我们感谢布莱恩Venema和埃里克·马丁森建设electroformation装置。这项工作是由补助国立普通医学科学研究所，美国国立卫生研究院（R01GM100718赛格）和美国国立卫生研究院国家眼科研究所（R01EY017564赛格）。

1。脂质沉积有机溶剂：古典协议<ol><li>删除脂质储存于-20℃或-80℃;温暖RT 注意：血脂极易吸湿，许多对氧气敏感。在干燥的氩气或氮气覆盖血脂，并在所有步骤尽量减少暴露在空气中 。 </li><li>如果有必要，在氯仿或环己烷暂停脂类在1-10毫克/毫升，注意制造商声明的浓度通常标称注意：在使用有机溶剂时，戴上适当的手套和其他个人防护装备。删除个人防护装备，很快，如果溶剂溅到他们，因为大多数材料的仍然是这些溶剂的渗透，它们只提供临时保护。 </li><li>清洁两侧的两个25 X 37.5毫米的ITO镀膜玻璃幻灯片用乙醇</li><li>脂类混合，以达到所期望的摩尔比。每1厘米2的面积的ITO滑动唇涂有ID，混合〜15-20微克血脂。例如，如果两个25×37.5毫米幻灯片是完全包覆，我们通常使用30微升10毫克/毫升脂质混合物。 注：添加0.1％（摩尔），德克萨斯红DPPE用于荧光成像，请参阅下面的警告荧光染料。 </li><li>验证用欧姆表或万用表测量的表面电阻ITO镀膜侧玻片朝上。 </li><li>吸血脂耐溶剂性注射器注意：无胶或塑料，聚四氟乙烯以外，应该联系的有机溶剂。 </li><li>用注射器针头不太接触的ITO表面，慢慢地来回移动针幻灯片应用脂质。表面覆盖均匀，寻找“彩虹光泽”在玻璃表面。 </li><li> &lt;1托（1毫米汞柱）真空下快速将幻灯片0.5-1小时，删除任何痕迹溶剂。用惰性气体的释放真空。 </li><li>应用硅胶垫片，一个一层薄薄的两侧硅真空润滑脂。留出至少5毫米的未覆盖的滑动的一端暴露在滑动。 </li><li> 立即着手第4节。 </li></ol> 2。小脂质体准备控制脱水<ol><li>准备的脂质混合物在步骤1.1到1.4。 </li><li>在10至15毫米直径的培养管中使用的氩气或氮气流干燥的脂质。对于小批量的脂质，≤0.5毫克，得到的膜可以直接水合后，将其放置在真空下0.5〜1小时，以除去残留的溶剂，进行步骤2.5。数额较大的脂质往往捕获有机溶剂在厚厚的凝胶，即使在真空条件下，更好地准备冻干见步骤2.3-4。 </li><li>干燥后的脂质膜悬挂在环己烷中，用氩气和用塞子密封的盖，将管子在寒冷的块，冷冻在-80℃下至少1小时。 </li><li>放置在真空系统中的冷块和脂质样品能够达到100毫托。应用真空的，它会在约1托作为溶剂升华的“失速”。一旦完全除去溶剂（约1小时），真空将下降低于这个水平。与惰性气体一起推出真空，立即密封管或水合物。 </li><li>脂类是在真空条件下，采用真空脱气水化缓冲和喷射15-17。 </li><li>水合物的脂质使用脱气缓冲液终浓度1-10毫克/毫升。允许脂类水合物缓慢。 30分钟，1小时后，涡分手剩余的团块。等待一个额外的0.5-1小时，让完全水化。使用一个osmoticant，如山梨糖醇或蔗糖至200mM，在下面的步骤中，以防止完全脱水。 </li><li>准备直径为100-200纳米的脂质体受到挤压。协议的详细信息，请参见制造商的挤压。请注意，该缓冲区必须有小于〜25 mM的离子强度，或的特殊electroformation电压协议第4节中，将需要。 </li></ol> 3。脂质沉积控制小脂质体脱水<ol><li> 注意：我们的协议需要被放置在多个小时，用饱和盐溶液的蒸气平衡的脂质。我们强烈建议在惰性气体气氛中进行的协议，使用的手套箱中，或类似的外壳。 </li><li> 注意：如果含有蛋白质样品制备，避免完全脱水。水合物中的气氛与温水的烧杯中，任何外壳，或基于超声波仪的加湿器。使用，以确保有至少30％的相对湿度的湿度计。在小脂质体缓冲使用山梨糖醇或蔗糖作为一个额外的预防措施，防止脱水。 </li><li>准备适当的相对湿度饱和盐溶液。目标湿度取决于囊泡制剂的渗透压。渗透压与生理准备，用30-45％RH，而低渗透压泡准备可以充分脱水75-90％相对湿度（另请参阅表1）的平衡状态。 </li><li>将饱和盐溶液和过量的盐，在一个紧密密封的容器的内部搁板。商业食品容器酸奶和燕麦的工作非常出色。后更换货架充盈，确保流体在货架下面是5-10毫米。 </li><li>清洁的ITO涂层第1幻灯片。用万用表测量，以确定哪一面是导电的，并与样品名称，标签的一面不导电。 </li><li>抹油的聚硅氧烷（USP级VI）垫片（次）与一个或多个孔的两侧。 </li><li>将幻灯片导电侧在板凳上，并应用硅胶垫片（次），确保有至少为5毫米的外露滑动一端连接到的electroformation装置的每个幻灯片，将应用脂质。光滑的垫片，以确保良好的密封。 </li><li>在低盐等渗缓冲〜1-2毫克/毫升稀释囊泡准备。我们发现较高concentr的ations产生不良结果。 </li><li>应用脂质在1-10微升滴幻灯片。一般较小的墨滴产生更好的结果。 </li><li>将饱和盐溶液之上的室内货架上滑动密封容器。在室温下离开3小时到O / N容器可以被放置在4℃下，但注意，一些盐，将RH强烈地依赖于温度，和那个寒冷会增加平衡时间。 </li><li>所得到的膜，可能有轻微的彩虹光泽，但在任何情况下，应该会出现接近干涸。高渗透压的起始溶液可以是不可能完全干燥的脂质膜，这将不会产生不利影响的结果。 </li></ol> 4。巨脂质体Electroformation <ol><li>脂片，特别是那些由脱水脂质体，容易脱落。 小心水合物的各孔中，放置一个27 G的注射器针头上面的垫片的边缘， 慢慢施加缓冲区。缓冲区可以包括水，≤200毫米蔗糖，山梨醇≤1 M≤5毫米肝素钠。超过10毫米的盐溶液可能需要备用electroformation电压协议。溢满每个垫片以及约10％。 </li><li> 注：一旦脂类滋润，迅速 ​​完成剩下的步骤进行，因为脂质膜立即开始分层。产量和规模最大如果开始electroformation及时。 </li><li>以平滑的动作，应用第二个ITO滑动，导电面，最佳的垫片。确保外悬有至少5毫米的垫片区和对面的第一悬。轻轻按压以保证良好的密封。 </li><li>清洁两个出挑，用乙醇和验证双方面临的垫片是导电使用万用表。 </li><li>室可进一步用封口膜或轻张力弹簧夹固定，如果需要的话。 </li><li>固定铝或铜条，“EMI GASK的的electroformation室连接到一个电压源等发泡体，或导电性粘合带的导电性的两个滑动表面，我们使用：EMI垫圈泡沫状物。计划的夹具和夹紧， 如图5所示。 </li><li>验证是没有电气短路之间的接触，而且接触正确连接到ITO的表面，使用万用表。 </li><li>加热腔室10℃以上的最高熔化温度的任何脂类本例如为DPPC，加热到52℃下最小，链的熔化温度的42℃以上10℃ </li><li>对于低盐缓冲器，适用于10赫兹正弦波〜0.7 V RMS每毫米两层ITO涂层表面之间的差距，为60-90分钟。对于高盐缓冲剂，应使用其他电压协议（请参阅参考资料）。 </li></ol> 5。成像和故障排除<ol><li>图片的脂质体，使用倒置显微镜配备罗丹明或得克萨斯州的红色染料过滤器的多维数据集。该脂质体是球形的，单层为主眼，无缺陷，如“弦”，挂脂质体。 </li><li>如果脂质体太小，使用较少的脂质。 </li><li>如果有很多缺陷或几个脂质体，这可能是由于凝胶相的脂质。考虑提高温度electroformation。 </li><li>如果很少或没有巨脂质体形成在从脱水的小脂质体的脂质体的缓冲液中，减少渗透强度，或，增加渗透强度的electroformation缓冲区。缓冲到浓间质性缓冲溶液可引起浪涌的分层沉积的脂质膜。 </li><li>如果泡产量，质量或尺寸差，包括盐或pH缓冲的electroformation或脂质体缓冲区，考虑替代electroformation的电压协议。比如，波特， 等。11，建议一个三个步骤的协议，使用一个500 Hz的正弦波，提高电压为50-1,300 Vpp /米在30分钟内，保持90分钟，N的减少30-60分钟频率为50 Hz。在这种情况下，铂金或钛的电极可能需要，但基本上不会改变协议。 </li></ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiphoton+polarization+imaging+of+steady-state+molecular+order+in+ternary+lipid+vesicles+for+the+purpose+of+lipid+phase+assignment.">Multiphoton polarization imaging of steady-state molecular order in ternary lipid vesicles for the purpose of lipid phase assignment.</a> J. Phys. Chem. B. 114 (47), 15512-15522 (2010).</li><li>Hauser, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+ultrasonic+irradiation+on+the+chemical+structure+of+egg+lecithin.">The effect of ultrasonic irradiation on the chemical structure of egg lecithin.</a> Biochem. Biophys. Res. Commun. 45 (4), 1049-1055 (1971).</li><li>Hauser, H., Cevc, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipid+vesicles.">Phospholipid vesicles.</a> Phospholipids Handbook. , (1993).</li><li>Veatch, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electro-formation+and+fluorescence+microscopy+of+giant+vesicles+with+coexisting+liquid+phases.">Electro-formation and fluorescence microscopy of giant vesicles with coexisting liquid phases.</a> Meth. Mol. Biol. 398, 59-72 (2007).</li><li>Kim, R. S., LaBella, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+analytical+methods+for+monitoring+autoxidation+profiles+of+authentic+lipids.">Comparison of analytical methods for monitoring autoxidation profiles of authentic lipids.</a> J. Lipid. Res. 28 (9), 1110-1117 (1987).</li><li>Veatch, S. L., Leung, S. S. W., Hancock, R. E. W., Thewalt, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+probes+alter+miscibility+phase+boundaries+in+ternary+vesicles.">Fluorescent probes alter miscibility phase boundaries in ternary vesicles.</a> J. Phys. Chem. B. 111 (3), 502-504 (2007).</li><li>Juhasz, J., Davis, J. H., Sharom, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+probe+partitioning+in+GUVs+of+binary+phospholipid+mixtures:+implications+for+interpreting+phase+behavior.">Fluorescent probe partitioning in GUVs of binary phospholipid mixtures: implications for interpreting phase behavior.</a> Biochim. Biophys. Acta. 1818 (1), 19-26 (2012).</li><li>Herold, C., Chwastek, G., Schwille, P., Petrov, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+electroformation+of+supergiant+unilamellar+vesicles+containing+cationic+lipids+on+ITO-coated+electrodes.">Efficient electroformation of supergiant unilamellar vesicles containing cationic lipids on ITO-coated electrodes.</a> Langmuir. 28 (13), 5518-5521 (2012).</li></ol>

作者宣称，他们有没有竞争经济利益。

发展成一个灵活的技术，兼容不同的血脂，准备和缓冲区巨脂质体Electroformation已。仔细的脂质沉积过程的控制是最关键的成功。我们已经提出了简单的工具，使小脂质体准备一个简单的过程控制血脂沉积。相对湿度是至关重要的适当初始脂质体脱水，将随溶质的初始浓度的脂质体悬浮液中的最佳值。较低的相对湿度是必需的，脱水更浓缩的样品，以适当的水平。 确切的的水?...

巨脂质体（通常称为巨室脂质体，或GUVs的）主要被用来研究的物理和物理化学的脂质双层，包括研究的双层变形，横向相共存（“木筏”），膜融合等 1-4。他们有一个严重的细胞样结构：膜周围的水的内部，比周围的水性缓冲液中，可以很容易作出不同的球壳。 ，顾名思义，他们是≈直径1-100微米，所以他们可以使用各种光镜方法成像。它们可使用渗透压梯度或机械施加张力拉紧，因此，虽然一般较软，它们的属性可以被操纵，易于处理。特别是控制的“刚性”的脂质体，使得它可以直接形成“脂质体连接”或切除电补丁。在过去，主要是在平面脂B进行离子通道重建ilayers。现在，巨脂质体形成的补丁，并使用相当颤动的常规电开发工具（荧光显微镜，微吸管，快速灌注和温度控制等 ）的能力，使得巨脂质体重建研究5,6越来越有吸引力。 巨脂质体已作出许多战略。事实上，巨脂质体膨胀过程自发形成，当干脂质膜水化4,7,8。的愿望更加迅速地准备更大，更均匀的脂质体的研究人员领导的其他方法，其中最主要的electroformation 1,9。 electroformation也依赖于水化干燥的脂质膜，但加速的过程中，通过应用一个振荡电场穿过脂质膜。施加电场时，通过两个电极，无论是铂丝或铟锡氧化物（ITO）涂层的载玻片，分离水或缓冲和脂质沉积其上。加快脂质体肿胀，达到较高的产量较大的脂质体。因此，electroformation已经成为默认的方式产生巨大的脂质体4。 的electroformation的机理还没有完全理解，大多数协议都是依靠经验（ 例如 10,11）。然而，我们可以学到一点什么期望考虑的理论和一些实证结果。人们普遍认为，的发生electroformation通过驱动电渗流的缓冲液层叠在沉积的脂质膜10,11之间的脂质双层。脂质双层静电耦合热波动也可能是涉及12。这些假说定性预测的电场的频率和强度，可用于10,12的上限。特别是，据预测，高导电性的解决方案（ <eM&gt;即生理盐溶液）减少电流体的力量，可能引发的脂质体electroformation的12。电渗流速率一般随盐的浓度增加，并经常达到高峰，一些电场振荡频率（ 例如，尽管在不同的几何形状，Green 等人 ）。因此，较高的磁场强度和更高的频率是合理的，高导电性的解决方案，在一定限度内10。 然而，膜蛋白的脂质沉积到电极上，即在有机溶剂中，然后蒸发掉，留下一个薄的脂质膜为electroswelling过程与通常的方法很可能是不相容的。解决这个困难主要有两个路径：巨脂质体形成后，将蛋白质或脂质沉积，以适应如何。我们的做法的基础上存入脂类和reconsti的别人5,11tuted膜蛋白从暂停或大或小的“蛋白脂质体”。我们描述了过程冗长，更具挑战性的脂蛋白体纯化的蛋白质和脂类（科林斯和戈登， 在审查）。这里，我们描述的协议，在没有任何蛋白质的情况下，但它是相同的蛋白结合时，我们包括结果显示，含有离子通道TRPV1受体的蛋白脂质体可以转化成GUVs用于膜片钳电生理。在任何electroformation方法中，脂质沉积过程过程中的脂质样品目视检查是成功的关键。 我们的做法可能超出了专门的应用程序相关的离子通道重建。在规定的时间，因为我们第一次开发了这个协议，现在也被证明的方式，脂质沉积在电极electroformation如何影响所产生的GUVs组成的异质性。 Baykal-恰拉尔的等14表明，，仔细脱水脂质体形成的GUVs有2.5倍的变化较小的混溶性化转变温度的各种磷脂和胆固醇的混合物形成的GUVs。他们的工作表明，脂质，尤其是胆固醇，可从脂质混合物沉淀在沉积有机溶剂时，导致沉积的脂质膜的组合物中的大的空间变化。脂质膜的相行为的研究，这是特别重要的，但也可以是离子通道功能的定量实验的关键。巴伊卡尔恰拉尔等人的协议是我们自己相似但不完全相同的，并鼓励读者研究。 此协议（见概述， 图1）是一个可以使用的许多。在原则electroformation成功依赖于脂质混合物，水化，温度，其他溶质（特别是离子），和当然，形成中使用的电压和频率。 ，由于electroformation变得更好理解，我们预期更加完善我们的协议。 最后，在电铸巨脂质体往往有一个陡峭的学习曲线。我们建议掌握学习脂质沉积脂质体悬浮液（2-5节）之前，传统的协议（第1和第4，和，如果有必要的话，第5节）。

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Digital Multimeter</td>
			<td>Agilent Technologies, <a href="http://www.agilent.com" target="_blank">www.agilent.com</a></td>
			<td>U1232A or similar</td>
			<td>Any multimeter will do, but avoid old style analog ohmmeters which apply much more current to the resistance under test.</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Fluke</td>
			<td>117 or 177</td>
			<td>Any multimeter will do, but avoid old style analog ohmmeters which apply much more current to the resistance under test.</td>
		</tr>
		<tr>
			<td>Function Generator</td>
			<td>Agilent Technologies, <a href="http://www.agilent.com" target="_blank">www.agilent.com</a></td>
			<td>33210A or similar</td>
			<td>Most function generators work for simple protocols. This programmable model is useful for advanced electroformation protocols. Make sure the generator can drive 10 V peak-to-peak into a 50 &Omega; load</td>
		</tr>
		<tr>
			<td>ITO coated glass slides</td>
			<td>Delta Technologies, Loveland, CO <a href="http://www.delta-technologies.com" target="_blank">www.delta-technologies.com</a></td>
			<td>CB-90IN-S107 or similar</td>
			<td>Break these in half to make two slides, 25 mm x 37 mm</td>
		</tr>
		<tr>
			<td>Temperature controller</td>
			<td>Omega Engineering Stamford, CT <a href="http://www.omega.com" target="_blank">www.omega.com</a></td>
			<td>CNi3233 or similar</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hygrometer</td>
			<td>Extech, Nashua, NH, <a href="http://www.extech.com" target="_blank">www.extech.com</a></td>
			<td>445815</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Silicone rubber sheet</td>
			<td>McMaster-Carr Elmhurst, IL <a href="http://www.mcmaster.com" target="_blank">www.mcmaster.com</a></td>
			<td><a href="http://www.mcmaster.com/#87315K64" target="_blank">87315K64</a></td>
			<td>Use USP Grade VI silicone for its high purity</td>
		</tr>
		<tr>
			<td>EMI gasket</td>
			<td>Laird Technologies <a href="http://www.lairdtech.com" target="_blank">www.lairdtech.com</a></td>
			<td>4202-PA-51H-01800 or similar</td>
			<td>Distributed by Mouser <a href="http://www.mouser.com" target="_blank">www.mouser.com</a></td>
		</tr>
		<tr>
			<td>TxR-DHPE</td>
			<td>Life Technologies, Carlsbad, CA <a href="http://www.lifetechnologies.com" target="_blank">www.lifetechnologies.com</a></td>
			<td>T1395MP</td>
			<td>Other fluorescently labeled lipids are available, but TxR-DHPE is one of the brightest and most photostable.</td>
		</tr>
		<tr>
			<td>POPC</td>
			<td>Avanti Polar Lipids, Alabaster, AL <a href="http://www.avantilipids.com" target="_blank">www.avantilipids.com</a></td>
			<td>850457P or 850457C</td>
			<td>Lipids can be ordered as powders (P) or in chloroform (C)</td>
		</tr>
		<tr>
			<td>POPS</td>
			<td>Avanti Polar Lipids</td>
			<td>840034P/C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Sigma-Aldrich</td>
			<td>C8667</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

giant liposome preparation for imaging and patch-clamp electrophysiology

离子通道电生理记录的已有化学定义的脂膜的重组一直是一个强大的技术确定并探讨这些重要的蛋白质的功能。然而，经典的制剂，如平面双层，限制了可被执行的操作和实验上的重建的信道和它的膜环境。巨脂质体的细胞状结构允许在不牺牲脂质环境控制的传统的膜片钳实验。 electroformation&gt;10μm的直径依赖于一个薄的，有序的脂质膜沉积在电极表面的交变电压的应用产生巨大的脂质体是一种高效的平均值。然而，由于经典的协议需要从有机溶剂中的脂质沉积，它是不兼容不太可靠的膜蛋白，如离子通道，并必须进行修改。近日，公关已经开发otocols部分脱水的小我们已经适应了在我们的实验室中的含蛋白质的脂质体的脂质体，脂质体从电铸巨。 我们在座的背景下，设备，技术，脂质体分散的小巨脂质体和陷阱electroformation。我们开始尝试更具挑战性的协议之前，应首先掌握经典的协议。我们证明了小脂质体饱和盐溶液的蒸汽平衡的过程中，控制部分脱水。最后，我们演示过程的electroformation本身的。我们将描述简单，价格低廉的设备，可在内部，以产生高质量的脂质体，并描述在每一个阶段的准备目视检查，以确保最佳的效果。

在我们的例子中，我们准备了脂质体的混合物约55％（摩尔）POPC（1 -棕榈酰-2 -油酰-SN-甘油-磷酸胆碱），15％（摩尔）持久性有机污染物（1 -棕榈酰-2 -油酰-SN-甘油磷酸丝氨酸摩尔，30％胆固醇和0.1％（摩尔）德州红色标记的1,2 -二棕榈酰-SN-磷酸乙醇胺（TXR DPPE），这个组合被选为约代表性的背根神经节血脂18，我们注意到，扣除15％（摩尔）脂质（POPS）的限制，可用于?...

Watch this Scientific Journal Video about Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology at JoVE.com

Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

重构功能膜蛋白组成界定成巨脂质体是一个功能强大的方法，结合膜片钳电。然而，传统的巨头脂质体产量可能与蛋白质的稳定性不相容。我们描述生产从单纯的脂质的巨脂质体或小脂质体含有离子通道的协议。

巨脂质体制剂成像和膜片钳电

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify ...

giant-liposome-preparation-for-imaging-patch-clamp

Research

JoVE Journal

Biology

22.0K 次观看.  University of Washington. 重构功能膜蛋白组成界定成巨脂质体是一个功能强大的方法，结合膜片钳电。然而，传统的巨头脂质体产量可能与蛋白质的稳定性不相容。我们描述生产从单纯的脂质的巨脂质体或小脂质体含有离子通道的协议。

视频: Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

Methods for Patch Clamp Capacitance Recordings from the Calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal.

膜片钳从花萼电容录音方法

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. ...

科研

生物学

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.

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 ...

Pressure-polishing Pipettes for Improved Patch-clamp Recording

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording. We first developed this method for fabricating pipettes suitable for recording from small (&lt;3 m) neuronal cell bodies. The basic principal is similar to glass-blowing and combines air pressure and heat to modify the shape of patch pipettes prepared by a conventional micropipette puller. It can be applied to so-called soft (soda lime) and hard (borosilicate) glasses. Generally speaking, pressure polishing can reduce pipette resistance by 25% without decreasing the diameter of the tip opening (Goodman and Lockery, 2000). It can be applied to virtually any type of glass and requires only the addition of a high-pressure valve and fitting to a microforge. This technique is essential for recording from ultrasmall cells (&lt;5 m) and can also improve single-channel recording by minimizing pipette resistance. The blunt shape is also useful for perforated-patch clamp recording since this tip shape results in a larger membrane bleb available for perforation.

This is a guide to modifying the shape of glass micropipettes. Specifically, by using heat and air pressure the taper is widened without increasing the tip opening, leading to lower pipette resistance. This is critical to obtain low noise recordings of small cells but is useful in many applications.

改进的膜片钳记录的压力抛光移液器

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording.  We first developed this method for fabricating pipettes suitable for ...

Preparation of Artificial Bilayers for Electrophysiology Experiments

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can be used for many different studies, such as the characterization of membrane-active peptides, the reconstitution of ion channels or investigations on how changes in lipid bilayer properties alter the function of bilayer-spanning channels.  Here, we show how to form a planar bilayer and how to isolate small patches from the bilayer, and in a second video  will also demonstrate a procedure for using gramicidin channels to determine changes in lipid bilayer elastic properties.   We also demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. Here, we demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

电生理实验人工双层膜的制备

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can ...

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

脊髓电

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

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 ...

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca2+]cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).
Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.
The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.

Measurements of Kv7 (KCNQ) potassium channel activity in isolated arterial myocytes (using patch clamp electrophysiological techniques) in parallel with measurements of constrictor/dilator responses (using pressure myography) can reveal important information about the roles of Kv7 channels in vascular smooth muscle physiology and pharmacology.

探讨动脉平滑肌的KV7钾离子通道功能使用膜片钳电生理和压力Myography

Contraction  or relaxation of smooth muscle cells within the walls of resistance arteries  determines the artery diameter and thereby controls flow of ...

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

视网膜平片准备成像和免疫组化优化方案

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

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.

单通道细胞贴附膜片钳记录

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

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

跨膜蛋白的重组，电压门控离子通道，KvAP，进入巨人单层囊的显微镜和膜片钳研究

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...