セントルイス心臓分子工学研究所のワシントン大学のすべてのメンバー。 （JSに）1010299 - バロウズは科学的なインターフェイスで、基金のキャリア賞を歓迎します。

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著者らは、開示することは何もありません。

カットオープン卵母細胞ワセリンギャップ電圧クランプ法は、データの迅速な解決を可能にし、低ノイズで、比較的長いプロトコル19に係る内部液と外液組成を制御し、安定した録画を増加させた。これらの利点は別として標準の2電極電圧クランプおよびパッチクランプ技術から、この手法を設定してください。特殊な装置が必要であり、プロトコルが比較的困難であるされていま?...

特殊な電圧クランプ技法を制御膜電位におけるイオン電流の記録を可能にする。広く使用される2つの電極電圧クランプ（TEVC）およびパッチクランプ技術は、多くのイオンチャネルの電気生理学的特性に関する信頼できる情報を提供する。しかしながら、これらの方法の両方は、高速電位依存性ナトリウムチャネルおよびアフリカツメガエル卵母細胞のもののような膜における他の高速活性化チャネルに対する信頼性のあるデータの取得を妨げる欠点を有する。 Bezanillaとステファニー·ラボラトリーズは、結果的に卵母細胞2のカットのオープンワセリンギャップ電圧クランプ法（COVG）を開発しました。技術をNa +、K +、およびCa 2 +チャネル3-8を記録するために広く適用されている。 COVG記録中に、異種タンパク質を発現する卵母細胞膜は、3つの領域に分割されている。イオン電流のデータは次のように卵母細胞の上部領域から記録されている上部領域を取り囲む浴を容易かつ迅速に変更することができるコマンド電位にクランプされる。中間領域の頂部領域9と同電位にクランプされることにより、リーク電流を保護します。卵母細胞開度（カットオープン）サポニン溶液またはカニューレの使用を介して発生する場所底部領域である。化学的または底部領域での膜のマニュアル開はグランドにクランプされた内部電位の制御を可能にし、下部チャンバ溶液で細胞内部の連続をレンダリングする。上部チャンバー内の溶液交換は、外部環境を変えるのに対して、下室への解決策の灌流は、内部環境の特性を調整することができます。 <img alt="図1" fo:content-width="5in" fo:src="/files/ftp_upload/51040/51040fig1highres.jpg" src="/files/ftp_upload/51040/51040fig1.jpg" width="500px" /> 図1。卵母細胞のカット開放電圧クランプバースセットアップ図（A）トップ互いから分離された3つの浴のダウン図である。 COVG用チャンバーの寸法は、図に表示されます。 （B）試験位置にある風呂セットアップの側面図。 <a href="http://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51040/51040fig1highres.jpg" target="_blank">拡大画像を表示するには、ここをクリックしてください</a> 。 COVG技術の利点は、低電流ノイズ（3 kHzでは1nA）、外部媒体のイオン組成、内部メディア、高速時間分解能（の減衰20-100秒の時定数を調節する能力の制御を含む容量過渡）、数時間9安定した録音。欠点は、それが特殊な装置を必要とし、2つの電極電圧クランプ（TEVC）10と比較して行うことはより困難であることである。 COVGアプローチは非常に特殊な装置や複雑な手順要素が必要ですが、それは貴重の取得を可能にすることができることができ、電気生理学的データ。このような高速の動態およびテール電流4に電流をゲートするように、このデータは、チャネルランダウンを含め、他の電圧クランププロトコルに関連する問題のいくつかがなく記録することができます。 COVGのセットアップへの軽微な変更は、温度コントローラと電圧クランプ蛍光法（VCF）の使用を可能にすることができる。 COVGアセンブリ内の電圧クランプ蛍光測定要素を含めることは、同時に、現在の11-13を記録しながら、タンパク質コンホメーション変化を監視する機能を付与することによりデータ出力を増大させることができる。

<table><tbody><tr><td>External Solution</td><td>Brand</td><td>Catalog Number</td><td>[Final], weight, or volume</td></tr><tr><td>N-methyl-D-glucamine (NMDG)</td><td>Sigma-Aldrich</td><td>M2004</td><td>25mM</td></tr><tr><td>MES Sodium Salt</td><td>Sigma-Aldrich</td><td>M5057</td><td>90mM</td></tr><tr><td>HEPES</td><td>Research Products International</td><td>H75030</td><td>20mM</td></tr><tr><td>Calcium hydroxide</td><td>Sigma-Aldrich</td><td>239232</td><td>2mM</td></tr><tr><td>MES Hydrate</td><td>Sigma-Aldrich</td><td>M8250</td><td>variable (pH to 7.4)</td></tr><tr><td>Internal Solution</td><td></td><td></td><td></td></tr><tr><td>N-methyl-D-glucamine (NMDG)</td><td>Sigma-Aldrich</td><td>M2004</td><td>105mM</td></tr><tr><td>MES Sodium Salt</td><td>Sigma-Aldrich</td><td>M5057</td><td>10mM</td></tr><tr><td>HEPES</td><td>Research Products International</td><td>H75030</td><td>20mM</td></tr><tr><td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#039;,N&#039;-tetraacetic acid (EGTA)</td><td>Sigma-Aldrich</td><td>E4378</td><td>2mM</td></tr><tr><td>MES Hydrate</td><td>Sigma-Aldrich</td><td>M8250</td><td>variable (pH to 7.4)</td></tr><tr><td>Depolarizing Solution</td><td></td><td></td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>221473</td><td>110mM</td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>M8266</td><td>1.5mM</td></tr><tr><td>Calcium Chloride</td><td>Caisson</td><td>C021</td><td>0.8mM</td></tr><tr><td>HEPES</td><td>Research Products International</td><td>H75030</td><td>10mM</td></tr><tr><td>Pipet Solution</td><td></td><td></td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>221473</td><td>3M</td></tr><tr><td>Saponin Solution</td><td></td><td></td><td></td></tr><tr><td>Saponin</td><td>Sigma-Aldrich</td><td>47036</td><td>0.125g</td></tr><tr><td>Internal Solution</td><td>See above</td><td></td><td>50mL</td></tr><tr><td>Agar Bridge Solution</td><td></td><td></td><td></td></tr><tr><td>N-methyl-D-glucamine (NMDG)</td><td>Sigma-Aldrich</td><td>M2004</td><td>100ml of 1M</td></tr><tr><td>HEPES</td><td>Research Products International</td><td>H75030</td><td>1.2g</td></tr><tr><td>MES Hydrate</td><td>Sigma-Aldrich</td><td>M8250</td><td>variable (pH to 7.4)</td></tr><tr><td>Granulated Agar</td><td>Research Products International</td><td>A20250</td><td>3%</td></tr><tr><td>NMDG Storage Solution</td><td></td><td></td><td></td></tr><tr><td>NMDG, HEPES, MES Hydrate solution</td><td>see above</td><td></td><td>40ml</td></tr><tr><td>Water</td><td></td><td></td><td>60ml</td></tr><tr><td>Name of Material/ Equipment</td><td>Company</td><td>Catalog Number</td><td>Comments/Description</td></tr><tr><td>High Performance Oocyte Clamp</td><td>Dagan</td><td>CA-1B</td><td></td></tr><tr><td>Data Acquisition System</td><td>Axon CNS</td><td>&amp;nbsp;Digidata 1440A</td><td></td></tr><tr><td>Oscilloscope</td><td>Tektronix&amp;nbsp;</td><td>TDS 210</td><td></td></tr><tr><td>Rack Power Filter</td><td>APC&amp;nbsp;</td><td>G5</td><td></td></tr><tr><td>Heating/Cooling Bath Temperature Controller</td><td>Dagan</td><td>HCC-100A</td><td></td></tr><tr><td>PC</td><td>Dell</td><td>Optiplex 990</td><td></td></tr><tr><td>pCLAMP 10.3 Voltage Clamp Software</td><td>Molecular Devices, LLC</td><td>pCLAMP10.3</td><td></td></tr><tr><td>TMC Vibration Control TableTop Platform</td><td>TMC</td><td>64 SERIES</td><td></td></tr><tr><td>TMC Vibration Control Air Table</td><td>TMC</td><td>&lt;a name=&quot;RANGE!C10&quot;&gt;20 Series&amp;nbsp;&lt;/a&gt;</td><td></td></tr><tr><td>V1/I Electrode Data Collector</td><td>Dagan</td><td>part of CA-1B</td><td></td></tr><tr><td>MX10L Micromanipulator</td><td>Siskiyou</td><td>MX10L</td><td></td></tr><tr><td>Bath/Guard (I/V) Headstage (with appropriate connectors)</td><td>Dagan</td><td>part of CA-1B</td><td></td></tr><tr><td>Microscope</td><td>Omano</td><td>OM2300S-JW11</td><td></td></tr><tr><td>Temperature Control Bath</td><td>Custom or Dagan</td><td>Custom or HE-204C</td><td>Custom chamber made from materials from Cool Polymers (D-series). Dagan also provides a prefeabricated stage (HE-204C).</td></tr><tr><td>Custom AgCl Pellet Container</td><td>Custom</td><td>Custom</td><td>Custom machined</td></tr><tr><td>Ag/AgCl electrode, pellet, 2.0 mm</td><td>Warner</td><td>E-206</td><td></td></tr><tr><td>External Oocyte Bath</td><td>Custom or Dagan</td><td>Custom or CC-1-T-LB</td><td>Custom machined or purchased from Dagan</td></tr><tr><td>Internal Oocyte Bath</td><td>Custom or Dagan</td><td>Custom or CC-TG-ND</td><td>Custom machined or purchased from Dagan</td></tr><tr><td>Capillaries for Agar Bridges and Pulled Electrodes</td><td>Warner</td><td>G150T-4</td><td></td></tr><tr><td>Rotatable Mounts for the Microscope, Micromanipulator, and Bath</td><td>Siskiyou</td><td>SD-1280P</td><td></td></tr><tr><td>Fiber-Lite</td><td>Dolan-Jenner</td><td>LMI-600</td><td></td></tr><tr><td>Regular Bleach</td><td>Clorox</td><td>470174-764</td><td></td></tr><tr><td>Xenopus laevis Oocytes</td><td>Nasco</td><td>LM535M (sexually mature females)</td><td></td></tr><tr><td>90 Na&lt;sup&gt;+&lt;/sup&gt; External Solution</td><td>See Solutions sheet</td><td></td><td></td></tr><tr><td>10 Na&lt;sup&gt;+&lt;/sup&gt; Internal Solution</td><td>See Solutions sheet</td><td></td><td></td></tr><tr><td>3 M KCL</td><td>See Solutions sheet</td><td></td><td></td></tr><tr><td>Saponin</td><td>Sigma-Aldrich</td><td>47036</td><td></td></tr><tr><td>NMDG Storage Solution</td><td>See Solutions sheet</td><td></td><td></td></tr><tr><td>5mL transfer pipets</td><td>SciMart</td><td>GS-52</td><td></td></tr><tr><td>Modified KCl electrode injector</td><td>BD</td><td>309659</td><td>Plastic syringe tip melted to allow for injection of solution into electrodes. Alternatively, a Microfil by WPI can be purchased.</td></tr><tr><td>Microvaccum</td><td>Custom</td><td>Custom</td><td></td></tr><tr><td>Forceps</td><td>VWR</td><td>63040-458</td><td></td></tr><tr><td>Oocyte Handling Tools (Pipette Pump)</td><td>VWR</td><td>53502-222</td><td></td></tr><tr><td>Deionized Water Squirt Bottle</td><td>VWR</td><td>16649-911</td><td></td></tr><tr><td>Vaseline Petroleum Jelly</td><td>Fisher Scientific</td><td>19-086-291&amp;nbsp;</td><td></td></tr><tr><td>Additional Materials Required for VCF Recordings:</td><td></td><td></td><td></td></tr><tr><td>VCF Microscope</td><td>Nikon</td><td>Eclipse FN1</td><td></td></tr><tr><td>Nikon CFI APO 40XW NIR Objective</td><td>Nikon</td><td>N40X-NIR</td><td></td></tr><tr><td>X-Y Translator System for Fixed-Stage Upright Microscopes</td><td>Sutter Instruments</td><td>MT500-586</td><td></td></tr><tr><td>External VCF Oocyte Bath</td><td>Custom</td><td></td><td>Custom machined. The chamber dimensions are 2.7 x 1.9 x 0.4 cm.</td></tr><tr><td>Internal VCF Oocyte Bath</td><td>Custom</td><td></td><td>Custom machined. The chamber dimensions are 1.6 x 1.6 x 0.4 cm.</td></tr><tr><td>Modified Temperature Control Bath</td><td>Custom</td><td></td><td>Custom chamber made from materials from Cool Polymers (D-series). The chamber dimensions of the modified temperature controller bath are 2.7 x 1.9 x 0.3 cm for the horizontal chamber, and 1 x 2.5 x 0.5 cm for the vertical chamber.</td></tr></tbody></table>

the xenopus oocyte cut-open vaseline gap voltage-clamp technique with fluorometry

カットオープン卵母ワセリンギャップ（COVG）電圧クランプ技術は、卵母細胞における異種イオンチャネルの電気生理学的および動力学的特性の分析を可能にする。カットオープンセットアップからの記録は、低振幅ゲート電流、急速なイオン電流の活性化、および非アクティブ化を解決するために特に有用である。 2電極電圧クランプ（TEVC）技術上の主な利点は、増加したクランプ速度、改善された信号対雑音比、および細胞内および細胞外環境を調節する能力が挙げられる。 ここでは、人間の心臓のナトリウムチャネル（HNA V 1.5）を採用し、切り開い設定やプロトコルだけでなく、電圧クランプ蛍光測定機能を追加するために必要な変更を示すために、 アフリカツメガエル卵母細胞で発現。 このようなHNA V 1.5の速度で活性化イオンチャネルの特性は、完全にwhicで、TEVCを用いて室温付近では解決できないhは、卵母細胞膜の全体が電圧制御を困難にクランプされる。しかしながら、カットオープン技術では、細胞膜の小部分のみの単離は、パッチクランプ技術に関連するチャネルランダウンを防止しつつ、正確に速い反応速度を記録するために必要な迅速な締付けを可能にする。 COVG法、イオンチャネル動態及び電気生理学的特性に関連してさらにタンパク質の動きが細胞外に適用さフルオロフォア、遺伝的にコード化された蛍光タンパク質の挿入、または非天然アミノ酸の組込みのシステインコンジュゲーションを介して追跡される電圧クランプ蛍光法を用いてアッセイすることができる関心1の領域へ。この追加データは、蛍光分子を取り巻く微小環境の変化を介してタンパク質の電位依存性のコンホメーションの再編成についての速度論的な情報が得られます。

図4のディスプレイは、サポニン溶液として卵母細胞の透過性の変化は、卵母細胞の底部に印加される。5サポニン透過化以下の拡散によって細胞内液交換の速度を示している図 。 20〜40分は、定常状態の2,18に来るために必要とされています。 記録プロトコルから生成された図6aショートレースします。図は、電?...

Watch this Scientific Journal Video about The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry at JoVE.com

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

カットオープンワセリンギャップアプローチは、高速チャネル動態の高解像度で、アフリカツメガエル卵母細胞で発現電位依存性イオンチャネルからのイオンおよびゲート電流の低ノイズの録音を得るために使用される。マイナーな変更を加えて、電圧クランプ蛍光法は、カットオープン卵母プロトコルに結合することができる。

ザ·<em&gt;アフリカツメガエル</em蛍光測定で&gt;卵母細胞のカットオープンワセリンギャップ電圧クランプ法

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

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Unit 1

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

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18.3K ビュー.  Washington University in St. Louis. カットオープンワセリンギャップアプローチは、高速チャネル動態の高解像度で、アフリカツメガエル卵母細胞で発現電位依存性イオンチャネルからのイオンおよびゲート電流の低ノイズの録音を得るために使用される。マイナーな変更を加えて、電圧クランプ蛍光法は、カットオープン卵母プロトコルに結合することができる。 

ビデオ: The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where real-time measurements of fluorescence and currents simultaneously report on local rearrangements and global function, respectively1. While high-resolution structural techniques such as cryo-electron microscopy or X-ray crystallography provide static images of the proteins of interest, VCF provides dynamic structural data that allows us to link the structural rearrangements (fluorescence) to dynamic functional data (electrophysiology). Until recently, the thiol-reactive chemistry used for site-directed fluorescent labeling of the proteins restricted the scope of the approach because all accessible cysteines, including endogenous ones, will be labeled. It was thus required to construct proteins free of endogenous cysteines. Labeling was also restricted to sites accessible from the extracellular side. This changed with the use of Fluorescent Unnatural Amino Acids (fUAA) to specifically incorporate a small fluorescent probe in response to stop codon suppression using an orthogonal tRNA and tRNA synthetase pair2. The VCF improvement only requires a two-step injection procedure of DNA injection (tRNA/synthetase pair) followed by RNA/fUAA co-injection. Now, labelling both intracellular and buried sites is possible, and the use of VCF has expanded significantly. The VCF technique thereby becomes attractive for studying a wide range of proteins and &#8211; more importantly &#8211; allows investigating numerous cytosolic regulatory mechanisms.

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

This article describes an enhancement of conventional Voltage-Clamp Fluorometry (VCF) where Fluorescent Unnatural Amino Acids (fUAA) are used instead of maleimide dyes, to probe structural rearrangements in ion channels. The procedure includes Xenopus oocyte DNA injection, RNA/fUAA coinjection, and simultaneous current and fluorescence measurements.

電圧クランプ<em&gt;アフリカツメガエル</em&gt;蛍光性の非天然アミノ酸を用いた卵母細胞

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where ...

生化学

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

アフリカツメガエルの卵母細胞で発現イオンチャネルのパッチクランプ記録

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

生物学

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

イオンチャネル研究のためのパッチクランプおよび灌流のテクニックで表さアフリカツメガエル卵母細胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

膜タンパク質の構造動力学を調べるその場で部位特異的蛍光標識を持つ

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

全細胞電圧クランプ記録のための電気生理学的方法<em&gt;ショウジョウバエ</em&gt;感光体

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

神経科学

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

ヒト人工多能性幹細胞由来心筋細胞における単細胞光学作用電位測定

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

生物工学

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+,K+-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+ or Li+ transport by Na+,K+- or gastric H+,K+-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb+ (Li+) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na+/2K+ transport stoichiometry of the Na+,K+-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+,K+-ATPase. In principle, the assay is not limited to K+-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

のNa、KとH、K-ATPaseのインによって陽イオン輸送を測定する<em&gt;アフリカツメガエル</em原子吸光法による&gt;卵母：アイソトープアッセイの代替

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating ...

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including by mechanical forces. Certain TRPV4 mutations cause complex hereditary bone or neuronal pathologies in human. Wild-type or mutant TRPV4 transgenes are commonly expressed in cultured mammalian cells and examined by Fura-2 fluorometry and by electrodes. In terms of the mechanism of mechanosensitivity and the molecular bases of the diseases, the current literature is confusing and controversial. To complement existing methods, we describe two additional methods to examine the molecular properties of TRPV4. (1) Rat TRPV4 and an aequorin transgene are transformed into budding yeast. A hypo-osmtic shock of the transformant population yields a luminometric signal due to the combination of aequorin with Ca2+, released through the TRPV4 channel. Here TRPV4 is isolated from its usual mammalian partner proteins and reveals its own mechanosensitivity. (2) cRNA of TRPV4 is injected into Xenopus oocytes. After a suitable period of incubation, the macroscopic TRPV4 current is examined with a two-electrode voltage clamp. The current rise upon removal of inert osmoticum from the oocyte bath is indicative of mechanosensitivity. The microAmpere (10-6 to 10-4 A) currents from oocytes are much larger than the subnano- to nanoAmpere (10-10 to 10-9 A) currents from cultured cells, yielding clearer quantifications and more confident assessments. Microscopic currents reflecting the activities of individual channel proteins can also be directly registered under a patch clamp, in on-cell or excised mode. The same oocyte provides multiple patch samples, allowing better data replication. Suctions applied to the patches can activate TRPV4 to directly assess mechanosensitivity. These methods should also be useful in the study of other types of TRP channels.

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

To complement commonly used methods to study TRPV4&#8217;s, two methods are described: Its mechanosensitivity can be studied by Ca2+-aequorin luminometry in transgenic yeast upon hypo-osmotic challenge. It can also be examined in TRPV4-RNA injected Xenopus oocytes by whole-cell two-electrode voltage clamp or patch clamp in on-cell or excised mode.

酵母発光測定と<em&gt;アフリカツメガエル</emTRPV4の分子機械的感受性の&gt;卵母細胞電気生理学的検定試験

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including ...

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery (References 2 - 3, and references therein). A characteristic that makes the oocyte attractive for foreign channel expression is the poor abundance of endogenous ion channels4. This expression system has proven useful for the characterization of many proteins, among them ligand-gated ion channels.
The expression of GABAA receptors in Xenopus oocytes and their functional characterization is described here, including the isolation of oocytes, microinjections with cRNA, the removal of follicular cell layers, and fast solution changes in electrophysiological experiments. The procedures were optimized in this laboratory5,6 and deviate from the ones routinely used7-9. Traditionally, denuded oocytes are prepared with a prolonged collagenase treatment of ovary lobes at RT, and these denuded oocytes are microinjected with mRNA. Using the optimized methods, diverse membrane proteins have been expressed and studied with this system, such as recombinant GABAA receptors10-12, human recombinant chloride channels13, Trypanosome potassium channels14, and a myo-inositol transporter15, 16.
The methods detailed here may be applied to the expression of any protein of choice in Xenopus oocytes, and the rapid solution change can be used to study other ligand-gated ion channels.

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Optimized procedures for the isolation of single follicles, cytoplasmic RNA microinjections, the removal of surrounding cell layers, and protein expression in Xenopus oocytes are described. In addition, a simple method for fast solution changes in electrophysiological experiments with ligand-gated ion channels is presented.

アフリカツメガエル卵母細胞：マイクロインジェクションのために最適化された方法、濾胞細胞層の除去、および電気生理学的実験における高速解法の変更

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery ...

Recording Gap Junction Current from Xenopus Oocytes

Heterologous expression of connexins and innexins in Xenopus oocytes is a powerful approach for studying the biophysical properties of gap junctions (GJs). However, this approach is technically challenging because it requires a differential voltage clamp of two opposed oocytes sharing a common ground. Although a small number of labs have succeeded in performing this technique, essentially all of them have used either homemade amplifiers or commercial amplifiers that were designed for single-oocyte recordings. It is often challenging for other labs to implement this technique. Although a high side current measuring mode has been incorporated into a commercial amplifier for dual oocyte voltage-clamp recordings, there had been no report for its application until our recent study. We have made the high side current measuring approach more practical and convenient by introducing several technical modifications, including the construction of a magnetically based recording platform that allows precise placement of oocytes and various electrodes, use of the bath solution as a conductor in voltage differential electrodes, adoption of a commercial low-leakage KCl electrode as the reference electrode, fabrication of current and voltage electrodes from thin-wall glass capillaries, and positioning of all the electrodes using magnetically based devices. The method described here allows convenient and robust recordings of junctional current (Ij) between two opposed Xenopus oocytes.

Recording Gap Junction Current from Xenopus Oocytes

Here we present a protocol to express gap junction proteins in Xenopus oocytes and record junctional current between two apposed oocytes using a commercial amplifier designed for dual oocyte voltage-clamp recordings in a high side current measuring mode.

アフリカツメガエル卵母細胞からのギャップ接合電流の記録

Heterologous expression of connexins and innexins in Xenopus oocytes is a powerful approach for studying the biophysical properties of gap junctions ...

ザ·

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電圧クランプ

アフリカツメガエルの卵母細胞で発現イオンチャネルのパッチクランプ記録

イオンチャネル研究のためのパッチクランプおよび灌流のテクニックで表さアフリカツメガエル卵母細胞

膜タンパク質の構造動力学を調べるその場で部位特異的蛍光標識を持つ

全細胞電圧クランプ記録のための電気生理学的方法

ヒト人工多能性幹細胞由来心筋細胞における単細胞光学作用電位測定

のNa、KとH、K-ATPaseのインによって陽イオン輸送を測定する

酵母発光測定と

アフリカツメガエル卵母細胞：マイクロインジェクションのために最適化された方法、濾胞細胞層の除去、および電気生理学的実験における高速解法の変更

アフリカツメガエル卵母細胞からのギャップ接合電流の記録