该<em>爪蟾</em>卵母细胞切割开凡士林隙电压钳技术与荧光
Summary
剖切开凡士林差距的方法来获得电压依赖性离子通道的离子和门控电流的表达在非洲爪蟾卵母细胞具有高分辨率的快速通道动力学低噪音录音。有轻微的修改，电压钳荧光可以被耦合到所述剖切开的卵母细胞的协议。
Abstract
剖切开的卵母细胞凡士林间隙（COVG）电压钳技术允许对异源的离子通道在卵母细胞的电生理和动力学特性分析。从开封设置录音是解决低幅度门控电流，快速离子电流的激活和失活时特别有用。通过双电极电压钳（TEVC）技术的主要好处包括提高夹紧的速度，提高了信号 – 噪声比，并以调制胞内和细胞外环境的能力。
在这里，我们采用人类心脏钠通道（HNA V 1.5），表达在非洲爪蟾卵母细胞，以展示开封的设置和协议，以及所需要的加电压钳位荧光能力的修改。
快速激活离子通道，如海航V 1.5的属性，不能完全接近室温解决使用TEVC，在whic卵母细胞的膜h的全部内容被夹紧，使电压控制困难。然而，在剖切开的技术中，在细胞膜中的一小部分的隔离允许为需要同时防止用膜片钳技术相关联的信道破落准确地记录快速动力学的快速装夹。
与COVG技术中，离子通道的动力学和电生理学特性相结合可以通过使用电压钳荧光，其中蛋白质运动经由的细胞外施加荧光团，插入基因编码荧光蛋白的，或者非天然氨基酸掺入半胱氨酸缀跟踪可以进一步测定入息1的区域。这个额外的数据产生关于该蛋白质的电压依赖性的构象重排通过改变周围的荧光分子的微环境动力学信息。
Introduction
专门的电压钳位技术允许离子流在控制膜电位的记录。广泛使用双电极电压钳（TEVC）和膜片钳技术提供许多离子通道的特性可靠的电生理信息。然而，这两种方法都有缺点，防止收购可靠的数据进行快速电压门控钠离子通道等快速激活通道，膜如爪蟾卵母细胞。该Bezanilla和史蒂芬妮因而实验室开发的剖切开凡士林隙电压钳技术（COVG）的卵母细胞2。该技术已被广泛应用于录音，的Na +，K +和Ca 2 +通道3-8。
在COVG记录，异源蛋白表达的卵母细胞的膜被分成三个区域。离子电流数据从卵母细胞作为的顶部区域记录浴周围的顶部区域被钳位到一个命令的潜力，这可以容易地和迅速地改变。中间区域防泄漏电流通过被钳位到电位相同的顶部区域9。底部区域是卵母细胞的开口（切开的）通过使用皂苷溶液或套管发生。化学或膜中的底部区域手册开口允许内部潜力，这被钳位到地的控制权，并呈现与下部腔室的溶液中的细胞内是连续的。灌注液进入下部腔室可以调节内部环境的属性，而在顶室溶液交换改变的外部环境。 
图1。卵母细胞剖切开的电压钳巴斯安装图（A）顶部往下看彼此分开了三次澡的。各室的COVG尺寸显示在图中。 （ 二）在测试位置的浴场设置的侧视图。 点击这里查看大图 。
该COVG技术的优点包括低电流噪声（1 nA在3千赫），外部媒体的离子组成，以调节内部媒体，快速的时间分辨率（衰变20-100微秒的时间常数的能力控制瞬态的能力），以及稳定的录音数小时9。的缺点是，它需要专门的设备，并且更难以进行对比2电极电压钳（TEVC）10。
而COVG方法需要高度专业化的设备和复杂的程序要素，它可以允许用于收购有价值的能电生理数据。这个数据，如门控电流，具有快速动力学和尾电流4，可以记录一些不与其他电压钳位协议，包括通道破败的相关问题。轻微的修改，COVG设置可以允许使用温度控制器和电压钳位荧光（VCF）的。列入COVG组件中的电压钳荧光元素可以通过赋予监察蛋白质构象的变化，同时记录电流11-13的能力，增强数据输出。
Protocol
1。最初的设备设置放置在振动隔离系统（ 例如一个空气表）的阶段和在微电极操纵器与周围的法拉第笼，以防止电气和机械噪声。 焊接6银/氯化银颗粒为24 AWG电线六英寸长。对于这些长度中的一个（也可以连接到P1），拼接在第二导线，以形成一个“Y”。对每根线的两端焊金BNC端子，它包含与放大器。 连接焊接到24 AWG电线的浴室/卫队探头（P1，P2，CC，GS1，GS2和）五?…
Representative Results
图4显示了在卵母细胞作为皂苷溶液的渗透性的变化施加到卵母细胞的底部部分， 图5展示了胞内溶液交 换的速率由扩散以下皂苷透化。 20-40分钟必须来一个稳态2,18。 从记录协议生成图6A表演的痕迹。图中示出了离子电流响应于所述电压协议（插图）（P/-8漏减之后）。图中的每个轨迹代表了不同的外加电压。与?…
Discussion
剖切开的卵母细胞凡士林隙电压钳技术，可以进行数据的快速高分辨率，低噪音，增加控制权的内部解决方案和外部解决方案组成，并记录稳定在相对 长的协议19。这些优势设置这项技术除了标准的双电极电压钳和膜片钳技术。虽然专门的设备是必要的，该协议是比较困难的，一旦系统进行了优化，非常少的问题发生。这使得钠（HNA V 1.5）和其它快速激活信道可重复的记录。…
Disclosures
The authors have nothing to disclose.
Acknowledgements
华盛顿大学圣路易斯分校心脏分子工程实验室的所有成员。一个伯勒斯欢迎基金事业奖在科学界面 – 1010299（JS的）。
Materials
| External Solution | Brand | Catalog Number | [Final], weight, or volume |
| N-methyl-D-glucamine (NMDG) | Sigma-Aldrich | M2004 | 25mM |
| MES Sodium Salt | Sigma-Aldrich | M5057 | 90mM |
| HEPES | Research Products International | H75030 | 20mM |
| Calcium hydroxide | Sigma-Aldrich | 239232 | 2mM |
| MES Hydrate | Sigma-Aldrich | M8250 | variable (pH to 7.4) |
| Internal Solution | |||
| N-methyl-D-glucamine (NMDG) | Sigma-Aldrich | M2004 | 105mM |
| MES Sodium Salt | Sigma-Aldrich | M5057 | 10mM |
| HEPES | Research Products International | H75030 | 20mM |
| Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) | Sigma-Aldrich | E4378 | 2mM |
| MES Hydrate | Sigma-Aldrich | M8250 | variable (pH to 7.4) |
| Depolarizing Solution | |||
| KCl | Sigma-Aldrich | 221473 | 110mM |
| Magnesium chloride | Sigma-Aldrich | M8266 | 1.5mM |
| Calcium Chloride | Caisson | C021 | 0.8mM |
| HEPES | Research Products International | H75030 | 10mM |
| Pipet Solution | |||
| KCl | Sigma-Aldrich | 221473 | 3M |
| Saponin Solution | |||
| Saponin | Sigma-Aldrich | 47036 | 0.125g |
| Internal Solution | See above | 50mL | |
| Agar Bridge Solution | |||
| N-methyl-D-glucamine (NMDG) | Sigma-Aldrich | M2004 | 100ml of 1M |
| HEPES | Research Products International | H75030 | 1.2g |
| MES Hydrate | Sigma-Aldrich | M8250 | variable (pH to 7.4) |
| Granulated Agar | Research Products International | A20250 | 3% |
| NMDG Storage Solution | |||
| NMDG, HEPES, MES Hydrate solution | see above | 40ml | |
| Water | 60ml | ||
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| High Performance Oocyte Clamp | Dagan | CA-1B | |
| Data Acquisition System | Axon CNS | Digidata 1440A | |
| Oscilloscope | Tektronix | TDS 210 | |
| Rack Power Filter | APC | G5 | |
| Heating/Cooling Bath Temperature Controller | Dagan | HCC-100A | |
| PC | Dell | Optiplex 990 | |
| pCLAMP 10.3 Voltage Clamp Software | Molecular Devices, LLC | pCLAMP10.3 | |
| TMC Vibration Control TableTop Platform | TMC | 64 SERIES | |
| TMC Vibration Control Air Table | TMC | 20 Series | |
| V1/I Electrode Data Collector | Dagan | part of CA-1B | |
| MX10L Micromanipulator | Siskiyou | MX10L | |
| Bath/Guard (I/V) Headstage (with appropriate connectors) | Dagan | part of CA-1B | |
| Microscope | Omano | OM2300S-JW11 | |
| Temperature Control Bath | Custom or Dagan | Custom or HE-204C | Custom chamber made from materials from Cool Polymers (D-series). Dagan also provides a prefeabricated stage (HE-204C). |
| Custom AgCl Pellet Container | Custom | Custom | Custom machined |
| Ag/AgCl electrode, pellet, 2.0 mm | Warner | E-206 | |
| External Oocyte Bath | Custom or Dagan | Custom or CC-1-T-LB | Custom machined or purchased from Dagan |
| Internal Oocyte Bath | Custom or Dagan | Custom or CC-TG-ND | Custom machined or purchased from Dagan |
| Capillaries for Agar Bridges and Pulled Electrodes | Warner | G150T-4 | |
| Rotatable Mounts for the Microscope, Micromanipulator, and Bath | Siskiyou | SD-1280P | |
| Fiber-Lite | Dolan-Jenner | LMI-600 | |
| Regular Bleach | Clorox | 470174-764 | |
| Xenopus laevis Oocytes | Nasco | LM535M (sexually mature females) | |
| 90 Na+ External Solution | See Solutions sheet | ||
| 10 Na+ Internal Solution | See Solutions sheet | ||
| 3 M KCL | See Solutions sheet | ||
| Saponin | Sigma-Aldrich | 47036 | |
| NMDG Storage Solution | See Solutions sheet | ||
| 5mL transfer pipets | SciMart | GS-52 | |
| Modified KCl electrode injector | BD | 309659 | Plastic syringe tip melted to allow for injection of solution into electrodes. Alternatively, a Microfil by WPI can be purchased. |
| Microvaccum | Custom | Custom | |
| Forceps | VWR | 63040-458 | |
| Oocyte Handling Tools (Pipette Pump) | VWR | 53502-222 | |
| Deionized Water Squirt Bottle | VWR | 16649-911 | |
| Vaseline Petroleum Jelly | Fisher Scientific | 19-086-291 | |
| Additional Materials Required for VCF Recordings: | |||
| VCF Microscope | Nikon | Eclipse FN1 | |
| Nikon CFI APO 40XW NIR Objective | Nikon | N40X-NIR | |
| X-Y Translator System for Fixed-Stage Upright Microscopes | Sutter Instruments | MT500-586 | |
| External VCF Oocyte Bath | Custom | Custom machined. The chamber dimensions are 2.7 x 1.9 x 0.4 cm. | |
| Internal VCF Oocyte Bath | Custom | Custom machined. The chamber dimensions are 1.6 x 1.6 x 0.4 cm. | |
| Modified Temperature Control Bath | Custom | 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. |
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Cite This Article
Rudokas, M. W., Varga, Z., Schubert, A. R., Asaro, A. B., Silva, J. R. The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry. J. Vis. Exp. (85), e51040, doi:10.3791/51040 (2014).