Name: whole-cell patch-clamp recordings for electrophysiological determination of ion selectivity in channelrhodopsins
Uploaded: 2017-05-22T14:00:00.000+00:00
Duration: 8 min 39 s
Description: Watch this Scientific Journal Video about Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins at JoVE.com

我们感谢Maila Reh，Tharsana Tharmalingam，特别是Altina Klein，为您提供卓越的技术帮助。这项工作得到德国研究基金会（DFG）（SFB1078 B2，FOR1279 SPP1665至PH）和催化，UniCat，BIG-NSE（JV）和E4（PH）的卓越统一概念支持。

<img alt="图1" src="/files/ftp_upload/55497/55497fig1.jpg" /> 图1:跳线钳设置。 （1）光源，（2）光纤，（3）可编程快门，（4）数字化仪，（5）快门驱动器，（6）放大器，（7）灌注系统，（8）个人计算机，（9） ，（10）法拉第笼，（11）显微镜台，（12）灌注入口，（13）灌注出口，（14）记录室，（15）液位传感器，（16）带琼脂桥的浴电极，（17）移液器支架，（18）头灯，（19）显微操纵器，（20）倒置显微镜，（21）显微镜灯罩，（22）显微镜灯电源，（23）充水U型管，（24）三通阀门，（25）防振台，（26）CCD摄像头。 （ A ）照片和（ B ）设置的示意图。 <a href="http://ecsource.jove.com/files/ftp_upload/55497/55497fig1large.jpg" target="_blank">请点击这里查看更大的版本</a>他的身影 1.录音前设置<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td>内。 </td><td> extra.1 </td><td> extra.2 </td></tr><tr><td></td><td>高氯化物</td><td>高氯化物</td><td>低氯化物</td></tr><tr><td></td><td> c [mM] </td><td> c [mM] </td><td> c [mM] </td></tr><tr><td> NA-ASP </td><td> 0 </td><td> 0 </td><td> 140 </td></tr><tr><td>氯化钠</td><td> 110 </td><td> 140 </td><td> 0 </td></tr><tr><td>氯化钾</td><td> 1 </td><td> 1 </td><td> 1 </td></tr><tr><td>氯化铯</td><td> 1 </td><td> 1 </td><td> 1 </td></tr><tr><td> CaCl 2 </td><td> 2 </td><td> 2 </td><td> 2 </td></tr><tr><td> MgCl 2 </td><td> 2 </td><td> 2 </td><td> 2 </td></tr><tr><td> HEPES </td><td> 10 </td><td> 10 </td><td> 10 </td></tr><tr><td> EGTA </td><td> 10 </td><td> 0 </td><td> 0 </td></tr><tr><td> pH值</td><td> 7.2 </td><td> 7.2 </td><td> 7.2 </td></tr><tr><td>的渗透压</td><td> 290 mOsm </td><td> 320 mOsm </td><td> 320 mOsm </td></tr></tbody></table> 表1:缓冲溶液的离子组成。在HEK细胞中用于氯化物选择性实验的细胞内（内）和胞外（额外）缓冲液的组成。使用的缩写:乙二醇四乙酸（EGTA），4-（2-羟基乙基）-1-哌嗪乙磺酸（HEPES）和天冬氨酸（ASP）。所有浓度均为mM。 <ol><li> 用离子浓度准备记录溶液，如表1所示。 <ol><li>在超纯水中制备15 mL 2M储备溶液，用于MgCl 2 ，CaCl 2 ，KCl和CsCl。 </li><li>称量100 mL细胞内固体组分将500mL根据表1的细胞外缓冲溶液分成单独的烧杯，然后加入相应量的制备的储备溶液。例如，对于细胞内缓冲液，使用0.3803g（10mM）EGTA，0.2383g（10mM）HEPES和0.6428g（110mM）NaCl，加入100μL2M MgCl 2和CaCl 2 ，加入50μL 2 M KCl和CsCl储备溶液。 </li><li>添加超纯水，并使用不干扰测量的酸和碱精心调节pH， 即不由ChR进行，如柠檬酸和N-甲基-D-葡萄糖胺（NMG + ）。 </li><li>调节渗透压为290 mOsm为细胞内和320 mOsm细胞外溶液与葡萄糖。 注意:稍微低渗透的细胞内溶液增加修补的成功率26 。 </li><li>无菌过滤所有溶液通过0.22-μm过滤器。将溶液在4°C储存2周或在-20°C下冷冻长时间储存​​。 </li></ol></li><li> 计算液体结电位并准备记录方案。 注意:建立全细胞配置后细胞外氯化物浓度的变化会导致必须校正的显着液体接合电位（LJP） 27,28 。可以直接测量或计算LJP，这里使用后一个选项。在该协议中，将对每个外部缓冲器使用一个记录协议进行在线校正。然而，对于更大的外部解决方案，建议对LJP进行后期校正，以避免错误纠正。 <ol><li>打开连接点计算器（JPC） 27并打开"新建实验"对话框。然后选择"贴片钳测量"。选择"全细胞测量"，"标准盐溶液电极"，输入温度25°C。在每个步骤之间，通过按"下一步"按钮接受选择。 注意:实验在室温25摄氏度的空调实验室进行。确保缓冲溶液在开始实验之前具有室温。为了改变样品温度，可以使用孵育阶段或室。浴温可以用温度计频繁测量。 </li><li>根据表1中列出的浓度添加吸移溶液和高氯缓冲液的所有单独阴离子和阳离子浓度。 注意:JPC将计算起始条件为-0.5 mV的LJP。 </li><li>点击"新浴溶液"，输入低氯化物缓冲液的阴离子和阳离子组成。 注意:根据改变的细胞外记录解决方案，JPC现在将计算一个+12.6 mV的新LJP。 </li><li>准备两个LJP校正的协议两个测量条件通过从所施加的保持电位中减去所计算的LJP;对于低氯化物外部溶液，高电平为-0.5 mV，+12.6 mV。因此，协议以-79.5mV（高氯化物）和-92.6mV（低氯化物）开始，以获得在两种情况下为-80mV的LJP校正的起始保持电位。 注意:以下步骤适用于材料列表中提到的数据采集软件。 </li><li>在采集软件中打开协议编辑器。切换到"模式"菜单，选择"特效刺激"，并包括7次扫描。 </li><li>切换到编辑器中的"波形"菜单，包括一个200毫秒的周期，0 mV的保持电位，然后是2秒的周期，对于高氯离子，变化的保持电位从-79.5 mV开始。在第二个周期内包括20 mV的"Delta电平"，以在每次扫描中增加20 mV的保持电位。 </li><li>在波形ed的电压阶段用540nm光（3.10mW / mm 2）施加500ms的照明周期。为此，请在上述第二个周期（参见1.2.6）后，将控制光源的所连接快门的"数字位模式"更改为有效500 ms。 注意:光强度可以影响通道电流的生物物理性质，如振幅或失活。因此，应始终报告光功率密度。要通过所有光学元件和盖玻片后测量光功率，请使用校准的光学计数器。要计算功率密度，通过对象测微计确定物镜的照明场，并将测得的光功率除以确定的照明场。 </li><li>保存高氯离子细胞外溶液的方案。修改协议，并将第一级保持电位替换-92.6 mV（低外部氯化物）。保存第二个方案来测量低氯化物细胞外条件。 </li></oL&gt; </li><li> 根据适用的方案29 ，玻璃盖滑盖的赖氨酸涂层。 <ol><li>将几百个盖玻片放在玻璃培养皿中，加入250 mL HCl（1 N）。 </li><li>将盖玻片放在线性摇床上的培养皿上，以120rpm振荡72小时以清洁盖子，然后用超纯水冲洗以中和pH。 </li><li>将其浸泡在少量95％乙醇中以进一步清洗。 注意:清洁盖子可以储存在70％乙醇中，然后再继续操作。在层流罩下工作以进行以下步骤。 </li><li>使用本生灯和镊子，每个盖子都会滑动并将其转移到新的培养皿中。 </li><li>在室温下用无菌的50μg/ mL聚-D-赖氨酸溶液孵育至少1小时（或过夜）的玻璃盖玻片，同时用线性振荡器以150rpm摇动。 </li><li>用超纯水清洗盖子以除去多余的聚-D-赖氨酸。 </li><li>将无盖纸上的盖子撕开干燥10分钟，并将其暴露于紫外线下进行灭菌（至少20分钟）。 </li><li>将盖子收集到铝箔上的无菌培养皿中，直到使用。 </li></ol></li><li> 使用标准分子生物学技术制备与mCherry融合的Ps ACR1基因的DNA。 <ol><li>使用限制酶或其他已建立的克隆方法，使用mCherry荧光团将编码Ps ACR1的人密码子优化的基因序列克隆到peGFP-C1质粒（CMV启动子，卡那霉素抗性）框架中。 注意:也可以使用其他荧光团，但请确保在设置中可以使用相应的过滤器组，以在显微镜下观察其荧光。 </li><li>根据标准方案，通过热休克将质粒DNA转化为化学活性的XL1Blue 大肠杆菌细胞，并将其置于琼脂平板（30μg/ mL卡那霉素） 30上 </SUP&gt;。在37°C将细菌培养过夜。 </li><li>第二天，将来自单个菌落的细胞的补充有30μg/ mL卡那霉素的4mL溶菌酵母培养基接种，并在37℃和180rpm下在培养箱中孵育过夜。 </li><li>孵育过夜后，使用市售的试剂盒（参见材料表 ），根据制造商的说明从培养物中纯化质粒DNA。 </li></ol></li><li> 种细胞 注意:在层流罩下执行步骤。 <ol><li>在35 mm的陪替氏培养皿中并排放置三根赖氨酸涂层的玻璃盖。 注意:轻轻地将它们压在盘子的底部，以防彼此滑动。 </li><li>在每个培养皿中补充有10％胎牛血清（FBS）的2mL标准Dulbecco&#39;s Modified Eagle培养基（DMEM）中种子1.5×10 5个 HEK细胞。 </li><li>补充全反式视网膜最终浓度为1μM。生长细胞至少一天，然后再进行步骤1.6。 </li></ol></li><li> 通过脂转染瞬时转染细胞31 。 <ol><li>对于每个细胞皿，在250μL不含FBS和6μL转染试剂的DMEM（参见材料表 ）中制备2μg质粒DNA（如步骤1.4中制备）的溶液 。 </li><li>孵育15分钟后，轻轻（滴加）将溶液加入细胞。 </li></ol></li><li> 准备琼脂桥 <ol><li>向50ml无菌氯化钠溶液（140mM）中加入0.75g琼脂糖，并通过在微波中加热溶解。 注意:2 - 3 M KCl用于琼脂桥制备由于较高的浓度（ 例如来自桥的恒定扩散）和K +和Cl -离子的相等迁移率而进一步降低了LJP，但是避免了使离子（特别是Cl - ）泄漏最小化进入t<html>他洗澡液32 。 </li><li>使用注射器用液体琼脂糖溶液填充10μL移液器吸头（或小直径软管）。 注意:避免桥梁中的气泡。 </li><li>琼脂桥已经冷却并固化后，将其储存在无菌的140mM氯化钠溶液中。 </li></ol></li><li> 准备录音和浴缸电极。 <ol><li>取出金属丝和贴片夹设置的记录电极。 </li><li>用砂纸擦拭线，以从以前的实验中除去氯化银。将干净的银线浸入3 M KCl中，并将其连接到1.5 V电池的正极上，用氯化银电解涂层。 注意:可以使用实验室电源代替电池。 </li></ol></li><li>用微量吸管拔出器从玻璃毛细管拉出低电阻贴片移液器（1.5-3.0MΩ），并按照Brown 等lass ="xref"&gt; 33。储存移液器无尘，用于测量当天。 </li><li>打开所有设置组件，加热到工作温度30分钟前录制。确保缓冲液在室温下。 </li></ol> 2.选择性测量<ol><li>在1.6中描述的细胞瞬时转染后24至48小时开始记录。 </li><li>将一个盖板放在测量室中，并用硅密封，以防止外部缓冲液泄漏。将培养皿与其他盖子一起存放在孵化器中进行下一组实验。 </li><li>小心地用细胞外缓冲液（高[Cl - ]）填充室以防止细胞分离，然后将其置于显微镜下，并使用40X物镜进行细胞可视化。 </li><li>将琼脂桥放在浴电极上，并与液位传感器和浴液处理器的灌注出口一起放入室内。 </li><li>交换前微量溶液用1 mL新鲜外部溶液（高[Cl - ]）洗涤两次，以除去残留的培养基和分离的细胞。 </li><li>使细胞聚焦并搜索需要从其他细胞分离的转染（荧光）细胞。使用三波段滤光片和橙色光（590nm;带宽15nm; 100％强度）来激发和可视化mCherry。 注意:HEK电池可以通过间隙连接34电耦合到它们的邻居，导致全电池配置中的低电阻。 </li><li>用细胞内溶液填充一个拉出的移液器，并通过翻转移液管多次去除气泡。 </li><li>将移液器安装在移液器支架上，并施加一些正压力以防止尖端堵塞。 </li><li>在显微镜下找到补片移液管的尖端，并使用显微操纵器将其靠近细胞。 注意:以下步骤适用于a材料清单中提到的数据采集和评估软件。 </li><li>在数据采集软件中启动"膜测试"，并在运行"膜测试"的同时，通过指定的软件使用"浴模式"手动或自动应用电压阶跃（测试脉冲， 例如 5 ms，10 ms，基线为0 mV）在数据采集软件中。 </li><li>检查移液器电阻是否在所需的范围内（1.5-3.0MΩ）。 </li><li>通过在"膜测试"的"浴模式"工作时，通过转动放大器上的移液器偏移旋钮来调整移液管电位来实现零偏移电流。 </li><li> 在全细胞配置26中建立补丁。 注意:以下步骤需要使用传递到使用过的移液器支架侧端的正压和负压。这可以通过使用注射器，通过口件或电子连接器来完成拖曳压力调节器。在这个协议中，正压和负压是通过一个口件施加的。 <ol><li>用顶部的补片移液器缓慢接近细胞，并在触摸之前缓解正压。 注意:当尖端靠近电池时，电阻将略微上升;这由测试脉冲的尺寸减小来表示。 </li><li>将"膜测试"从"浴模式"更改为"贴片模式"，从而保持细胞预期静息电位（-30至-40 mV）的潜力。 </li><li>在一些情况下，释放正压（2.13.1）后，细胞附着的形状本身就会形成，否则，轻轻地施加负压来协助气泡形成。 注意:当脉冲开始和结束时测试脉冲降低到两个小电容峰值，膜电阻处于GΩ范围内时，建立电池连接配置;负电压高达-60 mV m这样可以促进。al形成。 </li><li>通过转动"移液器电容补偿"旋钮来补偿移液管电容，以获得测试脉冲的平坦响应。 </li><li>通过用增加的强度施加负压或负压的短脉冲来破坏贴片而不破坏密封，以获得全细胞构象。 注意:从电池连接到全电池配置的成功转换由电容峰值的增加表示。 </li><li>通过设置全频率参数和使用的放大器的补偿调整来应用串联电阻补偿，以将膜电位精确夹紧到所需的保持电压。 <ol><li>将"膜测试"切换为"全电池"模式，在"串联电阻补偿"面板中，打开"预测"和"校正"达90％，可以避免电容响应的过大和振荡，补偿开关，并以相反的方式使用相应的前面板旋钮（"全单元容量"和"串联电阻"）调整整个单元参数，以获得理想的平面测试密封响应。 注:有关串联电阻补偿和校正的详细说明，请参考放大器手册或指南，因为不同制造商之间的程序不同。 </li></ol></li><li>建立全细胞配置后，在记录前至少等待2分钟，以确保通过贴片移液器35充分更换细胞内溶液。 </li></ol></li><li>通过使用以前设置的协议（1.2）记录不同保持电位的光诱导电流。 </li><li>将细胞外缓冲液至室内交换至低[Cl - ]至少五次，而不破坏贴片，并记录另一个电流 - 电压关系（ 参见步骤1.2）t他新氯化物浓度。 注意:用于自动缓冲区交换的灌注系统有助于此过程，但不是必需的。 </li><li>重复2.15。对于要测试的每个离子条件，但是通过上述"膜测试"重复检查测量之间的贴片的质量。 注意:为了保证精确的膜电压和稳定的细胞内离子组成，膜电阻应大于500MΩ，而访问电阻不应超过10MΩ 。 2.12.6）。不要忘记切换全单元参数补偿以进行膜测试。 </li><li>重复2.2。至2.15。对于几个细胞，但为每个测试系列采取新的封面，以确保细胞的健康。 </li></ol> 3.数据评估<ol><li>通过减去照明前的平均电流来调整每个电流曲线的基线。 </li><li>计算平均固定光电流I s （ <st图2A），通过平均过去40毫秒的照明中的电流。 注意:根据协议或科学问题，分析峰值光电流或改变平均长度以确定固定的光电流。 </li><li>对所有测试条件和不同细胞重复步骤3.1）和3.2）。 </li><li>绘制确定的光电流与相应的保持电位。 注意:如果将多个测量值进行平均，则可以将单个记录归一化为指定条件或单元格的电容。 </li><li>电流 - 电压关系与电压轴的交点表示反向电位（净电流为零）。通过两个相邻数据点之间的线性插值来确定它。 注意:如果光电流没有极性反转，则从接近零的最后两点线性推断，以获得交点的近似值。 </li><li>平均确定r在相同条件下的电位电位和它们对抗外部缓冲溶液的氯化物浓度（ 参见 图2B ）。 </li></ol>

<ol>
	<li>Hegemann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Algal+sensory+photoreceptors.">Algal sensory photoreceptors.</a> Annu. Rev. Plant Biol. 59, 167-189 (2008).</li><li>Nagel, G., 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=Channelrhodopsin-1:+a+light-gated+proton+channel+in+green+algae.">Channelrhodopsin-1: a light-gated proton channel in green algae.</a> Science. 296 (5577), 2395-2398 (2002).</li><li>Bruegmann, T., 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=Optogenetic+control+of+contractile+function+in+skeletal+muscle.">Optogenetic control of contractile function in skeletal muscle.</a> Nat. Commun. 6 (7153), 1-8 (2015).</li><li>Bruegmann, T., 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=Optogenetic+control+of+heart+muscle+in+vitro+and+in+vivo.">Optogenetic control of heart muscle in vitro and in vivo.</a> Nat. Methods. 7 (11), 897-900 (2010).</li><li>Zhang, F., 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=The+microbial+opsin+family+of+optogenetic+tools.">The microbial opsin family of optogenetic tools.</a> Cell. 147 (7), 1446-1457 (2011).</li><li>Nagel, G., 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=Light+activation+of+channelrhodopsin-2+in+excitable+cells+of+Caenorhabditis+elegans+triggers+rapid+behavioral+responses.">Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses.</a> Curr. Biol. 15 (24), 2279-2284 (2005).</li><li>Li, X., 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=Fast+noninvasive+activation+and+inhibition+of+neural+and+network+activity+by+vertebrate+rhodopsin+and+green+algae+channelrhodopsin.">Fast noninvasive activation and inhibition of neural and network activity by vertebrate rhodopsin and green algae channelrhodopsin.</a> Proc. Natl. Acad. Sci. U. S. A. 102 (49), 17816-17821 (2005).</li><li>Ishizuka, T., Kakuda, M., Araki, R., Yawo, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+evaluation+of+photosensitivity+in+genetically+engineered+neurons+expressing+green+algae+light-gated+channels.">Kinetic evaluation of photosensitivity in genetically engineered neurons expressing green algae light-gated channels.</a> Neurosci. Res. 54 (2), 85-94 (2006).</li><li>Wietek, J., 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=Conversion+of+channelrhodopsin+into+a+light-gated+chloride+channel.">Conversion of channelrhodopsin into a light-gated chloride channel.</a> Science. 344 (6182), 409-412 (2014).</li><li>Berndt, 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=Structural+foundations+of+optogenetics:+Determinants+of+channelrhodopsin+ion+selectivity.">Structural foundations of optogenetics: Determinants of channelrhodopsin ion selectivity.</a> Proc. Natl. Acad. Sci. U. S. A. 113 (4), 822-829 (2016).</li><li>Tsunoda, S. P., Hegemann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glu+87+of+Channelrhodopsin-1+Causes+pH-dependent+Color+Tuning+and+Fast+Photocurrent+Inactivation.">Glu 87 of Channelrhodopsin-1 Causes pH-dependent Color Tuning and Fast Photocurrent Inactivation.</a> Photochem. Photobiol. 85 (2), 564-569 (2009).</li><li>Schneider, F., Gradmann, D., Hegemann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+selectivity+and+competition+in+channelrhodopsins.">Ion selectivity and competition in channelrhodopsins.</a> Biophys. J. 105 (1), 91-100 (2013).</li><li>Govorunova, E. G., Spudich, E. N., Lane, C. E., Sineshchekov, O. A., Spudich, 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=New+Channelrhodopsin+with+a+Red-Shifted+Spectrum+and+Rapid+Kinetics+from+Mesostigma+viride.">New Channelrhodopsin with a Red-Shifted Spectrum and Rapid Kinetics from Mesostigma viride.</a> MBio. 2 (3), 1-9 (2011).</li><li>Govorunova, E. G., Sineshchekov, O. A., Li, H., Janz, R., Spudich, 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=Characterization+of+a+highly+efficient+blue-shifted+channelrhodopsin+from+the+marine+alga+Platymonas+subcordiformis.">Characterization of a highly efficient blue-shifted channelrhodopsin from the marine alga Platymonas subcordiformis.</a> J. Biol. Chem. 288 (41), 29911-29922 (2013).</li><li>Hou, S. -. Y., 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=Diversity+of+Chlamydomonas+Channelrhodopsins.">Diversity of Chlamydomonas Channelrhodopsins.</a> Photochem. Photobiol. 88 (1), 119-128 (2012).</li><li>Klapoetke, N. 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=Independent+optical+excitation+of+distinct+neural+populations.">Independent optical excitation of distinct neural populations.</a> Nat. Methods. 11 (3), 338-346 (2014).</li><li>Lin, J. Y., Lin, M. Z., Steinbach, P., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+engineered+channelrhodopsin+variants+with+improved+properties+and+kinetics.">Characterization of engineered channelrhodopsin variants with improved properties and kinetics.</a> Biophys. J. 96 (5), 1803-1814 (2009).</li><li>Yizhar, O., 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=Neocortical+excitation/inhibition+balance+in+information+processing+and+social+dysfunction.">Neocortical excitation/inhibition balance in information processing and social dysfunction.</a> Nature. 477 (7363), 171-178 (2011).</li><li>Berndt, A., Yizhar, O., Gunaydin, L. a., Hegemann, P., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bi-stable+neural+state+switches.">Bi-stable neural state switches.</a> Nat. Neurosci. 12 (2), 229-234 (2009).</li><li>Berndt, A., Lee, S. Y., Ramakrishnan, C., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-Guided+Transformation+of+Channelrhodopsin+into+a+Light-Activated+Chloride+Channel.">Structure-Guided Transformation of Channelrhodopsin into a Light-Activated Chloride Channel.</a> Science. 344 (6182), 420-424 (2014).</li><li>Govorunova, E. G., Sineshchekov, O. A., Spudich, 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=Proteomonas+sulcata+ACR1:+A+Fast+Anion+Channelrhodopsin.">Proteomonas sulcata ACR1: A Fast Anion Channelrhodopsin.</a> Photochem. Photobiol. 92 (2), 257-263 (2016).</li><li>Wietek, J., Broser, M., Krause, B. S., Hegemann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+Natural+Green+Light+Absorbing+Chloride+Conducting+Channelrhodopsin+from+Proteomonas+sulcata.">Identification of a Natural Green Light Absorbing Chloride Conducting Channelrhodopsin from Proteomonas sulcata.</a> J. Biol. Chem. 291 (8), 4121-4127 (2016).</li><li>Govorunova, E. G., Sineshchekov, O. A., Janz, R., Liu, X., Spudich, 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=Natural+light-gated+anion+channels:+A+family+of+microbial+rhodopsins+for+advanced+optogenetics.">Natural light-gated anion channels: A family of microbial rhodopsins for advanced optogenetics.</a> Science. 349 (6248), 647-650 (2015).</li><li>Neher, E., Sakmann, B., Steinbach, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+patch+clamp:+A+method+for+resolving+currents+through+individual+open+channels+in+biological+membranes.">The extracellular patch clamp: A method for resolving currents through individual open channels in biological membranes.</a> Pfl&#252;gers Arch. Eur. J. Physiol. 375 (2), 219-228 (1978).</li><li>Hamill, O. P., Marty, A., Neher, E., Sakmann, B., Sigworth, 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=Improved+patch-clamp+techniques+for+high-resolution+current+recording+from+cells+and+cell-free+membrane+patches.">Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.</a> Pfl&#252;gers Arch. Eur. J. Physiol. Arch. Eur. J. Physiol. 391 (2), 85-100 (1981).</li><li>Barry, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JPCalc,+a+software+package+for+calculating+liquid+junction+potential+corrections+in+patch-clamp,+intracellular,+epithelial+and+bilayer+measurements+and+for+correcting+junction+potential+measurements.">JPCalc, a software package for calculating liquid junction potential corrections in patch-clamp, intracellular, epithelial and bilayer measurements and for correcting junction potential measurements.</a> J. Neurosci. Methods. 51 (1), 107-116 (1994).</li><li>Amtmann, A., Sanders, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unified+procedure+for+the+correction+of+liquid+junction+potentials+in+patch+clamp+experiments+on+endo-+and+plasma+membranes.">A unified procedure for the correction of liquid junction potentials in patch clamp experiments on endo- and plasma membranes.</a> J. Exp. Bot. 48, 361-364 (1997).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+hippocampal+neurons.">Culturing hippocampal neurons.</a> Nat. Protoc. 1 (5), 2406-2415 (2006).</li><li>Sanders, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aseptic+laboratory+techniques:+plating+methods.">Aseptic laboratory techniques: plating methods.</a> J. Vis. Exp. (63), e1-e18 (2012).</li><li>Felgner, P. L., 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=Lipofection:+a+highly+efficient,+lipid-mediated+DNA-transfection+procedure.">Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure.</a> Proc. Natl. Acad. Sci. U. S. A. 84 (21), 7413-7417 (1987).</li><li>Halliwell, J., Whitaker, M., Ogden, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+microelectrodes.">Using microelectrodes.</a> Microelectrode Tech. Plymouth Work. Handb. 3, 1-15 (1949).</li><li>Brown, A. L., Johnson, B. E., Goodman, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+patch-pipettes+and+sharp+electrodes+with+a+programmable+puller.">Making patch-pipettes and sharp electrodes with a programmable puller.</a> J. Vis. Exp. (20), e1-e2 (2008).</li><li>Thomas, P., Smart, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HEK293+cell+line:+A+vehicle+for+the+expression+of+recombinant+proteins.">HEK293 cell line: A vehicle for the expression of recombinant proteins.</a> J. Pharmacol. Toxicol. Methods. 51 (3), 187-200 (2005).</li><li>Pusch, M., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rates+of+diffusional+exchange+between+small+cells+and+a+measuring+patch+pipette.">Rates of diffusional exchange between small cells and a measuring patch pipette.</a> Pfl&#252;gers Arch. Eur. J. Physiol. 411 (2), 204-211 (1988).</li><li>Prigge, 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=Color-tuned+channelrhodopsins+for+multiwavelength+optogenetics.">Color-tuned channelrhodopsins for multiwavelength optogenetics.</a> J. Biol. Chem. 287 (38), 31804-31812 (2012).</li><li>Gradmann, D., Berndt, A., Schneider, F., Hegemann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rectification+of+the+channelrhodopsin+early+conductance.">Rectification of the channelrhodopsin early conductance.</a> Biophys. J. 101 (5), 1057-1068 (2011).</li><li>Beppu, K., 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=Optogenetic+countering+of+glial+acidosis+suppresses+glial+glutamate+release+and+ischemic+brain+damage.">Optogenetic countering of glial acidosis suppresses glial glutamate release and ischemic brain damage.</a> Neuron. 81 (2), 314-320 (2014).</li><li>Wietek, J., Prigge, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+Channelrhodopsins:+An+Overview.">Enhancing Channelrhodopsins: An Overview.</a> Methods Mol. Biol. 1408, 141-165 (2016).</li><li>Ferenczi, E. 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=Optogenetic+approaches+addressing+extracellular+modulation+of+neural+excitability.">Optogenetic approaches addressing extracellular modulation of neural excitability.</a> Sci. Rep. 6, 1-20 (2016).</li><li>Zhang, F., 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=Red-shifted+optogenetic+excitation:+a+tool+for+fast+neural+control+derived+from+Volvox+carteri.">Red-shifted optogenetic excitation: a tool for fast neural control derived from Volvox carteri.</a> Nat. Neurosci. 11 (6), 631-633 (2008).</li><li>Sugiyama, Y., 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=Photocurrent+attenuation+by+a+single+polar-to-nonpolar+point+mutation+of+channelrhodopsin-2.">Photocurrent attenuation by a single polar-to-nonpolar point mutation of channelrhodopsin-2.</a> Photochem. Photobiol. Sci. 8 (3), 328-336 (2009).</li><li>Kleinlogel, 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=Ultra+light-sensitive+and+fast+neuronal+activation+with+the+Ca2+-permeable+channelrhodopsin+CatCh.">Ultra light-sensitive and fast neuronal activation with the Ca2+-permeable channelrhodopsin CatCh.</a> Nat. Neurosci. 14 (4), 513-518 (2011).</li><li>Wang, 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=Molecular+determinants+differentiating+photocurrent+properties+of+two+channelrhodopsins+from+chlamydomonas.">Molecular determinants differentiating photocurrent properties of two channelrhodopsins from chlamydomonas.</a> J. Biol. Chem. 284 (9), 5685-5696 (2009).</li><li>Gradinaru, V., Thompson, K. R., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=eNpHR:+a+Natronomonas+halorhodopsin+enhanced+for+optogenetic+applications.">eNpHR: a Natronomonas halorhodopsin enhanced for optogenetic applications.</a> Brain Cell Biol. 36 (1-4), 129-139 (2008).</li><li>Berglund, K., 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=Luminopsins+integrate+opto-+and+chemogenetics+by+using+physical+and+biological+light+sources+for+opsin+activation.">Luminopsins integrate opto- and chemogenetics by using physical and biological light sources for opsin activation.</a> Proc. Natl. Acad. Sci. 113 (3), 358-367 (2016).</li></ol>

The authors have nothing to disclose.

在确定的离子和电气条件下的反转电位的测定提供了关于在光激活ChR之后传输的离子物种的信息。如果在复杂的生理介质中仅排除一种离子种类，并且根据理论能斯特电位，获得的反转电势发生变化，则该离子种类是唯一传播的离子。 然而，对于ChRs，由于对不同离子物质的渗透性以及导电离子之间的竞争，反向电位偏移通常不如从恩斯特方程所预期的那么明显。在这种情况?...

通道视紫红质（Chr）是发生在运动性绿藻眼睛中的轻门控离子通道，可作为光敏感和恐惧反应的主要感光元件1 。自2002年第一次描述以来，ChR为新兴的光遗传学领域铺平了道路，可以应用于各种兴奋细胞， 如骨骼肌，心脏或大脑3,4,5 。 ChRs在靶细胞中的表达导致相应细胞的光可控离子渗透性。在神经元上下文中，这允许激活6,7,8或抑制作用电位（AP）的9,10（取决于导电离子）与空间和时间光的精度强调了ChR变体的离子选择性如何决定其光遗传应用。 第一个发现的Chlamydomonas reinhardtii和Volvox carteri的ChRs对质子是可渗透的，而且也可以是一钠阳离子，如钠，钾，二来阳离子如钙和镁11,12,13 。今天，超过70种天然阳离子导电通道视紫红质（CCR）14,15,16,17和几种具有不同性质的工程变体18,19,20具有 光电流尺寸，光谱灵敏度，动力学和阳离子选择性。而在神经科学中，CCRs a重新用于激活细胞并触发AP，光驱微生物泵是用于沉默神经元多年的唯一可用的拮抗剂。 2014年，两组同时表明，CCR可以通过分子工程9,21改变推定离子导电孔的极性，将其转化为阴离子导电通道视紫红质（ACR）。随后，在几种密类藻22,23,24中鉴定了自然ACR。最重要的是，ACR的光激活介导成年神经元中的氯化物电流，允许在比仅吸收光子仅传输单个电荷的微生物泵更低的光强度下抑制神经元活性。 ChR活性可以通过HEK293细胞中光诱导电流的电生理膜片钳记录直接解决。膜片夹技术最初是在20世纪70年代末开发的，而且由Hamill 等人进一步改进。允许从高电流分辨率的小电池（全电池模式）记录电流实体，并直接控制膜电压26 。应用于细胞培养，这种技术提供了离子和电记录条件的精确控制，并且能够研究离子选择性以及离子对总电流的相对贡献。这里我们举例说明通过记录各种细胞外氯化物浓度下的电流 - 电压关系来检测蛋白质组学（ Ps ACR1） 22,23的阴离子导电通道视紫红质的离子选择性，以证明高氯化物电导。

<table><tbody><tr><td>HEK293 cells</td><td>Sigma Aldrich</td><td>85120602</td><td>Human embryonic kidney cells</td></tr><tr><td>Retinal</td><td>Sigma Aldrich</td><td>R2500</td><td>all-trans retinal</td></tr><tr><td>FuGENE HD</td><td>Promega</td><td>E2312</td><td>Transfection reagent</td></tr><tr><td>DMEM</td><td>Biochrome</td><td>FG 0445</td><td>Dulbecco&#039;s Modified Eagle Medium</td></tr><tr><td>Agarose</td><td>Roth</td><td>3810</td><td>Agar bridges</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Roth</td><td>5239</td><td>CaCl&lt;sub&gt;2&lt;/sub&gt; 2H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>CsCl</td><td>Biomol</td><td>2452</td><td></td></tr><tr><td>EGTA</td><td>Roth</td><td>3054</td><td></td></tr><tr><td>FBS</td><td>Biochrome</td><td>S0615</td><td>Cell culture</td></tr><tr><td>Glucose</td><td>Roth</td><td>HN06</td><td>D(+)-Glucose</td></tr><tr><td>KCl</td><td>Roth</td><td>6781</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Roth</td><td>2189</td><td>MgCl&lt;sub&gt;2&lt;/sub&gt; 6H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>NaCl</td><td>Roth</td><td>3957</td><td></td></tr><tr><td>NMG</td><td>Sigma Aldrich</td><td>M2004</td><td>N-Methyl-D-glucamine</td></tr><tr><td>Na-Aspartate</td><td>Sigma Aldrich</td><td>A6683</td><td>L-Aspartic acid sodium salt monohydrate</td></tr><tr><td>Citric acid</td><td>Roth</td><td>6490</td><td></td></tr><tr><td>AgeI</td><td>ThermoFischerScientific</td><td>ER1462</td><td>Restriction enzyme</td></tr><tr><td>XhoI</td><td>ThermoFischerScientific</td><td>ER0695</td><td>Restriction enzyme</td></tr><tr><td>NheI</td><td>ThermoFischerScientific</td><td>ER0975</td><td>Restriction enzyme</td></tr><tr><td>XL1Blue &lt;em&gt;E. coli&lt;/em&gt;</td><td>Agilent Technologies</td><td>200249</td><td>Chemocompetent &lt;em&gt;E. coli&lt;/em&gt;</td></tr><tr><td>Kanamycin</td><td>Roth</td><td>T832</td><td></td></tr><tr><td>Lysogeny broth medium</td><td>Roth</td><td>X964</td><td></td></tr><tr><td>Agar-Agar</td><td>Roth</td><td>6494</td><td>Agar plates</td></tr><tr><td>Plasmid purification kit</td><td>Marchery-Nagel</td><td>740727.25</td><td></td></tr><tr><td>Penicilin/Streptomycin</td><td>Biochrome</td><td>A 2213</td><td>Cell culture</td></tr><tr><td>Poly-D-lysine hydrobromide</td><td>Sigma Aldrich</td><td>P6407-5MG</td><td>Cover slip coating</td></tr><tr><td>Microforge</td><td>Custom made</td><td></td><td>Fire polishing</td></tr><tr><td>Serological pipettes</td><td>TPP</td><td></td><td>Different sizes</td></tr><tr><td>Clean bench</td><td>Kojair</td><td>Biowizard SL130</td><td></td></tr><tr><td>Stirrer</td><td>IKA</td><td>RCT classic</td><td></td></tr><tr><td>Silver wire</td><td>Science Products</td><td>AG-T25; AG-T10</td><td>Electrodes, 0.64 mm (bath); 0.25 mm (electrode)</td></tr><tr><td>pH-meter</td><td>Knick</td><td>765 Calimetric</td><td></td></tr><tr><td>Osmometer</td><td>Vogel</td><td>OM 815</td><td></td></tr><tr><td>Microscope</td><td>Carl Zeiss</td><td>ID03</td><td>Fire polishing</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Binder</td><td>CB150</td><td></td></tr><tr><td>Cell culture dishes</td><td>TPP</td><td>93040</td><td>34 mm internal diameter</td></tr><tr><td>Cover slips</td><td>Roth</td><td>P232</td><td>15 mm diameter</td></tr><tr><td>Thermometer</td><td>R&amp;ouml;ssel Messtechnik</td><td>MTM12</td><td></td></tr><tr><td>Beamsplitter</td><td>Chroma</td><td>21011</td><td>90/10 transmission</td></tr><tr><td>Pipette holder</td><td>ALA Scientific Instruments</td><td>PPH-1P-AXU-0-1.5</td><td></td></tr><tr><td>Headstage</td><td>Molecular Devices</td><td>CV203BU</td><td></td></tr><tr><td>Amplifier</td><td>Molecular Devices</td><td>AxoPatch200B</td><td></td></tr><tr><td>Digitizer</td><td>Molecular Devices</td><td>DigiData1400</td><td>Digital analog converter</td></tr><tr><td>Lightsource</td><td>TILL Photonics</td><td>Polychrome V</td><td>Set to 540 nm full intensity</td></tr><tr><td>Microscope</td><td>Carl Zeiss</td><td>Axiovert 100</td><td></td></tr><tr><td>Shutter</td><td>Vincent Associates</td><td>VS25</td><td></td></tr><tr><td>Shutter driver</td><td>Vincent Associates</td><td>VCM-D1</td><td></td></tr><tr><td>Glass capilarries</td><td>Warner Instruments</td><td>G150F-3</td><td>Boresilicate capillaries with fire polished ends OD 1.5 mm ID&amp;nbsp; 0.86 mm</td></tr><tr><td>Micropipette puller</td><td>Sutter Instruments</td><td>P1000</td><td></td></tr><tr><td>Bath handler</td><td>Lorenz Messger&amp;auml;tebau</td><td>MPCU</td><td></td></tr><tr><td>Tripleband filterset</td><td>Chroma</td><td>69008</td><td>Fluorescence filter&amp;nbsp; ECFP/EYFP/mCherry&amp;nbsp;</td></tr><tr><td>CCD camera</td><td>Watec</td><td>Wat-221SCCD</td><td></td></tr><tr><td>Optometer</td><td>Gigahertz Optik</td><td>P9710</td><td>Measure light intensities</td></tr><tr><td>Objective</td><td>Carl Zeiss</td><td>421462-9900-000</td><td>W Plan-Apochromat 40X/1.0 DIC</td></tr><tr><td>Micromanipulator</td><td>Scientifica</td><td>PatchStar</td><td></td></tr><tr><td>Recording chamber</td><td>Custom made</td><td></td><td></td></tr><tr><td>Power supply</td><td>Manson</td><td>HCS-3202</td><td>Avoids electrical noise from microscope built-in power supply</td></tr><tr><td>Vibration isolated table</td><td>Newport</td><td>M-VW-3636-OPT-01</td><td></td></tr><tr><td>Faraday cage</td><td>Custom made or any commercial matching table</td><td></td><td></td></tr><tr><td>Hoses</td><td>Any comercial; e.g. Roth</td><td></td><td>Different sizes and materials for bath handling and application of pipette pressure; agar bridges</td></tr><tr><td>Linear shaker</td><td>Sunlab Instruments</td><td>SU 1000</td><td></td></tr><tr><td>Liquid junction potential calculator</td><td>Molecular Devices or directly from Peter H. Barry</td><td></td><td>Program is included in the Clampex aquisition software or can be obtained from&amp;nbsp; p.barry@unsw.edu.au</td></tr><tr><td>Data acquisition software</td><td>Molecular Devices</td><td>Clampex 10.X</td><td></td></tr><tr><td>Data evaluation software</td><td>Molecular Devices</td><td>Clampfit 10.X</td><td></td></tr><tr><td>&lt;em&gt;Ps&lt;/em&gt;ACR1</td><td>GenBank or Addgene</td><td>KF992074.1 or Addgene plasmid #85465</td><td>Gene encoding for &lt;em&gt;Ps&lt;/em&gt;ACR1</td></tr><tr><td>Amplifier guide</td><td>Molecular Devices</td><td>The Axon Guide</td><td></td></tr></tbody></table>

whole-cell patch-clamp recordings for electrophysiological determination of ion selectivity in channelrhodopsins

在过去十年中，通道观光素在神经科学研究中成为不可或缺的，它们被用作非侵入性操纵靶细胞电工作的工具。在这方面，通道视紫红质的离子选择性是特别重要的。本文介绍了通过HEK293细胞上的电生理膜片钳记录研究最近鉴定的蛋白质组培养基的阴离子导电通道视紫红质的氯选择性。用于测量光门控光电流的实验程序需要一个快速可切换的 - 理想的单色光源，耦合到另外传统的膜片钳设置的显微镜中。概述实验前的制备方法涉及缓冲溶液的制备，对液体接头电位的考虑，接种和细胞转染以及拉片移植管。实际记录电流 - 电压关系以确定不同氯化物浓度的逆转电位在转染后24小时至48小时。最后，关于氯化物传导的理论考虑分析电生理数据。

<html> 图2示出了根据所述方案从测量获得的代表性结果。在用绿光照明时， Ps ACR1具有快速衰减到稳定电流水平的快速瞬态电流。光线关闭后，光电流在几毫秒内就会衰减到零（ 图2A ）。交换细胞外氯离子浓度会导致在获得的光电流迹线中可以直接看到的反转电位的偏移。从多次测量的反转电位的评估量化了由外部氯化物浓度的变化引起的?...

Watch this Scientific Journal Video about Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins at JoVE.com

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

全细胞贴片记录用于电泳生理测定通道视紫质中的离子选择性

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate ...

whole-cell-patch-clamp-recordings-for-electrophysiological

Research

JoVE Journal

Neuroscience

17.0K 次观看.  Humboldt-Universität zu Berlin. This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

视频: Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

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 ...

科研

生物学

Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

One of the central tasks in retinal neuroscience is to understand the circuitry of retinal neurons and how those connections are responsible for shaping the signals transmitted to the brain. Photons are detected in the retina by rod and cone photoreceptors, which convert that energy into an electrical signal, transmitting it to other retinal neurons, where it is processed and communicated to central targets in the brain via the optic nerve. Important early insights into retinal circuitry and visual processing came from the histological studies of Cajal1,2 and, later, from electrophysiological recordings of the spiking activity of retinal ganglion cells - the output cells of the retina3,4.
A detailed understanding of visual processing in the retina requires an understanding of the signaling at each step in the pathway from photoreceptor to retinal ganglion cell. However, many retinal cell types are buried deep in the tissue and therefore relatively inaccessible for electrophysiological recording. This limitation can be overcome by working with vertical slices, in which cells residing within each of the retinal layers are clearly visible and accessible for electrophysiological recording.
Here, we describe a method for making vertical sections of retinas from larval tiger salamanders (Ambystoma tigrinum). While this preparation was originally developed for recordings with sharp microelectrodes5,6, we describe a method for dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells in which we manipulate the photoreceptor's membrane potential while simultaneously recording post-synaptic responses in horizontal or bipolar cells. The photoreceptors of the tiger salamander are considerably larger than those of mammalian species, making this an ideal preparation in which to undertake this technically challenging experimental approach. These experiments are described with an eye toward probing the signaling properties of the synaptic ribbon - a specialized synaptic structure found in a only a handful of neurons, including rod and cone photoreceptors, that is well suited for maintaining a high rate of tonic neurotransmitter release7,8 - and how it contributes to the unique signaling properties of this first retinal synapse.

We describe the preparation of thin retinal slices from aquatic tiger salamanders (Ambystoma tigrinum) and explain how we use these slices to study synaptic processing in the retina by obtaining dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells.

同时光感受器和二阶神经元在两栖类动物的视网膜切片制备全细胞记录

One of the central tasks in retinal neuroscience  is to understand the circuitry of retinal neurons and how those connections are  responsible for shaping ...

神经科学

Identification of Specific Sensory Neuron Populations for Study of Expressed Ion Channels

Sensory neurons transmit signals from various parts of the body to the central nervous system. The soma for these neurons are located in the dorsal root ganglia that line the spinal column. Understanding the receptors and channels expressed by these sensory afferent neurons could lead to novel therapies for disease. The initial step is to identify the specific subset of sensory neurons of interest. Here we describe a method to identify afferent neurons innervating the muscles by retrograde labeling using a fluorescent dye DiI (1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate). Understanding the contribution of ion channels to excitation of muscle afferents could help to better control excessive excitability induced by certain disease states such as peripheral vascular disease or heart failure. We used two approaches to identify the voltage dependent ion channels expressed by these neurons, patch clamp electrophysiology and immunocytochemistry. While electrophysiology plus pharmacological blockers can identify functional ion channel types, we used immunocytochemistry to identify channels for which specific blockers were unavailable and to better understand the ion channel distribution pattern in the cell population. These techniques can be applied to other areas of the nervous system to study specific neuronal groups.

Afferent sensory neurons signal sensory information from the periphery to the central nervous system. Identifying specific afferent neurons will help in understanding their physiology. We describe a method of retrograde labeling to identify afferent neurons, and study the voltage-gated ion channels in these neurons using patch clamp electrophysiology and immunocytochemistry.

具体的感觉神经元的人群离子通道的表达研究鉴定

Sensory neurons transmit signals from various parts of the body to the central nervous system. The soma for these neurons are located in the dorsal root ...

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 ...

Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due to contact with adjacent cells. Transfected channels must also express with high efficiency at the plasma membrane for whole-cell patch clamp recording of detectable currents above noise levels. Heterologous ion channels often require long incubation periods at 28&deg;C after transfection in order to achieve adequate membrane expression, but there are increasing losses of cell-coverslip adhesion and membrane stability at this temperature. To circumvent this problem, we developed an optimized strategy to transfect and plate HEK-293T cells. This method requires that cells be transfected at a relatively high confluency, and incubated at 28&deg;C for varying incubation periods post-transfection to allow for adequate ion channel protein expression. Transfected cells are then plated onto glass coverslips and incubated at 37&deg;C for several hours, which allows for rigid cell attachment to the coverslips and membrane restabilization. Cells can be recorded shortly after plating, or can be transferred to 28&deg;C for further incubation. We find that the initial incubation at 28&deg;C, after transfection but before plating, is key for the efficient expression of heterologous ion channels that normally do not express well at the plasma membrane. Positively transfected, cultured cells are identified by co-expressed eGFP or eGFP expressed from a bicistronic vector (e.g. pIRES2-EGFP) containing the recombinant ion channel cDNA just upstream of an internal ribosome entry site and an eGFP coding sequence. Whole-cell patch clamp recording requires specialized equipment, plus the crafting of polished recording electrodes and L-shaped ground electrodes from borosilicate glass. Drug delivery to study the pharmacology of ion channels can be achieved by directly micropipetting drugs into the recording dish, or by using microperfusion or gravity flow systems that produce uninterrupted streams of drug solution over recorded cells.

Reliable method for highly efficient in vitro expression and subsequent electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T).

优化转染的HEK - 293T细胞中的表达和重组的电压门控离子通道的电生理记录策略

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) ...

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

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

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

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

全细胞电压钳记录的电生理方法<em&gt;果蝇</em&gt;光感受器

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

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 ...

Optogenetic Electrophysiology Recording of Cells with Light-Dependent Ion Channels

Source: Grimm, C., et al. <a href="https://app.jove.com/v/55497">Whole-cell patch-clamp recordings for electrophysiological determination of ion selectivity in channelrhodopsins.</a> J. Vis. Exp. (2017)
In this video, embryonic kidney cell cultures expressing light-dependent ion channel proteins fused with a fluorescent reporter demonstrate the effect of light on ion movement. Whole-cell patch clamp is used to monitor the movement of ions.

具有光依赖性离子通道的细胞的光遗传学电生理学记录

1. Setup Prior to Recordings

	Prepare the recording solutions with ion concentrations as indicated in Table 1.&#8203;
	
		Prepare 15 mL of 2 M stock ...

JoVE Encyclopedia of Experiments

Neurophysiology

Imaging and Optical Techniques

Whole-Cell Voltage Clamp Recordings from Drosophila Photoreceptors

Source:&#160; Katz, B., et al., <a href="https://app.jove.com/v/55627">Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors.</a> J. Vis. Exp. (2017)
The video demonstrates recording electrical activity in the photoreceptor neurons of Drosophila&#39;s ommatidium by establishing a whole-cell configuration. It highlights the activation of transient receptor potential channels by light pulses, resulting in measurable quantum bumps.

来自果蝇光感受器的全细胞电压钳记录

NOTE: During all of the following steps, use only dim red light illumination and ensure that the ommatidia&#39;s exposure to light is minimal (i.e., work ...

Electrophysiology Techniques

Patch Clamp

Many fundamental cell functions such as muscle contraction and nerve transmission rely on the electrical signals produced by the movement of positively and negatively charged ions across the cell membrane. One competent method to record current flowing across the whole cell or single ion channel is the patch-clamp technique.
In this method, a glass micropipette containing electrolyte solution is tightly sealed against a small portion of the cell membrane. As a result, a patch of the cell membrane is electrically isolated, ensuring that the ions moving across the channels flow only into the micropipette. Further, this tight sealing of the cell membrane prevents ions from escaping into the bath solution in which the cell is suspended.
Different Patch-clamp Methods:
Depending upon the research interest, several variations of the patch-clamp method can be used for measuring the cell&#39;s biophysical properties. For example, researchers can clamp or control the membrane voltage and measure the current passing through it. Alternatively, they can clamp the current and measure any variation in the membrane voltage.
In cell-attached mode, the membrane patch containing a single or a few ion channels remains intact. As a result, the current flowing through the membrane patch alone can be measured. In contrast, the whole-cell method involves disruption of the membrane patch by briefly applying strong suction. Consequently, the interior of the pipette becomes continuous with the cytoplasm. This mode enables the measurement of electrical current and voltage from the entire cell.
Another patch-clamp method requires gentle retraction of the attached pipette. As a result, the membrane patch is excised without affecting the tight seal. In this inside-out configuration, the intracellular portion of the membrane is exposed to the bath solution, allowing the study of intracellular factors affecting the channel functions.

膜片钳

Many fundamental cell functions such as muscle contraction and nerve transmission rely on the electrical signals produced by the movement of positively ...