NOVEL ION CONDUCTING CHANNEL FUSION SUBUNITS AND METHODS OF USE THEREOF
20230160909 · 2023-05-25
Inventors
- Yann PERCHERANCIER (Villenave d'Ornon, FR)
- Vincent COMPAN (Combaillaux, FR)
- Francois RASSENDREN (Montpellier, FR)
- Matthias OLLIVIER (Myzieu, FR)
- Yann CHAPPE (Montréal, CA)
Cpc classification
G01N33/6872
PHYSICS
International classification
G01N33/542
PHYSICS
Abstract
The present invention is directed to a channel fusion subunit comprising 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, C-terminal, or within an intracellular or extracellular loop of said 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.
Claims
1-15. (canceled)
16. A channel fusion subunit comprising 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, C-terminal, or within an intracellular or extracellular loop of said 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 possible the non-radiative energy transfer between the bioluminescent energy donor and the fluorescent acceptor through nonradiative dipole-dipole coupling.
17. The channel fusion subunit of claim 16, wherein the ion conducting channel subunit is a voltage-dependent ion channel or a ligand-dependent ion channel.
18. The channel fusion subunit of claim 16, wherein the ion sensor is a sensor to calcium, potassium, sodium, chloride ions or a sensor to ionic strength.
19. The channel fusion subunit of claim 16, wherein the ion sensor is chosen among calcium binding proteins, including troponin C calcium sensitive domain and calmodulin calcium binding protein, potassium binding proteins (KBP), sodium binding proteins, including NhAs-1 protein, and chloride binding proteins.
20. The channel fusion subunit of claim 16, further comprising a linker between the ion conducting channel subunit and the probe.
21. The channel fusion subunit of claim 16, wherein the bioluminescent donor molecule is a luciferase chosen among Renilla luciferase, Firefly luciferase, Coelenterate luciferase, North American glow worm luciferase, click beetle luciferase, a railroad worm luciferase, Gaussia luciferase, Aequorin, Arachnocampa luciferase, Nanoluciferase derived from deep sea shrimp Oplophorus gracilirostris and biologically active variants or fragments thereof, GLuc, NanoLuc (NLuc), MLuc7, HtLuc, LoLuc, PaLuc1, PaLuc2, MpLucl, McLucl, MaLucl, MoLucl, MoLuc2, MLuc39, PsLucl, LocLucl-3, HtLuc2 Renilla, TurboLucl6 (TLuc) and homologs, orthologs, mutants or functional derivatives thereof.
22. The channel fusion subunit of claim 16, wherein the bioluminescent donor molecule is a non-luciferase bioluminescent protein chosen among β-galactosidase, lactamase, horseradish peroxidase, alkaline phosphatase, β-glucuronidase, and β-glucosidase.
23. The channel fusion subunit of claim 16, wherein the acceptor molecule is a protein chosen among green fluorescent protein (GFP), a variant of green fluorescent protein (GFP10), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, mAmetrine, LSS-mOrange, LSS-mKate, Emerald, Topaz, GFPuv, destabilised EGFP (dEGFP), destabilised ECFP (dECFP), destabilised EYFP (dEYFP), HcRed, t-HcRed, DsRed, DsRed2, mRFPl, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein, mNeonGreen derived from Branchiostoma lanceolatum, a Phycobiliprotein, TagCFP, mTagCFP2, Czurite, ECFP2, mKalamal, Sirius, Sapphire, T-Sapphire, ECFP, Cerulean, SCFP3C, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP, mTFPl, EGFP, Emerald, Superfolder GFP, Monomeric Czami Green, TagGFP2, mUKG, mWasabi, Clover, Citrine, Venus, SYFP2, TagYFP, Monomeric Kusabira-Orange, .Math.ηKOκ, mK02, mOrange, mOrange2, mRaspberry, mCherry, mStrawberry, mScarlet, mTangerine, tdTomato, TagRFP, TagRFP-T, mCpple, mRuby, mRuby2, mPlum, HcRed-Tandem, mKate2, mNeptune, NirFP, TagRFP657, IFP1.4, iRFP and biologically active variants or fragments thereof.
24. A recombinant cell expressing the channel fusion subunit of claim 16.
25. A method of using the recombinant cell of claim 24 for screening a candidate compound for its capability to modulate ion flux through a channel of said cell, comprising: (a) contacting the candidate compound with said recombinant cell; (b) providing a substrate of the bioluminescent donor molecule; and (c) measuring a variation of bioluminescent resonance energy transfer.
26. A nucleic acid comprising a nucleotide sequence encoding a channel fusion subunit comprising an ion conducting channel subunit 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 that the light energy delivered by the bioluminescent donor molecule is at a wavelength that is able to excite the acceptor molecule.
27. The nucleic acid of claim 26, wherein the ion conducting channel subunit is a voltage-dependent ion channel or a ligand-dependent ion channel, wherein the ion sensor is a sensor to calcium, potassium, sodium, chloride ions or a sensor to ionic strength, optionally comprising a linker sequence between the nucleotide sequence encoding the ion conducting channel subunit and the nucleotide sequence encoding the probe.
28. The nucleic acid of claim 26, wherein the ion sensor is chosen among troponin C calcium binding domain, calmodulin calcium binding protein, potassium binding proteins (KBP), sodium binding proteins, including the NhAs-1 protein, and chloride binding proteins.
29. The nucleic acid of claim 26, wherein the bioluminescent donor molecule is a luciferase chosen among Renilla luciferase, Firefly luciferase, Coelenterate luciferase, North American glow worm luciferase, click beetle luciferase, a railroad worm luciferase, Gaussia luciferase, Aequorin, Arachnocampa luciferase, Nanoluciferase derived from deep sea shrimp Oplophorus gracilirostris, or a biologically active variant or fragment thereof, GLuc, NanoLuc (NLuc), MLuc7, HtLuc, LoLuc, PaLuc1, PaLuc2, MpLucl, McLucl, MaLucl, MoLucl, MoLuc2, MLuc39, PsLucl, LocLucl-3, HtLuc2 Renilla, TurboLucl6 (TLuc) or homologs or orthologs thereof or mutants or functional derivatives thereof, or wherein the bioluminescent donor molecule is a non-luciferase bioluminescent protein chosen among β-galactosidase, lactamase, horseradish peroxidase, alkaline phosphatase, β-glucuronidase, or β-glucosidase.
30. The nucleic acid of claim 26, wherein the acceptor molecule is a protein chosen among green fluorescent protein (GFP), variant of green fluorescent protein (GFP10), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, mAmetrine, LSS-mOrange, LSS-mKate, Emerald, Topaz, GFPuv, destabilised EGFP (dEGFP), destabilised ECFP (dECFP), destabilised EYFP (dEYFP), HcRed, t-HcRed, DsRed, DsRed2, mRFPl, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein, mNeonGreen derived from Branchiostoma lanceolatum, a Phycobiliprotein, TagCFP, mTagCFP2, Czurite, ECFP2, mKalamal, Sirius, Sapphire, T-Sapphire, ECFP, Cerulean, SCFP3C, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP, mTFPl, EGFP, Emerald, Superfolder GFP, Monomeric Czami Green, TagGFP2, mUKG, mWasabi, Clover, Citrine, Venus, SYFP2, TagYFP, Monomeric Kusabira-Orange, .Math.ηKOκ, mK02, mOrange, mOrange2, mRaspberry, mCherry, mStrawberry, mScarlet, mTangerine, tdTomato, TagRFP, TagRFP-T, mCpple, mRuby, mRuby2, mPlum, HcRed-Tandem, mKate2, mNeptune, NirFP, TagRFP657, IFP1.4, iRFP and biologically active variants or fragments thereof.
Description
BRIEF DESCRIPTION OF THE FIGURES
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DETAILED DESCRIPTION
[0027] 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 & Sons (including all updates until present).
[0028] 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.
[0029] 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.
[0030] 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.
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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.sup.−5M, more preferably 10.sup.−7M, most preferably 10.sup.−9M or less.
[0035] 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).
[0036] 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.
[0037] 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.
[0038] The terms “more sensitive” refer to a greater change in resonance energy transfer ratio between the bioluminescent donor molecule and the fluorescent acceptor molecule.
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] 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.
[0048] As used herein, the terms “determining,” “measuring,” “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations.
[0049] 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.
[0050] 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.
[0051] 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.sup.+) channels are responsible for rapid spike-like action potentials. During action potentials the majority of Na.sup.+ channels open very briefly. These brief openings result in transient Na.sup.+ currents. However, a subset of voltage-gated Na.sup.+ channels does not close rapidly but remain open for relatively long intervals. These channels therefore generate sustained or persistent Na.sup.+ currents. The balance between transient and persistent Na.sup.+ current is crucial for maintaining normal physiological function and electrical signaling throughout the entire nervous system.
[0052] 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's disease, Alzheimer's disease, Huntington'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.
[0053] 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.
[0054] 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.
[0055] 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.sup.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.sup.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.sup.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.sup.2+, additional steps like internal Ca.sup.2+ buffering (1 mM EGTA) with elevated 10 mM extracellular Ca.sup.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.
[0056] 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.
[0057] 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.
[0058] 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. . . . .
[0059] 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.
[0060] 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.
[0061] 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.
[0062] 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.
[0063] 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.sup.+, K.sup.+, Ca.sup.2+, and/or Cl.sup.− to flow across the membrane. The pore is responsible for the permeability to the selected ion(s).
[0064] 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.
[0065] 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.sup.+, K.sup.+, and Ca.sup.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.sup.+ channels, or channels that are made of four homologous domains include Na.sup.+ and Ca.sup.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.sup.+, K.sup.+, and Ca.sup.2+ channels that underlie the action potentials and the rapid calcium signaling of electrically excitable cells. Fast voltage-dependent opening of Na.sup.+ 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.
[0066] 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.sup.+ 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.sup.+ channels.
[0067] 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.
[0068] 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.
[0069] A complete account of the voltage-gated ion channels that are within the scope of the present invention, are tabulated below.
TABLE-US-00001 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-US-00002 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-US-00003 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' 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-US-00004 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-US-00005 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-US-00006 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-US-00007 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-US-00008 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-US-00009 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-US-00010 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-US-00011 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
[0070] 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.sup.+, K.sup.+, Ca.sup.2+, and/or Cl.sup.− to pass through the membrane in response to the binding of a chemical messenger, i.e. a ligand.
[0071] 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.
[0072] 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. . . . .
[0073] 5-HT.sub.3 receptors or 5-hydroxytryptamine (serotonin) receptors exist as a pentamer of 4™ subunits that form an intrinsic cation selective. Five human 5-HT.sub.3 receptor subunits including 5-HT.sub.3A (https://www.uniprot.org/uniprot/P46098), 5-HT.sub.3B (https://www.uniprot.org/uniprot/P46098), 5-HT.sub.3c (https://www.unprot.org/uniprot/Q8WXA8), 5-HT3D (https://www.uniprot.org/uniprot/Q70Z44), 5-HT.sub.3E (https://www.uniprot.org/uniprot/A5X5Y0) have been cloned and homo-oligomeric assemblies of 5-HT.sub.3A and hetero-oligomeric assemblies of 5-HT.sub.3A and 5-HT.sub.3B subunits have been fully characterized (5-HT.sub.3A and 5-HT.sub.3AB receptors). The 5-HT.sub.3C subunit, as well as 5-HT.sub.3D and 5-HT.sub.3E subunits, like the 5-HT.sub.3B subunit, do not form functional homomers, but assemble with the 5-HT.sub.3A 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.
[0074] 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.sup.+, K.sup.+ and Cs.sup.+, but impermeable to Ca.sup.2+ and Mg.sup.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.sup.2+, although ZAC has also been found to activate spontaneously.
[0075] GABA.sub.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.sub.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.sub.A receptors in the CNS, followed by the α2β3γ2 and α3β3γ2 isoforms. Currently, eleven native GABA.sub.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.sub.A receptor selectively conducts chloride (Cl.sup.−) through its pore. Several inhibitory drugs have been identified including muscimol, gaboxadol, bicuculline, the benzodiazepines, nonbenzodiazepines, alprazolam, diazepam, and picrotoxin.
[0076] 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.
[0077] 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.
[0078] 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.
[0079] 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.sup.+, K.sup.+ and Ca.sup.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).
[0080] 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.sup.+ 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.sup.+ 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.
[0081] 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. . . . ).
[0082] 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.
[0083] 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.
[0084] 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.
[0085] 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.
[0086] 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.
[0087] 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.
[0088] All known kinds of luciferases, either naturally occurring luciferases or mutated luciferases are encompassed within the scope of the invention.
[0089] 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.
[0090] 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.
[0091] 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.
[0092] 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.
[0093] 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.
[0094] 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).
[0095] By way of examples, several bioluminescent donor molecules have been listed in the following Table 12.
TABLE-US-00012 TABLE 12 Examples of bioluminescent molecules MW Emission Species Name Organism kDa × 10.sup.−3 (nm) Substrate Insect FFluc Photinus pyralis ~61 560 D-(−)-2-(6'-hydroxybenzo- (North American Firefly) thiazolyl)-Δ.sup.2-thiazoline-4- carboxylic acid, HBTTCA, (C.sub.11H.sub.8N.sub.2O.sub.3S.sub.2)(Luciferin) Insect FF'luc Luciola cruciate 560-590 (many Luciferin (Japanese Firefly) mutants) Insect Phengodid beetles(railroad worms) Insect Arachnocampasp. 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 Obeliabiscuspidata ~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
[0096] 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.
[0097] 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.
[0098] 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.
[0099] Several acceptor molecules either protein or non-proteinaceous may be used in the intramolecular novel BRET probe according to the present invention.
[0100] 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, .Math.ηKOκ, 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.
[0101] 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.
[0102] 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.
[0103] 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.sup.2+ Dye; Calcium Green-2 Ca.sup.2+; Calcium Green-5N Ca.sup.2+; Calcium Green-C18 Ca.sup.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.
[0104] 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.
[0105] 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.
[0106] 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.
[0107] 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.
[0108] 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.
[0109] 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.
[0110] 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.
[0111] 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.
[0112] 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.
[0113] 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.
[0114] 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.sup.2+, and the binding of Ca.sup.2+ is required for the activation of calmodulin. Once bound to Ca.sup.2+, calmodulin acts as part of a calcium signal transduction pathway by modifying its interactions with various target proteins such as kinases or phosphatase.
[0115] 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.
[0116] 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.sup.2+ ions, rendering it inactive. The Ca.sup.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.
[0117] 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.
[0118] 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.sup.+ 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.sup.+ 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.sup.+ 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.sup.+ 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.
[0119] 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.
[0120] 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.
[0121] 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.
[0122] 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.
[0123] 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.
[0124] 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.
[0125] 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.
[0126] 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.
[0127] 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.
[0128] 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.
[0129] 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.
[0130] 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.
[0131] 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.
[0132] 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.
[0133] 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.
[0134] 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.SUP.nd .Generation
[0135] Intramolecular novel BRET probes as showed in
Example 1.1: Construction of Potassium Binding BRET Probe
[0136] 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
[0137] 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
[0138] The Ca.sup.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
[0139] 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
[0140] The expression of this protein in target mammals' 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.
[0141] 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 (
Example 3: Calcium Sensitive BRET Probe Targeting the TRPV1 Channel
[0142] 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 (
[0143] This activation was absolutely specific of the passage of the ions across TRPV1 for the following reasons: [0144] AMG517, which is a TRPV1 antagonist, shifted the dose-response to CAPS to the right indicating a competitive inhibition as expected (
[0148] 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.
[0149] 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.SUB.2.3
[0150] The novel BRET probe has been constructed to measure the activity of potassium channel SK3 (KCa.sub.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
[0151] 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.
[0152] 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 (
Example 5: Sensor-Based BRET Probe Targeting Receptor P2X2
[0153] 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.).
[0154] 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 (
[0155] 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 (
[0156] 2/ when the nLuc-Calflux-YFP probe is fused to the TRPV1 channel, this probe does not respond to the ATP stimulations (
[0157] 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
[0158] 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 (
[0159] 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 (
[0160] 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
[0161] 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
[0162] 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
[0163] 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
[0164] 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
[0165] 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
[0166] 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.).
[0167] 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 (
Example 13: Assessment of Calcium-Ion Flow Through TRPV4, TRPM8 and PIEZO1 Using Calcium Sensor-Based BRET Probe Targeting these Ion Channels
[0168] 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
[0169] 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
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
[0170] 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 (
Example 15: Sensor-Based BRET Probe Targeting all P2X Receptors
[0171] 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 (
Example 16: Sensor-Based BRET Probe Targeting AMPA (GluA1) Receptors
[0172] 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 (
[0173] 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 (
Example 17: Sensor-Based BRET Probe Targeting Cys-Loop Receptor Family
[0174] We have constructed second generation BRET probes allowing to measure the activity of cationic and anionic Cys-loop receptors.
[0175] 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 (
[0176] 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 (