Application of substances that increase the C-terminal content of Slack protein

Abstract

The present invention provides an application of a substance for increasing the C-terminal content of Slack protein, which relates to the field of pharmaceutical technology. The application is the application of a substance for increasing the C-terminal content of Slack protein in the preparation of a drug for treating, assisting in the treatment and / or preventing epilepsy. The drug for treating epilepsy is a combination of a Slack mutant and Na V 1.6 Interaction interface as target, Slack mutant C-terminus and voltage-gated sodium channel Na V 1.6 N and / or C-terminal interaction, the present invention found that the voltage-gated sodium channel Na V 1.6 mediates sodium influx and activates Slack, forming a channel complex. In vivo mouse and in vitro cell experiments have shown that substances that upregulate the C-terminus of the Slack protein can reduce the current amplitude of Slack mutants, reduce epilepsy susceptibility in mouse models, and have therapeutic effects on epilepsy.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to an application of a substance for increasing the C-terminal content of a Slack protein. Background Art

[0002] Epilepsy is a chronic neurological disorder that seriously impacts human health. It is not a disease characterized by a single phenotype or single etiology, but rather a complex disorder characterized by high genetic susceptibility and multiple risk factors. Epilepsy is associated with increased synchronous neuronal firing, abnormal firing, and elevated overall neuronal excitability. Based on its genetic basis, epilepsy can be divided into secondary epilepsy and hereditary epilepsy. Secondary epilepsy is primarily caused by brain trauma, stroke, infection, brain tumors, and other causes. Hereditary epilepsy is caused by mutations in epileptogenic genes and primarily develops during infancy. With the advancement of gene sequencing technology, hereditary epilepsy has garnered increasing attention. Whole-genome sequencing has revealed that 30-50% of epilepsy patients harbor genetic mutations in over 60 genes, including ion channels, synaptic proteins, and transcriptional regulators, most of which are de novo mutations. Ion channels control neuronal electrical signaling and regulate the excitation-inhibition balance of neural networks. They are the primary single-gene defects in hereditary epilepsy, including sodium, potassium, and calcium channels.

[0003] Among potassium channels, missense mutations in the KCNT1 gene, encoding the sodium-activated potassium channel Slack, can lead to a range of epilepsy syndromes, potentially accompanied by intellectual disability. Over 50 pathogenic mutations have been identified, most of which result in a gain-of-function (GOF) phenotype. The most common epileptic seizures are divided into two categories: autosomal dominant or sporadic sleep-related hypermotor epilepsy (AD)SHE and epilepsy of infancy with migrating focal seizures (EIMFS).

[0004] A high proportion of patients with KCNT1-related epilepsy suffer from refractory epilepsy, poor response to conventional antiepileptic drugs, and severe developmental impairments, necessitating the development of therapeutic agents. A therapeutic strategy targeting this enhanced-function phenotype is to screen for highly selective channel inhibitors. Quinidine, a Slack channel inhibitor, effectively reverses the increased current caused by various KCNT1 pathogenic mutations. It has subsequently been used in the clinical treatment of KCNT1-mutant epilepsy, reducing seizures in some patients. However, subsequent mutations have been identified that are ineffective against quinidine treatment, and clinically, many KCNT1 mutations are insensitive to quinidine treatment. As an antiarrhythmic drug, quinidine inhibits the cardiac hERG channel, potentially leading to QT interval prolongation. Some patients who fail to achieve seizure reduction during treatment are at risk for heart disease. Due to cardiac safety risks, such as QT interval prolongation, quinidine plasma concentrations must be monitored during treatment; concentrations exceeding 18.5 μM are prone to toxic effects.

[0005] Chinese patent CN202080013384.4 discloses the use of cannabidiol in the treatment of epilepsy caused by mutations in the KCNT1 gene. The CBD used in this patent is in the form of a highly purified extract of cannabis, such that CBD is present in more than 98% (w / w) of the total extract, and the other components of the extract are characterized. CBD can be used simultaneously with one or more other anti-epileptic drugs, or it can be provided in combination in a single dosage form. However, cannabidiol has certain toxic side effects on the human body.

[0006] Currently, there are no reports on substances that increase the C-terminal content of the Slack protein to treat epilepsy by reducing the current amplitude of Slack mutants and thus reducing epilepsy susceptibility. Summary of the Invention

[0007] Terms and Claims of the Present Invention:

[0008] As used herein, the articles "a," "an," and "the" include plural referents unless expressly limited to one or more referents otherwise.

[0009] As used herein, the term "protein" refers to at least two covalently linked amino acids, including proteins, polypeptides, oligopeptides, and peptides. The term also encompasses post-expression modifications of proteins, such as glycosylation, acetylation, and phosphorylation. The term also encompasses variants resulting from modifications to the amino acid sequence of native proteins or polypeptides, such as deletions, substitutions, and insertions.

[0010] In the present invention, the term "secondary epilepsy" refers to epilepsy with a clear etiology. The causes of secondary epilepsy include congenital diseases, prenatal and perinatal injuries, sequelae of febrile seizures, craniocerebral injury, intracranial infection, poisoning, intracranial tumors, cerebrovascular disease, nutritional and metabolic diseases, and other factors.

[0011] In the present invention, the term "genetic epilepsy" refers to a chronic neurological disease with no obvious organic or metabolic abnormalities in the brain, the mechanism of seizures may be related to genetic factors, and the main manifestations are varying degrees of impaired consciousness, autonomic nervous system symptoms and psychiatric symptoms.

[0012] In the present invention, the term "Slack protein" refers to a sodium ion-activated potassium ion channel protein.

[0013] In the present invention, the term "voltage-gated sodium ion channel Na V 1.6" is a voltage-gated ionic channel present in most neurons including the central nervous system and peripheral nervous system, and is one of the sodium channels in the central nervous system.

[0014] In the present invention, the term "KCNT1 gene" is located on chromosome 9q34.3, is involved in encoding sodium-gated potassium channels, is highly expressed throughout the central nervous system, plays an important role in regulating neuronal excitability, and in normal neuroelectrophysiology, is mainly involved in generating slow afterhyperpolarization after a single action potential or action potential burst.

[0015] In the present invention, the term "expression" refers to the process by which cells, during their life cycle, convert the genetic information stored in DNA sequences into protein molecules with biological activity through transcription and translation.

[0016] In the present invention, the term "antisense oligonucleotide" refers to a short-chain nucleic acid (consisting of about 15-25 nucleotides) that has undergone certain chemical modifications, whose base sequence is complementary to a specific target sequence, and after entering the cell, it can form a double-stranded structure with the target sequence according to the principle of Watson-Crick base complementary pairing.

[0017] In the present invention, the term "ribozyme" refers to an RNA molecule having the function of catalyzing a specific biochemical reaction.

[0018] In the present invention, the term "siRNA" refers to a ribonucleic acid (RNA) that can inhibit the expression of a target gene and includes a sense RNA segment region and an antisense RNA segment region.

[0019] In the present invention, the term "miRNA" refers to a ribonucleic acid (RNA) molecule of approximately 21 to 23 nucleotides in length that is widely present in eukaryotes and can regulate the expression of other genes.

[0020] As used herein, the term "therapeutically effective amount" refers to an amount of at least one agent or compound that, when administered, is sufficient to alleviate to some extent one or more symptoms of the disease or condition being treated. This can result in a reduction and / or alleviation of signs, symptoms, or causes of disease, or any other desired change in a biological system. Techniques such as dose escalation studies can be used to determine the effective amount appropriate for any individual case.

[0021] In the present invention, the term "pharmaceutically acceptable" means that a carrier, vehicle, diluent, excipient, and / or salt formed is generally chemically or physically compatible with other ingredients constituting a pharmaceutical dosage form and physiologically compatible with the receptor.

[0022] The technical solution of the present invention includes:

[0023] In a first aspect, the present invention provides use of a substance that increases the C-terminal content of a Slack protein in the preparation of a drug for treating, assisting in the treatment and / or prevention of epilepsy.

[0024] Preferably, the substance that increases the content of the C-terminus of the Slack protein is the C-terminus of the Slack protein and / or a substance that upregulates the expression of the C-terminus of the Slack protein.

[0025] Further preferably, the C-terminus of the Slack protein is the Slack 796-839 protein fragment.

[0026] In a second aspect, the present invention provides a use of a substance that upregulates the expression of the C-terminus of the Slack protein in the preparation of a drug for treating epilepsy.

[0027] Preferably, the Slack protein C-terminus includes the Slack protein C-terminal fragment 326-1238;

[0028] Further preferably, the C-terminus of the Slack protein is the Slack 796-839 protein fragment.

[0029] Preferably, the substance that upregulates the expression of the C-terminal end of the Slack protein includes a substance that upregulates the synthesis of the C-terminal end of the Slack protein, a substance that inhibits the degradation of the C-terminal end of the Slack protein, or a substance that promotes the protein function of the C-terminal end of the Slack protein.

[0030] Specifically, the substance that upregulates the expression of the C-terminal end of the Slack protein is a Slack mutant and a voltage-gated sodium channel Na V 1.6 The interaction interface is the target.

[0031] More specifically, the Slack mutant and the voltage-gated sodium channel Na V 1.6 Interaction between the C-terminus of the Slack mutant and the voltage-gated sodium channel NaV 1.6 N- and / or C-terminal interactions.

[0032] Specifically, the substance that upregulates the expression of the C-terminal end of the Slack protein is by destroying the Slack mutant and the voltage-gated sodium ion channel Na V 1.6 They work by interaction.

[0033] More specifically, the substance that upregulates the synthesis of the C-terminal end of the Slack protein reduces the amount of Na transported to the Slack mutant. + .

[0034] More specifically, the substance that upregulates the synthesis of the C-terminus of the Slack protein is used to reduce the current density of the Slack mutant.

[0035] Preferably, the drug comprises a first active ingredient, and the first active ingredient is selected from at least one of a synthetic small molecule, a chemical reagent, an antisense oligonucleotide, a siRNA, a miRNA, a ribozyme, a polypeptide, and a protein.

[0036] Further preferably, the polypeptide or protein includes hormones, cytokines, antibodies and fragments thereof.

[0037] Specifically, the epilepsy is secondary epilepsy or hereditary epilepsy.

[0038] More specifically, the epilepsy is hereditary epilepsy.

[0039] More specifically, the epilepsy is hereditary epilepsy associated with KCNT1 mutation.

[0040] Preferably, the drug further comprises a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier is selected from at least one of a diluent, a binder, a surfactant, a lubricant, a filler, a disintegrant, and a stabilizer.

[0041] Still further preferably, the diluent includes but is not limited to starch, lactose, glucose, sodium chloride, and urea.

[0042] Still further preferably, the binder includes but is not limited to dextrin, sucrose, gum arabic, ethyl cellulose, polyvinyl alcohol, pregelatinized starch, maltodextrin, polyethylene glycol, carboxymethyl cellulose, polyvinyl pyrrolidone, gelatin, hydroxypropyl cellulose and hydroxypropyl methylcellulose.

[0043] Still further preferably, the surfactant includes but is not limited to polyoxyethylene sorbitan fatty acid ester, stearic acid monoglyceride, sodium lauryl sulfate, and cetyl alcohol.

[0044] Still further preferably, the lubricant includes but is not limited to glyceryl monostearate, talc, zinc stearate, sodium stearyl fumarate, polyethylene glycol, monolauric sucrose ester, polyethylene glycol, sodium lauryl sulfate, magnesium lauryl sulfate, polyoxyethylene monostearate, and magnesium lauryl sulfate.

[0045] Still further preferably, the filler includes but is not limited to xylitol, maltose, sorbitol, lactose, sucrose, dextrin, mannitol, glucose, starch, sodium alginate, erythrose, laminarin powder, microcrystalline cellulose, agar powder, calcium carbonate and sodium bicarbonate.

[0046] Still further preferably, the disintegrant includes but is not limited to sodium carboxymethyl starch, cross-linked vinyl pyrrolidone, low-substituted hydroxypropyl methyl, and cross-linked sodium carboxymethyl cellulose.

[0047] Still further preferably, the stabilizer includes but is not limited to human serum protein, L-amino acids, sugars and cellulose derivatives.

[0048] In a third aspect, the present invention provides a drug for treating, assisting in the treatment and / or preventing epilepsy, characterized in that the drug contains a therapeutically effective amount of a substance that increases the C-terminal content of the Slack protein.

[0049] Preferably, the substance that increases the content of the C-terminus of the Slack protein is the C-terminus of the Slack protein and / or a substance that upregulates the expression of the C-terminus of the Slack protein.

[0050] Further preferably, the C-terminus of the Slack protein is the Slack 796-839 protein fragment.

[0051] Specifically, the drug for treating epilepsy is a combination of Slack mutants and Na V 1.6 The interaction interface is the target.

[0052] Specifically, the mechanism of action of the substance that upregulates the expression of the C-terminal end of the Slack protein is to interfere with the Slack mutant and Na V 1.6 Interaction.

[0053] Specifically, the substances that upregulate the expression of the C-terminal end of the Slack protein interfere with the expression of the Slack mutant and Na V 1.6 interaction, which in turn leads to a decrease in the current density of the Slack mutant.

[0054] Preferably, the drug comprises a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier is selected from at least one of a diluent, a binder, a surfactant, a lubricant, a filler, a disintegrant, and a stabilizer.

[0055] In a fourth aspect, the present invention provides the use of the above-mentioned drug and other drugs for treating epilepsy in the preparation of drugs for treating epilepsy.

[0056] Preferably, the other drugs for treating epilepsy are selected from at least one of carbamazepine, oxcarbazepine, clonazepam, diazepam, phenobarbital, phenytoin sodium, primidone, sodium valproate, gabapentin, lamotrigine, levetiracetam, and vigabatrin.

[0057] Preferably, the drug can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted drug reservoir. The drug of the present invention may contain any commonly used non-toxic pharmaceutically acceptable carrier, adjuvant or vehicle.

[0058] Preferably, the dosage forms of the drug include tablets, capsules, granules, pills, pellets, syrups, powders, granules, suppositories, drops, aerosols, emulsions, injections and suspensions.

[0059] In a fifth aspect, the present invention provides a method for treating epilepsy, comprising administering to a patient a substance that increases the C-terminal content of the Slack protein.

[0060] Preferably, the substance that increases the content of the C-terminus of the Slack protein is the C-terminus of the Slack protein and / or a substance that upregulates the expression of the C-terminus of the Slack protein.

[0061] Further preferably, the C-terminus of the Slack protein is the Slack 796-839 protein fragment.

[0062] Specifically, the substance that upregulates the expression of the C-terminal end of the Slack protein is a Slack mutant and a voltage-gated sodium ion channel Na V 1.6 The interaction interface is the target.

[0063] More specifically, the Slack mutant and the voltage-gated sodium channel Na V 1.6 Interaction between the C-terminus of the Slack mutant and the voltage-gated sodium channel Na V 1.6 N- and / or C-terminal interactions.

[0064] Specifically, the epilepsy is secondary epilepsy or hereditary epilepsy.

[0065] More specifically, the epilepsy is hereditary epilepsy.

[0066] More specifically, the epilepsy is hereditary epilepsy associated with KCNT1 mutation.

[0067] The beneficial effects of the present invention include:

[0068] The present invention discovered that voltage-gated sodium channel Na V 1.6 mediates sodium influx and activates Slack, which forms a channel complex. The C-terminus of the Slack channel and Na V In vitro cell models and in vivo mouse models have shown that overexpression of the Slack channel C-terminus can reduce the current amplitude of Slack mutants and reduce the Slack G288S Overexpression of Slack in mouse models of epilepsy has potential therapeutic effects. V 1.6 interaction is expected to become an innovative therapy for KCNT1-related epilepsy. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 To co-express Slack and Na V Current traces recorded in HEK293 cells at 1.6 with a 100ms prepulse of -90mV;

[0070] The figure shows the co-expression of Slack and Na V 1.6 Current traces recorded from HEK293 cells, extracellular sodium concentration [Na + ] out is 140 mM, and the intracellular sodium concentration [Na + ] in is 5mM.

[0071] Figure 2 To co-express Slack and Na V Current traces recorded in HEK293 cells at 1.6 with a 100ms prepulse of -40mV;

[0072] The figure shows the co-expression of Slack and Na V 1.6 Current traces recorded from HEK293 cells, extracellular sodium concentration [Na + ] out is 140 mM, and the intracellular sodium concentration [Na + ] in is 5mM.

[0073] Figure 3 This is the current-voltage curve of Slack;

[0074] The picture shows Slack and Na V 1.6 IV curves of Slack induced by a -90 mV or -40 mV prepulse during co-expression.

[0075] Figure 4 To co-express Slack and Na VCurrent traces recorded in HEK293 cells at 1.6;

[0076] Among them, there is no riluzole in the bath solution, and the extracellular sodium concentration [Na + ] out is 140 mM, and the intracellular sodium concentration [Na + ] in is 5mM.

[0077] Figure 5 To co-express Slack and Na V Current traces recorded in HEK293 cells at 1.6;

[0078] The concentration of riluzole in the bath solution was 20 μM, and the extracellular sodium concentration [Na + ] out is 140 mM, and the intracellular sodium concentration [Na + ] in is 5mM.

[0079] Figure 6 This is the current-voltage curve of Slack;

[0080] The figure shows the bath solution without riluzole and with 20 μM riluzole, Slack and Na V 1.6 IV curve of co-expression.

[0081] Figure 7 Figure 2 shows the afterhyperpolarization potentials generated in Scn8a+ / + neurons and Scn8a+ / - neurons when stimulated by 300 pA.

[0082] In the figures, **p<0.01; unpaired two-tailed Student's t-test.

[0083] Figure 8 Figure 3 shows the afterhyperpolarization potentials generated in Scn8a+ / + neurons and Scn8a+ / - neurons when stimulated by 100 Hz pulses.

[0084] In the figures, **p<0.01; unpaired two-tailed Student's t-test.

[0085] Figure 9 is the immunofluorescence image in hippocampal CA1;

[0086] In the figure, Slack, Na V 1.2 Na V 1.6 is green, AnkG is red, and DAPI is blue.

[0087] Figure 10 is the immunofluorescence image in the neocortex;

[0088] In the figure, Slack, NaV 1.2 Na V 1.6 is green, AnkG is red, and DAPI is blue.

[0089] Figure 11 Slack and Na in cell lysate V Figure 1.6 shows the immunoprecipitation results.

[0090] Figure 12 Slack and Na in rat brain tissue lysate V Figure 1.6 shows the immunoprecipitation results.

[0091] Figure 13 To show the fluorescent group labeled Slack and Na V Schematic diagram of 1.6;

[0092] In the figure, mTFP1 and mVenus are respectively associated with Slack (Slack-mTFP1) and Na V 1.6(Na V 1.6-mVenus) C-terminal region fusion.

[0093] Figure 14 Slack-mTFP1 and Na V FRET imaging of 1.6-mVenus co-expressed in HEK293T cells;

[0094] In the figure, the emission spectrum measured from the cell edge (red dashed arrow) was used for FRET efficiency calculation.

[0095] Figure 15 FRET efficiency statistics;

[0096] In the figure, mTFP+mVenus refers to the co-expression of mTFP1 and mVenus in HEK293T cells, and Slack-mTFP1+Nav1.6-mVenus refers to the co-expression of Slack-mTFP1 and Nav1.6-mVenus in HEK293T cells, ***p<0.0001, unpaired two-tailed Student's t-test.

[0097] Figure 16 Statistical graph of FRET efficiency measured from cells co-expressing ion channels labeled with fluorescent groups;

[0098] In the figure, the efficiency values ​​are plotted as a function of the fluorescence intensity ratio (Fc / Fy) between mTFP1 and mVenus. Each symbol represents a cell. The solid curve represents the FRET model that produces the best fit, and the dashed curve represents the model with FRET efficiency 5% higher or lower.

[0099] Figure 17 Statistical graph of FRET efficiency measured from cells co-expressing fluorophores;

[0100] In the figure, the efficiency values ​​are plotted as a function of the fluorescence intensity ratio (Fc / Fy) between mTFP1 and mVenus. Each symbol represents a cell. The solid curve represents the FRET model that produces the best fit, and the dashed curve represents the model with FRET efficiency 5% higher or lower.

[0101] Figure 18 Na for humans V Schematic diagram of the domain structure of the channel α subunit.

[0102] Figure 19 Schematic diagram of the domain structure of human Slack channel subunits.

[0103] Figure 20 For Slack and Na V 1.6 Immunoprecipitation results of the terminal domain;

[0104] In the figure, 3×Flag-tagged Slack (Slack-3×Flag) and 3×HA-tagged Na V 1.6 end (3×HA-Na V 1.6-N or 3×HA-Na V In HEK293T cell lysate of 1.6-C), 3×Flag tag was fused to the C-terminus of Slack, and 3×HA tag was fused to the C-terminus of Na V N-terminal fusion of 1.6 fragment.

[0105] Figure 21 The C-terminus of Slack and Na V 1.6 End-immunoprecipitation results diagram;

[0106] In the figure, the C-terminus of Myc-tagged Slack (Slack-C-Myc) and 3×HA-tagged Na V 1.6 end (3×HA-Na V 1.6-N or 3×HA-Na V 1.6-C) Immunoprecipitation of 3×HA tag and Na V The N-terminal region of the 1.6 fragment was fused, and the Myc tag was fused to the C-terminal region of the Slack fragment.

[0107] Figure 22 is a schematic diagram of the current trajectory;

[0108] The figure above shows co-expression of Slack G288S Mutants and Na V 1.5 / 6NC The current trajectory;

[0109] The figure shows co-expression of Slack in HEK293T cells G288S 、Na V 1.5 / 6 NC and Slack's C-terminal current trace example;

[0110] The figure below shows co-expression of Slack in HEK293T cells G288S 、Na V 1.5 / 6 NC and example current traces of overexpressing the Slack 796-839 fragment.

[0111] Figure 23 The current density statistics of each group at +100 mV are shown in the figure. **p<0.01, ***p<0.001, one-way analysis of variance, Bonferroni post hoc test.

[0112] Figure 24 is a schematic diagram of the current trajectory;

[0113] The picture above shows Slack co-expressed in HEK293T cells R398Q Mutants and Na V 1.5 / 6 NC Example of current traces;

[0114] The figure below shows co-expression of Slack in HEK293T cells R398Q Mutant, Na V 1.5 / 6 NC Example of current traces at the C-terminal of Slack.

[0115] Figure 25 Statistical graph of current density of each group at +100 mV. In the figure, *p<0.05, ***p<0.001, one-way analysis of variance, Bonferroni post hoc test.

[0116] Figure 26 Schematic diagram of the adeno-associated virus (AAV) expression system.

[0117] Figure 27 The figure shows the experimental flow chart and immunofluorescence image.

[0118] Figure 28 After KA injection, GFP control group, Slack G269S +GFP group and Slack G269SStatistical graph of the total seizure score in the +Slack-C group; in the figure, *p<0.05, **p<0.01; #p<0.05, ##p<0.01; two-way repeated measures ANOVA, Bonferroni post hoc test.

[0119] Figure 29 GFP control group, Slack G269S +GFP group and Slack G269S Statistical graph of the total seizure score of each mouse in the +Slack-C group within 2 hours after KA injection. In the figure, *p<0.05, **p<0.01; one-way ANOVA, Bonferroni post hoc test.

[0120] Figure 30 GFP control group, Slack G269S +GFP group and Slack G269S Statistical graph of the percentage of mice with grade IV and grade VI-IX epileptic seizures within 2 hours after KA injection in the +Slack-C group. In the figure, *p < 0.05; Fisher's exact test. DETAILED DESCRIPTION

[0121] Purchaser and item number of experimental materials:

[0122] Riluzole was purchased from Meilunbio, catalog number M1106A;

[0123] Kainic acid (KA) was purchased from Sigma-Aldrich, catalog number K0250;

[0124] C57BL / 6 mice and SD rats were purchased from Vital River Company;

[0125] Instrument: The confocal microscope was purchased from Carl Zeiss, model LSM 510META NLO.

[0126] Example 1

[0127] Experimental steps:

[0128] Co-expression of Slack and Na V 1.6 Recording currents in HEK293 cells:

[0129] (1) Co-expression of Slack and Na in HEK293 cells V 1.6;

[0130] (2) Perform whole-cell patch clamp recording on HEK293 cells to record the current traces of HEK293 cells.

[0131] Co-expression of Slack and Na in HEK293 cells V 1.6 includes the following steps:

[0132] Place an 8mm x 8mm glass slide in a 35mm dish for cell passage. 24 hours after passage, transfection should be performed when the cell density is approximately 30-50%. Prepare two 1.5mL EP tubes and add 100μL of Opti- I reduced serum medium, dilute the plasmid and Lipofectamine 2000 (Introvigen), let it stand for 5 minutes, then mix the two tubes and let it stand for 15 minutes. Replace the culture medium with reduced serum medium (1:1), add the plasmid and Lipofectamine mixture, and mix thoroughly. Place in an incubator and replace with complete medium after 3.5 hours. Perform patch clamp experiments 18-36 hours after expression.

[0133] Whole-cell patch clamp recordings in HEK293 cells involve the following steps:

[0134] Electrophysiological recordings were performed using an EPC10 amplifier (HEKA Electronic) and the corresponding PatchMaster software. 18–36 hours after cell transfection, extracellular solution was added to the chamber. A slide was removed and placed in the chamber, pressed firmly, and single cells were selected under an X-cite 120 Olympus fluorescence microscope (Olympus). Glass electrodes (BioSpikes) were pulled and polished using a DMZ universal electrode puller (Zeitz Instruments).

[0135] Subsequently, the intracellular fluid is injected into the electrode, and the air bubble at the tip of the electrode is gently ejected. The electrode is fixed to the holder, and the intracellular fluid contacts the recording electrode (silver wire). Use the micromanipulator MP-285 (Sutter Instrument) to control the electrode to move down to contact the extracellular fluid, and apply a slight positive pressure to expel impurities that may adhere to the tip of the electrode. Continue to lower the electrode, adjust the front, back, left and right positions, and when it contacts the cell surface and presses out a suitable depression, apply negative pressure to form a GΩ seal. Compensate for the electrode capacitance (C-fast), adjust the holding potential (HP), and then apply a short negative pressure to form a whole-cell recording mode. The inside-out recording method is slightly different. After forming a seal of more than 10GΩ, quickly lift the electrode away from the cell, even out of the liquid surface, briefly expose it to the air, and then quickly insert it into the extracellular fluid to maintain the GΩ seal. Afterwards, compensate for the cell membrane capacitance (C-slow) and leakage current (leak current), and the series resistance (R series , Rs ) Compensation 60%-90%.

[0136] Cellular currents were recorded by applying specific voltage pulses at a sampling frequency of 20-50 kHz. A gravity perfusion system (ALA Scientific Instruments) was used for extracellular riluzole administration. Electrode resistances ranged from 1.5-3.0 MΩ for whole-cell recordings of HEK293 cells, 3.5-5.0 MΩ for whole-cell recordings of primary cells, and 8.0-10.0 MΩ for inside-out recordings. All experiments were performed at room temperature.

[0137] The pulse program for measuring whole-cell current was as follows: the cell holding potential was -90 mV, and the measurement was performed at -90 mV for 100 ms, followed by a step voltage from -120 mV to +100 mV for 600 ms with an interval of 10 mV, and then back to the holding potential of -90 mV for 100 ms at a frequency of 0.2 Hz.

[0138] Na V 1.6 Heterozygous knockout includes the following steps:

[0139] The Na used in this study V 1.6 Heterozygous knockout C3HeB / FeJ mice were generously donated by Professor Shu Yousheng of Fudan University. V 1.6 Heterozygous knockout mice were obtained from Jackson Laboratory (Bar Harbor, Maine, USA).

[0140] The sAHP amplitude detection of mouse hippocampal CA1 pyramidal neurons includes the following steps:

[0141] Horizontal sections were obtained from the hippocampus of 6-8 week old C57BL / 6 mice. First, the mice were anesthetized with isoflurane and perfused cardiacally with a slicing solution that was pre-saturated with oxygen (95% O2 / 5% CO2, continuously ventilated, maintaining pH = 7.2) and pre-chilled to ice water. Approximately 15 mL was perfused into the left ventricle for approximately 2 minutes, until the organs and limbs turned white. The brain was quickly removed and immersed in pre-saturated with oxygen and pre-chilled slicing solution, and allowed to stand for 4-5 minutes. The mouse brain was placed on filter paper moistened with ice slicing solution, its shape was trimmed, and the cerebellum and brainstem were removed. Next, the mouse brain was fixed ventral side down in the slicing chamber (pre-chilled) of a vibrating microtome (WPI). Ice slicing solution was poured into the chamber and 95% O2 / 5% CO2 was continuously ventilated. The brain was then cut into horizontal sections 300 μm thick. The brain sections were incubated in oxygenated artificial cerebrospinal fluid (95% O2 / 5% CO2) for 30 minutes. After incubation, brain slices were transferred to a bath containing artificial cerebrospinal fluid (external fluid) maintained at 34–36° C. Hippocampal CA1 pyramidal neurons were recorded in whole-cell mode using a Multiclamp 700B amplifier (Molecular Devices) and a Digidata 1400A digital-to-analog converter under a ×60 water immersion objective on an Olympus BX51WI microscope (Olympus).

[0142] First, hippocampal CA1 pyramidal neurons were selected based on their morphology in brain slices. Pre-drawn electrodes (electrode resistance 5.0-8.0 MΩ) were filled with internal solution, and air bubbles were gently ejected. Using a micromanipulator (MPC-200, Sutter Instruments), the electrode was moved to the surface of the neuronal cell body. Once a suitable depression was formed, a GΩ seal was formed with gentle negative pressure. The clamp voltage was set to -70 mV to compensate for electrode capacitance. Subsequently, intermittent negative pressure was applied to rupture the membrane, compensating for membrane capacitance and series resistance (>80%). Whole-cell recording mode was established, and currents of varying duration and magnitude were injected into the cell to record changes in membrane potential. During recording, the external solution was supplemented with 50 μM (2R)-amino-5-phosphonovaleric acid (APV), 10 μM 6-cyano-7-nitro-quinoxaline-2,3-dione, 10 μM bicuculline, and 1 μM CGP 55845. The sampling frequency was 50 kHz, and the filtering frequency was 10 kHz. Data were collected and analyzed using pClamp 10.0 (Molecular Devices). Series resistance was controlled between 10 and 30 MΩ, with compensation between 60 and 80%. Data were discarded if series resistance increased by more than 20% during the recording.

[0143] The pulse program used to induce the slow afterhyperpolarization potential (sAHP) of neurons was: 300pA current pulse ( Figure 7 ) or 100Hz pulse train ( Figure 8 The sAHP amplitude was calculated as the difference between the action potential trough and the neuronal resting membrane potential.

[0144] Experimental results:

[0145] Slack channels are potassium ion channels activated by sodium ions, which generate outward potassium currents and contribute to the afterhyperpolarization potential (AHP) of the action potential, thus maintaining the firing rate of neurons. V 1.6 is a voltage-gated sodium channel that generates an inward sodium current. When induced by a 100ms prepulse (pre) at -90mV, the Slack and Na V Example current traces recorded from HEK293 cells in 1.6 Figure 1 When a 100ms pre-pulse from -40mV was induced to V 1.6 When the channel is rapidly inactivated, the current trace recorded from HEK293 cells is as follows Figure 2 When Slack and Na V 1.6 When co-expressed, the IV curves of the Slack current induced by -90mV and -40mV pre-pulses are as follows Figure 3 As shown. Figure 1-3 As can be seen, depolarizing voltage inactivation can block Na V 1.6-mediated sodium influx can significantly reduce the amplitude of Slack current.

[0146] Riluzole was not added to the bath solution, and Slack and Na were co-expressed. V The current traces recorded from HEK293 cells at 1.6 were as follows Figure 4 The current trace when 20 μM riluzole was added to the bath solution is shown in Figure 5 Slack and Na V 1.6 IV curve of co-expression Figure 6 As shown. Figure 4-6 As can be seen, riluzole can block Na V 1.6-mediated sodium influx reduces the Slack current amplitude. V 1.6 can activate the internal Slack channel.

[0147] from Figure 7As can be seen in Figure 3, action potentials are triggered at 300 pA, followed by sAHP in Scn8a+ / + (WT) and Scn8a+ / - neurons. The amplitude of sAHP in Scn8a+ / - neurons is reduced compared to Scn8a+ / +. Figure 8 As can be seen in the 100 Hz pulse, action potentials were evoked, followed by sAHPs in both Scn8a+ / + and Scn8a+ / - neurons. The amplitude of sAHPs in Scn8a+ / - neurons was reduced compared to Scn8a+ / + neurons. V 1.6 heterozygous knockout also significantly reduced the AHP amplitude of CA1 pyramidal neurons in the mouse hippocampus, indicating that Na V 1.6 can activate the internal Slack channel.

[0148] The above results show that Na V 1.6 channel-mediated sodium influx activates Slack channels.

[0149] Example 2

[0150] In Example 2, we further detected the voltage-gated sodium channel Na V 1.2 Na V The cellular distribution of 1.6 and Slack in the hippocampus and neocortex.

[0151] Detection of Na V 1.2 Na V 1.6 The detection of the cellular distribution of Slack in the hippocampus and neocortex includes the following steps:

[0152] Assemble the perfusion apparatus and connect the water pump outlet tube and the intravenous needle. Place the water inlet tube end in deionized water and PBS, respectively, and turn on the water pump to eliminate bubbles. Inject sodium pentobarbital intraperitoneally to anesthetize the mouse and secure it to the cage lid. Use scissors to cut the mouse's chest cavity and diaphragm to expose the heart, and secure the upper part with hemostats. Cut the right atrial appendage, insert the injection needle through the left ventricle, and rapidly perfuse 30 mL of PBS solution, followed by 10 mL of 0.5% paraformaldehyde + 0.5% sucrose, and then slowly perfuse 20 mL. After the perfusion is completed, remove the injection needle, remove the hemostats, and quickly remove the mouse brain. Place it in a 0.5% paraformaldehyde + 0.5% sucrose solution. After 2 hours, replace it with a 30% sucrose PBS solution and let it sit for 48 hours. Subsequently, remove the brain from the sucrose solution and soak it in n-hexane for approximately 20 seconds to harden it. The brain was then removed and placed in a homemade tinfoil container. Embedding medium was added to cover the brain, and the brain was placed in an ice box filled with dry ice and frozen at -80°C for 8 minutes. A cryostat was then used to obtain 20 μm thick coronal brain slices. Preparing for immunostaining:

[0153] 1) Permeabilization: Brain slices were circled for immunohistochemistry and washed three times with 0.01 M phosphate buffered saline (PBS) for 10 min each. Permeabilization was performed with 0.5% PBST (0.5% Triton X-100 in 0.01 M PBS) for 30 min. Subsequently, 0.01 M PBS was added and the slices were washed three times with 0.01 M PBS on a horizontal shaker for 10 min each.

[0154] 2) Blocking: Prepare blocking solution: 5% normal goat serum dissolved in 0.1% PBST, and block for 2 hours.

[0155] 3) Incubation with primary antibodies: 0.1% PBST (0.1% Triton X-100 dissolved in 0.01M PBS solution) diluted primary antibodies: AnkG (1:100, Santa Cruz, sc-31778), Slack (1:100, NeuroMab, 73-051), Na V 1.2 (1:200, Alomone, ASC-002), Na V 1.6 (1:200, Alomone, ASC-006). Incubate with primary antibody at 4°C overnight. After incubation, remove the plate and transfer to room temperature for 2 hours. Wash three times with 0.01M PBS for 10 minutes each time.

[0156] 4) Incubation with secondary antibodies: Dilute secondary antibodies (Alexa 488-conjugated goat anti-rabbit IgG (Abcam) and Alexa 594-conjugated goat anti-mouse IgG (Abcam) in 0.1% PBST. Incubate with secondary antibodies for 2 h at 20-25°C, then wash three times with 0.01 M PBS for 10 min each (protect from light).

[0157] 5) Mounting and photography: DAPI Fluoromount-G TM The sections were sealed with anti-fluorescence fading mounting medium (solarbio), covered with coverslips, and the edges were painted with nail polish. Images were taken with a laser scanning confocal microscope.

[0158] The extraction and homogenization of cerebral cortex and hippocampus tissues include the following steps:

[0159] Cortical and hippocampal tissues from adult Sprague-Dawley rats were placed in 1.5 mL EP tubes and ground on ice to obtain a tissue homogenate. The homogenate was then lysed for 30 minutes using GPCR Extraction Reagent (Pierce) and protease inhibitors. The homogenate was centrifuged at 16,000 g for 20 minutes at 4°C, and the supernatant was collected as the tissue protein lysate. Protein was quantified using a BCA protein quantification kit (Pierce).

[0160] Procedure for detecting immunoprecipitation:

[0161] Tissue or cell protein supernatant was mixed with 5 μg of Slack antibody (Neuromab) or Na V 1.6 antibody (Alomone) was incubated at 4°C with continuous rotation for 12 hours. 40 μL of Protein G Dynabeads (Invitrogen) was added and incubation continued until the next day. The beads were then washed three times with PBS. During the washes, the beads were collected using a DynaMag. The beads were then resuspended in 1× SDS-PAGE loading buffer and incubated at 37°C for 30 minutes to elute the remaining protein from the beads. Finally, samples from the immunoprecipitate or tissue were subjected to Western blot analysis:

[0162] 1) Prepare 8% separating gel and 4% stacking gel.

[0163] 2) Sample loading: Add the protein sample to the sample loading buffer, denature at 37°C for 30 min, centrifuge and then load the sample.

[0164] 3) Electrophoresis: Run at 90 V for 30 min, then at 130 V for 80 min, until bromophenol blue migrates to the bottom of the electrophoresis tank.

[0165] 4) Electrotransfer: Prepare two filter paper membranes and one nitrocellulose membrane (PALL) in advance and soak them in electrotransfer solution. Place the foam, paper membrane, and nitrocellulose membrane on the cathode (black) plate of the gel cassette in that order. Cut the separating gel and place it upside down on the nitrocellulose membrane. Place the paper membrane and foam membrane in that order, gently expel any air bubbles, and secure the gel cassette. Pour the electrotransfer solution into the electrotransfer chamber and add ice. Place the electrotransfer chamber on ice and electrotransfer at 300 mA for 100-120 minutes.

[0166] 5) Blocking: After electroporation, the NC membrane was removed and immersed in Tris-Tween buffered saline (TBST solution) (0.02 M Tris, 0.137 M NaCl, and 0.1% Tween 20) containing 5% skim milk powder. The membrane was shaken on a horizontal shaker for 15 minutes to block nonspecific binding sites.

[0167] 6) Incubation with Primary Antibody: After blocking, wash the blocking solution off the NC membrane with TBST. Prepare a plastic box with compartments, cut the NC membrane into strips of appropriate size, and place them into the compartments. Add 3 mL of diluted primary antibody to each compartment and incubate overnight at 4°C on a horizontal shaker. Then, remove the primary antibody and wash three times with TBST, each for 5-10 minutes.

[0168] 7) Incubation with secondary antibody: Dilute horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000) and add 3 mL per chamber. Incubate on a horizontal shaker for 1.5 hours. Recover the secondary antibody and wash three times with TBST solution, each for 5-10 minutes.

[0169] 8) Exposure: Immobilon Western chemiluminescent HRP substrate (Millipore) was used to prepare the luminescent solution. The plastic film was cut open, the strip was placed on it, the luminescent solution was evenly added, the plastic film was covered, and imaging was performed using the Tanon chemiluminescent image analysis system.

[0170] Experimental results:

[0171] Voltage-gated sodium channels Na V 1.2 Na V The distribution of 1.6 and Slack in the hippocampus is as follows Figure 9 As shown, Figure 9 The upper left is the immunofluorescence image of Slack protein. Figure 9 The middle is the immunofluorescence image of AnkG protein. Figure 9 The upper right is the immunofluorescence image of the fusion of Slack protein and AnkG protein. Figure 9 Na in the middle left V Immunofluorescence images of 1.2, Figure 9 Na in the middle right V 1.2 Immunofluorescence images of fusion with AnkG protein, Figure 9 Na is on the lower left V Immunofluorescence images of 1.6, Figure 9 Middle, lower right is Na V Immunofluorescence image of 1.6 and AnkG protein fusion. Voltage-gated sodium channel Na V 1.2 Na V The distribution of 1.6 and Slack cells in the neocortex is as follows Figure 10 shown. Figure 10 The upper left is the immunofluorescence image of Slack protein. Figure 10 The middle is the immunofluorescence image of AnkG protein. Figure 10 The upper right is the immunofluorescence image of the fusion of Slack protein and AnkG protein. Figure 10 Na in the middle left V Immunofluorescence images of 1.2, Figure 10 Na in the middle right V 1.2. Immunofluorescence image of AnkG protein and DAPI fusion. Figure 10 Na is on the lower left V Immunofluorescence images of 1.6, Figure 10 Middle, lower right is Na V 1.6. Immunofluorescence image of AnkG protein and DAPI fusion.

[0172] Na V Colocalization with AnkG indicates that Na V 1.2 and Na V1.6 is located in the initial segment of the axon of neurons. Slack channels are also located in the initial segment of the axon of these neurons. The above experimental results show that Slack channels are located in the Na V The 1.6 channel is close to the 1.6 channel, which supports the potential interaction between the two. Confocal microscopy images were obtained from coronary artery brain sections of C57BL / 6 mice.

[0173] also, Figure 11-12 The results showed that the homogenates of rat cortex and hippocampus, as well as Slack and Na V 1.6 In co-transfected HEK293T cells, Na V 1.6 produced immunoprecipitation with Slack, and the input amount was equivalent to 10% of the total immunoprecipitated lysate. V 1.6 There is a physical interaction in the body to form a channel complex.

[0174] To verify the Slack and Na V 1.6 Interactions in living cells We performed FRET assays in HEK293T cells.

[0175] Specific steps of FRET assay:

[0176] Emission spectroscopy imaging was performed using a Nikon TE2000-U microscope (Nikon). Excitation light was generated by an Ar laser. V 1.6 The fused fluorescent protein mVenus and the mTFP1 fused with Slack were excited by 500nm and 400-440nm laser lines, respectively. The computer-driven mechanical shutter (Uniblitz) controlled the exposure time. A spectrometer (Acton SpectraPro2150i) was coupled to a charge-coupled device (CCD) camera (Roper Cascade 128B), and spectral images were collected using two filters (excitation filter, dichroic filter) (Chroma) for each cell: cube I, D436 / 20, 455dclp; cube II, HQ500 / 20, Q515lp. Under these experimental conditions, the fluorescence generated by the untransfected cells themselves was negligible. MetaMorph software (Universal Imaging) was used for fluorescence imaging and analysis. User-designed macros automatically collected bright field cell images, fluorescent cell images, and spectral images. The emission spectrum was collected from the cell membrane area. The spectrometer slit was passed through the cell and the fluorescence intensity at the cell membrane position was recorded ( Figure 14 , red dashed line). This approach preserves spectral and positional information reliably, quantifies FRET efficiency at the cell membrane region, and corrects for background light estimated from a blank region of the same image.

[0177] FRET data were quantified as previously described, calculating the apparent FRET efficiency based on the increase in mVenus fluorescence emission. Briefly, the FRET efficiency was calculated by measuring Ratio A0 and Ratio A. Ratio A0 represents the ratio between the fluorescence emission intensities of mVenus when excited at the donor excitation wavelength and the acceptor excitation wavelength. In this study, it was calculated at the peak mVenus emission wavelength. Measuring Ratio A0 can offset variations in fluorescence intensity caused by many experimental factors. For mTFP1, the ratio between the fluorescence emission intensities of mTFP1 when excited at the donor excitation wavelength and the acceptor excitation wavelength, i.e., Ratio A, was calculated, similar to the Ratio A0 method. If FRET occurs, Ratio A should be higher than Ratio A0. The difference between Ratio A and Ratio A0 is proportional to the FRET efficiency, which depends on the ratio of the extinction coefficients of mTFP1 and mVenus.

[0178] Experimental results:

[0179] mTFP1 and mVenus were respectively associated with Slack and Na V The C-terminal region of 1.6 is fused as follows Figure 13 As shown, Figure 13 The left side of the middle image shows the gene fusion of mTFP1 and the C-terminal region of Slack. Figure 13 On the middle right are mVenus and Na V 1.6 C-terminal region was genetically fused. V The emission spectra of cells co-expressing 1.6-mVenus and Slack-mTFP1 were imaged (measured in the plasma membrane region). Figure 14 As shown, Figure 14 The above is the imaging result of the emission spectrum of cells co-expressing Slack-mTFP1. Figure 14 for Na V 1.6-mVenus co-expressed cells emission spectrum imaging results, from Figure 14 It can be seen that a positive FRET signal was detected. V The plasma membrane region of HEK293T cells expressing 1.6 and Slack showed higher FRET efficiency values ​​than the negative control co-expressing mVenus and mTFP1 proteins alone. Figure 15-17 As shown above, the Slack channels and Na V The 1.6 channels are very close in space (less than 10 nm), supporting the interaction between the two.

[0180] Example 3

[0181] In Example 3, we further detected Slack and Na V 1.6 The specific fragments that interacted with each other were co-expressed in HEK293T cells and immunoprecipitation experiments were performed.

[0182] The immunoprecipitation experiment involves the following steps:

[0183] The Slack protein fragment and Na were transfected into the 5-well plate using the transfection reagent Megatran 1.0 (Origene). V 1.6 The plasmids of the protein fragments were co-transfected into HEK293T cells, and the transfection operation was carried out according to the instructions. After 30 hours of transfection, the culture medium was discarded, the cells were washed twice with PBS, and the cells were lysed with GPCR extraction reagent for 30 minutes. Then, the cells were scraped and collected in a 1.5 mL EP tube. The cells were centrifuged at 16000g for 20 minutes at 4°C, and the supernatant was collected as the cell protein lysate. The protein was quantified using a BCA protein quantification kit (Pierce). The subsequent precipitation and detection steps were the same as in Example 2. The tag antibodies used were: Flag (1:500, Abbkine, ABT2010) and HA (1:500, Abbkine, ABT2040).

[0184] Experimental results:

[0185] Human Na V The domain structure of the channel α subunit is as follows Figure 18 As shown, Figure 18 In the N-terminus, Na V The N-terminus of the channel represents Na V At the C-terminus of the channel, DI, DI, DIII, and DIV represent the first, second, third, and fourth transmembrane domains, respectively. Figure 19 shown. Figure 19 In the figure, N-terminus represents the N-terminus of the Slack channel, C-terminus represents the C-terminus of the Slack channel, RCK1 and RCK2 are potassium ion conductance regulatory domains, and the Slack and Na V 1.6 The specific fragments that interacted with each other were co-expressed in HEK293T cells and immunoprecipitation experiments were performed. Figure 20-21 As shown, Figure 20 The results showed that Na V The N- and C-termini of channel 1.6 were immunoprecipitated with Slack. Figure 21 The Slack channel C-terminus and Na V Immunoprecipitation was observed at both the N and C termini of channel 1.6, indicating the existence of a physical interaction.

[0186] Example 4

[0187] Based on Na V 1.6 Na2O mediates sodium influx and activates endogenous Slack, Slack-Na V 1.6 complex increases the current of Slack mutants and increases the intracellular sodium ion concentration ([Na + ] in ) and aggravated the functional enhancement phenotype of the mutant. V In the 1.6 complex system, the C-terminus of Slack competes with the Slack channel itself, destroying the Slack-Na V 1.6 interaction, which may reduce Na delivery to Slack mutants + , thereby reducing the current of epilepsy-related Slack mutants.

[0188] To this end, the present invention expressed the C-terminus of Slack in HEK293T cells co-expressing an epilepsy-related Slack mutant (G288S, R398Q) and a sodium channel, and measured the whole-cell current density.

[0189] Slack mutants G288S , Slack mutant R398Q and sodium channel chimera Na V 1.5 / 6 NC The construction includes the following steps:

[0190] Plasmid construction was performed using the Gibson assembly method:

[0191] 1) Linearize the vector. Treat the vector plasmid with a restriction endonuclease to obtain a linearized vector. Measure the vector concentration.

[0192] 2) Design primers to amplify Slack sequence fragments. For the construction of Slack mutants, two pairs of primers were designed at the expected mutation site and the vector junction. Two DNA fragments were amplified by PCR reaction and the fragments were introduced into the mutation site and the homologous sequence required for Gibson assembly. The concentration of the two fragments was measured. V 1.5 / 6 NC (Na V The N-terminus and C-terminus of the 1.5 channel were replaced by Na V 1.6 channel N-terminus and C-terminus to replace the functional Na V 1.6 channel) construction, three pairs of primers were designed, and the PCR reaction and fragment concentration measurement were the same as above.

[0193] 3) Ligation of the fragment and vector. Incubate the vector, fragment, and Gibson mixture at 50°C for 40 minutes to form circular DNA. The Gibson assembly reaction system is as follows:

[0194]

[0195] 4) The ligation product is transformed into competent bacteria, single clones are picked, sequenced and verified, and plasmids are extracted to obtain the target plasmid.

[0196] Specific steps for measuring current density:

[0197] The whole-cell current recording procedure was the same as in Example 1. The current density (pA / pF) was calculated as the ratio of the whole-cell current (pA) to the cell membrane capacitance (pF).

[0198] AAV virus construction and injection include the following steps:

[0199] Adeno-associated viruses (AAVs) and negative EGFP control were from Shanghai GeneCare Gene Co., Ltd. G269S The sequence (1-1238) was ligated into a modified CV232 (CAG-MCS-HA-PolyA) adeno-associated virus vector. The C-terminal sequence of the Slack protein (residues 326-1238) and the negative control were ligated into a GV634 (CAG-MCS-3×Flag-T2A-EGFP-SV40-PolyA) adeno-associated virus vector. The virus titer used in this study was >10 11 TU / ml.

[0200] For viral injection in dorsal CA1, 3-week-old C57BL / 6J mice were anesthetized with isoflurane and placed on a stereotaxic apparatus (RWD Life Science Co., Ltd.). Using a 5 μL microsyringe (Hamilton) with a 30-gauge needle (RWD Life Science Co., Ltd.), 600 nL of virus was injected into the CA1 region on each side at a rate of 10 nL / min via a microsyringe pump (RWD Life Science Co., Ltd.). The stereotactic coordinates were: 2.5 mm (anterior-posterior), 2 mm (interior-exterior), ±1.5 mm (dorsal-ventral) from the anterior bregma. The syringe was left in place for 5 minutes after each injection and then slowly withdrawn. The exposed skin was surgically sutured and the mice were returned to their cages for recovery. All subsequent experiments were performed after at least 3 weeks of recovery.

[0201] The establishment of the kainic acid-induced temporal lobe epilepsy model includes the following steps:

[0202] Kainic acid (Sigma-Aldrich) was injected intraperitoneally into 6-8 week old mice to produce grade IV or higher seizures. The dose of kainic acid used was 28 mg / kg.

[0203] Specific classification standards for epileptic seizure levels:

[0204] To assess seizure susceptibility, seizures were graded using the modified Racine, Pinal, and Rovner scale. (1) Facial movements; (2) Nodding; (3) Forelimb contracture; (4) Dorsal extension (rolling over); (5) Loss of balance and falling; (6) Repeated rolling over and failure; (7) Vigorous jumping and running; (8) Grade 7 with a sense of frustration; (9) Death. Two hours after the onset of the seizure, sodium pentobarbital (30 mg / kg; Sigma-Aldrich) was used to terminate the seizure.

[0205] Experimental results:

[0206] Slack G288S The results are as follows Figure 22-23 As shown, from Figure 22-23 As can be seen, Slack G288S The current density increased when Slack was co-expressed G288S and Na V 1.5 / 6 NC In HEK293T cells, expression of the C-terminus of Slack or Slack 796-839 reduced the current density. R398Q The results are as follows Figures 24-25 As shown, from Figures 24-25 As can be seen, Slack R398Q The current density increased when Slack was co-expressed R398Q and Na V 1.5 / 6 NC The above results indicate that expression of the C-terminus of Slack can reverse the Slack G288S and Slack R398Q induced an increase in whole-cell current density.

[0207] In an in vivo epilepsy model, Slack was delivered using adeno-associated virus (AAV). G269S Introducing C57BL / 6N mice to mimic the human epilepsy-related mutation Slack G288S The architecture of adeno-associated virus is as follows Figure 26 shown. Figure 26 Among them, (1) is the Slack protein overexpressed with HA tag and carrying G269S mutation (Slack G269S-HA) schematic diagram of the AAV virus structure; (2) schematic diagram of the AAV virus structure overexpressing the C-terminus of Slack fused with 3×Flag tags (Slack-C-3×Flag); (3) schematic diagram of the negative control virus structure overexpressing GFP. G269S AAV9 or GFP negative control was stereotactically injected into the CA1 region of the hippocampus of 3-week-old C57BL / 6N mice. The viral transfection effect of mice injected with mixed viruses (1) and (2) was detected 5 weeks after AAV injection. Figure 27 As shown, Figure 27 Slack on the left G269S - Immunofluorescence image of HA, Figure 27 The middle is the immunofluorescence image of Slack-C-3×Flag, Figure 27 Slack on the right G269S - Immunofluorescence images of HA, Slack-C-3×Flag and DAPI fusion.

[0208] Three to five weeks after AAV injection, a kainic acid (KA)-induced temporal lobe epilepsy model was used to quantify epilepsy susceptibility in mice. In this model, mice were intraperitoneally injected with 28 mg / kg kainic acid to induce grade IV or higher seizures, and observation was recorded for 2 hours. The time course of KA-induced seizure severity was assessed at 10-minute intervals. Figure 28 As shown, it was found that overexpression of Slack G269S The seizure severity of the mice was significantly higher than that of the GFP control group. The total seizure score of each mouse was calculated to indicate the severity of the seizure. Figure 29 As shown, from Figure 29 As can be seen, the expression Slack G269S Mice expressing GFP had more severe seizures than mice expressing GFP. Figure 30 As can be seen, the proportion of mice with epileptic seizures of maximum grade VI-IX increased from 9.1% (GFP) to 58.3% (Slack G269S ). The above results show that Slack G269S Overexpression significantly increased the susceptibility to epilepsy in mice.

[0209] To evaluate the disruption of Slack-Na V 1.6 Potential therapeutic effects of interactions, Slack overexpression was simultaneously injected into the CA1 brain region of mice G269S The virus with the C-terminus of the Slack channel (residues 326-1238). G269S In overexpression mice, additional expression of the Slack channel C-terminus significantly reduced the grade of epileptic seizures, the severity of epileptic seizures, and the percentage of mice with grade VI-IX seizures. Figures 28-30 The above results indicate that overexpression of the C-terminus of the Slack channel can prevent Slack G269S Overexpression of this gene can inhibit epileptic seizures and reduce epilepsy susceptibility.

[0210] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. Use of a substance for increasing the C-terminal content of Slack protein in the preparation of a drug for treating, assisting in treating and / or preventing epilepsy, characterized in that: The substance for increasing the C-terminal content of the Slack protein is an AAV virus including the C-segment sequence of the Slack protein, and the C-segment sequence of the Slack protein is the Slack 326-1238 fragment.

2. Use of a substance that upregulates the expression of the C-terminus of the Slack protein in the preparation of a drug for treating, assisting in the treatment and / or preventing epilepsy, characterized in that: The substance that increases the content of the C-terminal end of the Slack protein is an AAV virus including the C-segment sequence of the Slack protein, and the C-segment sequence of the Slack protein is the Slack 326-1238 fragment.

3. The use according to claim 2, characterized in that The epilepsy is hereditary epilepsy associated with KCNT1 mutation.

4. The use according to claim 2, characterized in that The substance that upregulates the expression of the C-terminal end of the Slack protein is a Slack mutant and a voltage-gated sodium ion channel Na V 1.6 The interaction interface is the target.

5. The use according to claim 4, characterized in that The Slack mutant and the voltage-gated sodium channel Na V 1.6 Interaction between the C-terminus of the Slack mutant and the voltage-gated sodium channel Na V 1.6 N- and / or C-terminal interactions.

6. A drug for treating, assisting in the treatment and / or preventing epilepsy, characterized in that: The drug contains a therapeutically effective amount of a substance that increases the C-terminal content of the Slack protein. The substance that increases the C-terminal content of the Slack protein is an AAV virus including the C-segment sequence of the Slack protein, and the C-segment sequence of the Slack protein is the Slack 326-1238 fragment.

7. Use of the drug according to claim 6 and other drugs for treating epilepsy in the preparation of drugs for treating epilepsy.

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