CN1515680A - Application of ion channel inhibitor for curing arhythmia - Google Patents
Application of ion channel inhibitor for curing arhythmia Download PDFInfo
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Abstract
The present invention discloses a kind of mutant KCNQI, i.e. S140G-KCNQ1, also discloses a method for screening inhibitor of myocardial KCNQ1 potassium ion channel by utilizing KCNQ1. It includes the following steps: (a). transferring expression rector for expressing (i) KCNQI and expression vector for expressing (ii) KCNE1 or expression vector for expressing KCNE2 into mammalian cell; (2). in the culture medium of transferred mammalian cell adding candidate substance and determining electric physiological potassium ion flow before and after addition of the candidate substance. If the electric physiological potasisum flow after the candidate substance added is reduced, it shows that the substance is the inhibitor of myocardial KCNQ1 potassium ion channel.
Description
Technical field
The present invention relates to molecular biology and medical field, more specifically the present invention relates to the synergy sudden change and the application thereof of familial atrial fibrillation patient KCNQ1 gene.
Background technology
Definition according to the World Health Organization, atrial fibrillation (atrial fibrillation) is irregular, the rambling electrical activity in atrium, the P ripple disappears on the electrocardiogram(ECG, replace the f wave that shape on the baseline, size, direction and time-histories differ, Ventricular Rate does not wait fully under not height or dissociation situation.
Atrial fibrillation is one of common arrhythmia clinically, it inspires heart failure or haemodynamics obstacle, and fatal arrhythmia such as bring out to quiver in chamber speed or chamber causes thrombus and embolism incident, increase basic cardiopathic mortality ratio, atrial fibrillation itself can also cause cardiac dilatation.According to statistics, U.S.'s incidence of atrial fibrillation in patients is 0.9%.With advancing age, incidence of atrial fibrillation in patients swash to increase severely high, the over-65s person then up to 5.9%.According to Framinham research, the elderly (＞65 years old) palsy 1/3 is caused by atrial fibrillation.The Europe incidence of atrial fibrillation in patients and the U.S. are similar, and the Asia is lower slightly.Atrial fibrillation is seriously endangering human beings'health, has also brought suitable economical load to society.
As the specific type of atrial fibrillation, idiopathic atrial fibrillation refers to not have the atrial fibrillation on the heart change basis of bright bowl.Its name is relatively more chaotic, and other title has isolatism idiopathic atrial fibrillation, optimum idiopathic atrial fibrillation and familial idiopathic atrial fibrillation.Nearly 1/3rd atrial fibrillation classifies as idiopathic atrial fibrillation (Lip GYH, Beevers DG (1995) ABC of atrial fibrillation:history, epidemiology, and importance of atrial fibrillation. BMJ 311:1361).
Atrial fibrillation (AF) is as human a kind of common irregular pulse, and the pathogenic basis of its molecule is not well illustrated so far.
Summary of the invention
Purpose of the present invention just provides a kind of molecule pathogenesis that causes atrial fibrillation, and this mechanism is exactly that pathogenic mutation (S140G) has taken place the KCNQ1 gene, thereby causes the ionic current of myocardium potassium ion IKs ionic channel to increase.
Another object of the present invention has just provided the drug screening method based on this molecule pathogenesis.
In a first aspect of the present invention, a kind of method of screening the inhibitor of myocardium KCNQ1 potassium-channel is provided, comprise step:
(a) change α subunit expression carrier and β subunit expression carrier over to mammalian cell, obtain the mammalian cell of conversion, wherein α subunit expression carrier is the KCNQ1 expression vector, and β subunit expression carrier is selected from down group: the expression vector of KCNE1 expression vector or KCNE2;
(b) in the substratum of the mammalian cell of the conversion of step (a), add candidate substances, and measure the electric physiology potassium current that adds before and after the candidate substances,
Wherein, if the electric physiology potassium current that adds after the candidate substances reduces, show that then this active substance is the inhibitor of myocardium KCNQ1 potassium-channel.
In a preference, described KCNQ1 expression vector is expressed the KCNQ1 of wild-type.
In another preference, described KCNQ1 expression vector is expressed the KCNQ1 of mutant.More preferably, described KCNQ1 expression vector is expressed the KCNQ1 of S140G mutant.
In another preference, described β subunit expression vector expression KCNE1.
In another preference, described β subunit expression vector expression KCNE2.
In another preference, the measuring method in the step (b) is the patch clamp method.
In a second aspect of the present invention, a kind of method of screening the promotor of myocardium KCNQ1 potassium-channel is provided, comprise step:
(a) change α subunit expression carrier and β subunit expression carrier over to mammalian cell, obtain the mammalian cell of conversion, wherein α subunit expression carrier is the KCNQ1 expression vector, and β subunit expression carrier is selected from down group: the expression vector of KCNE1 expression vector or KCNE2;
(b) in the substratum of the mammalian cell of the conversion of step (a), add candidate substances, and measure the electric physiology potassium current that adds before and after the candidate substances,
Wherein, if the electric physiology potassium current that adds after the candidate substances increases, show that then this active substance is the promotor of myocardium KCNQ1 potassium-channel.
In a third aspect of the present invention, a kind of S140G mutant KCNQ1 is provided proteic purposes, it is used to screen the inhibitor or the promotor of myocardium KCNQ1 potassium-channel.
Description of drawings
Fig. 1 shows the ionic current of S140G-KCNQ1/KCNE1 ionic channel, than the ionic current of wild-type KCNQ1/KCNE1 ionic channel great increase is arranged.
Fig. 2 shows the ionic current of S140G-KCNQ1/KCNE2 ionic channel, than the ionic current of wild-type KCNQ1/KCNE2 ionic channel great increase is arranged.
Fig. 3 has shown the inhibition effect of chromanol 293B to the S140G-KCNQ1/KCNE1 ionic channel.
Fig. 4 has shown the inhibition effect of chromanol 293B to the S140G-KCNQ1/KCNE2 ionic channel.
Fig. 5 has shown the inhibition effect of chromanol 293B to the KCNQ1/KCNE2 ionic channel.
Embodiment
The inventor is through extensive and deep research, patient to a Chinese heredity atrial fibrillation family has carried out genetic analysis, by full genome scanning pathogenic sites is positioned at 11p15.5, and in this regional KCNQ1 gene, has found a pathogenic mutation S140G.KCNQ1 genes encoding cardiac muscle potassium-channel I
Ks(KCNQ1/KCNE1), the α subunit of KCNQ1/KCNE2, KCNQ1/KCNE3 ionic channel.This sudden change is present among all patients, is not present among the normal people.
In order to illustrate the pathogenesis of this sudden change, the inventor has further done the flow analysis of mutant ionic channel.Functional analysis shows that the S140G sudden change strengthens the ionic current of KCNQ1/KCNE1, KCNQ1/KCNE2 ionic channel.These are different with the previous KCNQ1 sudden change of finding that causes the LQT syndromes, cause that the KCNQ1 sudden change of LQT syndromes reduces the ionic current of KCNQ1/KCNE1, KCNQ1/KCNE2 ionic channel.
Experimentation on animals and clinical study result show that most of atrial fibrillations are to be formed by polynary the turning back of heart muscle conductivity, and action potential is held the shortening of journey and effective refractory period and helped the polynary formation of turning back.The atrial fibrillation sudden change S140G that the present invention finds causes I
KsThe enhancing of the function of ionic channel (KCNQ1/KCCNE1) and KCNQ1/KCNE2 passage, these enhancings can cause that the heart muscle action potential holds the shortening of journey and effective refractory period, thus the initiation atrial fibrillation.
Based on above result,, may alleviation be arranged, result of treatment to atrial fibrillation and other irregular pulse at the inhibitors of ion channels of KCNQ1/KCNE2 ionic current.Therefore, the present invention also provides a kind of method of screening the drug candidate of treatment atrial fibrillation, the method for promptly screening the inhibitor of myocardium KCNQ1 potassium-channel, and it comprises step:
(a) change α subunit expression carrier and β subunit expression carrier over to mammalian cell, obtain the mammalian cell of conversion, wherein α subunit expression carrier is the KCNQ1 expression vector, and β subunit expression carrier is selected from down group: the expression vector of KCNE1 expression vector or KCNE2;
(b) in the substratum of the mammalian cell of the conversion of step (a), add candidate substances, and measure the electric physiology potassium current that adds before and after the candidate substances,
Wherein, if the electric physiology potassium current that adds after the candidate substances reduces, show that then this active substance is the inhibitor of myocardium KCNQ1 potassium-channel.
As used herein, term " wild-type KCNQ1 " refers to have the albumen of aminoacid sequence shown in the SEQID NO:1.The KCNQ1 myocardium potassium ion I that encodes
KsIonic channel, the α subunit in KCNQ1/KCNE2 and the KCNQ1/KCNE3 ionic channel.
As used herein, term " S140G-KCNQ1 " refers to have the albumen of aminoacid sequence shown in the SEQ ID NO:2, and its corresponding codes sequence is shown in SEQID NO:9.When S140G took place, Nucleotide 418 was by A → G among the SEQ ID NO:9, and 140 amino acids are by S → G among the SEQ ID NO:2.Functional analysis shows that the S140G sudden change increases the ionic current of KCNQ1/KCNE2, KCNQ1/KCNE3, and this ionic current reduction effect with original sudden change of finding that causes the LQT syndromes is opposite.(Action Potentia1 Duration, APD) (Effective Refratory Period ERP) causes and keeps atrial fibrillation with effective refractory period so S140G sudden change may be held journey by the action potential that shortens heart muscle.
As used herein, term " KCNE1 " refers to the albumen of GenBank NM_000219 sequence encoding, promptly Potassium (
K)
CHa
NNel Class
E, Member 1.
As used herein, term " KCNE2 " refers to the albumen of GenBank NM_005136 sequence encoding, promptly Potassium (
K)
CHa
NNel Class
E, Member 2.
As used herein, term " KCNE3 " refers to the albumen of GenBank NM_005472 sequence encoding, promptly Potassium (
K)
CHa
NNel Class
E, Member 3.
The cell that can be used for screening method of the present invention is not particularly limited.Representational example comprises: mammalian cell, and as the COS-7 cell, Chinese hamster ovary celI, or the ovocyte of vertebrates Xenopus laevis (Xenopus).
The ionic current determination techniques that can be used for screening method of the present invention is not particularly limited.Representational example comprises: patch clamp method (being used for mammalian cell) or little two electrodes pressing tongs method (being used for xenopus leavis oocytes).
Major advantage of the present invention is to screen in a large number effectively the drug candidate of treatment atrial fibrillation.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1: wild-type KCNQ1 preparation of expression vectors
From people's kidney cDNA storehouse (Clontech Inc.), use isotropic substance
32The KCNQ1 dna probe of P mark (PCR fragment) screening positive clone.The gained positive colony directly checks order, and standard announcement sequence (Accession No.AF000571) definite and among the GenBank is identical, and has complete encoding sequence.
Primer of design before the initial son of cDNA sequence A TG, and have an ECoRI point of contact sequence (primers F: 5 '-tc gag aat tcc tca cca tgg ccg cgg cct cct c (SEQ ID NO:3)), primer of design behind terminator TGA, and have XbaI point of contact (primer R:5 '-ggt cga ctc tag acc cca tcc cct cct cagg (SEQ ID NO:4)).
With primer to (F R), is a template with people's kidney cDNA storehouse (Clontech Inc.), carries out pcr amplification (HotStarTaq test kit, Qiagen company), and gained PCR product is by QIAquick PCRPurification Kit purifying (Qiagen company).
PCR product behind the purifying, is connected with the pCI carrier of handling through same enzyme (Promega Inc.) after the XbaI mixed enzyme is cut through EcoRI, change the XL1-Blue competent cell over to, inoculation and cultivation on penbritin LB plate, picking resistance clone, order-checking confirms clone's exactness.
The result has obtained to insert the expression vector pCI-KCNQ1 of wild-type KCNQ1 encoding sequence.
Embodiment 2: mutant S140G-KCNQ1 preparation of expression vectors
In the present embodiment, wild-type KCNQ1 expression plasmid with preparation among the embodiment 1 is a template, utilize fixed point to cause 140th the change coding subsequence of change technology (Site-directed Mutagenesis) at coding, the Serine (Serine) that will the encode Glycin (glycine) that instead encodes makes S140G mutant expression plasmid.Detailed process is as follows:
The synthetic two pairs of pcr amplification primers of design.The first pair of primer (being made up of forward primer 1 and the reverse primer 1 that contains the S140G mutational site) is used for increasing containing from the ECoRI point of contact to the segmental product in S140G mutational site
5’-gct?agc?ctc?gag?aat?tcc?tca?c(SEQ?ID?NO：5)
5’-gga?cag?cac?gcc?gaa?gat?g(SEQ?ID?NO：6)
Second pair of primer (being made up of forward primer 2 that contains the S140G mutational site and reverse primer 2) is used to increase and contains fragment from the S140G mutational site to the XbaI point of contact, and sequence is as follows:
5’-cat?ctt?cgg?cgt?gct?gtc?c(SEQ?ID?NO：7)
5’-ggt?cga?ctc?tag?acc?cca?tcc(SEQ?ID?NO：8)
The primer sequence that contains the S140G mutational site is introduced corresponding sudden change Nucleotide in the sudden change position.Expression vector pCI-KCNQ1 with preparation among the embodiment 1 is a template, carries out pcr amplification with first pair of primer (SEQ ID NO:5 and 6) and second pair of primer (SEQ ID NO:7 and 8) respectively.Two fragments of pcr amplification are mixed, amplify complete encode fragment with forward primer 1 and reverse primer 2 (SEQ ID NO:5 and 8).Gained PCR product is by QIAquick PCR Purification Kit purifying (Qiagen Inc).PCR product behind the purifying, is connected with the pCI carrier of handling through same enzyme (PromegaInc.) after the XbaI mixed enzyme is cut through EcoRI, change the XL1-Blue competent cell over to, inoculation and cultivation on penbritin LB plate, picking resistance clone, order-checking confirms clone's exactness.
The result has obtained to insert the expression vector pCI-S140G-KCNQ1 of mutant KCNQ1 encoding sequence.
The S140G sudden change of embodiment 3:KCNQ1 causes the KCNQ1/KCNE1 ionic current to strengthen
The S140G-KCNQ1 expression vector pCI-S140G-KCNQ1 and the wild-type KCNE1 expression plasmid (pIRES-KCNE1-CD8) of preparation in embodiment 1 and 2 are changed in the cells of mamma animals COS-7 cell.In another control group experiment, wild-type KCNQ1 expression vector pCI-KCNQ1 and KCNE1 expression plasmid are changed in the cells of mamma animals COS-7 cell.After behind the cell transfecting about 48 hours, carry out electric physiology potassium current and measure (utilizing patch clamp technique).Method is as follows:
At a moistening 5%CO that contains
2In the environment, the COS-7 cell is cultured in (complementary element: 10% foetal calf serum, 100U/mL Streptomycin sulphate, 100U/mL penicillin) in Dulbecco ' the smodified Eagle nutrient chemical.With DEAE-Dextran precipitator method transfectional cell.With pCI-KCNQ1 or the pCI-S140G-KCNQ1 plasmid DNA of 0.75 μ g, the pIRES-KCNE1-CD8 plasmid DNA of μ g (this plasmid is to insert two fragments of encoded K CNE1 gene respectively and coding CD8 gene simultaneously and the plasmid that forms in pIRES carrier (Clontech Inc.)) O.25.The cell of transfection one dish 35mm.Transfected COS-7 cell is by discerning with anti--CD8 antibody.Ionic current is recorded in room temperature (～22 ℃) to carry out, and adopts full cell (Whole-Cell) logging mode (VP500 Amplifier, BioLogic Inc).Liquid contains (mM) in the electrode: 150mM KCl, 0.5mM MgCl
2, 5mM EDTA and 10mM HEPES, pH7.3.Water-bath contains (mM): 150mM NaCl, 5mM KCl, 2mM CaCl
2, 1mM MgCl
2And 10mMHEPES, pH7.3.Electrode resistance 2.5-5M Ω.By depolarize cause membrane current (the film depolarize is from-130mV to+50mV, keep voltage for-80mV).
The result as shown in Figure 1, the ionic current of S140G-KCNQ1/KCNE1 ionic channel has great increase than the ionic current of wild-type KCNQ1/KCNE1 ionic channel.
The S140G sudden change of embodiment 4:KCNQ1 causes the KCNQ1/KCNE2 ionic current to strengthen
Press embodiment 3 same procedure, S140G-KCNQ1 expression vector and KCNE2 expression plasmid (pIRES-KCNE2-CD8) are changed in the suitable cells of mamma animals COS-7 cell.In the experiment of another control group, expression vector and the KCNE2 expression plasmid of wild-type KCNQ1 changed in the suitable cells of mamma animals COS-7 cell.After behind the cell transfecting about 48 hours, carry out electric physiology potassium current and measure (utilizing patch clamp technique).
The result as shown in Figure 2, the ionic current of S140G-KCNQ1/KCNE2 ionic channel has great increase than the ionic current of wild-type KCNQ1/KCNE2 ionic channel.
Embodiment 5: mutant S140G-KCNQ1/KCNE1 (I
Ks) screening of inhibitors of ion channels
In the present embodiment, S140G-KCNQ1 expression vector (pCI-S140G-KCNQ1) and KCNE1 expression plasmid are changed in the suitable cells of mamma animals (as the COS-7 cell), after behind the cell transfecting about 48 hours, carry out electric physiology potassium current and measure.Relatively add before and after people's inhibitor to be selected, ionic current intensity and characteristic do not have not change, can judge whether this compound has the S140G-KCNQ1/KCNE1 ionic channel to suppress effect.In addition, present method also can be used to the relative inhibition effect of the different inhibitor of comparison.
Repeat the process of embodiment 3, difference is to add candidate substances, chromanol 293B (Chromanol293B) (chemical name is trans-6-cyano4-{N-ethylsulfonyl-N-methylamino}-3-hydroxy-2,2-dimethyl-chromanane) after.Found that ionic current is then suppressed (Fig. 3) fully.Therefore, chromanol 293B is myocardium potassium-channel I
Ks(KCNQ1/KCNE1) inhibitor.
Embodiment 6: the screening of mutant S140G KCNQ1/KCNE2 inhibitors of ion channels
In the present embodiment, S140G-KCNQ1 expression vector (pCI-S140G-KCNQ1) and KCNE2 expression plasmid are changed in the suitable cells of mamma animals (as the COS-7 cell), after behind the cell transfecting about 48 hours, carry out electric physiology potassium current and measure.Relatively add before and after people's inhibitor to be selected, ionic current intensity and characteristic do not have not change, can judge whether this compound has the S140G-KCNQ1/KCNE2 ionic channel to suppress effect.In addition, present method also can be used to the relative inhibition effect of the different inhibitor of comparison.
Repeat the process of embodiment 4, difference is to add candidate substances, chromanol 293B (Chromanol293B) (chemical name is trans-6-cyano4-{N-ethylsulfonyl-N-methylamino}-3-hydroxy-2,2-dimethyl-chromanane) after.Found that ionic current is then suppressed (Fig. 4) fully.Therefore, chromanol 293B is the inhibitor of myocardium potassium-channel KCNQ1/KCNE2.
Embodiment 7: the screening of wild-type KCNQ1/KCNE2 inhibitors of ion channels
In the present embodiment, expression vector (pCI-KCNQ1) and the KCNE2 expression plasmid of wild-type KCNQ1 changed in the suitable cells of mamma animals (as the COS-7 cell), after behind the cell transfecting about 48 hours, carry out electric physiology potassium current and measure.Relatively add before and after people's inhibitor to be selected, ionic current intensity and characteristic do not have not change, can judge whether this compound has the KCNQ1/KCNE2 ionic channel to suppress effect.In addition, present method also can be used to the relative inhibition effect of the different inhibitor of comparison.
Repeat the process of embodiment 4, difference is to add candidate substances, chromanol 293B (Chromanol293B) (chemical name is trans-6-cyano4-{N-ethyIsuIfonyl-N-methylamino}-3-hydroxy-2,2-dimethyl-chromanane) after.Found that ionic current is then suppressed (Fig. 5) fully.Therefore, chromanol 293B is the inhibitor of myocardium potassium-channel KCNQ1/KCNE2.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Tongji University
Nanfang Research Centre, State Human Gene Group
＜120〉application of inhibitors of ion channels in the treatment irregular pulse
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Trp?Gly?Arg?Leu?Pro?Gly?Ala?Arg?Arg?Gly?Ser?Ala?Gly?Leu?Ala?Lys
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Lys?Cys?Pro?Phe?Ser?Leu?Glu?Leu?Ala?Glu?Gly?Gly?Pro?Ala?Gly?Gly
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Ala?Leu?Tyr?Ala?Pro?Ile?Ala?Pro?Gly?Ala?Pro?Gly?Pro?Ala?Pro?His
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Val?Ser?Pro?Ala?Ala?Pro?Ala?Ala?Pro?Pro?Val?Ala?Ser?Asp?Leu?Gly
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Pro?Arg?Pro?Pro?Val?Set?Leu?Asp?Pro?Arg?Val?Ser?Ile?Tyr?Ser?Thr
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Arg?Arg?Pro?Val?Leu?Ala?Arg?Thr?His?Val?Gln?Gly?Arg?Val?Tyr?Asn
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Phe?Leu?Glu?Arg?Pro?Thr?Gly?Trp?Lys?Cys?Phe?Val?Tyr?His?Phe?Ala
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Val?Phe?Leu?Ile?Val?Leu?Val?Cys?Leu?Ile?Phe?Ser?Val?Leu?Ser?Thr
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Ile?Glu?Gln?Tyr?Ala?Ala?Leu?Ala?Thr?Gly?Thr?Leu?Phe?Trp?Met?Glu
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Ile?Val?Leu?Val?Val?Phe?Phe?Gly?Thr?Glu?Tyr?Val?Val?Arg?Leu?Trp
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Ser?Ala?Gly?Cys?Arg?Ser?Lys?Tyr?Val?Gly?Leu?Trp?Gly?Arg?Leu?Arg
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Phe?Ala?Arg?Lys?Pro?Ile?Ser?Ile?Ile?Asp?Leu?Ile?Val?Val?Val?Ala
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Ser?Met?Val?Val?Leu?Cys?Val?Gly?Ser?Lys?Gly?Gln?Val?Phe?Ala?Thr
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Ser?Ala?Ile?Arg?Gly?Ile?Arg?Phe?Leu?Gln?Ile?Leu?Arg?Met?Leu?His
225 230 235 240
Val?Asp?Arg?Gln?Gly?Gly?Thr?Trp?Arg?Leu?Leu?Gly?Ser?Val?Val?Phe
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Ile?His?Arg?Gln?Glu?Leu?Ile?Thr?Thr?Leu?Tyr?Ile?Gly?Phe?Leu?Gly
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Leu?Ile?Phe?Ser?Ser?Tyr?Phe?Val?Tyr?Leu?Ala?Glu?Lys?Asp?Ala?Val
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Asn?Glu?Ser?Gly?Arg?Val?Glu?Phe?Gly?Ser?Tyr?Ala?Asp?Ala?Leu?Trp
290 295 300
Trp?Gly?Val?Val?Thr?Val?Thr?Thr?Ile?Gly?Tyr?Gly?Asp?Lys?Val?Pro
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Gln?Thr?Trp?Val?Gly?Lys?Thr?Ile?Ala?Ser?Cys?Phe?Ser?Val?Phe?Ala
325 330 335
Ile?Ser?Phe?Phe?Ala?Leu?Pro?Ala?Gly?Ile?Leu?Gly?Ser?Gly?Phe?Ala
340 345 350
Leu?Lys?Val?Gln?Gln?Lys?Gln?Arg?Gln?Lys?His?Phe?Asn?Arg?Gln?Ile
355 360 365
Pro?Ala?Ala?Ala?Ser?Leu?Ile?Gln?Thr?Ala?Trp?Arg?Cys?Tyr?Ala?Ala
370 375 380
Glu?Asn?Pro?Asp?Ser?Ser?Thr?Trp?Lys?Ile?Tyr?Ile?Arg?Lys?Ala?Pro
385 390 395 400
Arg?Ser?His?Thr?Leu?Leu?Ser?Pro?Ser?Pro?Lys?Pro?Lys?Lys?Ser?Val
405 410 415
Val?Val?Lys?Lys?Lys?Lys?Phe?Lys?Leu?Asp?Lys?Asp?Asn?Gly?Val?Thr
420 425 430
Pro?Gly?Glu?Lys?Met?Leu?Thr?Val?Pro?His?Ile?Thr?Cys?Asp?Pro?Pro
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Glu?Glu?Arg?Arg?Leu?Asp?His?Phe?Ser?Val?Asp?Gly?Tyr?Asp?Ser?Ser
450 455 460
Val?Arg?Lys?Ser?Pro?Thr?Leu?Leu?Glu?Val?Ser?Met?Pro?His?Phe?Met
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Arg?Thr?Asn?Ser?Phe?Ala?Glu?Asp?Leu?Asp?Leu?Glu?Gly?Glu?Thr?Leu
485 490 495
Leu?Thr?Pro?Ile?Thr?His?Ile?Ser?Gln?Leu?Arg?Glu?His?His?Arg?Ala
500 505 510
Thr?Ile?Lys?Val?Ile?Arg?Arg?Met?Gln?Tyr?Phe?Val?Ala?Lys?Lys?Lys
515 520 525
Phe?Gln?Gln?Ala?Arg?Lys?Pro?Tyr?Asp?Val?Arg?Asp?Val?Ile?6lu?Gln
530 535 540
Tyr?Ser?Gln?Gly?His?Leu?Asn?Leu?Met?Val?Arg?Ile?Lys?6lu?Leu?Gln
545 550 555 560
Arg?Arg?Leu?Asp?Gln?Ser?Ile?Gly?Lys?Pro?Ser?Leu?Phe?Ile?Ser?Val
565 570 575
Ser?Glu?Lys?Ser?Lys?Asp?Arg?Gly?Ser?Asn?Thr?Ile?Gly?Ala?Arg?Leu
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595 600 605
Ile?Thr?Asp?Met?Leu?His?Gln?Leu?Leu?Ser?Leu?His?Gly?Gly?Ser?Thr
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Pro?Gly?Ser?Gly?Gly?Pro?Pro?Arg?Glu?Gly?Gly?Ala?His?Ile?Thr?Gln
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Pro?Cys?6ly?Ser?Gly?Gly?Ser?Val?Asp?Pro?Glu?Leu?Phe?Leu?Pro?Ser
645 650 655
Asn?Thr?Leu?Pro?Thr?Tyr?Glu?Gln?Leu?Thr?Val?Pro?Arg?Arg?Gly?Pro
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＜213〉homo sapiens (Homo sapiens)
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Met?Ala?Ala?Ala?Ser?Ser?Pro?Pro?Arg?Ala?Glu?Arg?Lys?Arg?Trp?Gly
l 5 10 15
Trp?Gly?Arg?Leu?Pro?Gly?Ala?Arg?Arg?Gly?Ser?Ala?Gly?Leu?Ala?Lys
20 25 30
Lys?Cys?Pro?Phe?Ser?Leu?Glu?Leu?Ala?Glu?Gly?Gly?Pro?Ala?Gly?Gly
35 40 45
Ala?Leu?Tyr?Ala?Pro?Ile?Ala?Pro?Gly?Ala?Pro?Gly?Pro?Ala?Pro?His
50 55 60
Val?Ser?Pro?Ala?Ala?Pro?Ala?Ala?Pro?Pro?Val?Ala?Ser?Asp?Leu?Gly
65 70 75 80
Pro?Arg?Pro?Pro?Val?Ser?Leu?Asp?Pro?Arg?Val?Ser?Ile?Tyr?Ser?Thr
85 90 95
Arg?Arg?Pro?Val?Leu?Ala?Arg?Thr?His?Val?Gln?Gly?Arg?Val?Tyr?Asn
100 105 110
Phe?Leu?Glu?Arg?Pro?Thr?Gly?Trp?Lys?Cys?Phe?Val?Tyr?His?Phe?Ala
115 120 125
Val?Phe?Leu?Ile?Val?Leu?Val?Cys?Leu?Ile?Phe?Gly?Val?Leu?Ser?Thr
130 135 140
Ile?Glu?Gln?Tyr?Ala?Ala?Leu?Ala?Thr?Gly?Thr?Leu?Phe?Trp?Met?Glu
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Ile?Val?Leu?Val?Val?Phe?Phe?Gly?Thr?Glu?Tyr?Val?Val?Arg?Leu?Trp
165 170 175
Ser?Ala?Gly?Cys?Arg?Ser?Lys?Tyr?Val?Gly?Leu?Trp?Gly?Arg?Leu?Arg
180 185 190
Phe?Ala?Arg?Lys?Pro?Ile?Ser?Ile?Ile?Asp?Leu?Ile?Val?Val?Val?Ala
195 200 205
Ser?Met?Val?Val?Leu?Cys?Val?Gly?Ser?Lys?Gly?Gln?Val?Phe?Ala?Thr
210 215 220
Ser?Ala?Ile?Arg?Gly?Ile?Arg?Phe?Leu?Gln?Ile?Leu?Arg?Met?Leu?His
225 230 235 240
Val?Asp?Arg?Gln?Gly?Gly?Thr?Trp?Arg?Leu?Leu?Gly?Ser?Val?Val?Phe
245 250 255
Ile?His?Arg?Gln?Glu?Leu?Ile?Thr?Thr?Leu?Tyr?Ile?Gly?Phe?Leu?Gly
260 265 270
Leu?Ile?Phe?Ser?Ser?Tyr?Phe?Val?Tyr?Leu?Ala?Glu?Lys?Asp?Ala?Val
275 280 285
Asn?Glu?Ser?Gly?Arg?Val?Glu?Phe?Gly?Ser?Tyr?Ala?Asp?Ala?Leu?Trp
290 295 300
Trp?Gly?Val?Val?Thr?Val?Thr?Thr?Ile?Gly?Tyr?Gly?Asp?Lys?Val?Pro
305 310 315 320
Gln?Thr?Trp?Val?Gly?Lys?Thr?Ile?Ala?Ser?Cys?Phe?Ser?Val?Phe?Ala
325 330 335
Ile?Ser?Phe?Phe?Ala?Leu?Pro?Ala?Gly?Ile?Leu?Gly?Ser?Gly?Phe?Ala
340 345 350
Leu?Lys?Val?Gln?Gln?Lys?Gln?Arg?Gln?Lys?His?Phe?Asn?Arg?Gln?Ile
355 360 365
Pro?Ala?Ala?Ala?Ser?Leu?Ile?Gln?Thr?Ala?Trp?Arg?Cys?Tyr?Ala?Ala
370 375 380
Glu?Asn?Pro?Asp?Ser?Ser?Thr?Trp?Lys?Ile?Tyr?Ile?Arg?Lys?Ala?Pro
385 390 395 400
Arg?Ser?His?Thr?Leu?Leu?Ser?Pro?Ser?Pro?Lys?Pro?Lys?Lys?Ser?Val
405 410 415
Val?Val?Lys?Lys?Lys?Lys?Phe?Lys?Leu?Asp?Lys?Asp?Asn?Gly?Val?Thr
420 425 430
Pro?Gly?Glu?Lys?Met?Leu?Thr?Val?Pro?His?Ile?Thr?Cys?Asp?Pro?Pro
435 440 445
Glu?Glu?Arg?Arg?Leu?Asp?His?Phe?Ser?Val?Asp?Gly?Tyr?Asp?Ser?Ser
450 455 460
Val?Arg?Lys?Ser?Pro?Thr?Leu?Leu?Glu?Val?Ser?Met?Pro?His?Phe?Met
465 470 475 480
Arg?Thr?Asn?Ser?Phe?Ala?Glu?Asp?Leu?Asp?Leu?Glu?Gly?Glu?Thr?Leu
485 490 495
Leu?Thr?Pro?Ile?Thr?His?Ile?Ser?Gln?Leu?Arg?Glu?His?His?Arg?Ala
500 505 510
Thr?Ile?Lys?Val?Ile?Arg?Arg?Met?Gln?Tyr?Phe?Val?Ala?Lys?Lys?Lys
515 520 525
Phe?Gln?Gln?AIa?Arg?Lys?Pro?Tyr?Asp?Val?Arg?Asp?Val?Ile?Glu?Gln
530 535 540
Tyr?Ser?Gln?Gly?His?Leu?Asn?Leu?Met?Val?Arg?Ile?Lys?Glu?Leu?Gln
545 550 555 560
Arg?Arg?Leu?Asp?Gln?Ser?Ile?Gly?Lys?Pro?Ser?Leu?Phe?Ile?Ser?Val
565 570 575
Ser?Glu?Lys?Ser?Lys?Asp?Arg?Gly?Ser?Asn?Thr?Ile?Gly?Ala?Arg?Leu
580 585 590
Asn?Arg?Val?Glu?Asp?Lys?Val?Thr?Gln?Leu?Asp?Gln?Arg?Leu?Ala?Leu
595 600 605
Ile?Thr?Asp?Met?Leu?His?Gln?Leu?Leu?Ser?Leu?His?Gly?Gly?Ser?Thr
610 615 620
Pro?Gly?Ser?Gly?Gly?Pro?Pro?Arg?Glu?Gly?Gly?Ala?His?Ile?Thr?Gln
625 630 635 640
Pro?Cys?Gly?Ser?Gly?Gly?Ser?Val?Asp?Pro?Glu?Leu?Phe?Leu?Pro?Ser
645 650 655
Asn?Thr?Leu?Pro?Thr?Tyr?Glu?Gln?Leu?Thr?Val?Pro?Arg?Arg?Gly?Pro
660 665 670
Asp?Glu?Gly?Ser
675
<210>3
<211>32
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(32)
＜223〉primer
<400>3
cgagaattcc?tcaccatggc?cgcggcctcc?tc 32
<210>4
<211>31
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(31)
＜223〉primer
<400>4
ggtcgactct?agaccccatc?ccctcctcag?g 31
<210>5
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(22)
＜223〉primer
<400>5
gctagcctcg?agaattcctc?ac 22
<210>6
<211>19
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(19)
＜223〉primer
<400>6
ggacagcacg?ccgaagatg 19
<210>7
<211>19
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(19)
＜223〉primer
<400>7
catcttcggc?gtgctgtcc 19
<210>8
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(21)
＜223〉primer
<400>8
ggtcgactct?agaccccatc?c 21
<210>9
<211>2031
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>9
atggccgcgg?cctcctcccc?gcccagggcc?gaaaggaagc?gctggggttg?gggccgcctg 60
ccaggcgccc?ggcggggcag?cgcgggcctg?gccaaaaaat?gccccttctc?gctggaactg 120
gcggagggcg?gcccggcggg?cggcgcgctc?tacgcgccca?tcgcgcccgg?cgccccaggt 180
cccgcgcccc?atgtgtcccc?ggccgcgccc?gccgcgcccc?cagttgcttc?cgaccttggc 240
ccgcggccgc?cggtgagcct?agacccgcgc?gtctccatat?acagcacgcg?ccgcccggtg 300
ttggcgcgaa?cccacgtcca?gggccgcgtc?tacaacttcc?tcgagcgtcc?caccggctgg 360
aaatgcttcg?tttaccactt?cgccgtcttc?ctcatcgtcc?tggtctgcct?catcttcagc 420
gtgctgtcca?ccatcgagca?gtatgccgcc?ctggccacgg?ggactctctt?ctggatggag 480
atcgtgctgg?tggtgttctt?csggacggag?tacgtggtcc?gcctctggtc?cgccggctgc 540
cgcagcaagt?acgtgggcct?ctgggggcgg?ctgcgctttg?cccggaagcc?catttccatc 600
atcgacctca?tcgtggtcgt?ggcctccatg?gtggtcctct?gcgtgggctc?caaggggcag 660
gtgtttgcca?cgtcggccat?caggggcatc?cgcttcctgc?agatcctgag?gatgctacac 720
gtcgaccgcc?agggaggcac?ctggaggctc?ctgggctccg?tggtcttcat?ccaccgccag 780
gagctgataa?ccaccctgta?catcggcttc?ctgggcctca?tcttctcctc?gtactttgtg 840
tacctggctg?agaaggacgc?ggtgaacgag?tcaggccgcg?tggagttcgg?cagctacgca 900
gatgcgctgt?ggtggggggt?ggtcacagtc?accaccatcg?gctatgggga?caaggtgccc 960
cagacgtggg?tcgggaagac?catcgcctcc?tgcttctctg?tctttgccat?ctccttcttt 1020
gcgctcccag?cggggattct?tggctcgggg?tttgccctga?aggtgcagca?gaagcagagg 1080
cagaagcact?tcaaccggca?gatcccggcg?gcagcctcac?tcattcagac?cgcatggagg 1140
tgctatgctg?ccgagaaccc?cgactcctcc?acctggaaga?tctacatccg?gaaggccccc 1200
cggagccaca?ctctgctgtc?acccagcccc?aaacccaaga?agtctgtggt?ggtaaagaaa 1260
aaaaagttca?agctggacaa?agacaatggg?gtgactcctg?gagagaagat?gctcacagtc 1320
ccccatatca?cgtgcgaccc?cccagaagag?cggcggctgg?accacttctc?tgtcgacggc 1380
tatgacagtt?ctgtaaggaa?gagcccaaca?ctgctggaag?tgagcatgcc?ccatttcatg 1440
agaaccaaca?gcttcgccga?ggacctggac?ctggaagggg?agactctgct?gacacccatc 1500
acccacatct?cacagctgcg?ggaacaccat?cgggccacca?ttaaggtcat?tcgacgcatg 1560
cagtactttg?tggccaagaa?gaaattccag?caagcgcgga?agccttacga?tgtgcgggac 1620
gtcattgagc?agtactcgca?gggccacctc?aacctcatgg?tgcgcatcaa?ggagctgcag 1680
aggaggctgg?accagtccat?tgggaagccc?tcactgttca?tctccgtctc?agaaaagagc 1740
aaggatcgcg?gcagcaacac?gatcggcgcc?cgcctgaacc?gagtagaaga?caaggtgacg 1800
cagctggacc?agaggctggc?actcatcacc?gacatgcttc?accagctgct?ctccttgcac 1860
ggtggcagca?cccccggcag?cggcggcccc?cccagagagg?gcggggccca?catcacccag 1920
ccctgcggca?gtggcggctc?cgtcgaccct?gagctcttcc?tgcccagcaa?caccctgccc 1980
acctacgagc?agctgaccgt?gcccaggagg?ggccccgatg?aggggtcctg?a 2031
Claims (9)
1. a method of screening the inhibitor of myocardium KCNQ1 potassium-channel is characterized in that, comprises step:
(a) change α subunit expression carrier and β subunit expression carrier over to mammalian cell, obtain the mammalian cell of conversion, wherein α subunit expression carrier is the KCNQ1 expression vector, and β subunit expression carrier is selected from down group: the expression vector of KCNE1 expression vector or KCNE2;
(b) in the substratum of the mammalian cell of the conversion of step (a), add candidate substances, and measure the electric physiology potassium current that adds before and after the candidate substances,
Wherein, if the electric physiology potassium current that adds after the candidate substances reduces, show that then this active substance is the inhibitor of myocardium KCNQ1 potassium-channel.
2. the method for claim 1 is characterized in that, described KCNQ1 expression vector is expressed the KCNQ1 of wild-type.
3. the method for claim 1 is characterized in that, described KCNQ1 expression vector is expressed the KCNQ1 of mutant.
4. method as claimed in claim 3 is characterized in that, described KCNQ1 expression vector is expressed the KCNQ1 of S140G mutant.
5. the method for claim 1 is characterized in that, described β subunit expression vector expression KCNE1.
6. the method for claim 1 is characterized in that, described β subunit expression vector expression KCNE2.
7. the method for claim 1 is characterized in that, the measuring method in the step (b) is the patch clamp method.
8. a method of screening the promotor of myocardium KCNQ1 potassium-channel is characterized in that, comprises step:
(a) change α subunit expression carrier and β subunit expression carrier over to mammalian cell, obtain the mammalian cell of conversion, wherein α subunit expression carrier is the KCNQ1 expression vector, and β subunit expression carrier is selected from down group: the expression vector of KCNE1 expression vector or KCNE2;
(b) in the substratum of the mammalian cell of the conversion of step (a), add candidate substances, and measure the electric physiology potassium current that adds before and after the candidate substances,
Wherein, if the electric physiology potassium current that adds after the candidate substances increases, show that then this active substance is the promotor of myocardium KCNQ1 potassium-channel.
9. the proteic purposes of S140G mutant KCNQ1 is characterized in that, is used to screen the inhibitor or the promotor of myocardium KCNQ1 potassium-channel.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03114730 CN1239713C (en) | 2003-01-06 | 2003-01-06 | Application of ion channel inhibitor for curing arhythmia |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03114730 CN1239713C (en) | 2003-01-06 | 2003-01-06 | Application of ion channel inhibitor for curing arhythmia |
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| Publication Number | Publication Date |
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| CN1515680A true CN1515680A (en) | 2004-07-28 |
| CN1239713C CN1239713C (en) | 2006-02-01 |
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| CN 03114730 Expired - Fee Related CN1239713C (en) | 2003-01-06 | 2003-01-06 | Application of ion channel inhibitor for curing arhythmia |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1306037C (en) * | 2004-10-19 | 2007-03-21 | 哈尔滨医科大学 | Novel use of cardiocyte M3 receptor and IkM3 in screening medicament for treating arrhythmia |
| CN102199197A (en) * | 2011-04-13 | 2011-09-28 | 安徽医科大学 | Polypeptides with function of self-assembled potassium channel and its application |
| CN102854176A (en) * | 2012-07-16 | 2013-01-02 | 河北工业大学 | Screening method of TMEM16A calcium-activated chloride channel inhibitor |
| CN103757091A (en) * | 2013-09-13 | 2014-04-30 | 广州市体育科学研究所 | Rapid gene detection kit and rapid gene detection method for sudden cardiac death |
| CN103820435A (en) * | 2012-11-19 | 2014-05-28 | 上海药明康德新药开发有限公司 | Host cell for expressing KCNQ1/KCNE1 protein and preparation method of host cell |
| CN103834616A (en) * | 2012-11-23 | 2014-06-04 | 苏州药明康德新药开发有限公司 | Cell model for stable expression of human myocardial cell Iks and construction method and application thereof |
-
2003
- 2003-01-06 CN CN 03114730 patent/CN1239713C/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1306037C (en) * | 2004-10-19 | 2007-03-21 | 哈尔滨医科大学 | Novel use of cardiocyte M3 receptor and IkM3 in screening medicament for treating arrhythmia |
| CN102199197A (en) * | 2011-04-13 | 2011-09-28 | 安徽医科大学 | Polypeptides with function of self-assembled potassium channel and its application |
| CN102854176A (en) * | 2012-07-16 | 2013-01-02 | 河北工业大学 | Screening method of TMEM16A calcium-activated chloride channel inhibitor |
| CN102854176B (en) * | 2012-07-16 | 2014-06-18 | 河北工业大学 | Screening method of TMEM16A calcium-activated chloride channel inhibitor |
| CN103820435A (en) * | 2012-11-19 | 2014-05-28 | 上海药明康德新药开发有限公司 | Host cell for expressing KCNQ1/KCNE1 protein and preparation method of host cell |
| CN103834616A (en) * | 2012-11-23 | 2014-06-04 | 苏州药明康德新药开发有限公司 | Cell model for stable expression of human myocardial cell Iks and construction method and application thereof |
| CN103757091A (en) * | 2013-09-13 | 2014-04-30 | 广州市体育科学研究所 | Rapid gene detection kit and rapid gene detection method for sudden cardiac death |
| CN103757091B (en) * | 2013-09-13 | 2016-09-07 | 广州市体育科学研究所 | Sudden cardiac death rapid gene detection kit and detection method |
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| Publication number | Publication date |
|---|---|
| CN1239713C (en) | 2006-02-01 |
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