been shown to bind preferentially to a specific gating state or states. For example, the voltage-gated sodium channel blocker Lamotrigine is thought to bind to the opened and inactivated states of the brain sodium channel protein. Preferential binding to a particular gating state may occur through an increase in channel affinity for the ion channel modulator, or simply through improved access of the drug to its binding site on the channel. 
     A complete account of the voltage-gated ion channels that are within the scope of the present invention, are tabulated below. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Sodium channels, voltage-gated 
               
            
           
           
               
               
               
               
               
               
            
               
                 Approved 
                   
                 Tissue 
                   
                   
                 Accession numbers on 
               
               
                 Symbol 
                 Approved Name 
                 distribution 
                 Synonyms 
                 Chromosome 
                 uniprot.org website 
               
               
                   
               
               
                 SCN1A 
                 sodium channel, 
                 CNS 
                 Nav1.1, 
                 2q24.3 
                 P35498 
               
               
                   
                 voltage-gated, 
                   
                 GEFSP2, 
                   
                 https://www.uniprot.org/uniprot/P35498 
               
               
                   
                 type I, alpha 
                   
                 HBSCI, 
                   
                 E9PG49 Q9C008, X03638 
               
               
                   
                 subunit 
                   
                 NAC1, SMEI 
                   
                 X65362, AF003372 
               
               
                 SCN1B 
                 sodium channel, 
                 CNS-brain, 
                   
                 19q13.12 
                 Q07699 
               
               
                   
                 voltage-gated, 
                 skeletal muscle, 
                   
                   
                 https://www.uniprot.org/uniprot/Q07699 
               
               
                   
                 type I, beta 
                 and heart 
                   
                   
                 Q5TZZ4, Q6TN97, M91808, 
               
               
                   
                 subunit 
                   
                   
                   
                 L10338 
               
               
                 SCN2A 
                 sodium channel, 
                 CNS 
                 Nav1.2, 
                 2q24.3 
                 Q99250, 
               
               
                   
                 voltage-gated, 
                   
                 HBSCII, 
                   
                 https://www.uniprot.org/uniprot/Q99250 
               
               
                   
                 type II, alpha 
                   
                 HBSCI 
                   
                 A6NC14, A6NC14, X03639, 
               
               
                   
                 subunit 
                   
                   
                   
                 X65361 M94055, X61149 
               
               
                 SCN2B 
                 sodium channel, 
                 CNS 
                   
                 11q23.3 
                 O60939 
               
               
                   
                 voltage-gated, 
                   
                   
                   
                 https://www.uniprot.org/uniprot/O60939 
               
               
                   
                 type II, beta 
                   
                   
                   
                 O75302, Q9UNN3, U37026, 
               
               
                   
                 subunit 
                   
                   
                   
                 U37147, AF007783 
               
               
                 SCN3A 
                 sodium channel, 
                 CNS 
                 Nav1.3 
                 2q24 
                 Q9NY46 
               
               
                   
                 voltage-gated, 
                   
                   
                   
                 https://www.uniprot.org/uniprot/Q9NY46 
               
               
                   
                 type III, alpha 
                   
                   
                   
                 Q16142 Q9Y6P4, Y00766 
               
               
                   
                 subunit 
               
               
                 SCN3B 
                 sodium channel, 
                 Heart 
                 HSA243396 
                 11q24.1 
                 Q9NY72 
               
               
                   
                 voltage-gated, 
                   
                   
                   
                 https://www.uniprot.org/uniprot/Q9NY72 
               
               
                   
                 type III, beta 
               
               
                   
                 subunit 
               
               
                 SCN4A 
                 sodium channel, 
                 skeletal 
                 Nav1.4, 
                 17q23.3 
                 P35499 
               
               
                   
                 voltage-gated, 
                 muscle- 
                 HYPP, SkM1 
                   
                 https://www.uniprot.org/uniprot/P35499 
               
               
                   
                 type IV, alpha 
                 denervated and 
                   
                   
                 Q15478, Q16447, Q7Z6B1 
               
               
                   
                 subunit 
                 innervated 
                   
                   
                 M26643, M81758 
               
               
                   
                   
                 skeletal muscle 
               
               
                 SCN5A 
                 sodium channel, 
                 denervated 
                 Nav1.5, 
                 3p21 
                 Q14524 
               
               
                   
                 voltage-gated, 
                 skeletal 
                 LQT3, HB1, 
                   
                 https://www.uniprot.org/uniprot/Q14524 
               
               
                   
                 type V, alpha 
                 muscle, heart 
                 HBBD, 
                   
                 M27902, M77235, Q86V90 
               
               
                   
                 subunit 
                   
                 PFHB1, IVF, 
                   
                 A5H1P8, A5H1P8 
               
               
                   
                   
                   
                 HB2, HH1, 
               
               
                   
                   
                   
                 SSS1, 
               
               
                   
                   
                   
                 CDCD2, 
               
               
                   
                   
                   
                 CMPD2, 
               
               
                   
                   
                   
                 ICCD 
               
               
                 SCN7A 
                 sodium channel, 
                 astrocytes PNS 
                 Nav2.1, 
                 2q21-q23 
                 Q01118 
               
               
                   
                 voltage-gated, 
                 (DRG) 
                 Nav2.2, NaG 
                   
                 https://www.uniprot.org/uniprot/Q01118 
               
               
                   
                 type VII, alpha 
                   
                   
                   
                 M96578 Y09164 
               
               
                   
                 subunit 
               
               
                 SCN8A 
                 sodium channel, 
                 CNS 
                 Nav1.6, 
                 12q13.1 
                 Q9UQD0 
               
               
                   
                 voltage gated, 
                   
                 NaCh6, PN4, 
                   
                 https://www.uniprot.org/uniprot/Q9UQD0 
               
               
                   
                 type VIII, alpha 
                   
                 CerIII 
                   
                 L39018 AF049239A 
               
               
                   
                 subunit 
                   
                   
                   
                 F049240 U26707 
               
               
                   
                   
                   
                   
                   
                 AF049617 AF050736 
               
               
                   
                   
                   
                   
                   
                 AF003373, 
               
               
                   
                   
                   
                   
                   
                 Q9UPB2, B9VWG8 
               
               
                 SCN9A 
                 sodium channel, 
                 medullary 
                 Nav1.7, PN1, 
                 2q24 
                 Q15858 
               
               
                   
                 voltage-gated, 
                 thyroid Ca 
                 NE-NA, 
                   
                 https://www.uniprot.org/uniprot/Q15858 
               
               
                   
                 type IX, alpha 
                 Schwann 
                 NENA, ETHA 
                   
                 A1BUH5, Q8WWN4, U79568 
               
               
                   
                 subunit 
                 Cells 
                   
                   
                 X82835, U35238 
               
               
                   
                   
                 PNS 
               
               
                 SCN10A 
                 sodium channel, 
                 PNS 
                 Nav1.8, 
                 3p22.2 
                 Q9Y5Y9 
               
               
                   
                 voltage-gated, 
                   
                 hPN3, SNS, 
                   
                 https://www.uniprot.org/uniprot/Q9Y5Y9 
               
               
                   
                 type X, alpha 
                   
                 PN3 
                   
                 A6NDQ1, X92184, U53833 
               
               
                   
                 subunit 
                   
                   
                   
                 Y09108 
               
               
                 SCN11A 
                 sodium channel, 
                   
                 Nav1.9, NaN, 
                 3p22.2 
                 Q9UI33 
               
               
                   
                 voltage-gated, 
                   
                 SNS-2 
                   
                 https://www.uniprot.org/uniprot/Q9UI33 
               
               
                   
                 type XI, alpha 
                   
                   
                   
                 A6NN05, Q9UHM0 
               
               
                   
                 subunit 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Calcium channels, voltage-gated 
               
            
           
           
               
               
               
               
               
               
            
               
                 Approved 
                   
                   
                   
                   
                 Accession numbers on 
               
               
                 Symbol 
                 Approved Name 
                 Tissue distribution 
                 Synonyms 
                 Chromosome 
                 uniprot.org website 
               
               
                   
               
               
                 CACNA1A 
                 calcium channel, 
                 Brain specific; mainly 
                 Cav2.1, EA2, 
                 19p13 
                 O00555 
               
               
                   
                 voltage-dependent, 
                 found in cerebellum, 
                 APCA, 
                   
                 https://www.uniprot.org/uniprot/O00555 
               
               
                   
                 P/Q type, alpha 
                 cerebral cortex, 
                 HPCA, FHM 
                   
                 J3KP41, Q9UDC4 
               
               
                   
                 1A subunit 
                 thalamus and 
               
               
                   
                   
                 hypothalamus. 
               
               
                   
                   
                 Expressed in the small 
               
               
                   
                   
                 cell lung carcinoma 
               
               
                   
                   
                 cell line SCC-9 
               
               
                 CACNA1B 
                 calcium channel, 
                 Isoform Alpha-1b-1 
                 Cav2.2, 
                 9q34 
                 Q00975 
               
               
                   
                 voltage-dependent, 
                 and isoform Alpha-1b- 
                 CACNN 
                   
                 https://www.uniprot.org/uniprot/Q00975 
               
               
                   
                 N type,alpha 1B 
                 2 are expressed in the 
                   
                   
                 B1AQK5 
               
               
                   
                 subunit 
                 central nervous system 
               
               
                 CACNA1C 
                 calcium channel, 
                 Expressed in brain, 
                 Cav1.2, 
                 12p13.3 
                 Q13936 
               
               
                   
                 voltage-dependent, 
                 heart, jejunum, ovary, 
                 CACH2, 
                   
                 https://www.uniprot.org/uniprot/Q13936 
               
               
                   
                 L type, alpha 1C 
                 pancreatic beta-cells 
                 CACN2, TS, 
                   
                 B2RUT3, Q99875 
               
               
                   
                 subunit 
                 and vascular smooth 
                 LQT8 
               
               
                   
                   
                 muscle. Overall 
               
               
                   
                   
                 expression is reduced 
               
               
                   
                   
                 in atherosclerotic 
               
               
                   
                   
                 vascular smooth 
               
               
                   
                   
                 muscle 
               
               
                 CACNA1D 
                 calcium channel, 
                 Expressed in pancreatic 
                 Cav1.3, 
                 3p14.3 
                 Q01668 
               
               
                   
                 voltage-dependent, 
                 islets and in brain, 
                 CACH3, 
                   
                 https://www.uniprot.org/uniprot/Q01668 
               
               
                   
                 L type, alpha 1D 
                 where it has been seen 
                 CACN4 
                   
                 B0FYA3, Q9UDC3 
               
               
                   
                 subunit 
                 in cerebral cortex, 
               
               
                   
                   
                 hippocampus, basal 
               
               
                   
                   
                 ganglia, habenula and 
               
               
                   
                   
                 thalamus. Expressed in 
               
               
                   
                   
                 the small cell lung 
               
               
                   
                   
                 carcinoma cell line 
               
               
                   
                   
                 SCC-9. 
               
               
                 CACNA1E 
                 calcium channel, 
                 Expressed in neuronal 
                 Cav2.3, BII, 
                 1q25.3 
                 Q15878 
               
               
                   
                 voltage-dependent, 
                 tissues and in kidney 
                 CACH6 
                   
                 https://www.uniprot.org/uniprot/Q15878 
               
               
                   
                 R type, alpha 1E 
                   
                   
                   
                 Q14581, B1AM12 
               
               
                   
                 subunit 
               
               
                 CACNA1F 
                 calcium channel, 
                 Expression in skeletal 
                 Cav1.4, JM8, 
                 Xp11.23 
                 O60840 
               
               
                   
                 voltage-dependent, 
                 muscle and retina 
                 JMC8, 
                   
                 https://www.uniprot.org/uniprot/Q60840 
               
               
                   
                 L type, alpha 1F 
                   
                 CSNBX2, 
                   
                 A6NI29, Q9UHB1 
               
               
                   
                 subunit 
                   
                 CORDX3, 
               
               
                   
                   
                   
                 CSNB2A, 
               
               
                   
                   
                   
                 OA2 
               
               
                 CACNA1G 
                 calcium channel, 
                 Highly expressed in 
                 Cav3.1, 
                 17q22 
                 O43497 
               
               
                   
                 voltage-dependent, 
                 brain, in particular in 
                 NBR13 
                   
                 https://www.uniprot.org/uniprot/O43497 
               
               
                   
                 T type, alpha 1G 
                 the amygdala, 
                   
                   
                 D6RA64, Q9Y5T3 
               
               
                   
                 subunit 
                 subthalamic nuclei, 
               
               
                   
                   
                 cerebellum and 
               
               
                   
                   
                 thalamus. Moderate 
               
               
                   
                   
                 expression in heart; 
               
               
                   
                   
                 low expression in 
               
               
                   
                   
                 placenta, kidney and 
               
               
                   
                   
                 lung. Also expressed in 
               
               
                   
                   
                 colon and bone marrow 
               
               
                   
                   
                 and in tumoral cells to 
               
               
                   
                   
                 a lesser extent. Highly 
               
               
                   
                   
                 expressed in fetal brain, 
               
               
                   
                   
                 but also in peripheral 
               
               
                   
                   
                 fetal tissues as heart, 
               
               
                   
                   
                 kidney and lung, 
               
               
                   
                   
                 suggesting a 
               
               
                   
                   
                 developmentally 
               
               
                   
                   
                 regulated expression 
               
               
                 CACNA1H 
                 calcium channel, 
                 Expressed in kidney, 
                 Cav3.2 
                 16p13.3 
                 O95180 
               
               
                   
                 voltage-dependent, 
                 liver, heart, brain. 
                   
                   
                 https://www.uniprot.org/uniprot/O95180 
               
               
                   
                 T type,alpha 1H 
                 Isoform 
                 2 seems to be 
                   
                 B5ME00, Q9NYY5 
               
               
                   
                 subunit 
                 testis-specific 
               
               
                 CACNA1I 
                 calcium channel, 
                 Brain specific 
                 Cav3.3 
                 22q13.1 
                 Q9P0X4 
               
               
                   
                 voltage-dependent, 
                   
                   
                   
                 https://www.uniprot.org/uniprot/Q9P0X4 
               
               
                   
                 T type, alpha 1I 
                   
                   
                   
                 B0QY12, Q9UNE6 
               
               
                   
                 subunit 
               
               
                 CACNA1S 
                 calcium channel, 
                 Skeletal muscle 
                 Cav1.1, 
                 1q32 
                 Q13698 
               
               
                   
                 voltage-dependent, 
                 specific. 
                 hypoPP 
                   
                 https://www.uniprot.org/uniprot/Q13698 
               
               
                   
                 L type, alpha 1S 
                   
                   
                   
                 A4IF51, Q13934 
               
               
                   
                 subunit 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Transient receptor potential cation channels 
               
            
           
           
               
               
               
               
               
               
            
               
                 Approved 
                   
                   
                   
                   
                 Accession numbers on 
               
               
                 Symbol 
                 Approved Name 
                 Tissue distribution 
                 Synonyms 
                 Chromosome 
                 uniprot.org website 
               
               
                   
               
               
                 TRPA1 
                 transient receptor 
                 Expressed at very low 
                   
                 8q13 
                 O75762 
               
               
                   
                 potential cation 
                 level in human 
                   
                   
                 https://www.uniprot.org/uniprot/O75762 
               
               
                   
                 channel, subfamily 
                 fibroblasts and at a 
                   
                   
                 A6NIN6 
               
               
                   
                 A,member1 
                 moderate level in 
               
               
                   
                   
                 liposarcoma cells 
               
               
                 TRPC1 
                 transient receptor 
                 Seems to be ubiquitous 
                 HTRP-1 
                 3q23 
                 P48995 
               
               
                   
                 potential cation 
                   
                   
                   
                 https://www.uniprot.org/uniprot/P48995 
               
               
                   
                 channel, subfamily 
                   
                   
                   
                 Q14CE4 
               
               
                   
                 C,member1 
               
               
                 TRPC2 
                 transient receptor 
                   
                   
                 11p15.4 
                 Q6ZNB5 
               
               
                   
                 potential cation 
               
               
                   
                 channel, subfamily 
               
               
                   
                 C,member2, 
               
               
                   
                 pseudogene 
               
               
                 TRPC3 
                 transient receptor 
                 Expressed 
                   
                 4q27 
                 Q13507, A7VJS1, Q5G1L5 
               
               
                   
                 potential cation 
                 predominantly in brain 
               
               
                   
                 channel, subfamily 
                 and at much lower 
               
               
                   
                 C, member 3 
                 levels in ovary, colon, 
               
               
                   
                   
                 small intestine, lung, 
               
               
                   
                   
                 prostate, placenta and 
               
               
                   
                   
                 testis 
               
               
                 TRPC4 
                 transient receptor 
                 Strongly expressed in 
                 HTRP4 
                 13q13.3 
                 Q9UBN4, B1ALE0, Q9UIB2 
               
               
                   
                 potential cation 
                 placenta. Expressed at 
                 TRP4, 
               
               
                   
                 channel, subfamily 
                 lower levels in heart, 
               
               
                   
                 C,member4 
                 pancreas, kidney and 
               
               
                   
                   
                 brain. Expressed in 
               
               
                   
                   
                 endothelial cells. 
               
               
                   
                   
                 Isoform alpha was 
               
               
                   
                   
                 found to be the 
               
               
                   
                   
                 predominant isoform. 
               
               
                 TRPC5 
                 transient receptor 
                 Expressed in brain 
                 PPP1R159 
                 Xq23 
                 Q9UL62, B2RP53 
               
               
                   
                 potential cation 
                 with higher levels in 
                   
                   
                 Q9Y514 
               
               
                   
                 channel, 
                 fetal brain. Found 
               
               
                   
                 subfamily C, 
                 in cerebellum and 
               
               
                   
                 member 5 
                 occipital pole 
               
               
                 TRPC6 
                 transient receptor 
                 Expressed primarily in 
                 TRP6 
                 11q22.1 
                 Q9Y210 Q52M59 Q9NQA9 
               
               
                   
                 potential cation 
                 placenta, lung, spleen, 
               
               
                   
                 channel, subfamily 
                 ovary and small 
               
               
                   
                 C,member6 
                 intestine. Expressed in 
               
               
                   
                   
                 podocytes and is a 
               
               
                   
                   
                 component of the 
               
               
                   
                   
                 glomerular slit 
               
               
                   
                   
                 diaphragm 
               
               
                 TRPC7 
                 transient receptor 
                 Highly expressed in 
                   
                 5q31.2 
                 O94759 D3DSL6 
               
               
                   
                 potential cation 
                 brain and peripheral 
                   
                   
                 Q96Q93_Q9HCX4 A1A4Z4 
               
               
                   
                 channel, subfamily 
                 blood cells, such as 
                   
                   
                 Q8IWP7 
               
               
                   
                 C,member7 
                 neutrophils. Also 
               
               
                   
                   
                 detected in bone 
               
               
                   
                   
                 marrow, spleen, heart, 
               
               
                   
                   
                 liver and lung.Isoform 
               
               
                   
                   
                 2 is found in neutrophil 
               
               
                   
                   
                 granulocytes 
               
               
                 TRPM1 
                 transient receptor 
                 Expressed in the retina 
                 LTRPC1, 
                 15q13.3 
                 Q7Z4N2 D9IDV2 Q7Z4N5 
               
               
                   
                 potential cation 
                 where it localizes to 
                 CSNB1C 
               
               
                   
                 channel, 
                 the outer plexiform 
               
               
                   
                 subfamily 
                 layer. Highly 
               
               
                   
                 M,member1 
                 expressed in 
               
               
                   
                   
                 benign melanocytic 
               
               
                   
                   
                 nevi and diffusely 
               
               
                   
                   
                 expressed in various in 
               
               
                   
                   
                 situ melanomas, but not 
               
               
                   
                   
                 detected in melanoma 
               
               
                   
                   
                 metastases. Also 
               
               
                   
                   
                 expressed in 
               
               
                   
                   
                 melanocytes and 
               
               
                   
                   
                 pigmented metastatic 
               
               
                   
                   
                 melanoma cell lines. In 
               
               
                   
                   
                 melanocytes expression 
               
               
                   
                   
                 appears to be regulated 
               
               
                   
                   
                 at the level of 
               
               
                   
                   
                 transcription and 
               
               
                   
                   
                 mRNA processing 
               
               
                 TRPM2 
                 transient receptor 
                 Detected in blood 
                 KNP3, 
                 21q22.3 
                 P10909 B2R9Q1 Q7Z5B9 
               
               
                   
                 potential cation 
                 plasma, cerebrospinal 
                 LTRPC2, 
               
               
                   
                 channel, subfamily 
                 fluid, milk, seminal 
                 NUDT9L1, 
               
               
                   
                 M,member2 
                 plasma and colon 
                 NUDT9H, 
               
               
                   
                   
                 mucosa. Detected in 
                 EREG1 
               
               
                   
                   
                 the germinal center of 
               
               
                   
                   
                 colon lymphoid 
               
               
                   
                   
                 nodules and in colon 
               
               
                   
                   
                 parasympathetic 
               
               
                   
                   
                 ganglia of the 
               
               
                   
                   
                 Auerbach plexus (at 
               
               
                   
                   
                 protein level). 
               
               
                   
                   
                 Ubiquitous. Detected in 
               
               
                   
                   
                 brain, testis, ovary, 
               
               
                   
                   
                 liver and pancreas, and 
               
               
                   
                   
                 at lower levels in 
               
               
                   
                   
                 kidney, heart, spleen 
               
               
                   
                   
                 and lung 
               
               
                 TRPM3 
                 transient receptor 
                   
                 KIAA1616, 
                 9q21.11 
                 Q504Y1 
               
               
                   
                 potential cation 
                   
                 LTRPC3, 
               
               
                   
                 channel, subfamily 
                   
                 GON-2 
               
               
                   
                 M, member 3 
               
               
                 TRPM4 
                 transient receptor 
                 Widely expressed with 
                 FLJ20041 
                 19q13.3 
                 Q8TD43 A2RU25 Q9NXV1 
               
               
                   
                 potential cation, 
                 a high expression in 
               
               
                   
                 channel, subfamily 
                 intestine and prostate. 
               
               
                   
                 M,member4 
                 In brain, it is both 
               
               
                   
                   
                 expressed in whole 
               
               
                   
                   
                 cerebral arteries and 
               
               
                   
                   
                 isolated vascular 
               
               
                   
                   
                 smooth muscle cells. 
               
               
                   
                   
                 Prominently expressed 
               
               
                   
                   
                 in Purkinje fibers. 
               
               
                   
                   
                 Expressed at higher 
               
               
                   
                   
                 levels in T-helper 2 
               
               
                   
                   
                 (Th2) cells as 
               
               
                   
                   
                 compared to T-helper 1 
               
               
                   
                   
                 (Th1) cells 
               
               
                 TRPM5 
                 transient receptor 
                 Strongly expressed in 
                 LTRPC5, 
                 11p15.5 
                 Q9NZQ8 A6NHS0, 
               
               
                   
                 potential cation 
                 fetal brain, liver and 
                 MTR1 
                   
                 Q52LU2, Q9NY34 
               
               
                   
                 channel, subfamily 
                 kidney, and in adult 
               
               
                   
                 M, member 5 
                 prostate, testis, ovary, 
               
               
                   
                   
                 colon and peripheral 
               
               
                   
                   
                 blood leukocytes. Also 
               
               
                   
                   
                 expressed in a large 
               
               
                   
                   
                 proportion of Wilms&#39; 
               
               
                   
                   
                 tumors and 
               
               
                   
                   
                 rhabdomyosarcomas. 
               
               
                   
                   
                 In monochromosomal 
               
               
                   
                   
                 cell lines shows 
               
               
                   
                   
                 exclusive paternal 
               
               
                   
                   
                 expression 
               
               
                 TRPM6 
                 transient receptor 
                 Highly expressed in 
                 CHAK2, 
                 9q21.13 
                 Q9BX84 Q6VPR8 Q6VPS2 
               
               
                   
                 potential cation 
                 kidney and colon. 
                 FLJ22628 
               
               
                   
                 channel, subfamily 
                 Isoform TRPM6a and 
               
               
                   
                 M,member6 
                 isoform TRPM6b, are 
               
               
                   
                   
                 coexpressed with 
               
               
                   
                   
                 TRPM7 in kidney, and 
               
               
                   
                   
                 testis, and are also 
               
               
                   
                   
                 found in several cell 
               
               
                   
                   
                 lines of lung origin. 
               
               
                   
                   
                 Isoform TRPM6c is 
               
               
                   
                   
                 detected only in testis 
               
               
                   
                   
                 and in NCI-H510A 
               
               
                   
                   
                 small cell lung 
               
               
                   
                   
                 carcinoma cells. 
               
               
                 TRPM7 
                 transient receptor 
                   
                 CHAK1, 
                 15q21 
                 Q96QT4 Q6ZMF5 Q9NXQ2 
               
               
                   
                 potential cation 
                   
                 LTRPC7, 
               
               
                   
                 channel, subfamily 
                   
                 TRP-PLIK 
               
               
                   
                 M,member7 
               
               
                 TRPM8 
                 transient receptor 
                 Expressed in prostate. 
                   
                 2q37 
                 Q7Z2W7, A0AVG2, 
               
               
                   
                 potential cation 
                 Also expressed in 
                   
                   
                 Q9BVK1 
               
               
                   
                 channel, subfamily 
                 prostate tumors and in 
               
               
                   
                 M,member8 
                 non-prostatic primary 
               
               
                   
                   
                 tumors such as colon, 
               
               
                   
                   
                 lung, breast and skin 
               
               
                   
                   
                 tumors 
               
               
                 MCOLN1 
                 mucolipin 1 
                 Widely expressed in 
                 TRPML1, 
                 19p13.2 
                 Q9GZU1 D6W647 Q9H4B5 
               
               
                   
                   
                 adult and fetal tissues 
                 ML4, MLIV, 
               
               
                   
                   
                   
                 MST080, 
               
               
                   
                   
                   
                 MSTP080, 
               
               
                   
                   
                   
                 TRPM-L1 
               
               
                 MCOLN2 
                 mucolipin 2 
                   
                 TRPML2, 
                 1p22 
                 Q8IZK6 A6NI99 Q8N9R3 
               
               
                   
                   
                   
                 FLJ36691, 
               
               
                   
                   
                   
                 TRP-ML2 
               
               
                 MCOLN3 
                 mucolipin 3 
                   
                 TRPML3, 
                 1p22.3 
                 Q8TDD5 Q5T4H5, Q5T4H6, 
               
               
                   
                   
                   
                 FLJ11006, 
                   
                 Q9NV09 
               
               
                   
                   
                   
                 TRP-ML3 
               
               
                 PKD1 
                 polycystic kidney 
                   
                 PBP, Pc-1, 
                 16p13.3 
                 P98161Q15140 Q15141 
               
               
                   
                 disease 1 
                   
                 TRPP1 
               
               
                   
                 (autosomal 
               
               
                   
                 dominant) 
               
               
                 PKD2 
                 polycystic kidney 
                 Strongly expressed in 
                 PKD4, PC2, 
                 4q22.1 
                 Q13563O60441 Q2M1Q5 
               
               
                   
                 disease 2 
                 ovary, fetal and adult 
                 Pc-2, TRPP2 
               
               
                   
                 (autosomal 
                 kidney, testis, and 
               
               
                   
                 dominant) 
                 small intestine. Not 
               
               
                   
                   
                 detected in peripheral 
               
               
                   
                   
                 leukocytes 
               
               
                 PKD2L1 
                 polycystic kidney 
                 Expressed in adult 
                 PCL, TRPP3 
                 10q24.31 
                 Q9P0L9 O75972 Q9UPA2 
               
               
                   
                 disease 2-like 1 
                 heart, skeletal muscle, 
               
               
                   
                   
                 brain, spleen, testis, 
               
               
                   
                   
                 retina and liver. 
               
               
                 PKD2L2 
                 polycystic kidney 
                 Testis, Brain and 
                 TRPP5 
                 5q31 
                 Q9NZM6 A6NK98 Q9UNJ0 
               
               
                   
                 disease 2-like 2 
                 Kidney 
               
               
                 TRPV1 
                 transient receptor 
                 Widely expressed at 
                   
                 17p13.2 
                 Q8NER1 A2RUA9 Q9NY22 
               
               
                   
                 potential cation 
                 low levels. Expression 
               
               
                   
                 channel, subfamily 
                 is elevated in dorsal 
               
               
                   
                 V,member1 
                 root ganglia. In skin, 
               
               
                   
                   
                 expressed in cutaneous 
               
               
                   
                   
                 sensory nerve fibers, 
               
               
                   
                   
                 mast cells, epidermal 
               
               
                   
                   
                 keratinocytes, dermal 
               
               
                   
                   
                 blood vessels, the inner 
               
               
                   
                   
                 root sheet and the 
               
               
                   
                   
                 infundibulum of hair 
               
               
                   
                   
                 follicles, differentiated 
               
               
                   
                   
                 sebocytes, sweat gland 
               
               
                   
                   
                 ducts, and the secretory 
               
               
                   
                   
                 portion of eccrine 
               
               
                   
                   
                 sweat glands (at protein 
               
               
                   
                   
                 level). 
               
               
                 TRPV2 
                 transient receptor 
                   
                 VRL, VRL-1, 
                 17p11.2 
                 Q9Y5S1, A6NML2, 
               
               
                   
                 potential cation 
                   
                 VRL1 
                   
                 A8K0Z0, Q9Y670 
               
               
                   
                 channel, subfamily 
               
               
                   
                 V,member2 
               
               
                 TRPV3 
                 transient receptor 
                 Abundantly expressed 
                 VRL3 
                 17p13.3 
                 Q8NET8, Q8NDW7, 
               
               
                   
                 potential cation 
                 in CNS. Widely 
                   
                   
                 Q8NET9, Q8NFH2 
               
               
                   
                 channel, subfamily 
                 expressed at low levels. 
               
               
                   
                 V, member 3 
                 Detected in dorsal root 
               
               
                   
                   
                 ganglion (at protein 
               
               
                   
                   
                 level). Expressed in the 
               
               
                   
                   
                 keratinocyte layers of 
               
               
                   
                   
                 the outer root sheath 
               
               
                   
                   
                 and, to lesser extent, to 
               
               
                   
                   
                 the matrix of the hair 
               
               
                   
                   
                 follicles (at protein 
               
               
                   
                   
                 level). 
               
               
                 TRPV4 
                 transient receptor 
                 Found in the 
                 OTRPC4, 
                 12q24.1 
                 Q9HBA0, 
               
               
                   
                 potential cation 
                 synoviocytes from 
                 TRP12, 
                   
                 B7ZKQ6, Q9HBC0 
               
               
                   
                 channel, subfamily 
                 patients with (RA) and 
                 VROAC, 
               
               
                   
                 V,member4 
                 without (CTR) 
                 VRL-2, VR- 
               
               
                   
                   
                 rheumatoid arthritis (at 
                 OAC, 
               
               
                   
                   
                 protein level). 
                 CMT2C 
               
               
                 TRPV5 
                 transient receptor 
                 Expressed at high 
                 CaT2 
                 7q34 
                 Q9NQA5, A4D2H7, 
               
               
                   
                 potential cation 
                 levels in kidney, small 
                   
                   
                 Q96PM6 
               
               
                   
                 channel, subfamily 
                 intestine and pancreas, 
               
               
                   
                 V, member 5 
                 and at lower levels in 
               
               
                   
                   
                 testis, prostate, 
               
               
                   
                   
                 placenta, brain, colon 
               
               
                   
                   
                 and rectum 
               
               
                 TRPV6 
                 transient receptor 
                 Expressed at high 
                 CaT1 
                 7q34 
                 Q9H1D0, A4D2I8, Q9H296 
               
               
                   
                 potential cation 
                 levels in the 
               
               
                   
                 channel, subfamily 
                 gastrointestinal tract, 
               
               
                   
                 V,member6 
                 including esophagus, 
               
               
                   
                   
                 stomach, duodenum, 
               
               
                   
                   
                 jejunum, ileum and 
               
               
                   
                   
                 colon, and in pancreas, 
               
               
                   
                   
                 placenta, prostate and 
               
               
                   
                   
                 salivary gland. 
               
               
                   
                   
                 Expressed at moderate 
               
               
                   
                   
                 levels in liver, kidney 
               
               
                   
                   
                 and testis. Expressed in 
               
               
                   
                   
                 locally advanced 
               
               
                   
                   
                 prostate cancer, 
               
               
                   
                   
                 metastatic and 
               
               
                   
                   
                 androgen-insensitive 
               
               
                   
                   
                 prostatic lesions but not 
               
               
                   
                   
                 detected in healthy 
               
               
                   
                   
                 prostate tissue and 
               
               
                   
                   
                 benign prostatic 
               
               
                   
                   
                 hyperplasia 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 CatSper channels 
               
            
           
           
               
               
               
               
               
               
            
               
                 Approved 
                   
                   
                   
                   
                 Accession numbers on 
               
               
                 Symbol 
                 Approved Name 
                 Tissue distribution 
                 Synonyms 
                 Chromosome 
                 uniport.org website 
               
               
                   
               
               
                 CATSPER1 
                 cation channel, 
                 Testis-specific 
                 CATSPER 
                 11q12.1 
                 Q8NEC5 Q96P76 
               
               
                   
                 sperm associated 1 
               
               
                 CATSPER2 
                 cation channel, 
                 Testis-specific 
                   
                 15q15.3 
                 Q96P56, Q8NHT9, 
               
               
                   
                 sperm associated 2 
                   
                   
                   
                 Q96P54, Q96P55 
               
               
                 CATSPER3 
                 cation channel, 
                 Testis-specific 
                 CACRC 
                 5q31.2 
                 Q86XQ3, Q86XS6 
               
               
                   
                 sperm associated 3 
               
               
                 CATSPER4 
                 cation channel, 
                 Testis-specific 
                   
                 1p35.3 
                 Q7RTX7, A1A4W6, 
               
               
                   
                 sperm associated 4 
                   
                   
                   
                 Q5VY71 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Two-pore channels 
               
            
           
           
               
               
               
               
               
               
            
               
                 Approved 
                   
                   
                   
                   
                 Accession numbers on 
               
               
                 Symbol 
                 Approved Name 
                 Tissue distribution 
                 Synonyms 
                 Chromosome 
                 uniport.org website 
               
               
                   
               
               
                 TPCN1 
                 two pore segment 
                   
                 KIAA1169, 
                 12q24.21 
                 B7Z3R2_A5PKY2_H0YIK4 
               
               
                   
                 channel 1 
                   
                 FLJ20612, 
                   
                 F8VV93 
               
               
                   
                   
                   
                 TPC1 
               
               
                 TPCN2 
                 two pore segment 
                 Widely expressed. 
                 TPC2 
                 11q13.1 
                 Q8NHX9 Q9NT82_E7ETX0 
               
               
                   
                 channel 2 
                 Expressed at high level 
               
               
                   
                   
                 in liver and kidney 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Cyclic nucleotide-regulated channels 
               
            
           
           
               
               
               
               
               
               
            
               
                 Approved 
                   
                 Tissue 
                   
                   
                 Accession numbers on 
               
               
                 Symbol 
                 Approved Names 
                 Distribution 
                 Synonyms 
                 Chromosome 
                 uniprot.org website 
               
               
                   
               
               
                 CNGA1 
                 cyclic nucleotide gated 
                 Rod cells in the 
                 RCNC1, 
                 4p12 
                 P29973 A8K7K6 Q4W5E, 
               
               
                   
                 channel alpha 1 
                 retina 
                 RCNCa, 
                   
                 D6RCF1_D6R978_, 
               
               
                   
                   
                   
                 CNG1, RP49 
                   
                 A0A024R9X3_, Q14028, 
               
               
                   
                   
                   
                   
                   
                 H3BN09 Q9UMG2 
               
               
                 CNGA2 
                 cyclic nucleotide gated 
                   
                 CNG2, 
                 Xq27 
                 Q16280, A0AVD0_, Q8IV77 
               
               
                   
                 channel alpha 2 
                   
                 OCNC1, 
               
               
                   
                   
                   
                 OCNCa, 
               
               
                   
                   
                   
                 OCNCALPHA, 
               
               
                   
                   
                   
                 OCNCalpha, 
               
               
                   
                   
                   
                 FLJ46312 
               
               
                 CNGA3 
                 cyclic nucleotide gated 
                 Prominently 
                 CCNC1, 
                 2q11.2 
                 Q16281, Q4VAP7, 
               
               
                   
                 channel alpha 3 
                 expressed in 
                 CCNCa, 
                   
                 Q53RD2, Q9UP64 
               
               
                   
                   
                 retina 
                 CNG3 
               
               
                 CNGA4 
                 cyclic nucleotide gated 
                   
                 OCNC2, 
                 11p15.4 
                 Q8IV77 
               
               
                   
                 channel alpha 4 
                   
                 OCNCb, 
               
               
                   
                   
                   
                 CNG5 
               
               
                 CNGB1 
                 cyclic nucleotide gated 
                   
                 RCNC2, 
                 16q13 
                 Q16280, _Q16280 
               
               
                   
                 channel beta 1 
                   
                 RCNCb, 
               
               
                   
                   
                   
                 GARP, 
               
               
                   
                   
                   
                 GAR1, 
               
               
                   
                   
                   
                 CNGB1B, 
               
               
                   
                   
                   
                 RP45 
               
               
                 CNGB3 
                 cyclic nucleotide gated 
                 Expressed 
                   
                 8q21.3 
                 Q9NQW8 C9JA51, 
               
               
                   
                 channel beta 3 
                 specifically in the 
                   
                   
                 Q9NRE9, H0YAZ4 
               
               
                   
                   
                 retina 
                   
                   
                 B9EK43, _Q0QD47 
               
               
                 HCN1 
                 hyperpolarization 
                 Detected in brain, 
                 BCNG-1, 
                 5p12 
                 O60741, _Q86WJ6 
               
               
                   
                 activated cyclic 
                 in particular in 
                 HAC-2 
               
               
                   
                 nucleotide-gated 
                 amygdala and 
               
               
                   
                 potassium 
                 hippocampus, 
               
               
                   
                 channel 1 
                 while expression in 
               
               
                   
                   
                 caudate nucleus, 
               
               
                   
                   
                 corpus callosum, 
               
               
                   
                   
                 substantia nigra, 
               
               
                   
                   
                 subthalamic 
               
               
                   
                   
                 nucleus and 
               
               
                   
                   
                 thalamus is very 
               
               
                   
                   
                 low or not 
               
               
                   
                   
                 detectable. 
               
               
                   
                   
                 Detected at very 
               
               
                   
                   
                 low levels in 
               
               
                   
                   
                 muscle and 
               
               
                   
                   
                 pancreas 
               
               
                 HCN2 
                 hyperpolarization 
                 Highly expressed 
                 BCNG-2, 
                 19p13 
                 Q9UL51, O60742, 
               
               
                   
                 activated cyclic 
                 throughout the 
                 HAC-1 
                   
                 Q9UBS2 
               
               
                   
                 nucleotide-gated 
                 brain. Detected at 
               
               
                   
                 potassium channel 2 
                 low levels in heart. 
               
               
                 HCN3 
                 hyperpolarization 
                 Detected in brain 
                 KIAA1535 
                 1q21.2 
                 Q9P1Z3, D3DV90, 
               
               
                   
                 activated cyclic 
                   
                   
                   
                 Q9BWQ2, _Q2T9L6 
               
               
                   
                 nucleotide-gated 
                   
                   
                   
                 Q86WJ5 
               
               
                   
                 potassium channel 3 
               
               
                 HCN4 
                 hyperpolarization 
                 Highly expressed 
                   
                 15q24.1 
                 Q9Y3Q4 Q9UMQ7 
               
               
                   
                 activated cyclic 
                 in thalamus, testis 
               
               
                   
                 nucleotide-gated 
                 and in heart, both 
               
               
                   
                 potassium channel 4 
                 in ventricle and 
               
               
                   
                   
                 atrium. Detected at 
               
               
                   
                   
                 much lower levels 
               
               
                   
                   
                 in amygdala, 
               
               
                   
                   
                 substantia nigra, 
               
               
                   
                   
                 cerebellum and 
               
               
                   
                   
                 hippocampus 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Potassium channels, calcium-activated 
               
            
           
           
               
               
               
               
               
               
            
               
                 Approved 
                   
                 Tissue 
                   
                   
                 Accession numbers on 
               
               
                 Symbol 
                 Approved Name 
                 distribution 
                 Synonyms 
                 Chromosome 
                 uniport.org website 
               
               
                   
               
               
                 KCNMA1 
                 potassium large 
                 Widely expressed. 
                 KCa1.1, mSLO1 
                 10q22 
                 Q12791 F8WA96 
               
               
                   
                 conductance calcium- 
                 Except in 
                   
                   
                 Q9UQK6 
               
               
                   
                 activated channel, 
                 myocytes, it is 
               
               
                   
                 subfamily M, alpha 
                 almost ubiquitously 
               
               
                   
                 member 1 
                 expressed 
               
               
                 KCNN1 
                 potassium 
                   
                 KCa2.1, hSK1 
                 19p13.1 
                 Q92952 
               
               
                   
                 intermediate/small 
                   
                   
                   
                 Q5KR10, Q6DJU4 
               
               
                   
                 conductance calcium- 
               
               
                   
                 activated channel, 
               
               
                   
                 subfamily N, member 1 
               
               
                 KCNN2 
                 potassium 
                 Expressed in atrial 
                 KCa2.2, hSK2 
                 5q22.3 
                 Q9H2S1 A6NF94 
               
               
                   
                 intermediate/small 
                 myocytes (at 
                   
                   
                 Q6X2Y2 
               
               
                   
                 conductance calcium- 
                 protein level). 
               
               
                   
                 activated channel, 
                 Widely expressed 
               
               
                   
                 subfamily N, member 2 
               
               
                 KCNN3 
                 potassium 
                   
                 KCa2.3, hSK3, 
                 1q21.3 
                 Q9UGI6 B1ANX0 
               
               
                   
                 intermediate/small 
                   
                 SKCA3 
                   
                 Q8WXG7 
               
               
                   
                 conductance calcium- 
               
               
                   
                 activated channel, 
               
               
                   
                 subfamily N, member 3 
               
               
                 KCNN4 
                 potassium 
                 Widely expressed 
                 KCa3.1, hSK4, 
                 19q13.2 
                 O15554 Q53XR4 
               
               
                   
                 intermediate/small 
                 in non-excitable 
                 hKCa4, hIKCa1 
               
               
                   
                 conductance calcium- 
                 tissues 
               
               
                   
                 activated channel, 
               
               
                   
                 subfamily N, member 4 
               
               
                 KCNT1 
                 potassium channel, 
                 Highest expression 
                 KCa4.1, 
                 9q34.3 
                 Q5JUK3 B3KXF7, 
               
               
                   
                 subfamily T, member 1 
                 in liver, brain and 
                 KIAA1422 
                   
                 Q9P2C5 
               
               
                   
                   
                 spinal cord. Lowest 
               
               
                   
                   
                 expression in 
               
               
                   
                   
                 skeletal muscle 
               
               
                 KCNT2 
                 potassium channel, 
                   
                 KCa4.2, SLICK, 
                 1q31.3 
                 Q6UVM3, Q3SY59, 
               
               
                   
                 subfamily T, member 2 
                   
                 SLO2.1 
                   
                 Q5VTN1, Q6ZMT3 
               
               
                 KCNU1 
                 potassium channel, 
                 Testis-specific 
                 KCa5.1, Slo3, 
                 8p11.2 
                 A8MYU2 
               
               
                   
                 subfamily U, member 1 
                   
                 KCNMC1, 
               
               
                   
                   
                   
                 Kcnma3 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Potassium channels, voltage-gated 
               
            
           
           
               
               
               
               
               
               
            
               
                 Approved 
                   
                 Tissue 
                   
                   
                 Accession numbers on 
               
               
                 Symbol 
                 Approved Name 
                 distribution 
                 Synonyms 
                 Chromosome 
                 uniport.org website 
               
               
                   
               
               
                 KCNA1 
                 potassium voltage-gated 
                   
                 Kv1.1, RBK1, 
                 12p13 
                 Q09470 A6NM83, 
               
               
                   
                 channel, shaker-related 
                   
                 HUK1, MBK1 
                   
                 Q3MIQ9 
               
               
                   
                 subfamily, member 1 
               
               
                   
                 (episodic ataxia with 
               
               
                   
                 myokymia) 
               
               
                 KCNA2 
                 potassium voltage-gated 
                   
                 Kv1.2, HK4 
                 1p13 
                 P16389 
               
               
                   
                 channel, shaker-related 
                   
                   
                   
                 Q86XG6 
               
               
                   
                 subfamily, member 2 
               
               
                 KCNA3 
                 potassium voltage-gated 
                   
                 Kv1.3, MK3, 
                 1p13.3 
                 P22001 Q5VWN2 
               
               
                   
                 channel, shaker-related 
                   
                 HLK3, HPCN3 
               
               
                   
                 subfamily, member 3 
               
               
                 KCNA4 
                 potassium voltage-gated 
                   
                 Kv1.4, HK1, 
                 11p14 
                 P22459 
               
               
                   
                 channel, shaker-related 
                   
                 HPCN2 
               
               
                   
                 subfamily, member 4 
               
               
                 KCNA5 
                 potassium voltage-gated 
                 Pancreatic islets 
                 Kv1.5, HK2, 
                 12p13 
                 P22460 Q4KKT8 
               
               
                   
                 channel, shaker-related 
                 and insulinoma 
                 HPCN1 
                   
                 Q9UDA4 
               
               
                   
                 subfamily, member 5 
               
               
                 KCNA6 
                 potassium voltage-gated 
                   
                 Kv1.6, HBK2, 
                 12p13 
                 P17658 
               
               
                   
                 channel, shaker-related 
                   
                 PPP1R96 
               
               
                   
                 subfamily, member 6 
               
               
                 KCNA7 
                 potassium voltage-gated 
                 Highly expressed 
                 Kv1.7, HAK6 
                 19q13.3 
                 Q96RP8, A1KYX7, 
               
               
                   
                 channel, shaker-related 
                 in skeletal 
                   
                   
                 Q9BYS4 
               
               
                   
                 subfamily, member 7 
                 muscle, heart and 
               
               
                   
                   
                 kidney. 
               
               
                 KCNA10 
                 potassium voltage-gated 
                 Detected in 
                 Kv1.8 
                 1p13.1 
                 Q16322 
               
               
                   
                 channel, shaker-related 
                 kidney, in 
               
               
                   
                 subfamily, member 10 
                 proximal tubules, 
               
               
                   
                   
                 glomerular 
               
               
                   
                   
                 endothelium, in 
               
               
                   
                   
                 vascular 
               
               
                   
                   
                 endothelium and 
               
               
                   
                   
                 in smooth muscle 
               
               
                   
                   
                 cells 
               
               
                 KCNB1 
                 potassium voltage-gated 
                   
                 Kv2.1 
                 20q13.2 
                 Q14721 Q14193 
               
               
                   
                 channel, Shab-related 
               
               
                   
                 subfamily, member 1 
               
               
                 KCNB2 
                 potassium voltage-gated 
                   
                 Kv2.2 
                 8q13.2 
                 Q92953, Q7Z7D0, 
               
               
                   
                 channel, Shab-related 
                   
                   
                   
                 Q9BXD3 
               
               
                   
                 subfamily, member 2 
               
               
                 KCNC1 
                 potassium voltage-gated 
                   
                 Kv3.1 
                 11p15 
                 P48547, K4DI87 
               
               
                   
                 channel, Shaw-related 
               
               
                   
                 subfamily, member 1 
               
               
                 KCNC2 
                 potassium voltage-gated 
                   
                 Kv3.2 
                 12q14.1 
                 Q96PR1, B7Z231, 
               
               
                   
                 channel, Shaw-related 
                   
                   
                   
                 Q96PR0 
               
               
                   
                 subfamily, member 2 
               
               
                 KCNC3 
                 potassium voltage-gated 
                   
                 Kv3.3 
                 19q13.33 
                 Q14003 
               
               
                   
                 channel, Shaw-related 
               
               
                   
                 subfamily, member 3 
               
               
                 KCNC4 
                 potassium voltage-gated 
                   
                 Kv3.4, 
                 1p21 
                 Q03721, Q3MIM4, 
               
               
                   
                 channel, Shaw-related 
                   
                 HKSHIIIC 
                   
                 Q5TBI6 
               
               
                   
                 subfamily, member 4 
               
               
                 KCND1 
                 potassium voltage-gated 
                 Widely 
                 Kv4.1 
                 Xp11.23 
                 Q9NSA2 B2RCG0, 
               
               
                   
                 channel, Shal-related 
                 expressed. 
                   
                   
                 O75671 
               
               
                   
                 subfamily, member 1 
                 Highly expressed 
               
               
                   
                   
                 in brain, in 
               
               
                   
                   
                 particular in 
               
               
                   
                   
                 cerebellum and 
               
               
                   
                   
                 thalamus; 
               
               
                   
                   
                 detected at lower 
               
               
                   
                   
                 levels in the other 
               
               
                   
                   
                 parts of the brain 
               
               
                 KCND2 
                 potassium voltage-gated 
                 Highly expressed 
                 Kv4.2, RK5, 
                 7q31 
                 Q9NZV8, O95012 
               
               
                   
                 channel, Shal-related 
                 throughout the 
                 KIAA1044 
                   
                 Q9UNH9 
               
               
                   
                 subfamily, member 2 
                 brain. Expression 
               
               
                   
                   
                 is very low or 
               
               
                   
                   
                 absent in other 
               
               
                   
                   
                 tissues. 
               
               
                 KCND3 
                 potassium voltage-gated 
                 Highly expressed 
                 Kv4.3, KSHIVB 
                 1p13.2 
                 Q9UK17, O60576, 
               
               
                   
                 channel, Shal-related 
                 in heart and 
                   
                   
                 Q9UK16 
               
               
                   
                 subfamily, member 3 
                 brain, in 
               
               
                   
                   
                 particular in 
               
               
                   
                   
                 cortex, 
               
               
                   
                   
                 cerebellum, 
               
               
                   
                   
                 amygdala and 
               
               
                   
                   
                 caudate nucleus. 
               
               
                   
                   
                 Detected at lower 
               
               
                   
                   
                 levels in liver, 
               
               
                   
                   
                 skeletal muscle, 
               
               
                   
                   
                 kidney and 
               
               
                   
                   
                 pancreas. Isoform 
               
               
                   
                   
                 1 predominates in 
               
               
                   
                   
                 most tissues. 
               
               
                   
                   
                 Isoform 1 and 
               
               
                   
                   
                 isoform 2 are 
               
               
                   
                   
                 detected at 
               
               
                   
                   
                 similar levels in 
               
               
                   
                   
                 brain, skeletal 
               
               
                   
                   
                 muscle and 
               
               
                   
                   
                 pancreas 
               
               
                 KCNF1 
                 potassium voltage-gated 
                 Detected in heart, 
                 Kv5.1, kH1, IK8 
                 2p25 
                 Q9H3M0, O43527, 
               
               
                   
                 channel, subfamily F, 
                 brain, liver, 
                   
                   
                 Q585L3 
               
               
                   
                 member 1 
                 skeletal muscle, 
               
               
                   
                   
                 kidney and 
               
               
                   
                   
                 pancreas. 
               
               
                 KCNG1 
                 potassium voltage-gated 
                 Detected in brain 
                 Kv6.1, kH2, K13 
                 20q13 
                 Q9UIX4, A8K3S4, 
               
               
                   
                 channel, subfamily G, 
                 and placenta, and 
                   
                   
                 Q9BRC1 
               
               
                   
                 member 1 
                 at much lower 
               
               
                   
                   
                 levels in kidney 
               
               
                   
                   
                 and pancreas 
               
               
                 KCNG2 
                 potassium voltage-gated 
                 Highly expressed 
                 Kv6.2, KCNF2 
                 18q23 
                 Q9UJ96 
               
               
                   
                 channel, subfamily G, 
                 in heart, liver, 
               
               
                   
                 member 2 
                 skeletal muscle, 
               
               
                   
                   
                 kidney and 
               
               
                   
                   
                 pancreas. 
               
               
                   
                   
                 Detected at low 
               
               
                   
                   
                 levels in brain, 
               
               
                   
                   
                 lung and placenta 
               
               
                 KCNG3 
                 potassium voltage-gated 
                 Detected in many 
                 Kv6.3 
                 2p21 
                 Q8TAE7, Q53SC1 
               
               
                   
                 channel, subfamily G, 
                 parts of the brain 
               
               
                   
                 member 3 
                 with the 
               
               
                   
                   
                 exception of the 
               
               
                   
                   
                 cerebellum, in 
               
               
                   
                   
                 testis, pancreas, 
               
               
                   
                   
                 lung, kidney, 
               
               
                   
                   
                 ovary, small 
               
               
                   
                   
                 intestine, colon, 
               
               
                   
                   
                 thymus, adrenal 
               
               
                   
                   
                 gland and spinal 
               
               
                   
                   
                 cord 
               
               
                 KCNG4 
                 potassium voltage-gated 
                 Highly expressed 
                 Kv6.4 
                 16q24.1 
                 Q8TDN1, Q96H24 
               
               
                   
                 channel, subfamily G, 
                 in brain, and at 
               
               
                   
                 member 4 
                 lower levels in 
               
               
                   
                   
                 liver, small 
               
               
                   
                   
                 intestine and 
               
               
                   
                   
                 colon. 
               
               
                 KCNH1 
                 potassium voltage-gated 
                 Highly expressed 
                 Kv10.1, eag, h- 
                 1q32.2 
                 O95259 B1AQ26, 
               
               
                   
                 channel, subfamily H 
                 in brain and in 
                 eag, eag1 
                   
                 O76035, Q14CL3 
               
               
                   
                 (eag-related), member 1 
                 myoblasts at the 
               
               
                   
                   
                 onset of fusion, 
               
               
                   
                   
                 but not in other 
               
               
                   
                   
                 tissues. Detected 
               
               
                   
                   
                 in HeLa (cervical 
               
               
                   
                   
                 carcinoma), SH- 
               
               
                   
                   
                 SY5Y 
               
               
                   
                   
                 (neuroblastoma) 
               
               
                   
                   
                 and MCF-7 
               
               
                   
                   
                 (epithelial tumor) 
               
               
                   
                   
                 cells, but not in 
               
               
                   
                   
                 normal epithelial 
               
               
                   
                   
                 cells. 
               
               
                 KCNH2 
                 potassium voltage-gated 
                 Highly expressed 
                 Kv11.1, HERG, 
                 7q36.1 
                 Q12809 A5H1P7 Q9H3P0 
               
               
                   
                 channel, subfamily H 
                 in heart and 
                 erg1 
               
               
                   
                 (eag-related), member 2 
                 brain. Isoforms 
               
               
                   
                   
                 USO are 
               
               
                   
                   
                 frequently 
               
               
                   
                   
                 overexpressed in 
               
               
                   
                   
                 cancer cells 
               
               
                 KCNH3 
                 potassium voltage-gated 
                 Detected only in 
                 Kv12.2, BEC1, 
                 12q13 
                 Q9ULD8 
               
               
                   
                 channel, subfamily H 
                 brain, in 
                 elk2 
                   
                 Q9UQ06 
               
               
                   
                 (eag-related), member 3 
                 particular in the 
               
               
                   
                   
                 telencephalon. 
               
               
                   
                   
                 Detected in the 
               
               
                   
                   
                 cerebral cortex, 
               
               
                   
                   
                 occipital pole, 
               
               
                   
                   
                 frontal and 
               
               
                   
                   
                 temporal lobe, 
               
               
                   
                   
                 putamen, 
               
               
                   
                   
                 amygdala, 
               
               
                   
                   
                 hippocampus and 
               
               
                   
                   
                 caudate nucleus 
               
               
                 KCNH4 
                 potassium voltage-gated 
                 Detected only in 
                 Kv12.3, elk1 
                 17q21 
                 Q9UQ05 
               
               
                   
                 channel, subfamily H 
                 brain, in 
               
               
                   
                 (eag-related), member 4 
                 particular in the 
               
               
                   
                   
                 telencephalon. 
               
               
                   
                   
                 Detected in 
               
               
                   
                   
                 putamen and 
               
               
                   
                   
                 caudate nucleus, 
               
               
                   
                   
                 and at lower 
               
               
                   
                   
                 levels in cerebral 
               
               
                   
                   
                 cortex, occipital 
               
               
                   
                   
                 and hippocampus 
               
               
                 KCNH5 
                 potassium voltage-gated 
                 Detected in brain, 
                 Kv10.2, H- 
                 14q23.1 
                 Q8NCM2, C9JP98 
               
               
                   
                 channel, subfamily H 
                 skeletal muscle, 
                 EAG2, eag2 
               
               
                   
                 (eag-related), member 5 
                 heart, placenta, 
               
               
                   
                   
                 lung and liver, 
               
               
                   
                   
                 and at low levels 
               
               
                   
                   
                 in kidney 
               
               
                 KCNH6 
                 potassium voltage-gated 
                 Expressed in 
                 Kv11.2, erg2, 
                 17q23.3 
                 Q9H252, Q9BRD7 
               
               
                   
                 channel, subfamily H 
                 prolactin- 
                 HERG2 
               
               
                   
                 (eag-related), member 6 
                 secreting 
               
               
                   
                   
                 adenomas 
               
               
                 KCNH7 
                 potassium voltage-gated 
                 Expressed in 
                 Kv11.3, HERG3, 
                 2q24.3 
                 Q9NS40, Q53QU4, 
               
               
                   
                 channel, subfamily H 
                 prolactin- 
                 erg3 
                   
                 Q8IV15 
               
               
                   
                 (eag-related), member 7 
                 secreting 
               
               
                   
                   
                 adenomas 
               
               
                 KCNH8 
                 potassium voltage-gated 
                 Nervous system 
                 Kv12.1, elk3 
                 3p24.3 
                 Q96L42, Q59GQ6 
               
               
                   
                 channel, subfamily H 
               
               
                   
                 (eag-related), member 8 
               
               
                 KCNQ1 
                 potassium voltage-gated 
                 Abundantly 
                 Kv7.1, KCNA8, 
                 11p15.5 
                 P51787, O00347 
               
               
                   
                 channel, KQT-like 
                 expressed in 
                 KVLQT1, 
                   
                 Q9UMN9 
               
               
                   
                 subfamily, member 1 
                 heart, pancreas, 
                 JLNS1, LQT1 
               
               
                   
                   
                 prostate, kidney, 
               
               
                   
                   
                 small intestine 
               
               
                   
                   
                 and peripheral 
               
               
                   
                   
                 blood leukocytes. 
               
               
                   
                   
                 Less abundant in 
               
               
                   
                   
                 placenta, lung, 
               
               
                   
                   
                 spleen, colon, 
               
               
                   
                   
                 thymus, testis and 
               
               
                   
                   
                 ovaries. 
               
               
                 KCNQ2 
                 potassium voltage-gated 
                 In adult and fetal 
                 Kv7.2, ENB1, 
                 20q13.33 
                 O43526, O43796, 
               
               
                   
                 channel, KQT-like 
                 brain. Highly 
                 BFNC, KCNA11, 
                   
                 Q99454 
               
               
                   
                 subfamily, member 2 
                 expressed in 
                 HNSPC 
               
               
                   
                   
                 areas containing 
               
               
                   
                   
                 neuronal cell 
               
               
                   
                   
                 bodies, low in 
               
               
                   
                   
                 spinal chord and 
               
               
                   
                   
                 corpus callosum. 
               
               
                   
                   
                 Isoform2 is 
               
               
                   
                   
                 preferentially 
               
               
                   
                   
                 expressed in 
               
               
                   
                   
                 differentiated 
               
               
                   
                   
                 neurons. Isoform 
               
               
                   
                   
                 6 is prominent in 
               
               
                   
                   
                 fetal brain, 
               
               
                   
                   
                 undifferentiated 
               
               
                   
                   
                 neuroblastoma 
               
               
                   
                   
                 cells and brain 
               
               
                   
                   
                 tumors 
               
               
                 KCNQ3 
                 potassium voltage-gated 
                 Brain 
                 Kv7.3 
                 8q24 
                 O43525, A2VCT8, 
               
               
                   
                 channel, KQT-like 
                   
                   
                   
                 B4DJY4, E7EQ89 
               
               
                   
                 subfamily, member 3 
               
               
                 KCNQ4 
                 potassium voltage-gated 
                 Expressed in the 
                 Kv7.4 
                 1p34 
                 P56696, O96025 
               
               
                   
                 channel, KQT-like 
                 outer, but not the 
               
               
                   
                 subfamily, member 4 
                 inner, sensory 
               
               
                   
                   
                 hair cells of the 
               
               
                   
                   
                 cochlea. Slightly 
               
               
                   
                   
                 expressed in 
               
               
                   
                   
                 heart, brain and 
               
               
                   
                   
                 skeletal muscle. 
               
               
                 KCNQ5 
                 potassium voltage-gated 
                 Strongly 
                 Kv7.5 
                 6q14 
                 Q9NR82, B4DS33. 
               
               
                   
                 channel, KQT-like 
                 expressed in 
                   
                   
                 Q9NYA6 
               
               
                   
                 subfamily, member 5 
                 brain and skeletal 
               
               
                   
                   
                 muscle. In brain, 
               
               
                   
                   
                 expressed in 
               
               
                   
                   
                 cerebral cortex, 
               
               
                   
                   
                 occipital pole, 
               
               
                   
                   
                 frontal lobe and 
               
               
                   
                   
                 temporal lobe. 
               
               
                   
                   
                 Lower levels in 
               
               
                   
                   
                 hippocampus and 
               
               
                   
                   
                 putamen. Low to 
               
               
                   
                   
                 undetectable 
               
               
                   
                   
                 levels in medulla, 
               
               
                   
                   
                 cerebellum and 
               
               
                   
                   
                 thalamus. 
               
               
                 KCNS1 
                 potassium voltage-gated 
                 Detected in all 
                 Kv9.1 
                 20q12 
                 A2RUL8, _Q96KK3, 
               
               
                   
                 channel, delayed- 
                 tissues tested 
                   
                   
                 A2RUL9, Q6DJU6, 
               
               
                   
                 rectifier, subfamily S, 
                 with the 
                   
                   
                 Q92953, Q7Z7D0, 
               
               
                   
                 member 1 
                 exception of 
                   
                   
                 Q9BXD3 
               
               
                   
                   
                 skeletal muscle. 
               
               
                   
                   
                 Highly expressed 
               
               
                   
                   
                 in adult and fetal 
               
               
                   
                   
                 brain, fetal 
               
               
                   
                   
                 kidney and lung, 
               
               
                   
                   
                 and adult prostate 
               
               
                   
                   
                 and testis. 
               
               
                 KCNS2 
                 potassium voltage-gated 
                   
                 Kv9.2 
                 8q22 
                 Q9ULS6, A8KAN1, 
               
               
                   
                 channel, delayed- 
                   
                   
                   
                 Q92953, Q7Z7D0, 
               
               
                   
                 rectifier, subfamily S, 
                   
                   
                   
                 Q9BXD3, _Q14721, 
               
               
                   
                 member 2 
                   
                   
                   
                 Q14193 
               
               
                 KCNS3 
                 potassium voltage-gated 
                 Detected in 
                 Kv9.3 
                 2p24 
                 Q9BQ31, D6W520 
               
               
                   
                 channel, delayed- 
                 whole normal 
                   
                   
                 Q96B56, _C9J187, 
               
               
                   
                 rectifier, subfamily S, 
                 term placental 
                   
                   
                 L8E8W4, _Q92953, 
               
               
                   
                 member 3 
                 and placental 
                   
                   
                 Q7Z7D0, Q9BXD3 
               
               
                   
                   
                 chorionic plate 
                   
                   
                 Q14721, Q14193 
               
               
                   
                   
                 arteries and 
               
               
                   
                   
                 veins. Detected in 
               
               
                   
                   
                 syncytiotrophoblast 
               
               
                   
                   
                 and in blood 
               
               
                   
                   
                 vessel 
               
               
                   
                   
                 endothelium 
               
               
                   
                   
                 within 
               
               
                   
                   
                 intermediate villi 
               
               
                   
                   
                 and chorionic 
               
               
                   
                   
                 plate (at protein 
               
               
                   
                   
                 level). Detected 
               
               
                   
                   
                 in most tissues, 
               
               
                   
                   
                 but not in 
               
               
                   
                   
                 peripheral blood 
               
               
                   
                   
                 lymphocytes. The 
               
               
                   
                   
                 highest levels of 
               
               
                   
                   
                 expression are in 
               
               
                   
                   
                 lung 
               
               
                 KCNV1 
                 potassium channel, 
                 Detected in brain 
                 Kv8.1 
                 8q23.2 
                 Q6PIU1, Q9UHJ4, 
               
               
                   
                 subfamily V, member 1 
                   
                   
                   
                 B2R8Q7, _B4DMC1 
               
               
                   
                   
                   
                   
                   
                 Q76FP2 
               
               
                 KCNV2 
                 potassium channel, 
                 Detected in heart, 
                 Kv8.2 
                 9p24.2 
                 Q8TDN2, Q5T6X0 
               
               
                   
                 subfamily V, member 2 
                 brain, liver, 
                   
                   
                 P48547, K4DI87, _Q14721, 
               
               
                   
                   
                 skeletal muscle, 
                   
                   
                 Q14193, _Q9H3M0 
               
               
                   
                   
                 spleen, thymus, 
                   
                   
                 O43527, Q585L3 
               
               
                   
                   
                 prostate, testis, 
               
               
                   
                   
                 ovary colon, 
               
               
                   
                   
                 kidney and 
               
               
                   
                   
                 pancreas 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Potassium channels, inwardly rectifying 
               
            
           
           
               
               
               
               
               
               
            
               
                 Approved 
                   
                   
                   
                   
                 Accession numbers on 
               
               
                 Symbol 
                 Approved Name 
                 Tissue distribution 
                 Synonyms 
                 Chromosome 
                 uniport.org website 
               
               
                   
               
               
                 KCNJ1 
                 potassium 
                 In the kidney and 
                 Kir1.1, 
                 11q24 
                 P48048, B2RMR4, 
               
               
                   
                 inwardly-rectifying 
                 pancreatic islets. Lower 
                 ROMK1 
                   
                 Q6LD67, A0A024R3K6 
               
               
                   
                 channel, subfamily 
                 levels in skeletal muscle, 
               
               
                   
                 J, member 1 
                 pancreas, spleen, brain, 
               
               
                   
                   
                 heart and liver 
               
               
                 KCNJ2 
                 potassium 
                 Heart, brain, placenta, lung, 
                 Kir2.1, IRK1, 
                 17q24.3 
                 P63252, O15110, 
               
               
                   
                 inwardly-rectifying 
                 skeletal muscle, and 
                 LQT7 
                   
                 P48049, Q9NPI9 
               
               
                   
                 channel, subfamily 
                 kidney. Diffusely 
               
               
                   
                 J, member 2 
                 distributed throughout the 
               
               
                   
                   
                 brain. 
               
               
                 KCNJ3 
                 potassium 
                   
                 Kir3.1, GIRK1, 
                 2q24.1 
                 P48549, B4DEW7, 
               
               
                   
                 inwardly-rectifying 
                   
                 KGA 
                   
                 Q8TBI0, D2XBF0, 
               
               
                   
                 channel, subfamily 
                   
                   
                   
                 D2X9V0 
               
               
                   
                 J, member 3 
               
               
                 KCNJ4 
                 potassium 
                 Heart, skeletal muscle, and 
                 Kir2.3, HIR, 
                 22q13.1 
                 P48050, Q14D44, 
               
               
                   
                 inwardly-rectifying 
                 several different brain 
                 HRK1, hIRK2, 
                   
                 A0A024R1L8, P63252, 
               
               
                   
                 channel, subfamily 
                 regions including the 
                 IRK3 
                   
                 O15110, P48049 
               
               
                   
                 J, member 4 
                 hippocampus 
               
               
                 KCNJ5 
                 potassium 
                 Islets, exocrine pancreas 
                 Kir3.4, CIR, 
                 11q24 
                 Q4QRJ2, _P48544, 
               
               
                   
                 inwardly-rectifying 
                 and heart. Expressed in the 
                 KATP1, 
                   
                 B2R744 Q92807, 
               
               
                   
                 channel, subfamily 
                 adrenal cortex, particularly 
                 GIRK4, LQT13 
                   
                 H9A8K9, H7CGH0, 
               
               
                   
                 J, member 5 
                 the zona glomerulosa 
                   
                   
                 A0A024R3K7 
               
               
                 KCNJ6 
                 potassium 
                 Widely expressed. 
                 Kir3.2, GIRK2, 
                 21q22.1 
                 P48051 Q3MJ74, 
               
               
                   
                 inwardly-rectifying 
                 Expressed in cells of 
                 KATP2, BIR1, 
                   
                 Q53WW6, _B2RA12, 
               
               
                   
                 channel, subfamily 
                 hematopoietic origin (at 
                 hiGIRK2 
                   
                 Q96L92, Q32Q36 
               
               
                   
                 J, member 6 
                 protein level). Most 
                   
                   
                 Q9H3K8 
               
               
                   
                   
                 abundant in cerebellum, 
               
               
                   
                   
                 and to a lesser degree in 
               
               
                   
                   
                 islets and exocrine 
               
               
                   
                   
                 pancreas 
               
               
                 KCNJ8 
                 potassium 
                 Predominantly detected in 
                 Kir6.1 
                 12p12.1 
                 Q15842, O00657, 
               
               
                   
                 inwardly-rectifying 
                 fetal and adult heart 
                   
                   
                 F5GY12, 
               
               
                   
                 channel, subfamily 
                   
                   
                   
                 A0A024RAV6 
               
               
                   
                 J, member 8 
               
               
                 KCNJ9 
                 potassium 
                 Widely expressed. 
                 Kir3.3, GIRK3 
                 1q23.2 
                 Q92806, Q5JW75, 
               
               
                   
                 inwardly-rectifying 
                 Expressed in cells of 
                   
                   
                 Q96L92, Q32Q36, 
               
               
                   
                 channel, subfamily 
                 hematopoietic origin (at 
                   
                   
                 Q9H3K8 
               
               
                   
                 J, member 9 
                 protein level). 
               
               
                 KCNJ10 
                 potassium 
                   
                 Kir4.1, Kir1.2 
                 1q23.2 
                 P78508, 
               
               
                   
                 inwardly-rectifying 
                   
                   
                   
                 A3KME7, Q92808, 
               
               
                   
                 channel, subfamily 
                   
                   
                   
                 Q547K1, Q9BXC5 
               
               
                   
                 J, member 10 
                   
                   
                   
                 Q9NPI9 
               
               
                 KCNJ11 
                 potassium 
                   
                 Kir6.2, BIR 
                 11p15.1 
                 Q14654, 
               
               
                   
                 inwardly-rectifying 
                   
                   
                   
                 B4DWI4, Q8IW96 
               
               
                   
                 channel, subfamily 
                   
                   
                   
                 E9PPF1, H0YES9, 
               
               
                   
                 J, member 11 
                   
                   
                   
                 D2K1F9 
               
               
                 KCNJ12 
                 potassium 
                 Heart, skeletal muscle, and 
                 Kir2.2, Kir2.2v, 
                 17p11.1 
                 Q14500, O43401, 
               
               
                   
                 inwardly-rectifying 
                 several different brain 
                 IRK2, hIRK1 
                   
                 Q15756, Q8NG63, 
               
               
                   
                 channel, subfamily 
                 regions including the 
                   
                   
                 B7U540, Q9HAP6, 
               
               
                   
                 J, member 12 
                 hippocampus 
                   
                   
                 P48050, Q14D44 
               
               
                 KCNJ13 
                 potassium 
                 Predominantly expressed in 
                 Kir7.1, Kir1.4, 
                 2q37 
                 O60928 A0PGH1 
               
               
                   
                 inwardly-rectifying 
                 small intestine. Expression 
                 LCA16 
                   
                 Q8N3Y4_C9JWD6 
               
               
                   
                 channel, subfamily 
                 is also detected in stomach, 
                   
                   
                 H7C4D1 
               
               
                   
                 J, member 13 
                 kidney, and all central 
               
               
                   
                   
                 nervous system regions 
               
               
                   
                   
                 tested with the exception of 
               
               
                   
                   
                 spinal cord 
               
               
                 KCNJ14 
                 potassium 
                 Expressed preferentially in 
                 Kir2.4, IRK4 
                 19q13 
                 Q9UNX9 Q99712 
               
               
                   
                 channel, subfamily 
                 retina 
                   
                   
                 D3DSH5 Q99446 
               
               
                   
                 J, member 14 
               
               
                 KCNJ15 
                 potassium 
                   
                 Kir4.2, Kir1.3, 
                 21q22.2 
                 Q99712 D3DSH5 
               
               
                   
                 inwardly-rectifying 
                   
                 IRKK 
                   
                 Q99446, A8K9U7 
               
               
                   
                 channel, subfamily 
               
               
                   
                 J, member 15 
               
               
                 KCNJ16 
                 potassium 
                 Highly expressed in 
                 Kir5.1, BIR9 
                 17q24.3 
                 Q9NPI9 
               
               
                   
                 inwardly-rectifying 
                 kidney, pancreas and 
               
               
                   
                 channel, subfamily 
                 thyroid gland 
               
               
                   
                 J, member 16 
               
               
                 KCNJ18 
                 potassium 
                 Specifically expressed in 
                 KIR2.6, TTPP2 
                 17p11.2 
                 B7U540 
               
               
                   
                 inwardly-rectifying 
                 skeletal muscle 
               
               
                   
                 channel, subfamily 
               
               
                   
                 J, member 18 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Potassium channels, two-P 
               
            
           
           
               
               
               
               
               
               
            
               
                 Approved 
                   
                   
                   
                   
                 Accession numbers on 
               
               
                 Symbol 
                 Approved Name 
                 Tissue distribution 
                 Synonyms 
                 Chromosome 
                 uniport.org website 
               
               
                   
               
               
                 KCNK1 
                 potassium channel, 
                 Widely expressed with 
                 K2p1.1, DPK, 
                 1q42-q43 
                 O00180, Q13307, 
               
               
                   
                 subfamily K, 
                 high levels in heart and 
                 TWIK-1 
                   
                 Q5T5E8 
               
               
                   
                 member 1 
                 brain and lower levels in 
               
               
                   
                   
                 placenta, lung, liver and 
               
               
                   
                   
                 kidney 
               
               
                 KCNK2 
                 potassium channel, 
                   
                 K2p2.1, TREK- 
                 1q41 
                 Q6ZW95, Q13303, 
               
               
                   
                 subfamily K, 
                   
                 1 
                   
                 A0AVM9, Q99411 
               
               
                   
                 member 2 
               
               
                 KCNK3 
                 potassium channel, 
                 Widespread expression in 
                 K2p3.1, TASK, 
                 2p23 
                 O14649, Q535U2, 
               
               
                   
                 subfamily K, 
                 adult. Strongest expression 
                 TASK-1 
                   
                 B9EIJ4 
               
               
                   
                 member 3 
                 in pancreas and placenta. 
               
               
                   
                   
                 Lower expression in brain, 
               
               
                   
                   
                 lung, prostate, heart, 
               
               
                   
                   
                 kidney, uterus, small 
               
               
                   
                   
                 intestine and colon 
               
               
                 KCNK4 
                 potassium channel, 
                   
                 K2p4.1, 
                 11q13 
                 Q2YDA1 
               
               
                   
                 subfamily K, 
                   
                 TRAAK 
               
               
                   
                 member 4 
               
               
                 KCNK5 
                 potassium channel, 
                 Abundant expression in 
                 K2p5.1, TASK- 
                 6p21 
                 O95279 B2RAQ6, 
               
               
                   
                 subfamily K, 
                 kidney, also detected in 
                 2 
                   
                 B5TJL2, Q5VV76 
               
               
                   
                 member 5 
                 liver, placenta and small 
               
               
                   
                   
                 intestine. In the kidney, 
               
               
                   
                   
                 expression is restricted to 
               
               
                   
                   
                 the distal tubules and 
               
               
                   
                   
                 collecting ducts. 
               
               
                 KCNK6 
                 potassium channel, 
                 Widespread expression, 
                 K2p6.1, TWIK- 
                 19q13.1 
                 Q9Y257 Q9HB47 
               
               
                   
                 subfamily K, 
                 detected in all tissues tested 
                 2 
               
               
                   
                 member 6 
                 except for skeletal muscle. 
               
               
                   
                   
                 Strongest expression in 
               
               
                   
                   
                 placenta, pancreas, heart, 
               
               
                   
                   
                 colon and spleen, lower 
               
               
                   
                   
                 levels detected in 
               
               
                   
                   
                 peripheral blood 
               
               
                   
                   
                 leukocytes, lung, liver, 
               
               
                   
                   
                 kidney and thymus. Lowest 
               
               
                   
                   
                 expression detected in 
               
               
                   
                   
                 brain 
               
               
                 KCNK7 
                 potassium channel, 
                   
                 K2p7.1 
                 11q13 
                 Q3MI97 
               
               
                   
                 subfamily K, 
               
               
                   
                 member 7 
               
               
                 KCNK9 
                 potassium channel, 
                 Mainly found in the 
                 K2p9.1, 
                 8q24.3 
                 Q9NPC2, Q2M290, 
               
               
                   
                 subfamily K, 
                 cerebellum. Also found in 
                 TASK3, 
                   
                 Q540F2 
               
               
                   
                 member 9 
                 adrenal gland, kidney and 
                 TASK-3 
               
               
                   
                   
                 lung 
               
               
                 KCNK10 
                 potassium channel, 
                 Abundantly expressed in 
                 K2p10.1, 
                 14q31 
                 P57789, B2R8T4, 
               
               
                   
                 subfamily K, 
                 pancreas and kidney and to 
                 TREK-2, 
                   
                 Q9HB59 
               
               
                   
                 member 10 
                 a lower level in brain, 
                 TREK2, 
               
               
                   
                   
                 testis, colon, and small 
                 PPP1R97 
               
               
                   
                   
                 intestine. Isoform b is 
               
               
                   
                   
                 strongly expressed in 
               
               
                   
                   
                 kidney (primarily in the 
               
               
                   
                   
                 proximal tubule) and 
               
               
                   
                   
                 pancreas, whereas isoform 
               
               
                   
                   
                 c is abundantly expressed 
               
               
                   
                   
                 in brain 
               
               
                 KCNK12 
                 potassium channel, 
                   
                 THIK-2, 
                 2p16.3 
                 Q9HB15 
               
               
                   
                 subfamily K, 
                   
                 THIK2, 
               
               
                   
                 member 12 
                   
                 K2p12.1 
               
               
                 KCNK13 
                 potassium channel, 
                   
                 K2p13.1, 
                 14q32.11 
                 Q9HB14, B5TJL8, 
               
               
                   
                 subfamily K, 
                   
                 THIK-1, 
                   
                 Q96E79 
               
               
                   
                 member 13 
                   
                 THIK1 
               
               
                 KCNK15 
                 potassium channel, 
                 Detected in pancreas, heart, 
                 K2p15.1, 
                 20q13.12 
                 Q9H427, Q52LL3, 
               
               
                   
                 subfamily K, 
                 placenta, lung, liver, 
                 dJ781B1.1, 
                   
                 Q9HBC8 
               
               
                   
                 member 15 
                 kidney, ovary, testis, 
                 KT3.3, 
               
               
                   
                   
                 skeletal muscle and adrenal 
                 KIAA0237, 
               
               
                   
                   
                 gland, and at lower levels 
                 TASK5, 
               
               
                   
                   
                 in prostate, spleen and 
                 TASK-5 
               
               
                   
                   
                 thyroid gland 
               
               
                 KCNK16 
                 potassium channel, 
                 Highly expressed in 
                 K2p16.1, 
                 6p21.2-p21.1 
                 Q96T55, B5TJL9, 
               
               
                   
                 subfamily K, 
                 pancreas. 
                 TALK-1, 
                   
                 Q9H591 
               
               
                   
                 member 16 
                   
                 TALK1 
               
               
                 KCNK17 
                 potassium channel, 
                   
                 K2p17.1, 
                 6p21 
                 Q96T54, E9PB46, 
               
               
                   
                 subfamily K, 
                   
                 TALK-2, 
                   
                 Q9H592, B2RCT9 
               
               
                   
                 member 17 
                   
                 TALK2, 
               
               
                   
                   
                   
                 TASK4, 
               
               
                   
                   
                   
                 TASK-4 
               
               
                 KCNK18 
                 potassium channel, 
                 Expressed specifically in 
                 K2p18.1, 
                 10q26.11 
                 Q7Z418, Q5SQQ8 
               
               
                   
                 subfamily K, 
                 dorsal root ganglion and 
                 TRESK-2, 
               
               
                   
                 member 18 
                 trigeminal ganglion 
                 TRESK2, 
               
               
                   
                   
                 neurons. Detected at low 
                 TRESK, TRIK 
               
               
                   
                   
                 levels in spinal cord. 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Hydrogen voltage-gated ion channels 
               
            
           
           
               
               
               
               
               
               
            
               
                 Approved 
                   
                   
                   
                   
                 Accession numbers on 
               
               
                 Symbol 
                 Approved Name 
                 Tissue distribution 
                 Synonyms 
                 Chromosome 
                 uniport.org website 
               
               
                   
               
               
                 HVCN1 
                 hydrogen voltage- 
                 Enriched in immune 
                 MGC15619, 
                 12q24.11 
                 Q96D96, A8MQ37, 
               
               
                   
                 gated channel 1 
                 tissues, such as lymph 
                 Hv1, VSOP 
                   
                 Q96IS5 
               
               
                   
                   
                 nodes, B-lymphocytes, 
               
               
                   
                   
                 monocytes and spleen 
               
               
                   
               
            
           
         
       
     
     Ligand-dependent ion channels also commonly referred to as ligand-gated ion channels (LICs, LGIC), or as ionotropic receptors are a group of integral membrane proteins which contain a pore which allows 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. 
     These channels thus comprise a fourth domain which is extracellular and includes a ligand binding domain. Such ligand may be for example a neurotransmitter. When a presynaptic neuron is excited, it releases a neurotransmitter from vesicles into the synaptic cleft. The neurotransmitter then binds to receptors located on the postsynaptic neuron, causing a conformational change of the ion channels, and leading, upon activation or inhibition, to a flow of ions across the cell membrane. This, in turn, results in either a depolarization, for an excitatory receptor response, or a hyperpolarization, for an inhibitory response. The function of these channels when located within postsynapses is to directly and very quickly convert the chemical signal of presynaptically released neurotransmitter into a postsynaptic electrical signal. 
     These ligand-gated ion channels are subdivided with respect to the type of ion that they conduct, e.g., anionic or cationic and further into families defined by the endogenous ligand. They comprise cation-selective receptors including acetylcholine receptors, 5-HT3 receptors, ionotropic glutamate receptors as well as P2X receptors and anion-selective receptors including GABAA receptors, glycine receptors and zinc-activated channels. In terms of configuration, nicotinic acetylcholine receptors, 5-HT3 receptors, GABAA (gamma-aminobutyric acid A) receptors, glycine receptors, and the additional zinc-activated channel are pentameric structures and are frequently referred to as the Cys-loop receptors due to the presence of a defining loop of residues formed by a disulphide bond between two cysteine residues in the N terminal extracellular domains of their constituent subunits. The ionotropic glutamate receptors and P2X receptors are tetrameric and trimeric structures, respectively. A binding site in the extracellular N-terminal ligand-binding domain gives them receptor specificity for specific endogenous ligands including acetylcholine (AcCh), serotonin, glycine, glutamate and γ-aminobutyric acid (GABA), etc. . . . . 
     5-HT 3  receptors or 5-hydroxytryptamine (serotonin) receptors exist as a pentamer of 4™ subunits that form an intrinsic cation selective. Five human 5-HT 3  receptor subunits including 5-HT 3A  (https://www.uniprot.org/uniprot/P46098), 5-HT 3B  (https://www.uniprot.org/uniprot/P46098), 5-HT 3 c (https://www.unprot.org/uniprot/Q8WXA8), 5-HT3D (https://www.uniprot.org/uniprot/Q70Z44), 5-HT 3E  (https://www.uniprot.org/uniprot/A5X5Y0) have been cloned and homo-oligomeric assemblies of 5-HT 3 A and hetero-oligomeric assemblies of 5-HT 3 A and 5-HT 3 B subunits have been fully characterized (5-HT 3 A and 5-HT 3 AB receptors). The 5-HT 3 C subunit, as well as 5-HT 3 D and 5-HT 3 E subunits, like the 5-HT 3 B subunit, do not form functional homomers, but assemble with the 5-HT 3 A subunit to influence its functional expression rather than pharmacological profile. The serotonin receptors are a group of G protein-coupled receptor and ligand-gated ion channels found in the central and peripheral nervous systems which mediate both excitatory and inhibitory neurotransmission. They are activated by the neurotransmitter serotonin, which acts as their natural ligand, and they 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 influence various biological and neurological processes such as aggression, anxiety, appetite, cognition, learning, memory, mood, nausea, and sleep. 
     Zinc-activated ion channel (ZAC) is also known as ZAC1, L2m LICZ, LICZ1 (https://www.uniprot.org/uniprot/Q401N2). ZAC forms a cation-permeable ligand-gated ion channel of the cys-loop family. This channel most likely exists as a homopentamer of four transmembrane subunits that form an intrinsic cation selective channel equipermeable to Na + , K +  and Cs + , but impermeable to Ca 2+  and Mg 2+ . ZAC are expressed in prostate, thyroid, trachea, lung, brain (adult and fetal), spinal cord, skeletal muscle, heart, placenta, pancreas, liver, kidney and stomach. The endogenous ligand for ZAC is thought to be Zn 2+ , although ZAC has also been found to activate spontaneously. 
     GABA A  (gamma-aminobutyric acid A) receptors or channels are ionotropic receptors. They exist as pentamers of four transmembrane subunits including six alpha subunits: α1 to α6 subunits (https://www.uniprot.org/uniprot/P14867, P47869, P34903, P48169, P31644, Q16445), three beta subunits: β1 to β3 subunits (https://www.uniprot.org/uniprot/P14867, P47870, P28472), three gamma subunits: γ1 to γ3 subunits (https://www.uniprot.org/uniprot/Q8N1C3, P18507, Q99928), one delta (δ) subunit (https://www.uniprot.org/uniprot/014764), 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: ρ1 to ρ3) receptor subunits (https://www.uniprot.org/uniprot/P24046, P28476, A8MPY1). Many GABA A  receptor subtypes contain α-, β- and γ-subunits with the stoichiometry 2α.2β.1γ. In the CNS however, α1β2γ2 heterooligomer constitutes the largest population of GABA A  receptors in the CNS, followed by the α2β3γ2 and α3β3γ2 isoforms. Currently, eleven native GABA A  receptors are classed as conclusively identified, i.e., α1β2γ2, α1βγ2, α3βγ2, α4βγ2, α4β2δ, α4β3δ, α5βγ2, α6βγ2, α6β2δ, α6β3δ and ρ. The endogenous ligand is γ-aminobutyric acid (GABA) which is the major inhibitory neurotransmitter in the central nervous system. Upon activation, the GABA A  receptor selectively conducts chloride (Cl − ) through its pore. Several inhibitory drugs have been identified including muscimol, gaboxadol, bicuculline, the benzodiazepines, nonbenzodiazepines, alprazolam, diazepam, and picrotoxin. 
     The glycine receptor also referred to as GlyR or GLRi is the receptor of the amino acid neurotransmitter glycine. GlyR is an ionotropic receptor that produces its effects through chloride current. The receptor is expressed either as a homo-pentamer of a subunits, or a complex of 2α and 3β subunits. Amino acid sequences of glycine receptor subunits α1 to α3 and β are provided in 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 has important roles in a variety of physiological processes, especially in mediating inhibitory neurotransmission in the spinal cord and brainstem. The receptor can be activated by a range 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. 
     The ionotropic glutamate receptors bind the neurotransmitter glutamate. They form tetramers with each subunit consisting of an extracellular amino terminal domain (ATD), which is involved in subunit assembly, an extracellular ligand binding domain (LBD) which binds glutamate, and a transmembrane domain (TMD) which forms the ion channel. Each subunit of the tetramer has a binding site for glutamate formed by the two LBD sections forming a clamshell like shape. Only two of these sites in the tetramer need to be occupied to open the ion channel. lonotropic glutamate receptors comprise members of the NMDA (N-methyl-D-aspartate), AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid) and kainate receptor classes. 
     NMDA receptors assemble as heteromers comprising from 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/060391) subunits. AMPA receptors assemble as homomers, or heteromers comprising GluA1, GluA2, GluA3 and GluA4 subunits. kainate receptors can be expressed as homomers of 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). GluK1-3 subunits are also capable of assembling into heterotetramers, e.g., GluK1/K2. 
     P2X receptors are ATP-gated channels which open in response to binding the extracellular nucleotide ATP. They form trimers with two transmembrane domains with C and N termini located on the intracellular side, gating Na + , K +  and Ca 2+ . Native P2X receptors may occur as either homotrimers or heterotrimers including from P2X1 to 7 subunits (https://www.uniprot.org/uniprot/P51575, Q9UBL9, P56373, Q99571, Q93086, O15547, Q99572). 
     Nicotinic acetylcholine receptors (nAChR) are members of the Cys-loop family, consisting of a pentamer of protein subunits (typically ααβγδ). Subunits may be of class alpha (α1 to α10) referred to as ACHRA (https://www.uniprot.org/uniprot/P02708), class beta (β1 to β4) referred to as ACHRB (https://www.uniprot.org/uniprot/P11230), class gamma(γ) or ACHRG (https://www.uniprot.org/uniprot/P07510), class delta(δ) or ACH RD (https://www.uniprot.org/uniprot/Q07001), class epsilon (ε) or ACHRE (https://www.uniprot.org/uniprot/Q04844). Binding sites for acetylcholine are on the interface of each alpha subunit. When the acetylcholine binds, it alters the receptors configuration and causes the constriction in the pore of approximately 3 angstroms to widen to approximately 8 angstroms so that ions can pass through. This pore allows Na +  ions to flow down their electrochemical gradient into the cell. With a sufficient number of channels opening at once, the inward flow of positive charges carried by Na +  ions depolarizes the postsynaptic membrane sufficiently to initiate an action potential. nAChRs respond to the neurotransmitter acetylcholine and to drugs such as the agonist nicotine. They are found in the central and peripheral nervous system, muscle, and many other tissues of many organisms. At the neuromuscular junction they are the primary receptor in muscle for motor nerve-muscle communication that controls muscle contraction. In the peripheral nervous system: they transmit outgoing signals from the presynaptic to the postsynaptic cells within the sympathetic and parasympathetic nervous system, and they are the receptors found on skeletal muscle that receive acetylcholine released to signal for muscular contraction. 
     Therefore, according to the present invention, the ion conducting channel fusion subunits are thus subunits of the ion conducting channels, either ligand-dependent ion channels or voltage-dependent ion channel as described above, which is bound or coupled in C-terminal or N-terminal to the probe comprising an ion sensor which is sandwiched between at least one bioluminescent donor molecule and at least one fluorescent acceptor molecule. Alternatively, the ion sensor sandwiched by the bioluminescent donor and the fluorescent acceptor molecule may also be inserted inside an intracellular or an extracellular loop of an ion conducting channel subunit. When the subunits of said ion conducting channels are expressed in cells, they may thus reassemble to a form an active ion conducting channel fusion protein which may be used for HTS method according to the present invention. Such channel fusion protein may be formed by the same types of subunits and is a homopolymer channel (homodimer, homotetramer, etc.) or by different types of subunits and is a heteropolymer channel (heterodimer, heterotetramer, etc. . . . ). 
     Bioluminescent donor molecule and fluorescent acceptor molecule are selected so that the emission spectrum of the bioluminescent donor molecule overlaps with the absorbance spectrum of the acceptor molecule, so the light energy delivered by the bioluminescent donor molecule is at a wavelength that is able to excite the acceptor molecule, and thus making it possible the non-radiative energy transfer between the bioluminescent energy donor and the fluorescent acceptor through nonradiative dipole-dipole coupling. 
     Bioluminescent donor refers to any moiety capable of acting on a suitable substrate to transform chemical energy into light energy. For example, it may refer to an enzyme which converts a substrate into an activated product which then releases energy as it relaxes. The activated product (generated by the activity of the bioluminescent protein on the substrate) is the source of the bioluminescent protein-generated luminescence that is transferred to the acceptor molecule. 
     Several bioluminescent donor molecules may be used in the present invention. Light-emitting systems have been known and isolated from many luminescent organisms including bacteria, protozoa, coelenterates, molluscs, fish, millipedes, flies, fungi, worms, crustaceans, and beetles, particularly click beetles of genus  Pyrophorus  and the fireflies of the genera  Photinus, Photuris , and  Luciola . Additional organisms displaying bioluminescence are listed in international publications WO 00/024878 and WO 99/049019. 
     One well characterized example is the class of proteins known as luciferases. Luciferase proteins catalyze an energy-yielding chemical reaction in which a specific biochemical substance, a luciferin (a naturally occurring substrate), is oxidized by an enzyme having a luciferase activity. Both prokaryotic and eukaryotic organisms including species of bacteria, algae, fungi, insects, fish and other marine forms can emit light energy in this manner and each has specific luciferase activities and luciferins, which are chemically distinct from those of other organisms. 
     Luciferin/luciferase systems are very diverse in form, chemistry and function. For example, there are luciferase activities which facilitate continuous chemiluminescence, as exhibited by some bacteria and mushrooms, and those which are adapted to facilitate sporadic, or stimuli induced, emissions, as in the case of dinoflagellate algae. As a phenomenon, which entails the transformation of chemical energy into light energy, bioluminescence is not restricted to living organisms, nor does it require the presence of living organisms. It is simply a type of chemiluminescent reaction that requires a luciferase activity, which at one stage or another had its origins from a biological catalyst. Hence the preservation or construction of the essential activities and chemicals suffices to have the means to give rise to bioluminescent phenomena. 
     Such bioluminescent donor molecules may be for example luciferases which are well-known in the art. Luciferases are enzymes or oxygenases that catalyze a light emitting reaction, e.g., a bioluminescent reaction. 
     All known kinds of luciferases, either naturally occurring luciferases or mutated luciferases are encompassed within the scope of the invention. 
     Examples of bioluminescent proteins with luciferase activity may be found in U.S. Pat. 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: (i)  Renilla  luciferase (from  R. reniformis ), a 35 kDa protein, which uses coelenterazine as a substrate and emits light at 480 nm; and (ii) Firefly luciferase (from  Photinus pyralis ), a 61 kDa protein, which uses luciferin as a substrate and emits light at 560 nm. 
     Bioluminescent donor molecule may be a luciferase chosen among  Renilla  luciferase, Firefly luciferase, Coelenterate luciferase, North American glow worm luciferase, click beetle luciferase, railroad worm luciferase,  Gaussia  luciferase, Aequorin,  Arachnocampa  luciferase, luciferase derived from the copepods like  Gaussia princeps, Pleuromamma abdominalis, Metridia pacifica, Metridia curticauda, Metridia asymmetrica, Metridia okhotensis, Metridia longa, Lucicutia ovaliformis, Heterorhabdus tanneri, Pleuromamma scutullata , or a biologically active variant or fragment thereof. 
     A preferred selection of molecular weight range of luciferases for use according to the present invention may be in the range of 15 to 70 kDa, or of 15 to 60 kDa, or of 15 to 50 kDa, or 15 to 40 kDa, or 15-30 kDa. 
     Among preferred luciferases, we may cite GLuc, NanoLuc (NLuc), MLuc7, HtLuc, LoLuc, PaLuc1, PaLuc2, MpLuc1, McLuc1, MaLuc1, MoLuc1, MoLuc2, MLuc39, PsLuc1, LocLucl-3, HtLuc2  Renilla , TurboLuc16 (TLuc) or homologs or orthologs thereof or mutants or functional derivatives thereof. In various embodiments, mutants or functional derivatives of the luciferases retain at least 100%, 90%, 80%, 70%, 60% or 50% of the bioluminescence activity from which the mutant or function derivative is derived. As described in Takenaka et al. (Mol Biol Evol 29(6): 1669-1681, 2012), the copepod luciferases comprise two domains and each domain includes conserved sequences across the various luciferases. In some embodiments, any luciferase comprising 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 as described by Takenaka et al., may be used in the methods described herein. 
     Most preferred luciferases may include nanoluciferase which is a protein derived from the 19 kDa subunit of a luciferase extracted from the deep-sea shrimp Oplophorus  gracilirostris , the second subunit being a 35 kDa protein as described by S. Inouye et al. (FEBS Letters, 481 (2000) 19-25). This nanoluciferase is marketed under the commercial name NanoLuc® by the company Promega, and it utilizes novel substrates (furimazine or bisdeoxycoelenterazine as well as 6h-f-coelenterazine) to produce high intensity, glow-type luminescence as described by Hall M. et al. (ACS Chem. Biol. 2012, 7, 1848-1857) and in international publication WO2012/061530. 
     Alternative, non-luciferase, bioluminescent molecules may be any enzymes which can act on suitable substrates to generate a luminescent signal. Specific examples of such enzymes are 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, Mass., USA). 
     By way of examples, several bioluminescent donor molecules have been listed in the following Table 12. 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Examples of bioluminescent molecules 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 MW 
                 Emission 
                   
               
               
                 Species 
                 Name 
                 Organism 
                 kDa × 10 −3   
                 (nm) 
                 Substrate 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Insect 
                 FFluc 
                 
                   Photinus pyralis 
                 
                 ~61 
                 560 
                 D-(−)-2-(6&#39;-hydroxybenzo- 
               
               
                   
                   
                 (North American Firefly) 
                   
                   
                 thiazolyl)-Δ 2 -thiazoline-4- 
               
               
                   
                   
                   
                   
                   
                 carboxylic acid, HBTTCA, 
               
               
                   
                   
                   
                   
                   
                 (C 11 H 8 N 2 O 3 S 2 )(Luciferin) 
               
               
                 Insect 
                 FF&#39;luc 
                 
                   Luciola cruciate 
                 
                   
                 560-590 (many 
                 Luciferin 
               
               
                   
                   
                 (Japanese Firefly) 
                   
                 mutants) 
               
               
                 Insect 
                   
                 Phengodid beetles(railroad 
               
               
                   
                   
                 worms) 
               
               
                 Insect 
                   
                   Arachnocampa sp. 
                   
                   
                 Luciferin 
               
               
                 Insect 
                   
                 
                   Orphelia fultoni 
                 
               
               
                   
                   
                 (North American glow 
               
               
                   
                   
                 worm) 
               
               
                 Insect 
                 Clluc 
                 
                   Pyrophorus 
                 
                   
                 546, 560 578 
                 Luciferin 
               
               
                   
                   
                 
                   plagiophthalamus 
                 
                   
                 and 593 
               
               
                 Jelly Fish 
                 Aequorea 
                 
                   Aequorea 
                 
                 44.9 
                 460-470 
                 Coelenterazine 
               
               
                 Sea Pansy 
                 Rluc 
                 
                   Renilla Reniformis 
                 
                 36 
                 480 
                 Coelenterazine 
               
               
                 Sea 
                 Rluc8 
                 
                   Renilla 
                 
                 36 
                 487(peak) 
                 Coelenterazine/Deep Blue C 
               
               
                 Pansy(modified) 
                   
                   Reniformis (modified) 
               
               
                 Sea Pansy 
                 Rmluc 
                 
                   Renilla mullerei 
                 
                 36.1 
                 ~480 
                 Coelenterazine 
               
               
                 Sea Pansy 
                   
                 
                   Renilla kollikeri 
                 
               
               
                 Crustacea(shrimp) 
                 Vluc 
                 
                   Vargula hilgendoifii 
                 
                 ~62 
                 ~460 
                 coelenterazine 
               
               
                 Crustacea 
                   
                   Cypridina (sea firefly) 
                 75 
                 460 
                 coelenterazine 
               
               
                 Dinofagellate 
                   
                 
                   Gonyaulax polyedra 
                 
                 130 
                 ~475 
                 Tetrapyrrole 
               
               
                 (marine algae) 
               
               
                 Mollusc 
                   
                   Latia  (Freshwater limpet) 
                 170 
                 500 
                 Enol formate, 
               
               
                   
                   
                   
                   
                   
                 terpene, aldehyde. 
               
               
                 Hydroid 
                   
                 Obelia biscuspidata   
                 ~20 
                 ~470 
                 Coelenterazine 
               
               
                 Shrimp 
                   
                 
                   Oplophorus gracilorostris 
                 
                 31 
                 462 
                 Coelenterazine 
               
               
                 Others 
                 Ptluc 
                 
                   Ptilosarcus 
                 
                   
                 ~490 
                 Coelenterazine 
               
               
                   
                 Glue 
                 
                   Gaussia 
                 
                 ~20 
                 ~475 
                 Coelenterazine 
               
               
                   
                 Plluc 
                 
                   Pleuromamma 
                 
                 22.6 
                 ~475 
                 Coelenterazine 
               
               
                   
               
            
           
         
       
     
     The choice of the substrate of the bioluminescent donor molecule can impact on the wavelength and the intensity of the light generated by the bioluminescent protein. A widely known substrate is coelenterazine which occurs in cnidarians, copepods, chaetognaths, ctenophores, decapod shrimps, mysid shrimps, radiolarians and some fish taxa. For  Renilla  luciferase for example, coelenterazine analogues/derivatives are available that result in light emission between 418 and 512 nm. A coelenterazine analogue/derivative (400A, DeepBlueC) has been described emitting light at 400 nm with  Renilla  luciferase (WO01/46691). Other examples of coelenterazine analogues/derivatives are EnduRen and ViviRen. 
     Luciferins are light-emitting biological pigments found in organisms capable of bioluminescence, which are 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, for example fish and squid, however, many have been identified for example in worms, beetles and various other insects. 
     There are at least five general types of luciferin, which are each chemically different and catalyzed by chemically and structurally different luciferases that employ a wide range of different cofactors. First, is firefly luciferin, the substrate of firefly luciferase, which requires ATP for catalysis (EC 1.13.12.7). Second, is bacterial luciferin, also found in some squid and fish, that consists of a long chain aldehyde and a reduced riboflavin phosphate. Bacterial luciferase is FMNH-dependent. Third, is dinoflagellate luciferin, a tetrapyrrolic chlorophyll derivative found in dinoflagellates (marine plankton), the organisms responsible for night-time ocean phosphorescence. Dinoflagellate luciferase catalyzes the oxidation of dinoflagellate luciferin and consists of three identical and catalytically active domains. Fourth, is the imidazolopyrazine vargulin, which is found in certain ostracods and deep-sea fish, for example,  Porichthys . Finally, coelanterazine (an imidazolpyrazine), the light-emitter of the protein aequorin, found in radiolarians, ctenophores, cnidarians, squid, copepods, chaetognaths, fish and shrimp. 
     Several acceptor molecules either protein or non-proteinaceous may be used in the intramolecular novel BRET probe according to the present invention. 
     Acceptor proteins may include for example green fluorescent protein (GFP), variant of green fluorescent protein (such as 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, destabilised EGFP (dEGFP), destabilised ECFP (dECFP), destabilised EYFP (dEYFP), HcRed, t-HcRed, DsRed, DsRed2, mRFPI, pocilloporin,  Renilla  GFP, Monster GFP, paGFP, Kaede protein, a Phycobiliprotein, TagCFP, mTagCFP2, Czurite, ECFP2, mKalamal, Sirius, Sapphire, T-Sapphire, ECFP, Cerulean, SCFP3C, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP, mTFPI, EGFP, Emerald, Superfolder GFP, Monomeric Czami Green, TagGFP2, mUKG, mWasabi, Clover, Citrine, Venus, SYFP2, TagYFP, Monomeric Kusabira-Orange, ιηKOκ, 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 a biologically active variant or fragment of any one thereof. 
     The most frequently used bioluminescent or fluorophore is the green fluorescent protein from the jellyfish  Aequorea victoria  and numerous other variants (GFPs) obtained for example by mutagenesis and chimeric protein technologies. GFPs are classified based on the distinctive component of their chromophores, each class having distinct excitation and emission wavelengths: class 1, wild-type mixture of neutral phenol and anionic phenolate: class 2, phenolate anion: class 3, neutral phenol: class 4, phenolate anion with stacked s-electron system: class 5, indole: class 6, imidazole: and class 7, phenyl. 
     Preferred fluorescent molecules or fluorescent acceptor molecules may be the mNeonGreen, which is a monomeric yellow-green fluorescent protein derived from a tetrameric fluorescent protein from the cephalochordate  Branchiostoma lanceolatum  and is described as the “brightest monomeric green or yellow fluorescent protein.” The previously developed mNeonGreen monomeric protein is called here the wild-type mNeonGreen. Also included herein are mNeonGreen variants that are improved compared to the wild-type protein in their photo stability and fluorescence. See for example mNeonGreen variants of a wild-type mNeonGreen of SEQ ID NO: 65 as reported in international publication WO2019/055498, which comprise one or more mutations selected from V52H, D53E, N65Y, E69M or E69Q, A110Q, S 131I or S 131C, T139R, S 143D, K152H, A158P, T164Q, S 166K, A169G, W172P, S 175E, K177M, T178L, T188D or T188E, K190T, T194K, G196K or G196E, N197D, 5203C, T204Q, T208H, N218D, and Y226F. 
     Other representative acceptor molecules may include, but are not limited to sgGFP, sgBFP, BFP blue shifted GFP (Y66H), Cyan GFP, DsRed, monomeric RFP, EBFP, ECFP, GFP (S65T), GFP red shifted (rsGFP), non-UV excitation (wtGFP), UV excitation (wtGFP), GFPuv, HcRed, rsGFP, Sapphire GFP, sgBFP™, sgBFP™ (super glow BFP), sgGFP™, sgGFP™ (super glow GFP), Yellow GFP, semiconductor nanoparticles (e.g., raman nanoparticles), 1,5 IAEDANS; 1,8-ANS; 4-Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein; 5-Carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5-Carboxyfluorescein); 5-HAT (Hydroxy Tryptamine); 5-Hydroxy Tryptamine (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 (Photoprotein); AFPs (AutoFluorescent Protein; Quantum Biotechnologies); Alexa Fluor 350™; Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™; Alexa Fluor 568™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor 660™; Alexa Fluor 680™; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; AMCA (Aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin; Aminomethylcoumarin (AMCA); Anilin Blue; Anthrocyl stearate; APC (Allophycocyanin); APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO-TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulphate; Beta Lactamase; Bimane; Bisbenzamide; Bisbenzimide (Hoechst); bis-BTC; Blancophor FFG; BlancophorSV; BOBO™-1; BOBO™-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 FI; Bodipy FL ATP; Bodipy FI-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™-1; BO-PRO™-3; Brilliant Sulphoflavin FF; BTC; BTC-5N; Calcein; Calcein Blue; Calcium Crimson™; Calcium Green; Calcium Green-1 Ca 2+  Dye; Calcium Green-2 Ca 2+ ; Calcium Green-5N Ca 2+ ; Calcium Green-C18 Ca 2+ ; Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX); Cascade Blue™; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; Chlorophyll; Chromomycin A; Chromomycin A; CL-NERF; CMFDA; Coumarin Phalloidin; C-phycocyanine; CPM Methylcoumarin; CTC; CTC Formazan; Cy2™; Cy3.18; Cy3.5™; Cy3™; Cy5.18; Cy5.5™; CyS™; Cy7™; cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3′ DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di-16-ASP); Dichlorodihydrofluorescein Diacetate (DCFH); DiD-Lipophilic Tracer; DiD (DiIC18(5)); DIDS; Dihydorhodamine 123 (DHR); DiI (DiIC18(3)); Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DiIC18(7)); DM-NERF (high pH); DNP; Dopamine; DTAF; DY-630-NHS; DY-635-NHS; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide; Ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight; Europium (III) chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FIF (Formaldehyd Induced Fluorescence); FITC; Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43™; FM 4-46; Fura Red™ (high pH); Fura Red™/Fluo-3; Fura-2; Fura-2/BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow SGF; GeneBlazer (CCF2); Gloxalic Acid; Granular blue; Haematoporphyrin; 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; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso Tracker Green; Lyso Tracker Red; Lyso TrackerYellow; LysoSensor Blue; LysoSensor Green; LysoSensorYellow/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; Merocyanin; Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant lavin EBG; Oregon Green; Oregon Green 488-X; Oregon Green™; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed [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; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primuline; Procion Yellow; Propidium Iodid (PI); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Red 613 [PE-TexasRed]; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); 565A; S65C; S65L; S65T; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; SITS; SITS (Primuline); SITS (Stilbene Isothiosulphonic Acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF1; Sodium Green; SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ (6-methoxy-N-(3-sulfopropyl)quinolinium); Stilbene; Sulphorhodamine B can C; Sulphorhodamine 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; SYTO 45; 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™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC TetramethylRodaminelsoThioCyanate; True Blue; TruRed; 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 (interchelating dyes), or combinations thereof. 
     Alternatively, the acceptor molecule may be a fluorescent nanocrystal. Nanocrystals have several advantages over organic molecules as fluorescent labels, including resistance to photodegradation, improved brightness, non-toxicity, and size dependent, narrow emission spectra that enables the monitoring of several processes simultaneously. Additionally, the absorption spectrum of nanocrystals is continuous above the first peak, enabling all sizes, and hence all colors, to be excited with a single excitation wavelength. 
     The acceptor molecule may also be a fluorescent microsphere. These are typically made from polymers and contain fluorescent molecules (for example fluorescein GFP or YFP) incorporated into the polymer matrix, which can be conjugated to a variety of reagents. Fluorescent microspheres may be labelled internally or on the surface. Internal labelling produces very bright and stable particles with typically narrow fluorescent emission spectra. With internal labelling, surface groups remain available for conjugating ligands (for example, proteins) to the surface of the bead. Internally labelled beads are used extensively in imaging applications, as they display a greater resistance to photobleaching. 
     Multiple combinations of bioluminescent donor molecule and the fluorescent acceptor molecule may be used to increase the sensitivity of the detection of tested inhibitor or activator candidates. Criteria which should be considered in determining suitable pairings for BRET is the relative emission/fluorescence spectrum of the acceptor molecule compared to that of the bioluminescent donor molecule. The emission spectrum of the bioluminescent protein should overlap with the absorbance spectrum of the acceptor molecule such that the light energy from the bioluminescent protein luminescence emission is at a wavelength that is able to excite the acceptor molecule and thereby promote acceptor molecule fluorescence when the two molecules are in a proper proximity and orientation with respect to one another. Good spectroscopic properties, e.g., brightness, photostability, may also be considered. 
     The original BRET system uses the  Renilla  luciferase as donor, EYFP (or Topaz) as the acceptor and coelenterazine derivative as the substrate. These components when combined in a BRET assay generate maximal light in the 475-485 nm range for the bioluminescent protein and the 525-535 nm range for the acceptor molecule, giving a spectral resolution of 40-60 nm.  Renilla  luciferase generates a broad emission peak overlapping substantially the GFP emission, which in turn contributes to decrease the signal to noise of the system. Various coelenterazine derivatives are known in the art, including coe1400a, that generate light at various wavelengths (distinct from that generated by the wild type coelenterazine) as a result of  Renilla  luciferase activity. A skilled person in the art would appreciate that because the light emission peak of the donor has changed, it is necessary to select an acceptor molecule which will absorb light at this wavelength and thereby permit efficient energy transfer. Spectral overlapping between light emission of the donor and the light absorption peak of the acceptor is one condition among others for an efficient energy transfer. Class 3 and 1 GFPs are known to absorb light at 400 nm and re-emit between 505-511 nm. This results in a wavelength difference between donor and acceptor emissions of approximately 111 nm. 
     Preferred selection of the bioluminescent donor and fluorescent acceptor molecules suitable for use in the intramolecular BRET probe according to the present invention include the combination NanoLuc and mNeonGreen. 
     As the skilled person in the art will appreciate, the bioluminescent donor molecule and the fluorescent acceptor molecule are inserted such that it makes the ion sensor and/or ion conducting channel subunit active as result in a spatial change to the location and/or dipole orientation of the bioluminescent donor molecule relative to the fluorescent acceptor molecule. The spatial positioning of the ion channel, ion sensor probe, bioluminescent donor molecule and the fluorescent acceptor molecule is such that the resultant fusion channel protein is functional. The energy transfer primarily depends on: (i) an overlap between the emission and excitation spectra of the donor and acceptor molecules, respectively and (ii) the proximity of about 100 Angstroms (A) between the donor and acceptor molecules. The donor molecule in BRET produces light via chemiluminescence, so it is amenable to small animal imaging. In addition, the BRET system does not use an external light excitation source, which provides potentially greater sensitivity in living subjects because of the low signal to noise ratio. 
     The ion sensor which is used in the probe according to the present invention is a sensor receptive to calcium, potassium, sodium, chloride ions or any sensor receptive to ionic strength. It represents a sensing domain or a sensitive domain which is responsive or sensitive or capable of detecting/sensing presence and levels of a signal ion including for example calcium, potassium, sodium, chloride ions and the like. The sensor or sensing domain then typically changes conformation in response to the binding of a signal ion or molecule or to a changed intracellular condition. 
     The intramolecular BRET probes according to the present invention are thus useful to monitor ion channel structural conformational changes and ion channel activation in living cells. 
     According to the present invention, the ion sensor may be chosen among a calcium binding protein, such as the troponin C calcium binding domain or the calmodulin calcium binding protein, a potassium binding protein (KBP), a sodium binding protein, such as the NhAs-1 protein, or a chloride binding protein. 
     Calcium-sensing proteins or calcium binding proteins are well-known in the art, and they may be any proteins that act in the second messenger system. For example, we can cite Calmodulin, Calnexin, Calreticulin and Gelsolin. 
     Calmodulin (CaM) (an abbreviation for calcium-modulated protein) is a multifunctional intermediate calcium-binding messenger protein expressed in all eukaryotic cells. It is an intracellular target of the secondary messenger Ca 2+ , and the binding of Ca 2+  is required for the activation of calmodulin. Once bound to Ca 2+ , calmodulin acts as part of a calcium signal transduction pathway by modifying its interactions with various target proteins such as kinases or phosphatase. 
     Calnexin (CNX) is a 67 kDa integral protein (that appears variously as a 90 kDa, 80 kDa, or 75 kDa band on western blotting depending on the source of the antibody) of the endoplasmic reticulum. It consists of a large (50 kDa)N-terminal calcium-binding lumenal domain, a single transmembrane helix and a short (90 residues) acidic cytoplasmic tail. 
     Calreticulin also known as calregulin, CRP55, CaBP3, calsequestrin-like protein, and endoplasmic reticulum resident protein 60 (ERp60) is a protein that in humans encoded by the CALR gene. Calreticulin is a multifunctional soluble protein that binds Ca 2+  ions, rendering it inactive. The Ca 2+  is bound with low affinity, but high capacity, and can be released on a signal. Calreticulin is located in storage compartments associated with the endoplasmic reticulum (ER) and is considered an ER resident protein. 
     Gelsolin is an actin-binding protein that is a key regulator of actin filament assembly and disassembly. Gelsolin is one of the most potent members of the actin-severing gelsolin/villin superfamily, as it severs with nearly 100% efficiency. 
     Potassium sensors or potassium binding proteins are also well-known in the art and it may include any potassium sensor proteins capable of binding positively charged potassium ion and generating a detectable signal upon binding of positively charged potassium ion to the potassium sensor. The binding of K +  may for example induce a conformational shift in the potassium sensor and then enable signaling domain to generate a detectable signal. Preferably, the binding of K +  to the potassium sensor may induce a conformational shift in the peptide and the signaling domain may then be capable to generate the detectable signal. In a preferred embodiment, the potassium sensor comprises an amino acid sequence of 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 by a soluble 16-kDa cytoplasmic protein from  Escherichia coli . It is a highly specific K +  binding protein and is required for normal growth in the presence of high levels of external K. The potassium ion binds exclusively to the BON domain, which upon binding undergoes a conformational change. KBP further comprises the LysM domain which may interact with the BON domain. 
     Sodium ion binding proteins or sodium sensors may include for example nhaS gene product, NhaS, a protein characterized by binding to and sequestering sodium ion (Na). Also included within the scope are functional fragments of sodium ion binding proteins including NhaS, which fragments are characterized by their ability to bind to sodium ion, see for example U.S. Pat. No. 5,346,815A. 
     Chloride binding proteins or chloride sensors may include for example the OEC, Chameleon and other chloride requiring enzymes. The polypeptide structure of the OEC has been extensively described inter alia by Coleman W. J. ( Photosynth Res  (1990) 23: 1. https://doi.org/10.1007/BF00030059). The chloride biosensors like YFP-based chloride biosensor Chameleon consists of two fluorescent proteins, CFP and Topaz joined 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/fnce1.2014.00258) and by Watts et al. (Apr. 10, 2012, https://doi.org/10.1371/journal.pone.0035373). In some cases, the acceptor presents chloride sensitivity and the chloride sensor may thus be, in such cases, the acceptor itself. To this regard, we may cite the YFP-H148Q, a genetically modified YFP photostable fluorescent protein which also presents enhanced chloride sensitivity as described by Zhong S et al. ( PLoS One.  2014 Jun. 5; 9(6):e99095. doi: 10.1371/journal.pone.0099095). Such modified YFP may thus be used as fluorescent acceptor molecule as well as ion chloride sensor for studying activation or inhibition of the ion conducting channels by chloride ion flux. 
     The present invention thus provides a useful tool to probe for the passage of ions and thus actual activation or inhibition of ion conducting channels as described above. As a result, by multiplexing different BRET-biosensors of such ion conducting channels, the intramolecular novel BRET probes thus offer the possibility to reliably and rapidly test various candidates. 
     High-throughput screening for ion-channel function requires sensitive, simple assays and instrumentation that will report ion channel activity in living cells. The present invention thus relates to a nucleic acid or polynucleotide comprising a nucleotide sequence encoding a channel fusion subunit comprising a subunit of an ion conducting channel bound to a probe, wherein the probe comprises an ion sensor bound in between to at least a bioluminescent donor molecule and to at least a 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 to the N-terminal, the C-terminal, or within an intracellular or extracellular loop of the channel subunit via either the bioluminescent donor molecule or the protein fluorescent acceptor molecule, and wherein said bioluminescent donor molecule and acceptor molecule are selected so that the emission spectrum of the bioluminescent donor molecule overlaps with the absorbance spectrum of the acceptor molecule, so the light energy delivered by the bioluminescent donor molecule is at a wavelength that is able to excite the acceptor molecule. 
     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), the pET-series of expression vectors (Novagen) or pCRTOPO (Invitrogen) and vectors compatible with an expression in mammalian cells like 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 pClNeo (Promega). Examples for plasmid vectors suitable for  Pichia pastoris  comprise e.g. the plasmids pAO815, pPIC9K and pPIC3.5K (all Intvitrogen). The nucleic acid or polynucleotide of the present invention referred to above may also be inserted into vectors such that a translational fusion with another polynucleotide is generated. The other polynucleotide may encode a protein which may e.g. increase the solubility and/or facilitate the purification of the fusion protein. Non-limiting examples include pET32, pET41, pET43. The vectors may also contain an additional expressible polynucleotide coding for one or more chaperones to facilitate correct protein folding. Suitable bacterial expression hosts comprise e.g. strains derived from BL21 (such as BL21(DE3), BL21(DE3)PlysS, BL21(DE3)RIL, BL21(DE3)PRARE) or Rosetta®. In preferred embodiments, expression vector according to the invention is a DNA or RNA vector, capable of transforming eukaryotic host cells and effecting stable or transient expression of said TRP channel fusion subunit, and wherein said vector is a plasmid, a virus like adenovirus, adeno associated virus (AVV), lentiviral, Epstein-Barr, Herpes Simplex, Papilloma, Polyoma, Retro, SV40, Vaccinia, any retroviral vector, influenza viral vector and other non-viral vectors including naked DNA, or liposomes. 
     Transcriptional regulatory elements (parts of an expression cassette) ensuring expression in prokaryotes or eukaryotic cells are well known to those skilled in the art. These elements comprise regulatory sequences ensuring the initiation of the transcription (e.g., translation initiation codon, promoters, enhancers, and/or insulators), internal ribosomal entry sites (IRES) and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally associated or heterologous promoter regions. Preferably, the polynucleotide of the invention is operatively linked to such expression control sequences allowing expression in prokaryotes or eukaryotic cells. The vector may further comprise nucleotide sequences encoding secretion signals as further regulatory elements. Such sequences are well known to the person skilled in the art. Furthermore, depending on the expression system used, leader sequences capable of directing the expressed polypeptide to a cellular compartment may be added to the coding sequence of the polynucleotide of the invention. Such leader sequences are well known in the art. 
     Possible examples for regulatory elements ensuring the initiation of transcription comprise the cytomegalovirus (CMV) promoter, SV40-promoter, RSV-promoter (Rous sarcome virus), the lacZ promoter, human elongation factor 1α-promoter, CMV enhancer, CaM-kinase promoter, the  Autographa californica  multiple nuclear polyhedrosis virus (AcMNPV) polyhedral promoter or the SV40-enhancer. For the expression in prokaryotes, a multitude of promoters including, for example, the tac-lac-promoter, the lacUV5 or the trp promoter, has been described. Examples for further regulatory elements in prokaryotes and eukaryotic cells comprise transcription termination signals, such as SV40-poly-A site or the tk-poly-A site or the SV40, lacZ and AcMNPV polyhedral polyadenylation signals, downstream of the polynucleotide. 
     The present invention is further directed to a recombinant host cell containing an expression vector for expression of the ion conducting channel fusion unit as described above, wherein said vector contains a polynucleotide comprising a nucleic acid comprising a nucleotide sequence encoding a channel fusion subunit comprising a subunit of an ion conducting channel bound to a probe, wherein the probe comprises an ion sensor bound in between to at least a bioluminescent donor molecule and to at least a 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 to the N-terminal, the C-terminal, or within an intracellular or extracellular loop of the channel subunit via either the bioluminescent donor molecule or the protein fluorescent acceptor molecule, and wherein said bioluminescent donor molecule and acceptor molecule are selected so that the emission spectrum of the bioluminescent donor molecule overlaps with the absorbance spectrum of the acceptor molecule, so the light energy delivered by the bioluminescent donor molecule is at a wavelength that is able to excite the acceptor molecule. 
     Recombinant cells according to the present invention are thus genetically engineered to contain the polynucleotide construct of the ion channel fusion subunit as described above. The cell or host may be any prokaryote or eukaryotic cell and may be preferably a stable cell line. Suitable eukaryotic host may be a mammalian cell, an amphibian cell, a fish cell, an insect cell, a fungal cell or a plant cell. Eukaryotic cell may be an insect cell such as a  Spodoptera frugiperda  cell, a yeast cell such as a  Saccharomyces cerevisiae  or  Pichia pastoris  cell, a fungal cell such as an  Aspergillus  cell or a vertebrate cell. Suitable prokaryotes may be  E. coli  (e.g.,  E coli  strains HB101, DH5a, XL1 Blue, Y1090 and JM101),  Salmonella typhimurium, Serratia marcescens, Burkholderia glumae, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas stutzeri, Streptomyces lividans, Lactococcus lactis, Mycobacterium smegmatis  or  Bacillus subtilis.    
     Recombinant host cells according to the present invention thus comprise an expression vector wherein said channel fusion subunit is expressed and is able to 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 process for the production of said channel fusion subunits as described above comprising culturing said recombinant cell according to the invention and expressing said channel fusion subunit. 
     The present invention also relates to cell-free composition and assays comprising at least ion channel subunit in combination or coupled or bound to an ion sensor nucleotide sequence placed between a nucleotide sequence encoding bioluminescent donor molecule and a nucleotide sequence encoding at least one fluorescent acceptor molecule. Said cell-free composition comprises the ion channel fusion unit or a functional fusion channel as described above, wherein said fusion subunit is embedded in a lipid bilayer, preferably in a bilayer of a liposome. Such lipid bilayer comprises two layers of, typically amphiphilic, with a common hydrophobic bilayer interior and two hydrophilic surfaces. The lipid bilayer can be naturally occurring or artificial. Such lipid bilayer may be a cellular or bio-membrane for example from mammalian or yeast cell membrane, into which voltage-dependent ion channel fusion subunit is inserted. 
     Methods for preparing cell-free compositions from cells are well-known in the art and consist in obtaining recombinant cells as described above and disrupting the membrane of the cells. These methods generally include repeated cycles of freezing and thawing, grinding, treatment of cells with ultrasound in a sonicator device, homogenization, and use of a French press, the addition of detergent and/or enzymes, glass-bead lysis, differential centrifugation, and several density gradient procedures using a variety of gradient media. These techniques are inter alfa described in details in “Current Protocols in Protein Science”; John E. Caligan; Ben M. Dunn; Hidde L. Ploegh; David W. Speicher; Paul T. Wingfield; Wiley and Sons). For isolating or preparing cell membrane extracts, a combination of these methods is usually employed. Generally, cells are lysed either by mechanical means, or using detergents, and the membrane fractions isolated via differential centritugation. Liposomes containing recombinant cell as per the invention may be created by disrupting the phospholipid membrane of cells expressing the protein in water, for example by sonication. The phospholipids would reassemble into liposomal spheres containing a core of aqueous solution. Low shear rates would create multilamellar liposomes, which have many layers. Continued high-shear sonication would form smaller unilamellar liposomes, more suited to the application of the present invention. 
     The present invention further provides a method of assessing whether a test compound functions as a ligand for an ion conducting channel, the method comprising: (i) providing a cell comprising a nucleotide sequence encoding an ion conducting channel subunit bound to a probe, wherein the probe comprises an ion sensor bound in between to at least one bioluminescent donor molecule and at least one fluorescent acceptor molecule, wherein the probe is bound to the N-terminal or the C-terminal, or within an intracellular or extracellular loop of the channel subunit via either the bioluminescent donor molecule or the fluorescent acceptor molecule, and wherein said bioluminescent donor molecule and fluorescent acceptor molecule are selected so that the emission spectrum of the bioluminescent donor molecule overlaps with the absorbance spectrum of the acceptor molecule, making it possible the non-radiative energy transfer between the bioluminescent energy donor and the fluorescent acceptor through nonradiative dipole-dipole coupling; (ii) contacting said cell with a test compound; and (iii) determining the resultant reaction or interaction and output. 
     The method of screening in real time a compound candidate allow measuring the ion flux and thus the activation or inhibition of ion conducting channel, comprising contacting the candidate with the recombinant host cell or a cell free composition according to the invention, providing a substrate of the bioluminescent donor molecule; (iii) measuring the variation of BRET signal. Such methods allow the screening in real time agonist or antagonist of the ion conducting channel. 
     In a further aspect, the present invention provides a kit for screening agonist or inhibitor compound candidate of ion conducting channel comprising a nucleic acid or a polynucleotide of the invention, a vector of the invention, a recombinant cell or a host cell of the invention, or a cell-free composition or a composition of the invention, and/or a biosensor of the invention. 
     Throughout this application, various references are referred to and disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. 
     EXAMPLES 
     Example 1: Construction of an Intramolecular Probe of 2 nd  Generation 
     Intramolecular novel BRET probes as showed in  FIG.  1    have been constructed to measure the ionic flux in the microenvironment of voltage-gated ion channel pore, using ion binding protein sandwiched by YFP and Luc and fused to the intracellular extremities of several ion conducting channel. 
     Example 1.1: Construction of Potassium Binding BRET Probe 
     The K+-sensitive genetically encoded, bioluminescence resonance energy transfer-(BRET)based probes has been constructed by cloning the cDNA coding the amino acid sequence of KBP, as described by Ashraf et al. (2016) (Structure 2016, May 3; 24(5) 741-9), without the Stop codon, in fusion to the cDNA coding the NanoLuciferase at its 5′ extremity and in fusion to the cDNA coding the mNeonGreen fluorescent protein at its 3′ extremity, yielding to the NanoLuc-KBP-mNeonGreen BRET probe, or in fusion to the cDNA coding mNeonGreen at its 5′ extremity and in fusion to the cDNA coding the NanoLuciferase protein at its 3′ extremity, yielding to the NanoLuc-KBP-mNeonGreen BRET probe. A peptide spacer between the N-terminal acceptor or donor protein is not crucial to the operability of the sensor. A RMQDA peptide spacer may thus be optionally added in-between the N-terminal acceptor or donor protein and the KBP protein and a PLAEL peptide spacer is added in-between the KBP protein and the C-terminal donor or acceptor protein. 
     Example 1.2: Construction of Ionic Strenght—Sensitive BRET Probe 
     The ionic strength-sensitive genetically encoded, bioluminescence resonance energy transfer-(BRET) based probes has been constructed by cloning the cDNA coding the ionic strength sensor sequence described by Liu B. et al., (2017) (ACS Chem. Biol. 2017, 12, 2510-2514) and here referenced as SOFI, in fusion to the cDNA coding the NanoLuciferase at its 5′ extremity and in fusion to the cDNA coding the mNeonGreen fluorescent protein at its 3′ extremity, yielding to the NanoLuc-SOFI-mNeonGreen BRET probe, or in fusion to the cDNA coding mNeonGreen at its 5′ extremity and in fusion to the cDNA coding the NanoLuciferase protein at its 3′ extremity, yielding to the NanoLuc-SOFI-mNeonGreen BRET probe. A peptide spacer between the N-terminal acceptor or donor protein is not crucial to the operability of the sensor. A RMQDA peptide spacer may thus be optionally added in-between the N-terminal acceptor or donor protein and the calflux protein and a PLAEL peptide spacer is added in-between the calflux protein and the C-terminal donor or acceptor protein. 
     Example 1.3: Construction of Calcium Binding BRET Probe 
     The Ca 2+ -sensitive genetically encoded, bioluminescence resonance energy transfer-(BRET) based probes has been constructed by cloning the cDNA coding the amino acid sequence of calflux, as described by Yang J. et al., (2016) (Nature Communication, 2016, 27; 7:13268. doi: 10.1038/ncomms13268), without the Stop codon, in fusion to the cDNA coding the NanoLuciferase at its 5′ extremity and in fusion to the cDNA coding the mNeonGreen fluorescent protein at its 3′ extremity, yielding to the NanoLuc-calflux-mNeonGreen BRET probe, or in fusion to the cDNA coding mNeonGreen at its 5′ extremity and in fusion to the cDNA coding the NanoLuciferase protein at its 3′ extremity, yielding to the NanoLuc-calflux-mNeonGreen BRET probe. A peptide spacer between the N-terminal acceptor or donor protein is not crucial to the operability of the sensor. A RMQDA peptide spacer may thus be optionally added in-between the N-terminal acceptor or donor protein and the calflux protein and a PLAEL peptide spacer is added in-between the calflux protein and the C-terminal donor or acceptor protein. 
     Example 1.4: Construction of Chloride Binding BRET Probe 
     The chloride-sensitive genetically encoded, bioluminescence resonance energy transfer-(BRET) based probes has been constructed by cloning the cDNA coding the amino acid sequence of YFP or YFP variant in fusion to the cDNA coding the NanoLuciferase at its 3′ extremity and by introducing point mutations conferring chloride sensitivity to YFP construct (for instance mutations H148Q and I152L, as described by Galietta et al., FEBS letters, 2001, Jun. 22; 499(3):220-4. D01:10.1016/s0014-5793(01)02561-3). 
     Example 2: Expression Vectors Carrying the Sensor-Based BRET Probes 
     The expression of this protein in target mammals&#39; cells is made possible by the transfection of a vector of expression comprising the cDNA encoding a fusion protein comprising the channel of interest and the ion sensor using the BRET technology. 
     The transitory or stable expression of the vector in targeted mammalian cells allowed the direct measuring of the activity of the targeted channel by the BRET probe according to the invention. This protein allowed the emission of signal (light) during the addition of the luciferase substrate, such as Ccelenterazine H. Luciferase catalyzed the oxidation of the substrate. This biochemical reaction generates a light emission at a wavelength which is characteristic of the couple luciferase/substrate. If the acceptor which has an excitation spectrum compatible to the emission spectrum of the couple luciferase/substrate, is in proximity with the proper orientation, a non-radiative transfer of energy (BRET signal) will occur between the donor and the acceptor. This transfer of energy will be characterized by the fluorescence of the acceptor at its wavelength of emission ( FIG.  2   ). Typically, in order to have a transfer of energy between a donor and compatible acceptor, the 2 groups must be at a distance of around 10 nm from each other&#39;s, which is a distance compatible for the interaction&#39;s proteins-proteins. 
     Example 3: Calcium Sensitive BRET Probe Targeting the TRPV1 Channel 
     The intramolecular BRET probe: hTRPV1-nLuc-Calflux-YFP has been prepared wherein the DNA sequence encoding the human protein TRPV1 is fused in C-terminal to a calcium sensor, said sensor being sandwiched between the nano luciferase and YFP or mNeonGreen (Calflux probe, Yang J. et al., Nature Communication, 2016, 27; 7:13268. doi: 10.1038/ncomms13268.). Applicant showed that the basal BRET signal of the probe hTRPV1-nLuc-Calflux-YFP (or mNeonGreen) is modulated in dose-dependent manner further to the activation of the cells by Capsaïcin (CAPS), which is the principal agonist of TRPV1 ( FIG.  4 A ). 
     This activation was absolutely specific of the passage of the ions across TRPV1 for the following reasons:
         AMG517, which is a TRPV1 antagonist, shifted the dose-response to CAPS to the right indicating a competitive inhibition as expected ( FIG.  4 A );   The nLuc-Calflux-YFP probe when expressed alone without being fused to TRPV1 did not respond to the stimulation by CAPS in absence of co-expressed TRPV1 ion channel ( FIG.  4 A );   The hTRPV1-nLuc-Calflux D13A/D51A-YFP probe containing a double-mutation making the Calflux probe insensitive to calcium, did not respond to CAPS stimulation. This control was important to show that conformational change induced by CAPS on the TRPV1 channel did not participate to the increase of the observed BRET signal ( FIG.  4 A );   The non-specific diffusion of calcium into cells by using ionomycin (a calcium ionophore), did not cause a dose-dependent increase of the BRET signal, as measured with the free nLuc-Calflux-mNeonGreen probe, but only caused a marginal increase of the signal obtained with the TRPV-nLuc-Calflux-mNeonGreen probe ( FIG.  4 B ).       

     These data clearly evidenced that the herein described BRET probe targeting the TRPV1 channel specifically reflected the passage of ions across the TRPV1 channel and thus reflected TRPV1 real activity. 
     Analysis of this dose-response curve showed that CAPS induced TRPV1 activation with an EC50 of 328±33 nM, indicating a sensitivity of the BRET probe to CAPS with pharmacological properties similar to those found in the scientific literature using patch-clamp. 
     Example 4: Sensor-Based BRET Probe Targeting Canal KCa 2.3    
     The novel BRET probe has been constructed to measure the activity of potassium channel SK3 (KCa 2.3 ) by coupling the cDNA of the SK3 channel to a potassium sensor (protein KBP, Ashrafet KU al., 2016, Structure 24, 741-749) sandwiched in between luciferase and mNeonGreen (SK3-nLuc-KBP-mNeonGreen, See  FIG.  5 A ). 
     The SK channels are a family of potassium channel activated by the increase of the cytoplasmic calcium concentration. Opening of the SK3 channels induces a small outward flux of potassium ions diffusing through the opened pore according to the potassium ion electrochemical gradient. 
     Following addition of ionomycin at different concentrations, in order to induce a cytosolic increase of calcium and subsequently activating the SK3 channel, a decrease of the BRET signal measured in HEK293T cells expressing the SK3-nLuc-KBP-mNeonGreen probe indicating an efflux of potassium outside the cells ( FIG.  5 B ). The BRET sensor is based on KBP which has been published (Bischof H et al, 2017, Nat Commun. 8(1):1422), but this sensor was never coupled to a channel in order to specifically measure the activity of the channel as presented in this invention. 
     Example 5: Sensor-Based BRET Probe Targeting Receptor P2X2 
     We have constructed a rP2X2-nLuc-Calflux-YFP probe wherein the DNA sequence encoding the rat protein P2X2 is fused in C-terminal to a calcium sensor sandwiched in between the nanoluciferase and YFP or mNeonGreen (Calflux probe, Yang J. et al., Nature Communication, 2016, 27; 7:13268. doi: 10.1038/ncomms13268.). 
     We were able to show that the basal BRET signal of the rP2X2-nLuc-Calflux-YFP probe (or mNeonGreen) was modulated in a dose-dependent manner following the activation of the cells by ATP, which is the natural agonist of P2X2 ( FIG.  6 A ). This activation is specific of the passage of the ions through P2X2 for the following reasons: 
     1/ when the nLuc-Calflux-YFP probe is expressed alone without being fused to P2X2, the BRET probe does not respond to the ATP stimulations ( FIG.  6 B ); 
     2/ when the nLuc-Calflux-YFP probe is fused to the TRPV1 channel, this probe does not respond to the ATP stimulations ( FIG.  6 C ) but respond to the capsaicine (CAPS) stimulation, which is the agonist of TRPV1. 
     Analysis of the dose-response curve indicated that ATP activated the P2X2-NLuc-CalFlux-YFP probe with an EC50 of 888±274 nM, indicating that the ATP potency to activate P2X2 BRET probe is similar to the ATP potency to activate native P2X2 as found in the scientific literature using conventional techniques. 
     Example 6: Sensor-Based BRET Probe Targeting NMDA Receptors 
     We have constructed a novel BRET probe allowing to measure the activity of NMDA receptors by coupling the cDNA of the NR1 subunit to the BRET sensor nLuc-Calflux-YFP ( FIG.  7   ). NMDA receptors are heterotetramers comprising 2 NR1 subunits and 2 NR2 or NR3 subunits. The subunits NR1, NR2 or NR3 expressed separately cannot form functional receptors. 
     We were able to show that the BRET signal generated when expressing the BRET probe NR1-nLuc-Calflux-YFP alone is stable and is not modulated by increasing dose of NMDA ( FIG.  7 A ). However, the coexpression of this probe with the subunits NR2A ( FIG.  7 B ) or NR2B ( FIG.  7 C ) generated functional receptors and an increase of the signal was observed in HEK293T cells co-expressing NR1-nLuc-Calflux-YFP and NR2A subunits following cells&#39; stimulation with NMDA. 
     Analysis of the dose-response curve indicated that NMDA activated the NR1-nLuc-Calflux-YFP/NR2A and NR1-nLuc-Calflux-YFP/NR2B ion channels with EC50 of 79.7 μM+/−17.2 μM and 12.3+/−7.32 μM respectively. These data were perfectly in line with the scientific literature thus showing that heteromeric receptors comprising NR2A subunits are less sensitive to NMDA than receptors comprising NR2B subunits. 
     Example 7: Sensor-Based BRET Probe Targeting Calcium-Permeable Cation Channel TRPM8 
     The intramolecular BRET probe: hTRPM8-nLuc-Calflux-YFP has been prepared wherein the DNA sequence encoding the human protein TRPM8 is fused in C-terminal to a calflux-based calcium BRET sensor, said sensor being sandwiched between the nano luciferase and YFP or mNeonGreen. 
     Example 8: Sensor-Based BRET Probe Targeting Calcium-Permeable Cation Channel TRPV4 
     The intramolecular BRET probe: hTRPV4-nLuc-Calflux-YFP is prepared wherein the DNA sequence encoding the human protein TRPV4 is fused in C-terminal to a calflux-based calcium BRET sensor. 
     Example 9: Sensor-Based BRET Probe Targeting Sodium Channel Nav1.7 
     The intramolecular BRET probe: Nav1.7-nLuc-SOFI-YFP is prepared wherein the DNA sequence encoding the human protein TRPV4 is fused in C-terminal to a SOFI-based ionic strength BRET sensor. 
     Example 10: Sensor-based BRET Probe targeting potassium channel Kv7.1 
     The intramolecular BRET probe: Kv7.1-nLuc-KBP-YFP is prepared wherein the DNA sequence encoding the human protein TRPV4 is fused in C-terminal to a KBP-based potassium BRET sensor. 
     Example 11: Sensor-Based BRET Probe Targeting One Chloride Channel LRRC8A 
     The intramolecular BRET probe: LRRC8A-nLuc-YFP H148Q; 1152L is prepared wherein the DNA sequence encoding the human protein LRRC8A is fused in C-terminal to a Chloride-based BRET sensor comprising NanoLuc and a chloride sensitive YFP mutant (YFP H148Q; 1152L). 
     Example 12: Assessment of Calcium-Ion Flow Through TRPV1 Using Calcium Sensor-Based BRET Probe Targeting TRPV1 Ion Channel 
     As described above in Example 3, the intramolecular BRET biosensor probe hTRPV1-nLuc-Calflux-mNeonGreen has been prepared wherein the DNA sequence encoding the human protein TRPV1 is fused in C-terminal to a calcium sensor, said sensor being sandwiched between the nano luciferase and mNeonGreen (Calflux probe, Yang J. et al., Nature Communication, 2016, 27; 7:13268. doi: 10.1038/ncomms13268.). 
     HEK293T cells expressing mNeonGreen-Calflux-nLuc-hTRPV1 BRET biosensor were processed for BRET analysis. The basal BRET, measured in resting condition, dramatically increased with first-order kinetics after exposure to the prototypic TRPV1 agonist Capsaïcin (CAPS) but only faintly and transiently increased following exposure to ATP, that is known to trigger calcium release from intracellular stores through activation of endogenous P2Y receptors ( FIG.  8 A ). These results indicate that mNeonGreen-Calflux-nLuc-hTRPV1 BRET biosensor can specifically probe the calcium entering the cell through TRPV1 and is only marginally activated by calcium rise inside the cells. To exclude that the measured CAPS-induced BRET increase is not related with a conformational change into the quaternary structure of TRPV1 ion channel, we used the hTRPV1-nLuc-Calflux D/A-sYFP2 BRET probe comprising a calcium insensitive mutated Calflux reporter (D13A and D51A). Since no BRET increase could be significantly detected following CAPS activation in cells expressing this mutated BRET probe ( FIG.  8 B ), we concluded that the Calflux-based BRET biosensor fused to TRPV1 is effectively responding to the variation of calcium ion quantity in the nano-environment of the TRPV1 pore into the cytoplasm. We next confirmed that the agonist-induced BRET increase was dose dependent with half-maximal responses ( FIG.  8 C ) consistent with those reported in the literature, using patch-clamp or calcium-flux measurements on cells expressing endogenous or overexpressed TRPV1. The pharmacological selectivity of the ligand-promoted BRET changes was further demonstrated by the competitive nature of the effects, as AMG517, a well-known TRPV1 competitive antagonist, inhibited the CAPS-induced BRET increase, as illustrated by the shift in CAPS potency to higher values ( FIG.  8 C ). We also addressed whether mNeonGreen-Calflux-nLuc-hTRPV1 BRET biosensor could discriminate between known TRPV1 agonists and antagonists that are expected to display different potencies. In these experiments, HEK293T cells transiently expressing the mNeonGreen-Calflux-nLuc-hTRPV1 BRET biosensor were challenged with increasing quantities of four TRPV1 agonists ( FIG.  8 D ) or with increasing quantities of various TRPV1 antagonists ( FIG.  8 E ). For each tested compound we could measure a dose-dependent BRET modulation that correlate with the known ability of the tested ligand to either activate TRPV1 or inhibit Caps-induced TRPV1 activation. Importantly, RN1734, a known specific antagonist of TRPV4 failed to inhibit CAPS-activated TRPV1. Altogether, these data strongly suggest that the agonist-promoted BRET changes in both probe configurations correspond to an activation of TRPV1 channels in live cells. 
     Example 13: Assessment of Calcium-Ion Flow Through TRPV4, TRPM8 and PIEZO1 Using Calcium Sensor-Based BRET Probe 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 have been prepared wherein the DNA sequence encoding the human protein TRPM8 is fused in C-terminal to a calflux-based calcium BRET sensor, said sensor being sandwiched between the nano luciferase and mNeonGreen. In an effort to exemplify and validate the concept of Calcium sensor-based BRET probes fused to other ion channels, we first assessed whether the activity of two other TRPs ion channels, TRPV4 and TRPM8, could also be measured using such BRET probes. As shown in  FIGS.  9 A and  9 B , BRET signals measured on HEK293T cells transiently expressing mNeonGreen-Calflux-nLuc-hTRPV4 and mNeonGreen-Calflux-nLuc-hTRPM8 BRET probes were dose-dependently increased following addition in the cell culture medium of GSK1016790A and WS12, two specific agonists of TRPV4 and TRPM8 respectively. The measured half-maximal responses were consistent with those reported in the literature using patch-clamp or calcium-flux measurements on cells transiently expressing TRPV4 or TRPM8. The pharmacological selectivity of the ligand-promoted BRET changes was further demonstrated by the competitive nature of the effects, as both HC060747 and MBB, two well-known competitive antagonists of TRPV4 and TRPM8, respectively, right-shifted the corresponding agonist potency to higher values. Altogether, these data strongly suggest that the agonist-promoted BRET changes in TRPV4 and TRPM8 BRET probes correspond to an activation of these two ion channels in live cells. 
     We then constructed the mNeonGreen-Calflux-nLuc-PIEZO1 BRET probes that, according to the results obtained with TRPV1, TRPV4 and TRPM8, should be useful to assess calcium flow through the PIEZO1 channel if the N-terminus extremity of PIEZO1 is located inside the cytoplasm as suggested by others. As shown in  FIG.  9 C , BRET signal measured on HEK293T cells transiently expressing the mNeonG-Calflux-n Luc-PIEZO1 BRET probe was dose-dependently increased following addition in the cell culture medium of increasing quantities of YODA1, a known agonist of PIEZO1. This confirms the functionality of our calcium sensor-based BRET Probe targeting PIEZO1. 
     Example 14: Assessment of Potassium and Sodium-Ion Flow Through Non-TRPs Ion Channels Using Calcium Sensor-Based BRET Probe Targeting TREK1, KiR6.1 and NaV1.8 Channels 
     To go further into the demonstration that ion sensor-based BRET probes can be useful to probe other type of ion channels, we constructed mNeon-KBP-nLuc-TREK1 and mNeon-KBP-nLuc-KiR6.1, where the KBP potassium sensor was sandwiched in between mNeonGreen and NanoLuc and fused to the N-terminus of both TREK1 and KiR6.1 potassium channels, and mNeon-SOFI-nLuc-NaV1.8 BRET probe, where an ionic strength sensor was sandwiched in-between mNeonGreen and nanoLuc and fused to the N-terminus of the tetrodotoxin (TTX)-resistant voltage-gated sodium channels NaV1.8. TREK1 BRET probe was activated using the chemical activator BL1249. Kir6.1 BRET probe was activated using KCl. Following depolarization, a strong outward potassium current is known to occur in HEK293T cells. Extracellular potassium ions would therefore be expected to re-enter the cells through ectopically-expressed inward 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 that is known to prolong the open state of tetrodotoxin-resistant sodium channels. Repeated activation of TREK1 with increasing quantities of BL1249 dose-dependently increased the basal BRET measured with mNeon-KBP-nLuc-TREK1 BRET probe while no such effect could be detected using mock-activated conditions ( FIG.  10 A ). The increase of the BRET signal indicates that, following repeated activation of TREK1 with BL1241, the potassium concentration in the cytoplasmic microenvironment of the TREK1 pore increases. While surprising, this observation could nonetheless be explained knowing that TREK1 trigger hyperpolarisation of the cell membrane by allowing potassium to leak outside cells, thus lowering the intracellular potassium concentration which will tend to balance with the extracellular potassium concentration. In that conditions, it has been shown that potassium will inwardly flow through TREK1 opened pore, which is in line with our observations. As expected, mock activation with DMSO alone failed to induce TREK1 BRET probe activation ( FIG.  10 A ). We moreover could assess that KCl depolarisation could trigger an increase of the BRET signal measured on HEK293T cells expressing mNeon-KBP-nLuc-KiR6.1 ( FIG.  10 B ), again in agreement with the known inward rectifying activity of KiR6.1 ion channel. Finally, Deltamethrin successfully trigger a decrease of the BRET signal measured on HEK293T cells expressing mNeon-SOFI-nLuc-NaV1.8 ( FIG.  10 C ), indicating that ionic strength is increasing in the NaV1.8 pore microenvironment on the cytoplasmic side, in agreement with the notion that sodium is inwardly flowing through the NaV1.8 ion channel pore following its opening. Altogether, these results confirm that intramolecular BRET biosensors can probe the conformational changes occurring during the gating of ion channels belonging to various ion channels families and not just the TRP-ion channel family. 
     Example 15: Sensor-Based BRET Probe Targeting all P2X Receptors 
     We constructed human and murine P2X-NeonGreen-Calflux-nLuc probes wherein the DNA sequence encoding the seven human or mouse P2X subunits are individually fused in C-terminal to the calflux calcium sensor sandwiched in between the nanoluciferase and mNeonGreen ( FIG.  11    left panel). We were able to show that the basal BRET signal of the hP2X2, hP2X4, hP2X5, hP2X7 and mP2X4-NeonGreen-Calflux-nLuc probes were modulated in a dose-dependent manner following the activation of the cells by ATP (or Bz-ATP for hP2X7,  FIG.  11    right panel). Analysis of the dose-response curves indicated that ATP or BzATP activated the P2X-NeonGreen-Calflux-nLuc probes with 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 to activate native P2X as found in the scientific literature and measured using conventional techniques. 
     Example 16: Sensor-Based BRET Probe Targeting AMPA (GluA1) Receptors 
     We have constructed a second-generation BRET probe allowing to measure the activity of GluA1 receptors by coupling the cDNA of the subunit GluA1 to the BRET sensor nLuc-Calflux-YFP ( FIG.  12   , left panel). In order to block AMPA receptor desensitization by glutamate, we introduced 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). 
     We were able to show that the BRET signal generated when expressing the BRET probe GluA1-L497Y-nLuc-Calflux-YFP alone is stable and is modulated by increasing dose of glutamate ( FIG.  12   , right panel). Analysis of the dose-response curve indicated that glutamate activated the biosensor with EC50 of 8.98+/−1.019 μM. These data are in line with scientific literature reporting affinity of homomeric GluA1-L497Y receptors for glutamate obtained by patch clamp recording in HEK293 cells. 
     Example 17: Sensor-Based BRET Probe Targeting Cys-Loop Receptor Family 
     We have constructed second generation BRET probes allowing to measure the activity of cationic and anionic Cys-loop receptors. 
     For cationic receptors we inserted the cDNA of the sensor NLuc-CalFlux-sYFP2 (NL-CF-YFP) or NeonGreen-Calflux-nLuc (NG-CF-NL) in the second intracellular loop of the 5-HT3A receptor or the alpha-4 and beta-2 nicotinic receptor, respectively ( FIG.  13    A). We were able to show that the BRET signal generated when expressing the BRET probe 5-HT3A-NL-CF-YFP ( FIG.  13 C ) or a combination of alpha-4 and beta-2 nicotinic subunit fused to NG-CF-NL ( FIG.  13 B ) is stable and is modulated by the respective agonist stimulation. 
     We also developed a BRET probe allowing to sense anionic flux by fusing the cDNA of a chloride sensitive variant of YFP (YFP Chlore or YPetCI) to the cDNA of nLuc ( FIG.  13 D ). When expressed in HEK cells this probe is able to detect the anionic influx generated by VRAC channel opening following a hypotonic challenge ( FIG.  13 F ). Based on this probe, we generated a biosensor allowing to measure the activity of the anionic GABA receptors. We inserted the cDNA of the YPetCI sensor in the second intracellular loop of the GABARho1 subunit ( FIG.  13 E ). We were able to show that the BRET signal generated when expressing the BRET probe in combination or not with untagged GABARho1 subunit is stable and is modulated by GABA stimulation ( FIG.  13 G ).