这项工作是由F31NS086765（KAC）的支持，F31NS076235（MAP）和R01 NS052669（GKP）和EIA9100012。作者感谢艾琳Kasperek的专业知识，并与分子生物学和组织培养的援助;和杰森迈尔斯分享从早期的前额叶皮层神经元获得的数据。

1，细胞培养和蛋白表达<ol><li>保持HEK293细胞通道22和40之间（ATCC编号CRL-1573），它包括由ATCC执行的，在DMEM中单层培养在补充有10％胎牛血清（FBS）的通路，并在5％CO 2的1％青霉素/链霉素的混合物和37°C。实验之间，传代细胞进T25烧瓶在10毫升的最终体积5-20倍稀释液。注意：在这些通道使用的细胞可以保证良好的健康细胞，这将使以获得最佳的密封形成和补丁的稳定性。 </li><li>对于转染，板的HEK293细胞于35 mm培养皿中以约10 5细胞/皿的密度，它对应于〜0.5-0.6毫升每皿的细胞悬浮液，并于2ml培养基中生长的细胞18-24小时。 </li><li>在无菌1.5毫升离心管中，加入（按顺序）四个35mm培养皿准备转染混合物：（1）1微克每一个基因（GluN1，GluN2A，和GFP）的; （2）315微升双蒸水（双蒸2 O）; （3）350微升42毫4 - （2 -羟乙基）-1 -哌嗪乙磺酸（HEPES），和（4）35微升的2.5M的CaCl 2滴，这是用于形成沉淀物。 </li><li>涡流管，持续5秒，并在每四个35mm培养皿中添加175微升转染悬浮液的镀覆的前一天，现在包含的细胞在50-60％汇合，并于室温孵育在37℃下搅拌2小时。 </li><li>抽吸掉转染培养基，洗净，用PBS并用2ml生长培养基补充了2mM MgCl 2的更换，以防止NMDA受体介导的兴奋性中毒9。注意：该细胞可用于电生理记录24-48小时后的转染。 </li></ol> 2，电极的制备<ol><li>通过与垂直车夫拉着硼硅玻璃管产生2对称录音移液器。根据所得到的电极头的尺寸和几何形状，还形状移液器用抛光机。注：针尖大小可以直观和电气进行评估。从外观上看，外直径应在1.4-5.6微米范围内。电，最佳大小尖端，当充满细胞外溶液时，应在12-24MΩ范围产生的电阻（ 见图2B）。 </li><li>用移液管的解决方案，这对于细胞附着实验代表了细胞外环境，将产生最大通道活性和高电流幅值。注意：对于NMDA受体，这对应于包含激动剂（谷氨酸和甘氨酸），1mM EDTA中，其中螯合二价阳离子抑制剂和阻断剂的饱和浓度的溶液中，并具有钠的生理浓度（150毫米）作为唯一的透膜物离子（以mM：1谷氨酸，0.1甘氨酸，150氯化钠，氯化钾2.5，1 EDTA和10 HEPBS，缓冲在pH 8.0用1N NaOH）。 </li></ol> 3，细胞贴附膜片钳记录<ol><li>打开QUB数据采集softwa重（www.qub.buffalo.edu），并选择在&#39;布局&#39;的采集窗口。通过点击下的下拉菜单名为“文件”“新数据”，打开一个新的QUB数据文件（QDF），并输入以下初始参数：采样率，40千赫; A / D转换比例，3000元;输出信道，1;和A / D数据大小，2。通过右键单击数据文件，选择属性，并单击“数据”选项卡中调整幅度缩放到0.1 V / PA。注：这些参数可以细化为使用中的细胞贴附模式的典范，每个单元格设置。数据采集​​与QUB软件和QDF属性的其他指令都在网上www.qub.buffalo.edu。 </li><li>选择含有表达细胞的离子通道在35毫米培养皿，并用2毫升的PBS含有钙和镁取代生长培养基;安装盘安装于显微镜舞台，用相差显微镜来评估细胞是健康的，以及附着在培养皿集中在蜂窝领域上在单层时尚（ 图1A）。切换到荧光检测来验证转染成功（ 图1B）。注意：荧光强度的范围可被用来作为粗测量蛋白表达的影响。 </li><li>对于单声道录音，设置放大器的输出增益为x10，补丁配置，β= 1，模拟滤波器为10 kHz，模式，电压钳和施加电压+100 mV的。在OFF位置的电压保持命令，选择“密封试验”按钮。 </li><li>基于荧光和细胞的健康，请选择一个单元格修补。根据相衬确认电池是否装的菜，有细胞表面暴露的修补，很大一部分否则看起来很健康。注意：保持相衬照明，以避免做法和膜片钳漂白过程中的单元格。 </li><li>填写新鲜抛光的吸管只有足够的解决方案，因此它与electrod接触e和轻轻摇晃，以清除可能被困在尖气泡。通过拧紧吸液管保持密封螺钉，并确保该银线浸渍在吸移管溶液固定记录吸管到放大器探头。 </li><li>使用小的（5毫升）塑料注射器，其通过管道连接到所述吸液管保持器，轻轻地施加正压力，以防止杂质进入和方法时堵塞的前端（ 图2A）。 </li><li>用显微操作器，直接吸入熔池，并直接在选定的修补（ 图1C）的细胞定位，闭合电路。采取了示波器的说明，其中应说明对应于放大器的密封测试信号（ 图2B）的矩形波。注意：一旦进入浴，在最佳范围内的移液管阻力为12-24MΩ。对于一个5毫伏的测试信号，测得的电流R昂热对应〜20-40 pA的。 </li><li>而在示波器上监测吸移管的位置在视觉上通过显微镜和移液管电阻电，则继续该方法以较小的增量，直到吸液管轻轻地撞击在细胞上，并在测试信号略有下降，表明增加的抗性（ 图2B）。 </li><li>以形成密封，拿起注射器，通过横向管道拉动针筒柱塞，其中拉细胞膜到记录滴管的尖端，并启动一个GΩ电阻密封件10的形成施加轻微的负压。就拿示波器上的测试信号波形，其中密封的形成是由它的完全压扁表示，只有电容瞬态可见（ 图2B）的注意。注意：如果信号不完全压平或基线变得嘈杂，这表明弱密封与周围的密封圈大量电流泄漏。这将防止Řesolving一个通道电流。如果是这样的话，从细胞抽出吸管和远离浴，从头部阶段将其取出并丢弃它。重复上述过程，用新鲜抛光的吸管，直到获得一个密封在适当的范围内（≥1GΩ）。 </li><li>在放大器，开关由“封测&#39;为&#39;off&#39;的外部命令切换;切换电压保持命令阳性（这是以前设置为“关”）;并增加增益以X100。观察示波器通道活性，其中，如果存在的话，将显示为从以前平坦的基线（ 图2C）的平方向上偏转。 </li><li>如果显示在示波器通道活性，获取到的数据在QUB先前打开的数字文件，按“播放”按钮，然后按“记录”按钮。要停止采集数据，按在QUB停止按钮，QDF文件保存到一个容易retriev通过在下拉菜单中题为“文件”中选择“将数据保存为...”计算机硬盘驱动器上能位置。 </li></ol> 4，数据预处理和理想化注：重要信息可以从单通道记录进行统计分析，每个数据点分配到合适的电导类（在简单的情况下，关闭或打开）中提取。这个过程被称为数据理想化和数据理想化与节段性k均值（SKM）在QUB方法11在下面描述的简要描述。 <ol><li>打开QUB数据文件，并查看当前的痕迹在“预”界面下的“布局”。显示录制的文件未过滤（除去数字滤波器）通过取消标记框&#39;的Fc。“ </li><li>视觉扫描记录发现违规行为和文物（ 图4A）。正确的短暂电流尖峰occur中的微量内（ 图4A）通过选择同一电导类的相邻洁净区，突出该区域中，右键单击并选择“设置擦除缓冲区。”放大秒杀到各个采样点是可见的，选择要替换的区域，并通过突出显示只有这个区域，然后用鼠标右键单击“擦除”擦除。 </li><li>通过选择记录，其中基线是稳定的早期部分定义了零电流基准为整个记录，高亮显示，单击鼠标右键并选择“设置基准”。验证从而准确地出现在指引代表基线水平（紫图4B） </li><li>在原始数据的基线明显偏离从设置基线记录识别点。在偏离区域内选择基线的一小部分纠正此问题，请右键单击并选择“添加一个基准节点的命令。 </li><li>识别吨地区他录制含有过量的噪音或文物，不能轻易纠正（ 图4C）。突出显示要被丢弃的区域，单击鼠标右键，并选择“删除”。注意：在这种情况下的动力学信息将会丢失：将处理结果将缩短并在拼接点之后产生的点将显示为连续的，预噪声区域。 </li><li>要使用SKM算法11理想化的记录，彰显含微量的开放和封闭事件的部分，然后输入下的“国防部”接口“布局”。在“模式”面板，彰显一丝干净的部分是代表基线的整个文件中，用鼠标右键单击黑色正方形（闭合状态），然后选择“抢”。通过突出开放电导做同样的通道开口上，右键单击“模型”面板中的红色方块，然后选择“抢”。 </li><li>执行idealization的由下选择“文件”按钮，整个记录“数据源”。在“理想化”的“建模”部分下面选项卡上单击鼠标右键，以验证分析所需的参数是否正确，然后单击“运行”。理想化的结果，随着振幅直方图整个文件，重叠的数据，以允许对理想化的目视检查（ 图5）。注：理想化不足可能导致的“假事件”的一代，其中QUB检测通道开口和不实际发生倒闭。内记录的分辨率，它是由放大器的模拟滤波器和采样率设置，与该SKM检测打开和关闭事件的分辨率可以通过设置死区时间的分析来控制。最佳死区时间必须通过试验和错误被选中，将取决于采样率，但是，一个好的经验法则是选择一个死区时间是2-3个样品12。 </li><li>要验证理想化准确表示原始数据，手动扫描理想化的痕迹。经鉴定理想化的错误，突出跟踪过假事件，点击右键，选择“加入用idl。”注：如需其他选项和QUB各种命令和函数的详细说明，请参考QUB手动在线（www.qub.buffalo.edu）。 </li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+Conductance+Levels+of+Native+AMPA+Receptors.">Heterogeneous Conductance Levels of Native AMPA Receptors.</a> The Journal of Neuroscience. 20, 2073-2085 (2000).</li><li>Coste, B., 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=Piezo1+and+Piezo2+Are+Essential+Components+of+Distinct+Mechanically+Activated+Cation+Channels.">Piezo1 and Piezo2 Are Essential Components of Distinct Mechanically Activated Cation Channels.</a> Science. 330, 55-60 (2010).</li><li>Coste, B., 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=Piezo+proteins+are+pore-forming+subunits+of+mechanically+activated+channels.">Piezo proteins are pore-forming subunits of mechanically activated channels.</a> Nature. 483, 176-181 (2012).</li><li>Benndorf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> chapter in Single-channel recording. 2nd edn eds B. Sakmann and E Neher. , (1995).</li></ol>

这个手稿的作者宣称，他们有没有竞争的财务权益。

在离子通道字段，一个重要的研究领域致力于了解的事件，导致信道的开口或通道的门控机制的序列。对于大多数信道，该过程是复杂的，涉及不能从宏观的多声道信号推导出几个反应步骤。相比之下，实验可设计其中观察在单通道记录打开/闭合事件的顺序可以产生约门控机制的更详细的信息。在这里所描述的方法，由NMDA受体所具有的位于外部的配体结合域允许将在恒定条件下执行微创的膜片?...

过生物膜的快速通信几乎完全依赖于低聚孔形成的膜蛋白，通常被称为信道。这些蛋白质在活化信号，门控机制，传导性能有很大的出入。通道蛋白的毛孔是有选择性的离子被列为离子通道;它们的活化产生的离子电流穿过细胞膜，并且它们的响应可以使用电生理技术来记录具有高分辨率的实时性。激活信号跨越的化学和物理输入，包括浓度梯度，机械和电气的力量和温度浩如烟海;因此，进一步的分类离子通道进入配体门控，机械敏感性，电压门控，或热敏感的类型。在这篇文章中，协议被描述为从一个配体门控通道，NMDA受体和机械敏感性离子通道，PIEZO1记录单声道活动，用膜片钳技术。0; 膜片钳电是最早，最广泛使用的实验方法足够灵敏，以允许观察单个分子1，2。除了这个精致的灵敏度，它已大大扩展了生物制剂适合于电生理记录，并允许离子通道在细胞膜完整的观察。首先，因为这两个电压钳位与当前记录被完成与同一电极，它可以用来通过小的细胞或膜的补丁记录的信号。该技术表明，离子通道并不局限于蛙肌肉，鳗鱼electroplaques或乌贼巨轴突3，4，而是它们所代表的跨膜信号传导机制无处不装置及有固有的单向或所有细胞膜类型的可兴奋膜多细胞有机体，并且还细胞内膜。进口antly，通过简单的连接玻璃吸管，以完整的细胞，记录跨膜电流的能力提供了前所未有的机会，从离子通道在家乡不间断的膜录制活动。因此，细胞贴附式膜片钳技术，这是在本协议中所述，允许连续监测离子通道的活性数十分钟或在他们的母语环境的时间。 在正常温度波动，所有的蛋白质，包括离子通道蛋白，经过了广泛的时间尺度上的结构变化，与代表最有可能通过侧链运动和整个的定位表示要慢得多，那么频繁变化最快和最频繁的重排结构域或亚基，或在某些情况下，通过翻译后修饰或蛋白质-蛋白质相互作用5,6。观察活动由一个分子产生长时间可以帮助了解功能离子通道的完好生理膜际动力学，并提供关于所观察到的分子的运行机制的有价值的信息。 与此相反的离子通道穿过细胞类型和发育阶段，知识的离子通道在原生膜的分子组成的多样性，人们越来越认识仍然有限。所有离子通道是多聚体蛋白，大部分天然离子通道的从几种类型的亚基产生的宽分子多样性，这是经常伴随着不同的电导和门控性质的蛋白质的组装。出于这个原因，定义分子组成的离子通道表达时在异源系统的研究。特别地，HEK293细胞，这是永生化人胚胎肾细胞7的品系，获得广泛接受，作为重组的离子通道的异源表达的优选系统。其中男子该升高的HEK293细胞的选择系统用于离子通道电Ÿ优点是易于进行培养的承受能力和保持长期稳定的培养，其进行翻译后折叠，加工和贩卖的哺乳动物蛋白质的能力，并且在许多情况下，其低的水平，甚至不存在内源性表达的感兴趣7,8的通道。表达重组的离子通道，并研究在HEK293细胞中其功能特性仍然是一个有价值的方法，以获得关于离子通道的结构 - 功能特性以及离子通道亚型和它们在天然组织角色的特定属性的信息。本文中所描述的协议同样可以很好地应用于表达在HEK293细胞重组离子通道和原生的离子通道。 综上所述，膜片钳技术，通过其前所未有的能力来解决信号从1分子遗体，迄今为止，最直接的方法，观察单个分子的行为。在其细胞贴附模式下，膜片钳记录允许长期观察期间，当一个分子中完成的，可以提供出色的洞察离子通道的操作。下面给出一个协议，用于获得高分辨率当前记录从含有一种离子通道蛋白的细胞贴附式膜片。

<table border="0" cellpadding="0" cellspacing="0" width="695">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:189px;">Chemicals</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:128px;">Various</td>
			<td style="width:205px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Borosillicate Glass</td>
			<td>Sutter</td>
			<td>BF-150-86-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bright field inverted microscope</td>
			<td>Olympus</td>
			<td>1x51</td>
			<td>Nikon also has similar microscopes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluroescent box</td>
			<td>X-cite</td>
			<td>Series 120</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Liquid Light Guide</td>
			<td>X-cite</td>
			<td>OEX-LG15</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micromanipulator</td>
			<td>Sutter Instruments</td>
			<td>MP-225</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oscilloscope</td>
			<td>Tektronix</td>
			<td>TDS1001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Amplifier</td>
			<td>Molecular Devices</td>
			<td colspan="2">Axon Axopatch 200B&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Table</td>
			<td>TMC</td>
			<td align="right">63561</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NIDAQ card</td>
			<td>National Instruments</td>
			<td>776844-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Puller</td>
			<td>Narishige</td>
			<td>PC-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polisher</td>
			<td>Narishige</td>
			<td>Microforge MF-830</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Faraday Cage</td>
			<td>TMC</td>
			<td align="right">8133306</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High Speed Pressure Clamp</td>
			<td>ALA Scientific Instruments</td>
			<td>ALA HSPC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pressue/Vaccuum Pump</td>
			<td>ALA Scientific Instruments</td>
			<td>ALA PV-PUMP</td>
			<td>For HSPC-1</td>
		</tr>
	</tbody>
</table>

one-channel cell-attached patch-clamp recording

离子通道蛋白是通过生物膜快速通信的通用设备。离子通量它们产生的瞬时签名取决于固有的每个通道蛋白，以及由其所产生和控制的机制的属性和表示当前研究的重要领域。关于离子通道蛋白的经营动态信息，可通过观察当前很长一段由单个分子制造来获得。这里描述的是一种协议，用于获取一条通道细胞贴附膜片钳记录电流为配体门控离子通道，NMDA受体，异源表达在HEK293细胞中或在本地皮层神经元。本发明还提供关于如何通过呈现在机械性敏感通道PIEZO1的例子中，方法适应其他感兴趣的离子通道的指示。这种方法可以对于该通道的电导特性和氧化聚乙烯的时间序列数据提供正封闭构象组成通道的激活机制，从而有助于了解他们在健康和疾病的功能。

重组NMDA受体 NMDA受体结合并以两个共激动剂随之而来的行动作出回应：谷氨酸和甘氨酸。他们组装成两个甘氨酸异源结合GluN1亚基和两个谷氨酸结合GluN2亚基。 GluN2亚基是由这四个基因（AD）和编码在大脑的最广泛形式的转录是GluN2A在成年和GluN2B在幼龄动物。由于NMDA受体亚型在原生的准备，表达受体在HEK293细胞（ 图1A）允许控制的亚基组成的，以及?...

Watch this Scientific Journal Video about One-channel Cell-attached Patch-clamp Recording at JoVE.com

One-channel Cell-attached Patch-clamp Recording

这里描述的是获得当前记录的很长一段距离与细胞贴附膜片钳技术一种离子通道的过程。这种方法允许观察，实时的背后的生物信号开闭通道构象的图案。这些数据的通知关于在不受干扰的生物膜通道属性。

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 ...

one-channel-cell-attached-patch-clamp-recording

Research

JoVE Journal

Biology

24.2K 次观看.  University at Buffalo, SUNY. 这里描述的是获得当前记录的很长一段距离与细胞贴附膜片钳技术一种离子通道的过程。这种方法允许观察，实时的背后的生物信号开闭通道构象的图案。这些数据的通知关于在不受干扰的生物膜通道属性。 

视频: One-channel Cell-attached Patch-clamp Recording

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 ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

在电生理记录果蝇胚胎

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Ambulatory ECG Recording in Mice

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although the surface ECG is utilized for short-term measurements of waveform intervals, it is not practical for long-term studies of heart rate variability or the capture of rare episodes of arrhythmias. Implantable ECG telemeters offer the advantages of simple surgical implantation, long-term recording of electrograms in ambulatory mice, and scalability with simultaneous recordings of multiple animals. Here, we present a step-by-step guide to the implantation of telemeters for ambulatory ECG recording in mice. Careful adherence to aseptic technique is required for favorable survival results with the possibility of implantation and recording from weeks to months. Thus, implantable ECG telemetry is a valuable tool for detection of critical information on cardiac electrophysiology in ambulatory animal models such as the mouse. 

Telemetric ECG has emerged as an essential tool in evaluating animal models for cardiac arrhythmias and sudden cardiac death. Here, we present a stepwise guide to telemetric ECG recordings for application in long-term ambulatory ECG monitoring in mice.

在小鼠体内的动态心电图记录

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although ...

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 ...

Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify and explore the function of these important proteins. However, classical preparations, such as planar bilayers, limit the manipulations and experiments that can be performed on the reconstituted channel and its membrane environment. The more cell-like structure of giant liposomes permits traditional patch-clamp experiments without sacrificing control of the lipid environment.
Electroformation is an efficient mean to produce giant liposomes &gt;10 &mu;m in diameter which relies on the application of alternating voltage to a thin, ordered lipid film deposited on an electrode surface. However, since the classical protocol calls for the lipids to be deposited from organic solvents, it is not compatible with less robust membrane proteins like ion channels and must be modified. Recently, protocols have been developed to electroform giant liposomes from partially dehydrated small liposomes, which we have adapted to protein-containing liposomes in our laboratory.
We present here the background, equipment, techniques, and pitfalls of electroformation of giant liposomes from small liposome dispersions. We begin with the classic protocol, which should be mastered first before attempting the more challenging protocols that follow. We demonstrate the process of controlled partial dehydration of small liposomes using vapor equilibrium with saturated salt solutions. Finally, we demonstrate the process of electroformation itself. We will describe simple, inexpensive equipment that can be made in-house to produce high-quality liposomes, and describe visual inspection of the preparation at each stage to ensure the best results.

Reconstituting functional membrane proteins into giant liposomes of defined composition is a powerful approach when combined with patch-clamp electrophysiology. However, conventional giant liposome production may be incompatible with protein stability. We describe protocols for producing giant liposomes from pure lipids or small liposomes containing ion channels.

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 ...

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 ...

Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal fluid accumulation and extracellular matrix formation. Activity of ion channels and intracellular calcium signaling are key physiologic parameters which determine functions of tubular epithelium. We developed a method suitable for real-time observation of ion channels activity with patch-clamp technique and registration of intracellular Ca2+ level in epithelial monolayers freshly isolated from renal cysts. PCK rats, a genetic model of autosomal recessive polycystic kidney disease (ARPKD), were used here for ex vivo analysis of ion channels and calcium flux. Described here is a detailed step-by-step procedure designed to isolate cystic monolayers and non-dilated tubules from PCK or normal Sprague Dawley (SD) rats, and monitor single channel activity and intracellular Ca2+ dynamics. This method does not require enzymatic processing and allows analysis in a native setting of freshly isolated epithelial monolayer. Moreover, this technique is very sensitive to intracellular calcium changes and generates high resolution images for precise measurements. Finally, isolated cystic epithelium can be further used for staining with antibodies or dyes, preparation of primary cultures and purification for various biochemical assays.

Ion channels expressed in renal tubular epithelium play a significant role in the pathology of polycystic kidney disease. Here we describe experimental protocols used to perform patch-clamp analysis and intracellular calcium level measurements in cystic epithelium freshly isolated from rodent kidneys.

实施膜片钳和活荧光显微镜监视新鲜分离的PKD上皮细胞的功能特性

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal ...

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Subtitles

单通道细胞贴附膜片钳记录