Name: recording gap junction current from xenopus oocytes
Uploaded: 2022-01-21T14:15:00
Description: Watch this Scientific Journal Video about Recording Gap Junction Current from Xenopus Oocytes at JoVE.com

我们感谢詹海英，钱戈参与技术开发的初始阶段，Kiranmayi Vedantham帮助处理数字，以及Camillo Peracchia博士对卵母细胞配对室的建议。

手术按照康涅狄格大学医学院机构动物护理委员会批准的协议进行。
1.青蛙手术和去毛化卵母细胞的制备
<ol><li>通过浸泡在凉爽（加冰）的三卡因溶液（〜300mg / L）中麻醉成年雌性非洲爪蛙（非洲爪蟾）（材料表）。</li>
	<li>等待（约15分钟），直到青蛙对挤压其蹼脚表现出很少或没有反应。将青蛙放在手术台上，腹部朝上。</li>
	<li>在左下腹...

<ol>
	<li>Oshima, A., Matsuzawa, T., Murata, K., Tani, K., Fujiyoshi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hexadecameric+structure+of+an+invertebrate+gap+junction+channel.">Hexadecameric structure of an invertebrate gap junction channel.</a> Journal of Molecular Biology. 428 (6), 1227-1236 (2016).</li><li>Maeda, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+connexin+26+gap+junction+channel+at+3.5+A+resolution.">Structure of the connexin 26 gap junction channel at 3.5 A resolution.</a> Nature. 458 (7238), 597-602 (2009).</li><li>Flores, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexin-46/50+in+a+dynamic+lipid+environment+resolved+by+CryoEM+at+1.9+A.">Connexin-46/50 in a dynamic lipid environment resolved by CryoEM at 1.9 A.</a> Nature Communications. 11 (1), 4331 (2020).</li><li>Sohl, G., Willecke, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+junctions+and+the+connexin+protein+family.">Gap junctions and the connexin protein family.</a> Cardiovascular Research. 62 (2), 228-232 (2004).</li><li>Starich, T., Sheehan, M., Jadrich, J., Shaw, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innexins+in+C.+elegans.">Innexins in C. elegans.</a> Cell Communication &amp; Adhesion. 8 (4-6), 311-314 (2001).</li><li>Phelan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innexins:+members+of+an+evolutionarily+conserved+family+of+gap-junction+proteins.">Innexins: members of an evolutionarily conserved family of gap-junction proteins.</a> Biochimica et Biophysica Acta. 1711 (2), 225-245 (2005).</li><li>Shui, Y., Liu, P., Zhan, H., Chen, B., Wang, Z. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+junctional+current+rectification+at+an+electrical+synapse.">Molecular basis of junctional current rectification at an electrical synapse.</a> Science Advances. 6 (27), (2020).</li><li>Starich, T. A., Xu, J., Skerrett, I. M., Nicholson, B. J., Shaw, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+innexins+UNC-7+and+UNC-9+mediate+electrical+synapse+specificity+in+the+Caenorhabditis+elegans+locomotory+nervous+system.">Interactions between innexins UNC-7 and UNC-9 mediate electrical synapse specificity in the Caenorhabditis elegans locomotory nervous system.</a> Neural Development. 4, 16 (2009).</li><li>Chen, B., Liu, Q., Ge, Q., Xie, J., Wang, Z. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UNC-1+regulates+gap+junctions+important+to+locomotion+in+C.+elegans.">UNC-1 regulates gap junctions important to locomotion in C. elegans.</a> Current Biology. 17 (15), 1334-1339 (2007).</li><li>Jang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+neuronal+gap+junction+circuits+that+regulate+social+behavior+in+Caenorhabditis+elegans.">Dissection of neuronal gap junction circuits that regulate social behavior in Caenorhabditis elegans.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (7), 1263-1272 (2017).</li><li>Ebihara, L., Beyer, E. C., Swenson, K. I., Paul, D. L., Goodenough, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+expression+of+a+Xenopus+embryonic+gap+junction+protein.">Cloning and expression of a Xenopus embryonic gap junction protein.</a> Science. 243 (4895), 1194-1195 (1989).</li><li>Barrio, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+junctions+formed+by+connexins+26+and+32+alone+and+in+combination+are+differently+affected+by+applied+voltage.">Gap junctions formed by connexins 26 and 32 alone and in combination are differently affected by applied voltage.</a> Proceedings of the National Academy of Sciences of the United States of America. 88 (19), 8410-8414 (1991).</li><li>Spray, D. C., Harris, A. L., Bennett, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage+dependence+of+junctional+conductance+in+early+amphibian+embryos.">Voltage dependence of junctional conductance in early amphibian embryos.</a> Science. 204 (4391), 432-434 (1979).</li><li>Spray, D. C., Harris, A. L., Bennett, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Equilibrium+properties+of+a+voltage-dependent+junctional+conductance.">Equilibrium properties of a voltage-dependent junctional conductance.</a> The Journal of General Physiology. 77 (1), 77-93 (1981).</li><li>Qu, Y., Dahl, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+the+voltage+gate+of+gap+junction+channels:+selective+exclusion+of+molecules.">Function of the voltage gate of gap junction channels: selective exclusion of molecules.</a> Proceedings of the National Academy of Sciences of the United States of America. 99 (2), 697-702 (2002).</li><li>Swenson, K. I., Jordan, J. R., Beyer, E. C., Paul, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+gap+junctions+by+expression+of+connexins+in+Xenopus+oocyte+pairs.">Formation of gap junctions by expression of connexins in Xenopus oocyte pairs.</a> Cell. 57 (1), 145-155 (1989).</li><li>Tong, J. J., Liu, X., Dong, L., Ebihara, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exchange+of+gating+properties+between+rat+cx46+and+chicken+cx45.6.">Exchange of gating properties between rat cx46 and chicken cx45.6.</a> Biophysical Journal. 87 (4), 2397-2406 (2004).</li><li>Landesman, Y., White, T. W., Starich, T. A., Shaw, J. E., Goodenough, D. A., Paul, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innexin-3+forms+connexin-like+intercellular+channels.">Innexin-3 forms connexin-like intercellular channels.</a> Journal of Cell Science. 112, 2391-2396 (1999).</li><li>Skerrett, I. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applying+the+Xenopus+oocyte+expression+system+to+the+analysis+of+gap+junction+proteins.">Applying the Xenopus oocyte expression system to the analysis of gap junction proteins.</a> Methods in Molecular Biology. 154, 225-249 (2001).</li><li>Nielsen, P. A., Beahm, D. L., Giepmans, B. N., Baruch, A., Hall, J. E., Kumar, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning,+functional+expression,+and+tissue+distribution+of+a+novel+human+gap+junction-forming+protein,+connexin-31.9.+Interaction+with+zona+occludens+protein-1.">Molecular cloning, functional expression, and tissue distribution of a novel human gap junction-forming protein, connexin-31.9. Interaction with zona occludens protein-1.</a> Journal of Biological Chemistry. 277 (41), 38272-38283 (2002).</li><li>Kotsias, B. A., Salim, M., Peracchia, L. L., Peracchia, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+cystic+fibrosis+transmembrane+regulator+and+gap+junction+channels+made+of+connexins+45,+40,+32+and+50+expressed+in+oocytes.">Interplay between cystic fibrosis transmembrane regulator and gap junction channels made of connexins 45, 40, 32 and 50 expressed in oocytes.</a> The Journal of Membrane Biology. 214 (1), 1-8 (2006).</li><li>Peracchia, C., Peracchia, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inversion+of+both+gating+polarity+and+CO2+sensitivity+of+voltage+gating+with+D3N+mutation+of+Cx50.">Inversion of both gating polarity and CO2 sensitivity of voltage gating with D3N mutation of Cx50.</a> American Journal of Physiology. 288 (6), 1381-1389 (2005).</li><li>del Corsso, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+of+mammalian+cells+with+connexins+and+measurement+of+voltage+sensitivity+of+their+gap+junctions.">Transfection of mammalian cells with connexins and measurement of voltage sensitivity of their gap junctions.</a> Nature Protocols. 1 (4), 1799-1809 (2006).</li><li>Peracchia, C., Wang, X. G., Peracchia, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+gating+of+gap+junction+channels.">Chemical gating of gap junction channels.</a> Methods. 20 (2), 188-195 (2000).</li><li>Levine, E., Werner, R., Neuhaus, I., Dahl, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetry+of+gap+junction+formation+along+the+animal-vegetal+axis+of+Xenopus+oocytes.">Asymmetry of gap junction formation along the animal-vegetal axis of Xenopus oocytes.</a> Developmental Biology. 156 (2), 490-499 (1993).</li></ol>

系统优化似乎是双卵母细胞电压钳夹实验所必需的。没有它，录音可能非常不稳定，放大器可能不得不注入过多的电流才能到达目标 Vm，从而导致卵母细胞损伤和记录失败。有几个因素对于使用高压侧电流测量方法获得稳定的双卵母细胞记录至关重要。首先，电流和电压电极必须具有适当的电阻（~1 MΩ），并且其支架必须清洁。其次， VDIFF 电极必须具有低电阻（<150 kΩ）并?...

GJ是细胞间通道，可能允许相邻细胞之间小细胞质分子的电流流动和交换。它们存在于许多细胞类型中，并执行不同的生理功能。脊椎动物中的GJ由连接蛋白形成，而无脊椎动物中的GJ由连接蛋白形成。每个GJ由两个并列的半透明细胞组成，每个半汉奈尔有6个或8个亚基，这取决于它们是连接蛋白还是连接蛋白1，2，3。人类有21个连接蛋白基因4个，而常用的无脊椎动物模型 秀丽隐杆线虫 和 黑腹果蝇 有25个和8个连接蛋白基因，分别为5，6个。基因转录本的替代剪接可能进一步增加GJ蛋白的多样性，至少对于连接蛋白7，8。
GJs可以根据分子组成分为三类：同型，异型和异构。同型GJ的所有亚基都是相同的。异型GJ具有两个同构半透明质，但两个半质由两种不同的GJ蛋白形成。异构体GJ含有至少一种异构半构体。GJ的分子多样性可能赋予对其生理功能很重要的独特生物物理特性。GJ生物物理性质也由调节蛋白9调节。要了解GJ如何执行其生理功能，重要的是要了解它们的分子组成，生物物理性质以及调节蛋白在其功能中的作用。
异源表达系统通常用于研究离子通道（包括GJ）的生物物理性质以及调节蛋白对它们的影响。由于异源表达系统允许特定蛋白质的表达，因此它们通常比天然组织更适合解剖蛋白质功能，其中具有冗余功能的蛋白质会使分析复杂化，并且可能无法实现Ij的记录。不幸的是，除了Neuro-2A细胞外，最常用的细胞系由于内源性连接蛋白的并发症而不适合研究GJ的生物物理特性。即使是Neuro-2A细胞也并不总是适合这种分析。例如，在不存在或存在UNC-1（未发表）的情况下，我们无法在转染有连接蛋白UNC-7和UNC-9的Neuro-2A细胞中检测到任何Ij，这是秀丽隐杆线虫9，10中UNC-9 GJs功能所必需的。另一方面，非洲爪蟾卵母细胞是GJs电生理学分析的有用替代系统。尽管它们表达内源性GJ蛋白Connexin 38（Cx38）11，但通过注射特定的反义寡核苷酸12可以很容易地避免潜在的并发症。然而，使用非洲爪蟾卵母细胞分析GJ需要两个并置细胞的差分电压钳位，这在技术上具有挑战性。青蛙爆破粒双电压钳的最早成功是在大约40年前的13，14。从那时起，许多研究使用这种技术来记录配对非洲爪蟾卵母细胞中的Ij。然而，基本上所有以前的研究都是使用自制放大器12，15，16或设计用于单个卵母细胞记录的商业放大器（GeneClamp 500，AxoClamp 2A或AxoClamp 2B，Axon Instruments，Union City，CA）8，17，18，19，20.由于即使是商用放大器也不提供双卵母细胞电压钳位的指令，因此对于新的或不太复杂的电生理学实验室来说，实施这种技术通常具有挑战性。
只有一种商用放大器是为双卵母细胞电压钳位开发的，即华纳仪器的OC-725C（材料表，图1A）。该放大器可以以标准模式（对于单个卵母细胞）或高侧电流测量模式（对于单或双卵母细胞）使用，具体取决于其电压探头中的两个插座是否连接（图1B，C）。然而，直到我们最近的研究7，还没有一份出版物描述该放大器在其高端电流测量模式下的使用。尽管该放大器已被另一个实验室用于双卵母细胞记录，但它用于标准而不是高边模式21，22。在高端电流测量模式下使用放大器缺乏报告可能是由于技术困难造成的。我们无法按照制造商的说明使用高边模式获得稳定的双卵母细胞记录。多年来，我们尝试了三种不同的双卵母细胞记录方法，包括在高压侧电流测量模式下使用两个OC-725C放大器，在标准模式下使用两个OC-725C放大器，以及两个来自其他制造商的放大器。经过广泛的反复试验，我们最终仅通过第一种方法成功获得了稳定的录音。本出版物描述并演示了我们用于表达非洲爪蟾卵母细胞中GJ蛋白的程序，使用高边电流测量模式记录Ij，并使用流行的商业软件分析电生理数据。关于双电压钳位技术的其他信息可以在其他出版物19，23中找到。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agar Bridge Magnetic Holder</td>
			<td>ALA Scientific Instruments</td>
			<td>MPSALT-H</td>
			<td>More stable than the Narishige tube clamper due to its larger magnetic base but it requires modification to accmmodate a 2-mm female socket.</td>
		</tr>
		<tr>
			<td>Auto Nanoliter Injector</td>
			<td>Drummond Scientific Company, Broomall, PA, USA</td>
			<td>Nanoject II</td>
			<td>Automated nanoliter injector</td>
		</tr>
		<tr>
			<td>Collagenase, Type II</td>
			<td>Gibco-USA, Langley, OK, USA</td>
			<td>17101-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond Scriber</td>
			<td>Electron Microscopy Sciences, Hatfield, PA, USA</td>
			<td>62108-ST</td>
			<td></td>
		</tr>
		<tr>
			<td>Differential Voltage Probe</td>
			<td>Warner Instruments, Hamden, CT, USA</td>
			<td>7255DI</td>
			<td></td>
		</tr>
		<tr>
			<td>Analog-to-Digital Signal Converter</td>
			<td>Molecular Devices, San Jose,CA, USA</td>
			<td>Digidata 1440A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Tweezers</td>
			<td>World Precision Instruments, Sarasota, FL, USA</td>
			<td>500341</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Capillaries</td>
			<td>Drummond Scientific Company, Broomall, PA, USA</td>
			<td>3-000-203-G/X</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot Wire Cutter</td>
			<td>Amazon.com</td>
			<td>Proxxon 37080</td>
			<td>An alternative is Hercules 8500 DHWT, which has a foot control pedal.</td>
		</tr>
		<tr>
			<td>Hyaluronidase, Type I-S</td>
			<td>MilliporeSigma, Burlington, MA, USA</td>
			<td>H3506</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Holder Base</td>
			<td>Kanetec USA Corp. , Bensenville, IL, USA</td>
			<td>MB-L-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrode Beveler</td>
			<td>Sutter Instrument, Novato, CA, USA</td>
			<td>BV-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrode Holder</td>
			<td>World Precision Instruments, , Sarasota, FL, USA</td>
			<td>MEH1S15</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette Puller</td>
			<td>Sutter Instrument, , Novato, CA, USA</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>mMESSAGE mMACHINETM T3</td>
			<td>Invitrogen-FisherScientific</td>
			<td>AM1348</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc MicroWell MiniTray</td>
			<td>Nalge Nunc International, Rochester, NY, USA</td>
			<td>438733</td>
			<td>Microwell Minitray</td>
		</tr>
		<tr>
			<td>Nylon mesh</td>
			<td>Component Supply Company, Sparta, TN, USA</td>
			<td>U-CMN-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Oocyte Clamp Amplifier</td>
			<td>Warner Instruments, , Hamden, CT, USA</td>
			<td>OC-725C</td>
			<td></td>
		</tr>
		<tr>
			<td>OriginPro</td>
			<td>OriginLab Corporation, Northampton, MA, USA</td>
			<td>2020b</td>
			<td></td>
		</tr>
		<tr>
			<td>pClamp</td>
			<td>Molecular Devices, , San Jose,CA, USA</td>
			<td>Version 10</td>
			<td></td>
		</tr>
		<tr>
			<td>Reference Electrode</td>
			<td>World Precision Instruments, Sarasota, FL, USA</td>
			<td>DRIREF-2SH</td>
			<td>Specifications: https://www.wpiinc.com/blog/post/compare-dri-ref-reference-electrodes</td>
		</tr>
		<tr>
			<td>RNaseOUT (ribonuclease inhibitor)</td>
			<td>Invitrogen-FisherScientific</td>
			<td>10777-019</td>
			<td></td>
		</tr>
		<tr>
			<td>Silk Suture 5-0</td>
			<td>Covidien, North Haven, CT, USA</td>
			<td>VS890</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer NanoDrop Lite</td>
			<td>Thermo Scientific</td>
			<td>ND-LITE-PR</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin Wall Glass Capallaries</td>
			<td>World Precision Instruments,Sarasota, FL, USA</td>
			<td>TW150F-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube Clamper</td>
			<td>Narishige International USA, Amityville, NY, USA</td>
			<td>CAT-1</td>
			<td>Ready to use but its position is prone to shift due to the small magnetic base.</td>
		</tr>
		<tr>
			<td>Xenopus laevis</td>
			<td>Xenopus Express, Brooksville, FL, USA</td>
			<td>IMP-XL-FM</td>
			<td></td>
		</tr>
	</tbody>
</table>

recording gap junction current from xenopus oocytes

非洲爪蟾卵母细胞中连接蛋白和连接蛋白的异源表达是研究间隙连接（GJs）生物物理性质的有力方法。然而，这种方法在技术上具有挑战性，因为它需要两个对立的卵母细胞共享一个共同点的差分电压钳位。虽然少数实验室已经成功地执行了这种技术，但基本上他们都使用了自制放大器或专为单卵母细胞记录而设计的商业放大器。对于其他实验室来说，实施这种技术通常具有挑战性。尽管高边电流测量模式已被整合到用于双卵母细胞电压钳位记录的商用放大器中，但在我们最近的研究之前，还没有关于其应用的报告。我们通过引入一些技术修改，使高端电流测量方法更加实用和方便，包括构建基于磁性的记录平台，允许精确放置卵母细胞和各种电极，使用浴溶液作为电压差分电极中的导体，采用商用低泄漏KCl电极作为参比电极， 从薄壁玻璃毛细管制造电流和电压电极，并使用磁性器件定位所有电极。这里描述的方法允许方便和可靠地记录两个对立的非洲爪蟾卵母细胞之间的结电流（Ij）。

UNC-7和UNC-9是秀丽隐杆线虫的内联体。虽然UNC-9只有一种亚型，但UNC-7具有多种亚型，其主要区别在于其氨基末端7，8的长度和氨基酸序列。当在非洲爪蟾卵母细胞7，8中表达时，这些连接蛋白可能形成同型和异型（UNC-7和UNC-9）GJ。代表Ij迹线以及由此产生的UNC-7b和UNC-9?...

Watch this Scientific Journal Video about Recording Gap Junction Current from Xenopus Oocytes at JoVE.com

Recording Gap Junction Current from Xenopus Oocytes

在这里，我们提出了一种方案，用于表达 非洲爪蟾 卵母细胞中的间隙连接蛋白，并使用商业放大器记录两个附着的卵母细胞之间的连接电流，该放大器设计用于在高压侧电流测量模式下记录双卵母细胞电压钳位记录。

记录来自 非洲爪蟾 卵母细胞的间隙结电流

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

非洲爪蟾卵母细胞中连接蛋白和连接蛋白的异源表达是分析间隙连接生物物理性质的有力方法。我们技术的主要优点是使用适用于双站点电压钳位的高边电流测量方法。该程序将由我实验室的博士后研究员袁水演示。一旦70%至80%的卵母细胞落叶，通过轻轻倾斜管来倾倒酶溶液。通过用ND96溶液填充管子，然后倾析溶液来洗涤卵母细胞五次。最后洗涤后，将卵母细胞转移到含有ND96溶液的60毫米培养皿中。使用干净的玻璃巴斯德移液管挑选大而健康的卵母细胞并将其转移到含有ND96溶液的60毫米培养皿中，并补充有丙酮酸钠和青霉素链霉素。通过将一小块尼龙网粘合到35毫米培养皿的底部来制备卵母细胞注射皿。用补充丙酮酸和青霉素链霉素的ND96溶液填充卵母细胞注射皿约一半。然后，在培养皿内排放置25至30个卵母细胞。用轻质矿物油向注射微量移液管后填充，然后将其插入注射器的微量移液器支架中。通过移液将cRNA从其中一个储存的等分试样转移到培养皿盖或底部的内表面。按下注射器控制器中的填充按钮，将cRNA液滴吸入微量移液管的尖端。然后，按下注射按钮注射cRNA。将注射的卵母细胞转移到含有补充丙酮酸和青霉素链霉素的ND96溶液的新培养皿中。将它们保持在15至18摄氏度的环境室内一至三天。每天通过将卵母细胞转移到新的培养皿中来更换溶液。使用两个细镊子轻轻剥离包裹卵母细胞的透明绒毛膜。然后，将每孔两个卵母细胞置于含有ND96溶液的卵母细胞配对室中。将差分电压探头和电流电缆连接到模型单元。然后，使用随附的跳线将红色和黑色插座连接到差分电压探头，并将跳线的任一支连接到模型单元中的任一引脚。将模型单元中的地线连接到放大器接地电路插座的电缆。将 VM 偏移和 VE 偏移旋钮调至膜电压和膜电流表归零。将箝位从关闭切换为快速或慢速，并将增益拨盘顺时针旋转至允许适当电压钳位将放大器的电压电极和浴电极计归零的水平。运行一个简单的采集协议，其中包含几个电压步骤，以确认膜电压表中显示的电压根据采集协议而变化，并且电压和电流迹线在 Clampex 中正确显示。将含有配对卵母细胞的培养皿放入记录平台的培养皿容器中。选择一对卵母细胞，必要时旋转培养皿，使两个卵母细胞在左右方向上。通过磁性底座将载物台锁定到位。将参比电极靠近培养皿的边缘朝向用户侧。然后，将参比电极仅连接到两个差分电压探头之一中的黑色插座。取一对玻璃微量移液器，用钻石划线器掰开一点尖端，然后通过火抛光使尖端边缘光滑。将微量移液器保持在酒精燃烧器的火焰上方，并将其弯曲成距离尖端约一厘米的平滑角度。用ND96溶液将玻璃移液器完全填充，然后将其插入预填充的微电极支架中。确保系统中没有气泡。将微电极支架的两毫米引脚插入差分电压电极连接线的两毫米插座中。将两毫米插座夹在基于磁性的钳夹器上，并将差分电压电极的尖端对准两个卵母细胞中的一个。将差分电压电极连接线的一毫米引脚插入同一侧差分电压探头的红色插座中。从玻璃毛细管制备电压和电流电极，并用氯化钾溶液填充它们。然后，将电极插入放大器随附的电极支架中。将电极降低到浴液中，将VM偏移和VE偏移拨盘转至膜电压和膜电流计零。通过按 VM 电极测试和 VE 电极测试来检查电极电阻。将电流和电压电极插入卵母细胞并观察负膜电位。接下来，在放大器的箝位部分，确认直流增益处于到位位置。顺时针旋转增益旋钮至允许适当电压箝位的水平，并将箝位开关从关断调至快速。最后，运行采集协议。这里显示了卵母细胞实验的示意图。负膜电压和正膜电压步骤从负30毫伏的保持膜电压施加到卵母细胞1，而卵母细胞2保持在负30毫伏的恒定膜电压下。经结电压或Vj定义为卵母细胞2的膜电压减去卵母细胞1的膜电压。代表性图像显示了样品Lj迹线和所得UNC-9同型间隙结的归一化结电导跨结电压关系。示例Lj迹线和由此产生的UNC-7b同型间隙结的归一化Gj-Vj关系如下所示。结果表明，这两种类型的Gj在Vj依赖性IJ失活率，Vj依赖性和残余Gj量方面存在差异.在尝试该过程时，确保差分电压电极系统没有任何气泡并且其尖端对准非常靠近卵母细胞并且电压和电流电极具有大约一微欧姆的电阻非常重要。我们的技术易于实施。对于那些最近对使用非洲爪蟾卵母细胞研究间隙连接感兴趣的实验室来说，这可能特别感兴趣。

recording-gap-junction-current-from-xenopus-oocytes

Research

JoVE Journal

Biology

Recording Gap Junction Current from Xenopus Oocytes

Retrieval of Mouse Oocytes

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus Genetic Stock Center (http://stkctr.biol.sc.edu), we are characterizing the salient differences needed to develop this system.  A primary goal has been to optimize oocyte/early embryo retrieval.

This protocol illustrates the technique for extracting oocytes or early-stage fertilized embryos from the oviduct of mice. The ability to identify the infindibulum and insert a blunt end needle into it is essential to correctly performing the procedure.

检索小鼠卵母细胞

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus ...

科研

生物学

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

从非洲爪蟾女性获得卵子

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

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.

补丁钳爪蟾卵母细胞中表达的离子通道

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

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

非洲爪蟾卵母细胞的微量注射

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

线虫神经肌肉交界处的解剖和录制

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

可视化的RNA中的本地化爪蟾卵母细胞

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

施肥爪蟾卵母细胞使用主机传输方法

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

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.

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

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

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.

测量离子转运的Na，K，H，K-ATP酶活性的<em&gt;爪蟾</em卵母细胞的原子吸收分光光度法：另一种放射性同位素检测

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

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

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

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

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

该<em&gt;爪蟾</em&gt;卵母细胞切割开凡士林隙电压钳技术与荧光

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

记录来自 非洲爪蟾 卵母细胞的间隙结电流

记录来自非洲爪蟾卵母细胞的间隙结电流