アルコール乱用の国民の協会からとPAのプリ博士NIH NRSAフェローシップ（F31AA017042）、私たちの活動は、神経科学（PAS）、国立薬物乱用研究所（PAS R01 DA019022）のためのマックナイト基金基金からの財政支援によって可能となったとアルコール依存症。 GIRK2 - L257W変異体は、博士プラフルラAryalによって提供されていました。 親切にライカインスツルメンツが提供する蛍光顕微鏡とスポンサーシップ。

<ol>
	<li>Aryal, P., Dvir, H., Choe, S., Slesinger, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+discrete+alcohol+pocket+involved+in+GIRK+channel+activation.">A discrete alcohol pocket involved in GIRK channel activation.</a> Nature Neurosci. 12, 988-995 (2009).</li><li>Panchenko, V. A., Glasser, C. R., Mayer, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+similarities+between+glutamate+receptor+channels+and+K++channels+examined+by+scanning+mutagenesis.">Structural similarities between glutamate receptor channels and K+ channels examined by scanning mutagenesis.</a> J Gen Phys. 117, 345-360 (2001).</li><li>Doyle, D. A., Cabral, M. o. r. a. i. s., Pfuetzner, J., Kuo, R. A., Gulbis, A., Cohen, J. M., Chait, S. L., &amp;amp, B. T., MacKinnon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+potassium+channel:+molecular+basis+of+K++conduction+and+selectivity.">The structure of the potassium channel: molecular basis of K+ conduction and selectivity.</a> Science. 280, 69-77 (1998).</li><li>Nishida, M., Cadene, M., Chait, B. T., MacKinnon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+a+Kir3.1-prokaryotic+Kir+channel+chimera.">Crystal structure of a Kir3.1-prokaryotic Kir channel chimera.</a> EMBO J. 26, 4005-4015 (2007).</li><li>Tao, X., Avalos, J. L., Chen, J., MacKinnon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+eukaryotic+strong+inward-rectifier+K++channel+Kir2.2+at+3.1+&amp;Aring;+resolution.">Crystal structure of the eukaryotic strong inward-rectifier K+ channel Kir2.2 at 3.1 &amp;Aring; resolution.</a> Science. 326, 1668-1674 (2009).</li></ol>

利害の衝突は宣言されません。

部位特異的突然変異誘発法は、イオンチャネルの構造と機能の関係を研究する古典的なアプローチです。それはイオンチャネルの重要な残基を変更することにより、一つのチャネルの機能に顕著な変化を観察することができるはずという前提に基づいています。対照的に、非クリティカルな位置に多くの変異は、チャネルの機能に大きな摂動なしで導入することができる。通常は、問題の残留物が最初にアラニン、トリプトファン、アミノ酸の最大の2〜突然変異が続く小さな非極性アミノ酸に変異している。残留物は、チャネルの機能に重要な場合は、両方の変異がチャネル活性を変更します。あまり本質的な残基が変異している場合、特にアラニンへの変異の影響は、しばしば平凡です。トリプトファンの変異は、トリプトファンのサイズに関連付けられている立体的な支障に起因する変化を引き起こす可能性が高くなります。多くの場合、チャンネル（アラニンやトリプトファンスキャンと呼ばれる）の断片の中アラニンやトリプトファンの構造情報が不足して、体系的な変異が重要な残基を特定するために使用することができます。別の点突然変異を持つ複数のクローンを並列比較的短い時間内に複数の残基をテストするためにできるように製造することができることに注意することは重要です。最近では、チャネルの複数の結晶構造は3,4,5利用できるようになりました。彼らは、このプロトコルで説明されているアプローチを使用してテストすることがキーとなる可能性がある残基を提案するために使用することができます。 野生型チャネル上の測定値は常にトランスフェクションし、電気生理学的実験が正しく行われたことを確実にするためのポジティブコントロールとして機能する変異体チャネルの測定を添付してください。点突然変異は、機能的な現在の不足にチャネル活性の野生型または改変された規制と同様の電流を導くことができる。測定可能な電流の欠如は、不適切な折りたたみまたは変更された人身売買に起因する可能性があります。このようなケースでは、チャネルがセルの内部留保の非機能的な細胞膜上のチャンネルとチャンネルを区別するために細胞外タグに対するビオチン化アッセイまたは免疫蛍光染色を（細胞浸透性のない）を使用して表面に正確にトラヒックのかどうかを確認すると便利な場合があります。最後に、チャネルだけでなく、チャネルの規制上の変異の影響の基底活性だけでなく、を調査することをお勧めします。チャネル機能の調節機序の異なるが、Gタンパク質、PIP2、イオン、ATP濃度、リン酸化（チロシン、セリンまたはスレオニンの変異を使用して）、ユビキチン化やSUMO化（リジンの突然変異を使用して）による規制を含む、対象のイオンチャネルの種類により研究される可能性とダイレクトチャネルオープナーまたはブロッカー。

<table><tbody><tr><td>Chemicals</td><td>Sigma</td><td>Varies</td><td></td></tr><tr><td>Mini Prep Kit</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td>Radiacwash</td><td>Biodex</td><td>005-100</td><td></td></tr><tr><td>Poly-D-lysine hydrobromide</td><td>BD Biosciences</td><td>CB40210</td><td>Poly-L-lysine can also be used</td></tr><tr><td>Maxi Prep Kit</td><td>Qiagen</td><td>12263</td><td></td></tr><tr><td>QuickChange XL II Site Directed Mutagenesis Kit</td><td>Stratagene</td><td>200512-5</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen</td><td>11668-027</td><td></td></tr><tr><td>Cover slips &amp;#710;&amp;#8230; 12 mm</td><td>Warner Instruments</td><td>CS-12R</td><td></td></tr><tr><td>Capillary Glass Tubing Borosilicate-standard wall</td><td>Warner Instruments</td><td>G150-3</td><td></td></tr><tr><td>&lt;a href=&quot;http://www.leica-microsystems.com/products/light-microscopes/clinical/inverted-microscopes/details/product/leica-dmi3000-b/&quot; target=&quot;blank&quot;&gt;Inverted fluorescence microscope&lt;/a&gt;</td><td><a href="http://www.leica-microsystems.com/products/light-microscopes/clinical/inverted-microscopes/details/product/leica-dmi3000-b/" target="_blank"><img src="/img/new_window.png" /> Leica</a></td><td>DMI3000 B</td><td></td></tr><tr><td>Electrophysiology</td><td>Nikon/&lt;br/&gt;Leica/&lt;br/&gt;Zeiss</td><td>Any inverted DIC&lt;br/&gt;(Hoffmann)&lt;br/&gt;microscope with fluorescence</td><td></td></tr><tr><td>Amplifier</td><td>Molecular Devices</td><td>Axon Axopatch 200B amplifier</td><td></td></tr><tr><td>Perfusion system</td><td>Warner Instruments</td><td>VC-6 Perfusion MM-6</td><td></td></tr><tr><td>Digitizer</td><td>Molecular Devices</td><td>Digidata 1320 digitizer</td><td></td></tr><tr><td>Manipulators</td><td>Sutter Instruments</td><td>Manipulator: MP-85</td><td></td></tr><tr><td>Electrode Puller</td><td>Narishige</td><td>PC10 vertical electrode puller</td><td></td></tr><tr><td>Isolation Table</td><td>Technical Manufacturing Corporation</td><td>Air table</td><td></td></tr></tbody></table>

mutagenesis and functional analysis of ion channels heterologously expressed in mammalian cells

我々は、イオンチャネルの点突然変異を導入することの機能的効果を研究する方法を説明します。我々は、ニューロンの興奮性を調節するための重要なGタンパク質依存性内向き整流カリウム（GIRKと呼ばれる）チャンネルを、研究しています。つの異なる哺乳類のGIRKチャネルのサブユニット（GIRK1 - GIRK4）があります - それはホモ四量体を形成するので、我々はGIRK2に焦点を当てる。このようなムスカリン性受容体（M2R）のようなGタンパク質共役受容体（GPCR）、、のさまざまな種類の刺激は、GIRKチャネルの活性化につながる。アルコールはまた、直接GIRKチャネルをアクティブにします。我々は、特にGIRKチャネルのcDNAで1つ以上のヌクレオチドを​​変更することにより、一つのアミノ酸を変異させる方法を紹介します。この変異cDNA配列は、精製された、細菌中で増幅され、点突然変異の存在はDNAシークエンシングによって確認されます。変異と野生型GIRKチャネルのcDNAは、in vitroで培養したヒト胚性腎HEK293T細胞にトランスフェクトされます。最後に、ホールセルパッチクランプ電気生理学は、異所性に発現した野生型または変異GIRKチャネルを介して巨視的なカリウム電流を研究するために使用されます。この実験では、M2R依存とアルコール依存性活性化に関するGIRK2チャンネルでL257Wの突然変異の効果を検討する。

Watch this Scientific Journal Video about Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells at JoVE.com

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

我々は、イオンチャンネルの機能上の単一の点変異の効果を研究する方法を説明します。

異種哺乳動物細胞で発現したイオンチャネルの変異導入と機能解析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

mutagenesis-functional-analysis-ion-channels-heterologously-expressed

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

17.4K ビュー.  Salk Institute for Biological Studies. 我々は、イオンチャンネルの機能上の単一の点変異の効果を研究する方法を説明します。

ビデオ: Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

誘導性一過性トランスフェクションを介して制御可能なイオンチャネルの発現

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

遺伝学

Expression and Purification of Mammalian Bestrophin Ion Channels

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- channel (CaCC) predominantly expressed in retinal pigment epithelium (RPE). The physiological and pathological significance of BEST1 is highlighted by the fact that over 200 distinct mutations in the BEST1 gene have been genetically linked to a spectrum of at least five retinal degenerative disorders, such as Best vitelliform macular dystrophy (Best disease). Therefore, understanding the biophysics of bestrophin channels at the single-molecule level holds tremendous significance. However, obtaining purified mammalian ion channels is often a challenging task. Here, we report a protocol for the expression of mammalian bestrophin proteins with the BacMam baculovirus gene transfer system and their purification by affinity and size-exclusion chromatography. The purified proteins have the potential to be utilized in subsequent functional and structural analyses, such as electrophysiological recording in lipid bilayers and crystallography. Importantly, this pipeline can be adapted to study the functions and structures of other ion channels.

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels.

哺乳類 Bestrophin イオン チャネルの発現と精製

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

生化学

固形腫瘍を治療するためのイオンチャネルの薬理学的標的化

Screening Ion Channels in Cancer Cells

Ion channels are critical for cell development and maintaining cell homeostasis. The perturbation of ion channel function contributes to the development of a broad range of disorders or channelopathies. Cancer cells utilize ion channels to drive their own development, as well as to improve as a tumor and to assimilate in a microenvironment that includes various non-cancerous cells. Furthermore, increases in levels of growth factors and hormones within the tumor microenvironment can result in enhanced ion channel expression, which contributes to cancer cell proliferation and survival. Thus, the pharmacological targeting of ion channels is potentially a promising approach to treating solid malignancies, including primary and metastatic brain cancers. Herein, protocols to characterize the function of ion channels in cancerous cells and approaches to analyze modulators of ion channels to determine their impact on cancer viability are described. These include staining a cell(s) for an ion channel(s), testing the polarized state of mitochondria, establishing ion channel function using electrophysiology, and performing viability assays to assess drug potency.

著者スポットライト:がんにおけるイオンチャネルの役割の探索:特性評価と潜在的な治療アプローチ 

The pharmacological targeting of ion channels is a promising approach to treating solid tumors. Detailed protocols are provided for characterizing ion channel function in cancer cells and assaying the effects of ion channel modulators on cancer viability.

がん細胞におけるイオンチャネルのスクリーニング

Ion channels are critical for cell development and maintaining cell homeostasis. The perturbation of ion channel function contributes to the development ...

がん研究

Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due to contact with adjacent cells. Transfected channels must also express with high efficiency at the plasma membrane for whole-cell patch clamp recording of detectable currents above noise levels. Heterologous ion channels often require long incubation periods at 28&deg;C after transfection in order to achieve adequate membrane expression, but there are increasing losses of cell-coverslip adhesion and membrane stability at this temperature. To circumvent this problem, we developed an optimized strategy to transfect and plate HEK-293T cells. This method requires that cells be transfected at a relatively high confluency, and incubated at 28&deg;C for varying incubation periods post-transfection to allow for adequate ion channel protein expression. Transfected cells are then plated onto glass coverslips and incubated at 37&deg;C for several hours, which allows for rigid cell attachment to the coverslips and membrane restabilization. Cells can be recorded shortly after plating, or can be transferred to 28&deg;C for further incubation. We find that the initial incubation at 28&deg;C, after transfection but before plating, is key for the efficient expression of heterologous ion channels that normally do not express well at the plasma membrane. Positively transfected, cultured cells are identified by co-expressed eGFP or eGFP expressed from a bicistronic vector (e.g. pIRES2-EGFP) containing the recombinant ion channel cDNA just upstream of an internal ribosome entry site and an eGFP coding sequence. Whole-cell patch clamp recording requires specialized equipment, plus the crafting of polished recording electrodes and L-shaped ground electrodes from borosilicate glass. Drug delivery to study the pharmacology of ion channels can be achieved by directly micropipetting drugs into the recording dish, or by using microperfusion or gravity flow systems that produce uninterrupted streams of drug solution over recorded cells.

Reliable method for highly efficient in vitro expression and subsequent electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T).

発現とHEK - 293T細胞における組換え電位依存性イオンチャネルの電気生理学的記録用に最適化されたトランスフェクション戦略

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) ...

生物学

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

GIRKチャネルのモジュレーターを同定するための蛍光スクリーニングアッセイ

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

構造と機能研究のための脂質膜へのKvチャネルの再構築

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

一チャネルセル付パッチクランプ法

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

異種哺乳動物細胞で発現したイオンチャネルの変異導入と機能解析

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誘導性一過性トランスフェクションを介して制御可能なイオンチャネルの発現

哺乳類 Bestrophin イオンチャネルの発現と精製

がん細胞におけるイオンチャネルのスクリーニング

がん細胞におけるイオンチャネルのスクリーニング

がん細胞におけるイオンチャネルのスクリーニング

発現とHEK - 293T細胞における組換え電位依存性イオンチャネルの電気生理学的記録用に最適化されたトランスフェクション戦略

GIRKチャネルのモジュレーターを同定するための蛍光スクリーニングアッセイ

構造と機能研究のための脂質膜へのKvチャネルの再構築

一チャネルセル付パッチクランプ法

精製された野生型および変異型CFTRタンパク質の機能的再構成とチャネル活性の測定

異種哺乳動物細胞で発現したイオンチャネルの変異導入と機能解析

要約

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誘導性一過性トランスフェクションを介して制御可能なイオンチャネルの発現

哺乳類 Bestrophin イオン チャネルの発現と精製

がん細胞におけるイオンチャネルのスクリーニング

がん細胞におけるイオンチャネルのスクリーニング

がん細胞におけるイオンチャネルのスクリーニング

発現とHEK - 293T細胞における組換え電位依存性イオンチャネルの電気生理学的記録用に最適化されたトランスフェクション戦略

GIRKチャネルのモジュレーターを同定するための蛍光スクリーニングアッセイ

構造と機能研究のための脂質膜へのKvチャネルの再構築

一チャネルセル付パッチクランプ法

精製された野生型および変異型CFTRタンパク質の機能的再構成とチャネル活性の測定

哺乳類 Bestrophin イオンチャネルの発現と精製