CN102854176A - Screening method of TMEM16A calcium-activated chloride channel inhibitor - Google Patents
Screening method of TMEM16A calcium-activated chloride channel inhibitor Download PDFInfo
- Publication number
- CN102854176A CN102854176A CN2012102453651A CN201210245365A CN102854176A CN 102854176 A CN102854176 A CN 102854176A CN 2012102453651 A CN2012102453651 A CN 2012102453651A CN 201210245365 A CN201210245365 A CN 201210245365A CN 102854176 A CN102854176 A CN 102854176A
- Authority
- CN
- China
- Prior art keywords
- tmem16a
- inhibitor
- chloride channel
- drug candidate
- calcium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a screening method of a TMEM16A calcium-activated chloride channel inhibitor; with the screening method of the invention, a TMEM16A calcium-activated chloride channel inhibitor can be screened, which can be developed into a medicament for treating hypertension, CF disease, asthma, stomach and intestine dynamic dysfunction diseases, and diseases of some types of tumors.
Description
Technical field
The present invention relates to biology field, particularly a kind of TMEM16A calcium activates the screening technique of chloride channel inhibitor.
Background technology
Calcium activates chloride channel (Calcium-activated Chloride Channels, CaCCs) gain the name owing to being activated by the cell interior calcium ion, be found in the earliest in the Xenopus Oocytes, in succession have subsequently report to show among epithelial cell, vascular endothelial cell, neuron, smooth muscle and the cardiac muscle cell and all exist.The signal transduction pathway of calcium-mediated is the important component part of eukaryotic signal transduction, therefore CaCCs carries out several functions, the adjusting of comprise that fertilization, transepithelial ion/fluid transport, cardiac muscle cell's repolarization and the action potential of egg cell occurs, sense of smell conduction and smooth muscle are flexible etc.
Because technology, the molecular basis problem of CaCCs fail to solve, and cause Related Drug pharmacological research process very slow always.Until have three research groups to report that independently the molecular basis of CaCCs is transmembrane protein 16A(transmembrane16A the end of the year 2008, be called for short TMEM16A) (Schroeder BC etc., Cell, 2008,134:1019-1029; Caputo A etc., Science, 2008,322:590-594; Yang YD etc., Nature, 2008,455:1210-1215).This problem that is found to be the aspect such as the research physiology of CaCCs and pharmacology in specific cells and tissue provides new research platform.
Recent findings TMEM16A passage and multiple major disease are closely related.Bibliographical information, the TMEM16A ion channel may be the drug target of pulmonary cystic fibrosis (CF) disease, activate the TMEM16A passage and can play chloride channel cystic fibrosis transmembrane conductance regulator (cystic fibrosis transmembrane conductance regulator on the compensation epithelial tissue, abbreviation CFTR) ion due to the gene mutation/uneven (Rock JR etc. of fluid transport, J.Biol.Chem., 2009,284:14875-14880).In addition, 2012, Wang etc. (Wang M etc., Circulation, 2012,125:697-707) proof TMEM16A is the molecular basis of cerebrovascular smooth muscle cells CaCCs, and finds that activity and the blood pressure of TMEM16A ion channel are negative correlation.On the other hand, the inhibitor of CaCCs is in the news and can be used for tumour for the treatment of asthma, stomach and intestine dynamics disorder disease and some type etc.This shows, the treatment that screening TMEM16A ion channel specificity correctives can be the CaCCs relevant disease provides new drug target.
The Invitrogen of U.S. Life Sci-Tech company
TMKit Premo
TMThe fluorescence of the Trimutant protein YFP-F46L/H148Q/I152L of halogenide sensor utilization yellow fluorescence protein (Yellow Fluorescence Protein, YFP) screens the correctives of CaCCs/TMEM16A ion channel as index.Use the method, 2010, Namkung etc. (Namkung W etc., The FASEB J., 2010,24:4178-4186) use Premo
TMThe screening of halogenide sensor reagent box obtains tannic acid (tannic acid) and relevant nutgall tannin (gallotannin) is the inhibitor of TMEM16A/CaCCs.
Regrettably, lack at present the screening technique that simple and easy to do TMEM16A calcium activates the chloride channel inhibitor, cause the applied research slower development in the diseases such as tumour of hypertension, CF disease, asthma, stomach and intestine dynamics obstacle and some type take TMEM16A/CaCCs as drug target.
Summary of the invention
The purpose of this invention is to provide the screening technique that a kind of TMEM16A calcium activates the chloride channel inhibitor, the TMEM16A calcium activation chloride channel inhibitor that filters out by this screening technique provides the basis for the application of research TMEM16A/CaCCs in the diseases such as tumour of hypertension, CF disease, asthma, stomach and intestine dynamics disorder disease and some type.
The invention provides the screening technique that a kind of TMEM16A calcium activates the chloride channel inhibitor, use TMEM16A calcium to activate chloride channel activator chitosan oligosaccharide as the positive control of screening inhibitor, comprise the steps:
(a) TMEM16A plasmid stable transfection is entered mammalian cell HEK293 cell;
(b) yellow fluorescence protein YFP Trimutant YFP-F46L/H148Q/I152L is imported the HEK293 cell that stable transfection TMEM16A calcium in the step (a) activates chloride channel;
(c) cell after drug candidate and the described importing of step (b) is hatched, adding the solution that contains iodide ion observes again, the discovery fluorescence intensity is unchanged, it is still unchanged that add the chitosan oligosaccharide fluorescence intensity this moment again, determine that then drug candidate may be the inhibitor of TMEM16A passage, and adopt step (d) further to verify;
(d) measure drug candidate to the influence of peak current in the step (a) with electrophysiological method, if wherein adding the drug candidate after-current reduces than contrast, determine that then drug candidate is the inhibitor that TMEM16A calcium activates chloride channel, if the current ratio contrast does not reduce, determine that then this drug candidate is not the inhibitor that TMEM16A calcium activates chloride channel.
Further preferred, in the described step (b), YFP Trimutant YFP-F46L/H148Q/I152L is by the baculovirus vector transfered cell.
Further preferred, the fluorescence intensity described in the step (d) is detected by laser microscope, and wherein excitation wavelength is 470-540nm, and the detection wavelength is 540-590nm.
Further preferred, described drug candidate is tannic acid or other inhibitor molecules.
Adopt screening technique of the present invention can filter out TMEM16A calcium and activate the chloride channel inhibitor, can be developed as the medicine of the diseases such as tumour for the treatment of hypertension, CF disease, asthma, stomach and intestine dynamics disorder disease and some type.
Description of drawings
Fig. 1: according to TMEM16A calcium of the present invention activate chloride channel inhibitor tannic acid to the fluorescence intensity of yellow fluorescence protein without the obvious trend map of humidification.
Fig. 2: activating chloride channel inhibitor tannic acid according to TMEM16A calcium of the present invention has inhibiting trend map for the TMEM16A electric current.
Embodiment
The invention provides the screening technique that a kind of TMEM16A calcium activates the chloride channel inhibitor, use TMEM16A calcium to activate chloride channel activator chitosan oligosaccharide as the positive control of screening inhibitor, comprise the steps:
(a) TMEM16A plasmid stable transfection is entered mammalian cell HEK293 cell;
(b) yellow fluorescence protein YFP Trimutant YFP-F46L/H148Q/I152L is imported the HEK293 cell that stable transfection TMEM16A calcium in the step (a) activates chloride channel;
(c) cell after drug candidate and the described importing of step (b) is hatched, adding the solution that contains iodide ion observes again, the discovery fluorescence intensity is unchanged, it is still unchanged that add the chitosan oligosaccharide fluorescence intensity this moment again, determine that then drug candidate may be the inhibitor of TMEM16A passage, and adopt step (d) further to verify;
(d) measure drug candidate to the influence of peak current in the step (a) with electrophysiological method, if wherein adding the drug candidate after-current reduces than contrast, determine that then drug candidate is the inhibitor that TMEM16A calcium activates chloride channel, if the current ratio contrast does not reduce, determine that then this drug candidate is not the inhibitor that TMEM16A calcium activates chloride channel.
The cell that can be used for screening technique of the present invention is not particularly limited, and representational example comprises: mammalian cell, and such as the HEK293 cell, Chinese hamster ovary celI or vertebrate Africa xenopus (Xenopus laevis) egg mother cell.
The electrophysiology assay method that can be used for screening technique of the present invention is not particularly limited.Representational example comprises: patch-clamp method (being used for mammalian cell) or bipolar electrode voltage clamp method (being used for Xenopus Oocytes).
The fluorescence intensity detection technique that can be used for screening technique of the present invention is not particularly limited.Representational example comprises: laser confocal microscope (being used for the fluorescence real time imagery) or multi-functional microplate reader (being used for the fluorescence intensity real time record) etc.
Yellow fluorescence protein (YFP) is a kind of fluorescin that derives from green fluorescent protein (GFP), can be excited under wavelength 515nm and send yellow fluorescence.Iodide ion can be combined with YFP and be made fluorescent quenching, and suddenly change two site H148Q and I152L can make YFP that the susceptibility of iodide ion is strengthened.The CaCCs passage is not only a kind of chloride channel, and it has permeation to the most of negative ion that comprises iodide ion.In the YFP gene importing HEK293 cell of method with external source of this drug screening test with virus infections, make YFP great expression in born of the same parents; Drug candidate and cell being hatched fully acts on itself and passage again; Observe at last the cancellation degree that adds the later YFP fluorescence of solution that contains iodide ion.Can make YFP can be considered to the inhibitor of CaCCs passage without the medicine of obviously cancellation with the method.
Chitosan oligosaccharide (chitosan oligosaccharide, COS) is the linear homogeneous polysaccharide that is formed by the chitin dehydration.COS is naturally occurring alkaline amino-oligosacchride, has good water solubility, safety non-toxic, easily by advantages such as animal body absorptions, so its biologic activity receives much concern, and comprises antibiotic (Choi BK etc., International Journal of Antimicrobial Agents, 2001:18:553 – 557), antiviral (Bacon A etc., Infection and Immunity, 2000,68:5764 – 5770), antitumor (Xiong C etc., Carbohydrate Research, 2009,344:1975 – 1983), reduce Blood Cholesterol (Se-Kwon K, Carbohydr.Polym., 2005,62:357 – 368), immunological regulation (Okamoto Y etc., Macromol.Biosci.2003,3:587-590), and to asthma (Chung MJ etc., Int.Immunopharmacol., 2012,12::43-459), diabetes (Lee HW etc., Biol.Pharm.Bull., 2003, the therapeutic action such as 26:1100-1103), but its action site and pharmacological characteristics are still not unclear.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention 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, such as Sambrook etc., 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:
One, carry out first the scalping of inhibitor by the fluorescence experiments method, concrete steps are as follows:
First day is transfected into expression plasmid pEGFP-N1-TMEM16A among the mammalian cell HEK293, cultivates the HEK293 cell of stable transfection YFP in the burnt special-purpose ware of copolymerization; Second day washes the HEK293 cell of transfection YFP and overnight incubation 3 times with D-PBS, stays at last 800 μ l D-PBS; Add tannic acid and hatch 10min, the record fluorescence intensity is the baseline fluorescence intensity; With laser confocal microscope real time record fluorescence intensity, as shown in Figure 1, add the solution 800 μ l that contain 150mM I-this moment, make I concentration reach 75mM, the YFP fluorescence intensity adds chitosan oligosaccharide again without significant change, and the fluorescence intensity in this process is always without significant change (Fig. 1 a, 1b), the result shows that tannic acid may be the inhibitor that TMEM16A calcium activates chloride channel.
Two, use the electrophysiology assay method to carry out determining of inhibitor, concrete steps are as follows:
Expression plasmid pEGFP-N1-TMEM16A is changed among the mammalian cell HEK293.Within 24-72h behind the cell transfecting, carry out electrophysiology detection (utilizing patch clamp technique).Concrete grammar is as follows: the HEK293 cell is with the high sugar of the DMEM(that contains 10% hyclone) nutrient solution cultivations (penicillin and the 100 μ g/ml streptomysins of adding 100UI/ml) of going down to posterity.Stable transfection process Lipofectamine2000(Invitrogen company) liposome carries out.Cell is cultured to exponential phase and is used for experiment in 37 ℃ in the 5%CO2 saturated humidity incubator.The electricity physiological detection is at room temperature carried out (approximately 22 ℃), adopts inside-out (Inside-Out) logging mode (EPC-10Amplifier, HEKA company, Germany), and the composition of the outer liquid of interior liquid and basis is (unit: mM): NaCl140, MgCl
26H
2O, HEPES10, EGTA5 transfers to pH7.4 with NaOH.The concentration of tannic acid is made as 1 in the outer liquid of adding medicine, 10,50,100,500 μ g/ml, whether express with 500nM free calcium ions test TMEM16A passage, do negative control with 10mM EGTA without calcium solution, and make comparisons with the electric current under the 1 μ M free calcium condition, as shown in Figure 2, add the tannic acid after-current and reduce than contrast, determine that then tannic acid is the inhibitor that TMEM16A calcium activates chloride channel.
Claims (4)
1. the screening technique of a TMEM16A calcium activation chloride channel inhibitor uses TMEM16A calcium to activate chloride channel activator chitosan oligosaccharide as the positive control of screening inhibitor, it is characterized in that, comprises the steps:
(a) TMEM16A plasmid stable transfection is entered mammalian cell HEK293 cell;
(b) yellow fluorescence protein YFP Trimutant YFP-F46L/H148Q/I152L is imported the HEK293 cell that stable transfection TMEM16A calcium in the step (a) activates chloride channel;
(c) cell after drug candidate and the described importing of step (b) is hatched, adding the solution that contains iodide ion observes again, the discovery fluorescence intensity is unchanged, it is still unchanged that add the chitosan oligosaccharide fluorescence intensity this moment again, determine that then drug candidate may be the inhibitor of TMEM16A passage, and adopt step (d) further to verify;
(d) measure drug candidate to the influence of peak current in the step (a) with electrophysiological method, if wherein adding the drug candidate after-current reduces than contrast, determine that then drug candidate is the inhibitor that TMEM16A calcium activates chloride channel, if the current ratio contrast does not reduce, determine that then this drug candidate is not the inhibitor that TMEM16A calcium activates chloride channel.
2. screening technique according to claim 1 is characterized in that, in the described step (b), YFP Trimutant YFP-F46L/H148Q/I152L is by the baculovirus vector transfered cell.
3. screening technique according to claim 2 is characterized in that, the fluorescence intensity described in the step (d) is detected by laser microscope, and wherein excitation wavelength is 470-540nm, and the detection wavelength is 540-590nm.
4. arbitrary described screening technique is characterized in that according to claim 1-3, and described drug candidate is tannic acid or other inhibitor molecules.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210245365.1A CN102854176B (en) | 2012-07-16 | 2012-07-16 | Screening method of TMEM16A calcium-activated chloride channel inhibitor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210245365.1A CN102854176B (en) | 2012-07-16 | 2012-07-16 | Screening method of TMEM16A calcium-activated chloride channel inhibitor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102854176A true CN102854176A (en) | 2013-01-02 |
| CN102854176B CN102854176B (en) | 2014-06-18 |
Family
ID=47400952
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210245365.1A Expired - Fee Related CN102854176B (en) | 2012-07-16 | 2012-07-16 | Screening method of TMEM16A calcium-activated chloride channel inhibitor |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102854176B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102854236A (en) * | 2012-07-16 | 2013-01-02 | 河北工业大学 | Screening method of TMEM16A calcium-activated chloride channel activator |
| CN103898059A (en) * | 2014-03-05 | 2014-07-02 | 吉林医药学院 | Cell model and screening method for screening calcium-activated chloride ion channel inhibitor |
| CN108004198A (en) * | 2016-10-28 | 2018-05-08 | 华中农业大学 | The method for building up of the Dominant Plat based on ICAM-1 signal paths |
| CN110167588A (en) * | 2016-12-22 | 2019-08-23 | 南京大学 | Reduce the smooth muscle contraction response induced by inflammatory contracting agent |
| CN111450237A (en) * | 2020-03-17 | 2020-07-28 | 中国人民解放军63919部队 | Novel target for developing osteoporosis treatment drug |
| CN112359088A (en) * | 2020-10-09 | 2021-02-12 | 吉林医药学院 | FRT cell strain and application thereof in preparation of preparation or kit for detecting concentration of free calcium ions in cells |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1515680A (en) * | 2003-01-06 | 2004-07-28 | 同济大学 | Application of ion channel inhibitor for curing arhythmia |
-
2012
- 2012-07-16 CN CN201210245365.1A patent/CN102854176B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1515680A (en) * | 2003-01-06 | 2004-07-28 | 同济大学 | Application of ion channel inhibitor for curing arhythmia |
Non-Patent Citations (3)
| Title |
|---|
| WAN NAMKUNG等: "TMEM16A Inhibitors Reveal TMEM16A as a Minor Component of Calcium-activated Chloride Channel Conductance in Airway and Intestinal Epithelial Cells", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》, vol. 286, no. 3, 31 December 2011 (2011-12-31) * |
| 吴庆田等: "mCLCA3增强型绿色荧光蛋白真核表达系统的构建及功能分析", 《CHINESE JOURNA L OFM ICROECOLOGY》, vol. 20, no. 3, 31 December 2008 (2008-12-31) * |
| 陈娅斐等: "跨膜蛋白16A：钙激活氯通道的最新进展", 《生物化学与生物物理进展》, vol. 37, no. 11, 31 December 2010 (2010-12-31) * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102854236A (en) * | 2012-07-16 | 2013-01-02 | 河北工业大学 | Screening method of TMEM16A calcium-activated chloride channel activator |
| CN102854236B (en) * | 2012-07-16 | 2015-01-21 | 河北工业大学 | Screening method of TMEM16A calcium-activated chloride channel activator |
| CN103898059A (en) * | 2014-03-05 | 2014-07-02 | 吉林医药学院 | Cell model and screening method for screening calcium-activated chloride ion channel inhibitor |
| CN103898059B (en) * | 2014-03-05 | 2016-08-17 | 吉林医药学院 | The cell model of screening calcium-activated potassium current inhibitor and screening technique |
| CN108004198A (en) * | 2016-10-28 | 2018-05-08 | 华中农业大学 | The method for building up of the Dominant Plat based on ICAM-1 signal paths |
| CN108004198B (en) * | 2016-10-28 | 2021-07-16 | 华中农业大学 | Method for establishing high-throughput drug screening model based on ICAM-1 signal channel |
| CN110167588A (en) * | 2016-12-22 | 2019-08-23 | 南京大学 | Reduce the smooth muscle contraction response induced by inflammatory contracting agent |
| CN111450237A (en) * | 2020-03-17 | 2020-07-28 | 中国人民解放军63919部队 | Novel target for developing osteoporosis treatment drug |
| CN112359088A (en) * | 2020-10-09 | 2021-02-12 | 吉林医药学院 | FRT cell strain and application thereof in preparation of preparation or kit for detecting concentration of free calcium ions in cells |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102854176B (en) | 2014-06-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102854176B (en) | Screening method of TMEM16A calcium-activated chloride channel inhibitor | |
| CN102854236B (en) | Screening method of TMEM16A calcium-activated chloride channel activator | |
| Gomes et al. | ATP release through connexin hemichannels in corneal endothelial cells | |
| Margrie et al. | Targeted whole-cell recordings in the mammalian brain in vivo | |
| Arendt et al. | Chronic inactivation of a neural circuit enhances LTP by inducing silent synapse formation | |
| Lu et al. | ER-localized Hrd1 ubiquitinates and inactivates Usp15 to promote TLR4-induced inflammation during bacterial infection | |
| Kasymov et al. | Differential sensitivity of brainstem versus cortical astrocytes to changes in pH reveals functional regional specialization of astroglia | |
| Yan et al. | Il-1β and reactive oxygen species differentially regulate neutrophil directional migration and basal random motility in a zebrafish injury–induced inflammation model | |
| Kobelt et al. | Short duration electrical stimulation to enhance neurite outgrowth and maturation of adult neural stem progenitor cells | |
| Thompson et al. | Calcium influx affects intracellular transport and membrane repair following nanosecond pulsed electric field exposure | |
| Harmsen et al. | Amphotericin B is cytotoxic at locally delivered concentrations | |
| Petrilli et al. | LIM domain kinases as potential therapeutic targets for neurofibromatosis type 2 | |
| Nualart‐Marti et al. | Role of connexin 32 hemichannels in the release of ATP from peripheral nerves | |
| Fowlkes et al. | Mechanical loading promotes mast cell degranulation via RGD-integrin dependent pathways | |
| Lan et al. | Aquaporin 4 in astrocytes is a target for therapy in Alzheimer's disease | |
| Yang et al. | Expression of a dominant-negative Rho-kinase promotes neurite outgrowth in a microenvironment mimicking injured central nervous system | |
| Wu et al. | Lamotrigine reduces β-site AβPP-cleaving enzyme 1 protein levels through induction of autophagy | |
| Wen et al. | Piezoelectric ceramic (PZT) modulates axonal guidance growth of rat cortical neurons via RhoA, Rac1, and Cdc42 pathways | |
| Robertson et al. | Whole-cell patch-clamp recording of nicotinic acetylcholine receptors in adult Brugia malayi muscle | |
| Jin et al. | Acquisition of contextual discrimination involves the appearance of a RAS-GRF1/p38 mitogen-activated protein (MAP) kinase-mediated signaling pathway that promotes long term potentiation (LTP) | |
| Lautner et al. | In vitro sarcoma cells release a lipophilic substance that activates the pain transduction system via TRPV1 | |
| Seegers et al. | A pulsed DC electric field affects P2-purinergic receptor functions by altering the ATP levels in in vitro and in vivo systems | |
| Ramírez-Ponce et al. | Mast cell changes the phenotype of microglia via histamine and ATP | |
| Zeng et al. | In vitro analyses of mild heat stress in combination with antifungal agents against Aspergillus fumigatus biofilm | |
| Kanda et al. | A novel single-strand RNAi therapeutic agent targeting the (Pro) renin receptor suppresses ocular inflammation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140618 Termination date: 20210716 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |