娜功能分析<sup> +</sup> / H<sup> +</sup>细胞内的隔间使用质子打死选择器来表达它们的质膜
Summary
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.
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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 Pouysségur 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.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Standard cell culture equipement | Used for H+ killing selection composed of many devices with different catalog numbers | ||
| Incubator with CO2 | Sanyo | MCO15A | |
| Incubator without CO2 | Heraeus instrument | BB6220 | |
| Laminar flow hood PSM1200NF | Fisher | 52010120 | |
| DMEM medium | sigma | D5796 | |
| FBS gold | GE Healthcare | A15-151 | |
| Penicilin/streptomycin | PAA | P11-010 | |
| Trypsin 10X | PAA | L11-003 | |
| Atomic Absorption spectrometer with Zeeman furnace system | Thermo Scientific | ICE 3500 GFZ | |
| LiCl | sigma | L4408 | |
| Nitric Acid | sigma | 438073 | |
| Fluorescence videomicroscopy set | Leica | Composed of many devices with different catalog numbers | |
| Inverted automated microscope | Leica | DMI6000B | |
| microscope stand | leica | 11888906 | |
| 11888911 | |||
| 11505180 | |||
| 11888377 | |||
| incident fluorescence | leica | 11888901 | |
| 11504166 | |||
| motor bracket | leica | 11888379 | |
| 11505234 | |||
| 11521505 | |||
| 11522106 | |||
| LED transmission light | leica | 8097321 | |
| 8102034 | |||
| 11521580 | |||
| motorized plate | leica | 11522068 | |
| 11531172 | |||
| 11521734 | |||
| 11521719 | |||
| 11888423 | |||
| 11888424 | |||
| camera output | leica | 11888373 | |
| 11507807 | |||
| 11888393 | |||
| 11888259 | |||
| 11888258 | |||
| 11541510 | |||
| images acquisition/analysis software | leica | 11888375 | |
| optics | leica | 11506507 | |
| 11506243 | |||
| 11506203 | |||
| fluorescence Xenon lamp | leica | DMI6000 | |
| camera | hamamatsu | 8100601 | |
| metafluor/Mmfluor software | 11640905 | ||
| pH sensitive probe, BCECF-AM | life technologies | B1170 | |
| Nigericin | Sigma | N7143 |
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