The authors are deeply indebted to all the members of the scientific community working on pH and ion transport, who have originated and improved the measurements described here. They particularly thank Dr. Jacques Pouyss&#233;gur who originated the H+-killing selection technique used here. They acknowledge the University of Nice-Sophia Antipolis, the CNRS, the ANR (JCJC SVSE1 NHEint) and the ICST Labex for support.

1. H +杀戮的选择<ol><li>细胞系<ol><li>稳定地转染NHE缺陷型细胞（ 例如 ，CCL39衍生PS120细胞系16），使用哺乳动物表达载体和转染方法的任意组合，将产生有效的转染率和选择在一个成纤维细胞系。 注:多年来磷酸钙沉淀19与良好的收益率转用。这已被替换最近被商业试剂如Lipofectamine2000，正在执行按照制造商的说明转染。 </li></ol></li><li>解决方案<ol><li>制备描述如下3无菌溶液。通过过滤（0.22微米）灭菌这些溶液。 <ol><li>制备NH + 4加载溶液（pH7.4）中含有50mM的氯化铵 ，70毫氯化胆碱，5mM的氯化钾，1mM的MgCl 2的，2mM的氯化钙 ，5mM的GLucose和15mM MOPS pH为7.4。 </li><li>制备由120毫氯化胆碱，5mM的氯化钾，1mM的MgCl 2的，2mM的氯化钙 ，5mM葡萄糖和15mM HEPES，在pH 7.0漂洗溶液（pH 7.0）中。 </li><li>制备包含120mM氯化钠，5mM的氯化钾，1mM的MgCl 2的，2mM的氯化钙 ，5mM葡萄糖和15mM HEPES，在pH 7.4的回收溶液（pH7.4）中。 </li></ol></li></ol></li><li>在开始选择之前，获得细胞培养孵化器，可用于在37℃下，没有额外的CO 2，从外部罐（称为CO 2 - "自由"孵化），5％的CO 2将导致缓冲可能损害酸化。扩增表达感兴趣向上NHE至约2×10 8个细胞（通常为约20汇合100毫米平板）的细胞系。 </li><li> H +查杀方法: <ol><li>孵育表达目的细胞内NHE的细胞1小时，在加载溶液中，在37°C，在无二氧化碳排放的孵化器。 </li><li>吸出装载溶液，然后漂洗两次它们在上述漂洗溶液。有效地执行这一步，以消除残余细胞外氯化铵 ，将损害创造一个陡峭的NH 4 +梯度，将产生的酸化。 </li><li>消除通过抽吸漂洗介质，并在回收溶液中的CO 2培养箱自由再次孵育细胞一小时，在37℃。 </li><li>替换常规的培养基（通常的DMEM以7.5％FCS）中的恢复中，生长中的细胞的标准培养条件下（37℃，在5％的CO 2和95％空气的潮湿气氛中）。 </li></ol></li><li>每周重复选择两次这样的循环，直到平稳克隆出现。 注:要特别注意有关细胞培养和选择条件。工作几个月可以通过任何的污染是比较容易Ø毁经过文化和重复单元操作长时间CCur函数。 <ol><li>在这里，选择是否放大克隆并分别在第2-3为进一步说明他们的特点，或集中在一起，生成特征之前，细胞群体。 </li></ol></li><li>适用偶尔选择（每两周一次左右）通过反选择那些可能已经恢复到亲酸敏感的表型（ 图1B）保留酸化抗性细胞。 </li></ol> 2.细胞内pH测量 2.1）荧光pH值成像<ol><li>使用比例PH敏感荧光染料BCECF / AM，这是pH敏感当在490nm激发，并具有一个445纳米的等吸光点，按照制造商的协议。 <ol><li>可替代地，可以使用其他pH敏感的探针，按照制造商的协议。 </li></ol></li><li>使用的成像设置CONsisting倒置显微镜耦合到一个高灵敏度的摄像机的。装备这套450 nm和搭配激发适当的石英中性密度滤光片490 nm的窄带干涉滤光片。 <ol><li>如果可能的话，可以使用适当的一组过滤器和照明条件设置可比荧光值λ1和λ2，使得比接近1。此外，还要避免被设置或者饱和或过低，都为λ1和/或λ2基础荧光值。这可能会产生重要的文物和当时一样的比例不得被检测到，虽然细胞内pH值发生变化。 注意:此系统必须在适当的发射波长（535纳米的BCECF）使快速灌注，延时激励在两个上述的波长和采集。装备该系统用软件实现自动数据采集和存储。 </li></ol></li></ol> NHE转发活动2.2）测量（挤压从胞浆质子） <ol><li>酸化的细胞用1小时温育中的NH + 4加载溶液（参见步骤1.4），在不存在的CO 2。 </li><li>添加BCECF-AM（5μM的终浓度），以将溶液的最后五分钟，然后用NH冲洗4 +加载溶液，以消除外探头。 </li><li>安装在显微镜的细胞，并采取若干图像来记录一个稳定的基线。灌注冲洗液（见上文）。这导致了降荧光对应于酸化。 </li><li> pH值稳定后，灌注任何下面给出的解决方案，以测量由锂+ / H +，的Na + / H +或K + / H +交换介导的pH值回收率。每个溶液含有潜在耦合阳离子为H +交换进行测试。如果这些中的一个被传送，这将导致荧光增加，在495nm处，这将correspOND在细胞内pH加薪: 120毫氯化锂，5mM的氯化钾，1mM的MgCl 2的，2mM的氯化钙 ，5mM葡萄糖和15mM HEPES pH 7.4的 120mM氯化钠，5mM的氯化钾，1mM的MgCl 2的，2mM的氯化钙 ，5mM葡萄糖和15mM HEPES pH 7.4的 125毫米氯化钾，1mM的MgCl 2的，2mM的氯化钙 ，5mM葡萄糖和15mM HEPES pH 7.4的</li></ol>对NHE反向激活2.3）测量注:此处的原理是反转跨膜离子梯度。 <ol><li>在测量之前，加载细胞与感兴趣的细胞内阳离子（见下文）。 </li><li>它们孵育5分钟，BCECF / AM（见步骤2.2.2）;冲洗一下，将它们安装在视频显微镜。 </li><li>灌注的加载溶液，并采取若干图像来记录一个稳定的基线。 </li><li>基线稳定，图像当细胞灌注细胞外酸性介质中含有120米pH值变化后，M氯化胆碱，5mM的氯化钾，1mM的MgCl 2的，2mM的氯化钙 ，5mM葡萄糖和15mM MES pH为6.5。 </li><li>为氯化锂装载，在一个在pH7.4组成的120毫氯化锂，5mM的氯化钾，1mM的MgCl 2的，2mM的氯化钙 ，5mM葡萄糖和15mM HEPES溶液孵育细胞2小时。用原子吸收光谱测定胞内锂产生的约60mM的值。 </li><li>对Na +的负荷，在1mM的哇巴因的存在下孵育细胞两个小时在120 mM氯化钠，5mM的氯化钾，1mM的MgCl 2的，2mM的氯化钙 ，5mM葡萄糖和15mM HEPES组成的溶液在pH7.4挡住的Na / K ATP酶。在这些条件下，细胞内的Na +浓度为40毫摩尔的范围内。 注:K +负荷是不必要的，因为细胞内K +浓度为140mM的范围内和向外定向的K +梯度，因此容易实现。 </li></ol> 2.4）数据处理和校准: 注意:此步骤既适用于正向和反向测量。 <ol><li>在每个实验结束时，灌注细胞的140mM的氯化钾，20mM的HEPES和5μM的尼日利亚菌素溶液6.5和7.4之间调节至pH值。 </li><li>收集（代表亮度级的灰度级）的数据作为从图像中荧光测量和出口下的文本格式和下方使用电子表格程序当作说明。 </li><li>计算每个单独的细胞或使用以下等式关心区域内pH值: pH值= pKa值+（日志（R-Rmin的）/（R 最大 -R））X F 分钟（λ2）/ F MAX（λ2） </li><li>使用F 分钟（λ2）/ F 毫安×（λ2）的比例，如果有探针漂白或渗漏，从而导致在整个实验中的450纳米的荧光的减少。然而，这是非常罕见的，在沙漠中使用的条件下观察cribed实验。 </li><li>作为校准数据存在于每个pH测量结束时，检查相应的计算值是否与用于校准的pH值不同。如果是这种情况，则通过以下步骤调整所有实验确定的pH值下与校准: <ol><li>计算以下商数（Q，校准值之间的测量值/差分之间的差）。乘以整个数据集由Q设为通过加法或减法的最终调整，以使计算出的值将等于相应的校准值。 </li></ol></li></ol> 3.测量初始价格NHE7通过快速锂离子吸收<ol><li>多孔板种子细胞（6至24个孔），并使用任一所述的NH 4上方或交替的尼日利亚菌素/牛血清白蛋白（BSA）在20中所述酸化描述+加载技术酸化它们。 </li><lI&gt;冲洗板两次快速使用上述冲洗溶液，然后孵育细胞在含1-10毫锂（略低于Km值）的吸收溶液和阳离子的所需浓度和/或感兴趣的抑制剂所需量的时间。 </li><li>保持短和一致的吸收的持续时间（一般为一分钟或以下），以确保运输工作在初始速率条件下。另外，还要确保所有用于摄取的解决方案是等渗。 </li><li>在摄取时间结束时，小心地消除吸收介质并用冰冷的磷酸盐缓冲盐水（PBS）洗涤细胞4次。作为快速执行这些洗涤越好（低于10秒，其中的四个是理想的），以防止锂流出。 </li><li>裂解的细胞在25％的硝酸。应用250微升每25％的硝酸井并让它静置至少1小时，然后废每个孔与吸头的端部和在一个新的英里传送整个井悬浮crocentrifuge管。 </li><li>转移溶解物在1.5ml微量离心管中，离心它们在15000×g离心（室温）5分钟以除去细胞碎片。 </li><li>测量上清液用原子吸收光谱21的锂含量。 注意:不同的公司提供的原子吸收光谱仪，其中必须配备有一个锂空心阴极灯。按照制造商的说明，因为这些机器非常精确，但也相当细腻。 </li></ol>

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<li>Quentin, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RhBG+and+RhCG%2C+the+putative+ammonia+transporters%2C+are+expressed+in+the+same+cells+in+the+distal+nephron%5BTitle%5D)+AND+%22J+Am+Soc+Nephrol%22%5BJournal%5D)">RhBG and RhCG, the putative ammonia transporters, are expressed in the same cells in the distal nephron.</a> J Am Soc Nephrol. 14 (3), 545-554 (2003).</li>
<li>Geyer, R. R., Musa-Aziz, R., Enkavi, G., Mahinthichaichan, P., Tajkhorshid, E., Boron, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Movement+of+NH3+through+the+human+urea+transporter+B%3A+a+new+gas+channel%5BTitle%5D)+AND+%22Am+J+Physiol+Renal+Physiol%22%5BJournal%5D)">Movement of NH3 through the human urea transporter B: a new gas channel.</a> Am J Physiol Renal Physiol. 304 (12), F1447-F1457 (2013).</li>
</ol>

The authors have nothing to disclose.

这个协议描述如何选择细胞表达细胞内Na + / H +交换器在质膜而不通过定点诱变改变其一级序列。这些换热器现在可以表征。 该方法是基于细胞内的质子的细胞毒性来选择细胞系，将表达水疱的Na + / H +交换器在质膜。它是可能的，在原则上，以使用其它阳离子可怀疑被运输，例如Li +，K +或Cs +。然而，这种不确定性增加了一个?...

大部分胞内隔室显示酸性腔的pH值，这对于成熟，贩卖，回收蛋白质或激素和神经递质加载的一个关键参数。它已被证明是受液泡H + ATP酶1产生胞质溶胶和水泡性内容之间的pH梯度，耦合到水疱的ClC氯化物转运2。无论是在敲除（KO）小鼠和人患者，这些转运的重要性已经被强调在他们的基因3-6突变引起的重表型。 钠氢交换SLC9A家族的成员，也称为NHEs其Na + / H +交换剂，已被证明是关键效应物在细胞内的pH和调节细胞体积，以及在酸-碱当量的通过上皮的向量转运。除了 ​​质膜NHEs，三高度保守的Na + / H +交换剂，NHE 6,7和图9表示在反式高尔基网络和在早期内涵体7。在他们的基因突变已与安琪儿状或玮综合征8-9，以家庭为基础的自闭症10和多动症11-12。这些交换机还参与了神经退行性问题，如阿尔茨海默病的易感性13和X连锁智力低下基因连续证候14。两者合计，这些研究突出在脑发育和/或功能的这些细胞内NHEs的重要性。 这些交换机的细胞内定位防止其离子选择性精确的测量，传输方向，动力学参数，和监管。作为用于表达的细胞内区室的所有转运的情况下，这是非常困难的，以评估其生化活性，因此，以充分理解其生理作用和mechan主义的基本病理意义。基于高细胞内K +浓度时，最普遍接受的假设是，他们的工作为K +加上质子外流转运。这样的质子泄漏的存在已经被推测的，因为它可能会以维持稳定状态的pH水疱抗衡质子泵通过V-ATP酶。这种视觉本文的目的是（i）至展示出一种方法，其允许基因筛选表达这样水疱转运在它们的质膜，和（ii），以显示两个独立的方法来测量这些转运体的功能的细胞系。 三十年前，Pouysségur和弗兰基已经开创了一个，使该NHE家族15成员的分子克隆和鉴定遗传方式。这是基于对细胞内的质子作为筛选方法的毒性。第一步是获得的细胞系缺乏在任何的Na + / H +交换表示在质膜，使用这种转运的可逆性。成纤维细胞（CCL39细胞系）预装的Na +或Li +，然后置于酸性胞外培养基（pH6.5）中2小时。这导致了细胞的表达的功能性的Na + / H +交换的死亡，并逆向转运缺陷细胞（PS120细胞系）16的选择。当在碳酸氢盐的培养基培养，这些细胞是急性细胞酸化非常敏感。因此，在质膜的任何官能质子外排机制的表达将阳性选择（见17），如果这样的细胞被提交到急性细胞内acidifications。这样酸化技术可用于分离细胞系贩卖缺陷有利的WT细胞内NHEs的强制表达在细胞膜。 作为真核生物的Na + / H+交换是电中性的，他们是无法衡量由已经使用了巨大的成功来衡量通道电生理的方法。因此本手稿演示了如何通过细胞内pH值测量和锂的吸收快速动力学测量此热交换器的活性。作为基本概念是相同的，但有趣的是注意到，许多供选择部分开发的进程，也可直接用于功能测量。 有趣的是，我们已观察到，贩卖缺陷存在于使用本手稿描述的方法选择的细胞系将导致其它水泡性蛋白质在质膜的更大的表达，如水泡性钾通道TWIK1 18。这指出了朝向的一般保留缺陷机制水泡性跨膜蛋白的选择。因此，本次评选程序，它生成5月细胞构成一个有前途的工具，科学界上工作的细胞内区室的膜蛋白。以及这里介绍的测量技术可能适用于研究其他非电的转运。

<table><tbody><tr><td>Standard cell culture equipement</td><td></td><td></td><td>Used for H+ killing selection composed of many devices with different catalog numbers</td></tr><tr><td>Incubator with CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Sanyo</td><td>MCO15A</td><td></td></tr><tr><td>Incubator without CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Heraeus instrument</td><td>BB6220</td><td></td></tr><tr><td>Laminar flow hood PSM1200NF</td><td>Fisher&amp;nbsp;</td><td>52010120</td><td></td></tr><tr><td>DMEM medium</td><td>sigma</td><td>D5796</td><td></td></tr><tr><td>FBS gold</td><td>GE Healthcare</td><td>A15-151</td><td></td></tr><tr><td>Penicilin/streptomycin</td><td>PAA</td><td>P11-010</td><td></td></tr><tr><td>Trypsin 10x</td><td>PAA</td><td>L11-003</td><td></td></tr><tr><td>Atomic Absorption spectrometer with Zeeman furnace system</td><td>Thermo Scientific</td><td>ICE 3500 GFZ</td><td></td></tr><tr><td>LiCl</td><td>sigma</td><td>L4408</td><td></td></tr><tr><td>Nitric Acid</td><td>sigma</td><td>438073</td><td></td></tr><tr><td>Fluorescence videomicroscopy set</td><td>Leica</td><td></td><td>Composed of many devices with different catalog numbers</td></tr><tr><td>Inverted automated microscope</td><td>Leica</td><td>DMI6000B</td><td></td></tr><tr><td>microscope stand</td><td>leica</td><td>11888906</td><td></td></tr><tr><td></td><td></td><td>11888911</td><td></td></tr><tr><td></td><td></td><td>11505180</td><td></td></tr><tr><td></td><td></td><td>11888377</td><td></td></tr><tr><td>incident fluorescence</td><td>leica</td><td>11888901</td><td></td></tr><tr><td></td><td></td><td>11504166</td><td></td></tr><tr><td>motor bracket</td><td>leica</td><td>11888379</td><td></td></tr><tr><td></td><td></td><td>11505234</td><td></td></tr><tr><td></td><td></td><td>11521505</td><td></td></tr><tr><td></td><td></td><td>11522106</td><td></td></tr><tr><td>LED transmission light&amp;nbsp;</td><td>leica</td><td>8097321</td><td></td></tr><tr><td></td><td></td><td>8102034</td><td></td></tr><tr><td></td><td></td><td>11521580</td><td></td></tr><tr><td>motorized plate</td><td>leica</td><td>11522068</td><td></td></tr><tr><td></td><td></td><td>11531172</td><td></td></tr><tr><td></td><td></td><td>11521734</td><td></td></tr><tr><td></td><td></td><td>11521719</td><td></td></tr><tr><td></td><td></td><td>11888423</td><td></td></tr><tr><td></td><td></td><td>11888424</td><td></td></tr><tr><td>camera output</td><td>leica</td><td>11888373</td><td></td></tr><tr><td></td><td></td><td>11507807</td><td></td></tr><tr><td></td><td></td><td>11888393</td><td></td></tr><tr><td></td><td></td><td>11888259</td><td></td></tr><tr><td></td><td></td><td>11888258</td><td></td></tr><tr><td></td><td></td><td>11541510</td><td></td></tr><tr><td>images acquisition/analysis software</td><td>leica</td><td>11888375</td><td></td></tr><tr><td>optics</td><td>leica</td><td>11506507</td><td></td></tr><tr><td></td><td></td><td>11506243</td><td></td></tr><tr><td></td><td></td><td>11506203</td><td></td></tr><tr><td>fluorescence Xenon lamp</td><td>leica</td><td>DMI6000</td><td></td></tr><tr><td>camera</td><td>hamamatsu</td><td>8100601</td><td></td></tr><tr><td>metafluor/Mmfluor software</td><td></td><td>11640905</td><td></td></tr><tr><td>pH sensitive probe, BCECF-AM</td><td>life technologies</td><td>B1170</td><td></td></tr><tr><td>Nigericin</td><td>Sigma</td><td>N7143</td><td></td></tr></tbody></table>

functional characterization of na+/h+ exchangers of intracellular compartments using proton-killing selection to express them at the plasma membrane

内含体酸化是对广泛的过程，如蛋白质的回收和降解，受体脱敏，并装载神经递质在突触小泡的关键。此酸化被描述为通过质子ATP酶，其耦合到的ClC氯化物转运介导。高度保守的电中性的质子转运，所述的Na + / H +交换器（NHE）6,7和9还表示在这些隔室中。在他们的基因中的突变已与人类认知和神经变性疾病。奇怪的是，他们的角色仍然是难以捉摸的，因为他们的细胞内定位阻止详细的功能特性。这个手稿表示的方法来解决这个问题。这包括突变体的细胞系，能够通过在质膜保持细胞内NHEs幸存急性胞质酸化的选择。然后，它描绘了两个互补的协议来测量离子选择性和活性这些交换器:（i）一种基于使用荧光视频显微镜的细胞内pH值的测量，以及（ii）一种基于锂摄取的快速动力学。这样的协议可以外推到测量其他非电的转运。此外，这里介绍的选择过程生成的细胞与细胞内保留有缺陷的表型。因此，这些细胞也将表达其它水泡性膜蛋白在细胞膜。因此此处所描述的试验策略可能构成潜在有力工具，研究其它细胞内蛋白质，这将在质膜被再表达连同水疱的Na + / H +用于选择热交换器。

选择: 在H +杀选择是基于所述铵弱碱的扩散，如在图1A中所描绘。弱碱和酸对细胞内pH值扩散的影响已率先由Walter硼和合作者22。优雅的主意，使用这种现象产生一种致命的酸化积极基因筛选，然后由雅克Pouysségur17开发的。在这样的协议中，细胞内pH值降低至约5.5的漂洗步骤之后。细胞不表达功能性NHE在质膜不能恢复中性pH值，并显示具有平...

Watch this Scientific Journal Video about Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane at JoVE.c

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

娜功能分析<sup&gt; +</sup&gt; / H<sup&gt; +</sup&gt;细胞内的隔间使用质子打死选择器来表达它们的质膜

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and ...

functional-characterization-na-h-exchangers-intracellular

Research

JoVE Journal

Biology

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

9.2K 次观看.  Université Nice-Sophia Antipolis, Laboratoire de Physiomédecine Moléculaire, CNRS UMR7370, and Laboratories of Excellence Ion Channel Science and Therapeutics. The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers. 

视频: Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Expression, Solubilization, and Purification of Eukaryotic Borate Transporters

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also known as Band 3), the most abundant membrane protein in the red blood cells. The SLC4 family is homologous with borate transporters, which have been characterized in plants and fungi. It remains a significant technical challenge to express and purify membrane transport proteins to homogeneity in quantities suitable for&#160;structural or functional studies. Here we describe detailed procedures for the overexpression of borate transporters in Saccharomyces cerevisiae, isolation of yeast membranes, solubilization of protein by detergent, and purification of borate transporter homologs from S. cerevisiae, Arabidopsis thaliana, and Oryza sativa. We also detail a glutaraldehyde cross-linking experiment to assay multimerization of homomeric transporters. Our generalized procedures can be applied to all three proteins and have been optimized for efficacy. Many of the strategies developed here can be utilized for the study of other challenging membrane proteins.

Here we present a protocol to express, solubilize, and purify several eukaryotic borate transporters with homology to the SLC4 transporter family using yeast. We also describe a chemical cross-linking assay to assess the purified homomeric proteins for multimeric assembly. These protocols can be adapted for other challenging membrane proteins.

真核硼酸背液转运体的表达、增溶及纯化

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also ...

科研

生物化学

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine exchangers in plants have not been identified. Here, we outline a method to characterize polyamine antiporters using membrane vesicles generated from the lysis of Escherichia coli cells heterologously expressing a plant antiporter. First, we heterologously expressed AtBAT1 in an E. coli strain deficient in polyamine and arginine exchange transporters. Vesicles were produced using a French press, purified by ultracentrifugation and utilized in a membrane filtration assay of labeled substrates to demonstrate the substrate specificity of the transporter. These assays demonstrated that AtBAT1 is a proton-mediated transporter of arginine, &#947;-aminobutyric acid (GABA), putrescine and spermidine. The mutant strain that was developed for the assay of AtBAT1 may be useful for the functional analysis of other families of plant and animal polyamine exchangers. We also hypothesize that this approach can be used to characterize many other types of antiporters, as long as these proteins can be expressed in the bacterial cell membrane. E. coli is a good system for the characterization of novel transporters, since there are multiple methods that can be employed to mutagenize native transporters.

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

We describe a method for the characterization of proton-driven membrane transporters in membrane vesicle preparations produced by heterologous expression in E. coli and lysis of cells using a French press.

大肠杆菌中异构表达对膜迁移者的特性与膜囊泡的产生

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine ...

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.

This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as F&#246;rster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid.

实时测量核苷酸与完整功能性膜蛋白的结合

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

Selection of Transporter-Targeted Inhibitory Nanobodies by Solid-Supported-Membrane (SSM)-Based Electrophysiology

Single domain antibodies (nanobodies) have been extensively used in mechanistic and structural studies of proteins and they pose an enormous potential as tools for developing clinical therapies, many of which depend on the inhibition of membrane proteins such as transporters. However, most of the methods used to determine the inhibition of transport activity are difficult to perform in high-throughput routines and depend on labeled substrates availability thereby complicating the screening of large nanobody libraries. Solid-supported membrane (SSM) electrophysiology is a high-throughput method, used for characterizing electrogenic transporters and measuring their transport kinetics and inhibition. Here we show the implementation of SSM-based electrophysiology to select inhibitory and non-inhibitory nanobodies targeting an electrogenic secondary transporter and to calculate nanobodies inhibitory constants. This technique may be especially useful for selecting inhibitory nanobodies targeting transporters for which labeled substrates are not available.

Selection of Transporter-Targeted Inhibitory Nanobodies by Solid-Supported-Membrane SSM-Based Electrophysiology

Nanobodies are important tools in structural biology and pose a great potential for the development of therapies. However, the selection of nanobodies with inhibitory properties can be challenging. Here we demonstrate the use of solid-supported-membrane (SSM)-based electrophysiology for the classification of inhibitory and non-inhibitory nanobodies targeting electrogenic membrane transporters.

通过基于固体支持膜（SSM）的电生理学选择转运蛋白靶向抑制纳米抗体

Single domain antibodies (nanobodies) have been extensively used in mechanistic and structural studies of proteins and they pose an enormous potential as ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

生物学

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+,K+-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+ or Li+ transport by Na+,K+- or gastric H+,K+-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb+ (Li+) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na+/2K+ transport stoichiometry of the Na+,K+-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+,K+-ATPase. In principle, the assay is not limited to K+-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

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

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

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

重组脂膜的结构和功能研究的钾离子通道

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl- channels or transporters is described. The assay is based on the measure of the efflux rate of Cl- from proteoliposomes following the addition of the K+ ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H+/Cl- exchanger, as a model system. The efflux assay can be utilized to study any Cl- conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

一个脂蛋白体为基础的外排检测，以确定氯的单分子性质<sup&gt; -</sup&gt;通道和转运

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as ...

Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable efforts have been made in this field, but techniques enabling automated high-throughput transport analysis in combination with solvent-free lipid bilayer techniques required for the analysis of membrane transporters are rare. This class of transporters however is crucial in cell homeostasis and therefore a key target in drug development and methodologies to gain new insights desperately needed.
The here presented manuscript describes the establishment and handling of a novel biochip for the analysis of membrane protein mediated transport processes at single transporter resolution. The biochip is composed of microcavities enclosed by nanopores that is highly parallel in its design and can be produced in industrial grade and quantity. Protein-harboring liposomes can directly be applied to the chip surface forming self-assembled pore-spanning lipid bilayers using SSM-techniques (solid supported lipid membranes). Pore-spanning parts of the membrane are freestanding, providing the interface for substrate translocation into or out of the cavity space, which can be followed by multi-spectral fluorescent readout in real-time. The establishment of standard operating procedures (SOPs) allows the straightforward establishment of protein-harboring lipid bilayers on the chip surface of virtually every membrane protein that can be reconstituted functionally. The sole prerequisite is the establishment of a fluorescent read-out system for non-electrogenic transport substrates.
High-content screening applications are accomplishable by the use of automated inverted fluorescent microscopes recording multiple chips in parallel. Large data sets can be analyzed using the freely available custom-designed analysis software. Three-color multi spectral fluorescent read-out furthermore allows for unbiased data discrimination into different event classes, eliminating false positive results.
The chip technology is currently based on SiO2 surfaces, but further functionalization using gold-coated chip surfaces is also possible.

The presented protocol describes the analysis of membrane protein mediated transport on the single transporter level using pore-spanning solvent-free lipid bilayers. This is achieved by the creation of bulk produced nanopore array chips, combined with highly parallel data acquisition and analysis, enabling the future establishment of membrane protein effector screenings.

膜运输过程由单一的蛋白质解析一个高度并行的纳米孔芯片分析系统

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable ...

Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. These transport mechanisms are mediated by the activities of specialized transporter proteins. Specifically,pH dynamics are finely controlled by plasma membrane proton (H+) extrusion systems, such as the Na+/H+ exchanger (NHE) protein family. Despite extensive efforts to study the mechanisms underlying NHE regulation, our current understanding of the biophysical and molecular properties of the NHE family is inadequate because of the limited availability of methods to effectively measure NHE activity. In this manuscript, we used H+-selective electrodes during whole-cell patch clamping recording to measure NHE-induced H+ flux. We proposed this approach to overcome some limitations of typically used methods to measure NHE activity, such as radioactive uptake and fluorescent membrane permeants. Measurement of NHE activity using the described method enables high sensitivity and time resolution and more efficient control of intracellular H+ concentrations. H+-selective electrodes are based on the fact that transporter activity creates an ion gradient in close proximity to the cell membrane. An H+-selective electrode moving up to and away from the cell membrane in a repetitive, oscillatory fashion records a voltage difference that is dependent on H+ flux. While H+-selective electrodes are used to detect H+ flux moving out of the cell, the patch clamp method in the whole-cell configuration is used to control the intracellular ion composition. Moreover, application of the giant patch clamp technique allows modification of the intracellular composition of not only ions but also lipids. The transporter activity of NHE isoform 3 (NHE3) was measured using this technical approach to study the molecular basis of NHE3 regulation by phosphoinositides.

This manuscript describes the applications of proton-selective electrodes and patch clamping methods to measure the activity of proton transport systems. These methods overcome some limitations of techniques commonly used to study proton transport activity, such as moderate sensitivity, time resolution and insufficient intracellular milieu control.

电生理测量在中性转运体活性研究中的应用

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. ...

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