This work was supported by NIH grant GM085232 and an Irma T. Hirschl/ Monique Weill-Caulier Scholar Award (to A.A.).

1.脂质制备<ol><li>分装所需量的脂质成透明玻璃试管中。利用E.大肠杆菌极性脂质提取物，但大部分脂质组合物都可以使用。如果脂质是粉末形式，悬浮它们的氯仿至20毫克/毫升的浓度。干燥的脂质在室温下N 2气，直到所有溶剂蒸发。 </li><li>悬浮在戊烷的脂类，再晾干气体N 2源源不断除去氯仿遗留的痕迹。干，直到所有溶剂蒸发。而干燥，轻轻旋转管，使得所述脂质分布在均匀的膜覆盖所述管的底部第三而非形成致密质在底部。 </li><li>添加含洗涤剂重建缓冲液（300mM的氯化钾，50mM的柠檬酸，25mM的K 2 HPO 4，pH为4.5，35毫CHAPS）溶解脂质。使用其他温和的清洁剂进行这一步，如果蛋白质是很差容忍CHAPS。 </li><li> ResuspenD使用水浴超声波仪中的脂质对清晰度。浸入管在水中超声发生器浴中的最大功率的中心在短（30秒-1分钟）脉冲短（30秒-1分钟）暂停。该解决方案应该变得清晰。 注：清晰的最终程度实现取决于所使用的特定的脂质组合物。一些批与批变异在该步骤中，甚至在名义上相同脂质，也是可能的。残留雾度是由于光散射的大颗粒在悬浮液中，这可通过离心除去。 </li><li>在室温下孵育的悬浮脂类为20-60分钟。 </li></ol> 2.脂蛋白体的形成注：几种策略可以用来插入洗涤剂增溶的蛋白质到脂质体中。对于CLC-EC1透析效果很好，因此是选择6,9,10的方法。 <ol><li>添加纯化的蛋白质的期望的蛋白质与脂质（P / L）的比值（表示为微克Ò<html> F蛋白/毫克脂质）。的P / L的比例的选择取决于实验的目的。 <ol><li>如果目标是评估所关注的蛋白质的活性，重组在高的P / L的最大化信号，因为每个脂质体含有的蛋白质的多个副本。量化的活性和/或蛋白的单一传输速率，重建在泊松稀释制度低的P / L的，以使每个小泡平均含有1蛋白质。见第7步和讨论进行了较深入的分析时，使用每个政权。 </li></ol></li><li>来确定用于各种治疗方案的最佳P / L的值，执行活动的完整滴定作为蛋白质浓度8,11-13的函数。然而，对于激活单元中的任何蛋白质的量，分子量（MW，以kDa表示）被称为后的P / L的值可以被用作初始瞎猜： 皮克“/&gt; <img alt="公式2" fo:content-width="1.5in" src="/files/ftp_upload/52369/52369eq2.jpg" /></li><li>在预润湿的透析管或盒加载的蛋白质和脂质的混合物。将透析设备在含有500-1,000x脂质重建缓冲液的体积的烧杯中（ 即 ，进行1 ml类脂再悬浮的1升缓冲液）下缓慢搅拌。 <ol><li>改变缓冲液的3-4倍的间隔&gt; 3小时。在每一个解决方案的变化，洗磁带/管和用蒸馏水的烧杯中，注入新鲜的重建缓冲液的烧杯中。包括1-2 O / N区间的解决方案转变。确切的数目的溶液的变化和持续时间取决于所用的确切的脂质和去污剂。 </li></ol></li><li>在最后一步完成后，从透析器去除脂质体，分装在其中管和闪光灯在-80℃冻结，在液体N 2或冻结。 </li></ol> 3.记录的建立 &lt;P类=“jove_content”&gt;注意：记录设置（ 图2A）包括两个腔室（平底缸，〜3-4毫升容积）中，Cl -的（见下文）的电极，pH计用模拟或数字电输出，数字转换器，和一个计算机用适当的采集软件。 <ol><li>连接Cl -的电极pH计，pH计，以被连接到计算机的数字化板的输出。放置在一个腔室中的参考电极和重新编码导线中的另一个（ 图2B）。 注：导线的极性是正确的，如果ΔV 校准 （见步骤6.4和7.2）是正的。 </li><li>放置室上的轰动板录音室（ 图1B）的搅拌棒。确保搅拌棒不接触电极。 </li><li>连接使用琼脂桥（ 图2B）（100毫米氯化钾和2％琼脂糖）的腔室。存储在100毫米氯化钾琼脂桥梁。 </LI&gt; </ol> 4.制备的Cl -电极<ol><li>以一个老-possibly-非正常pH电极。打破玻璃涂层完全显示出来的银线。 </li><li>小心地从使用手术刀刀片的银线去除涂层。清洗用H 2 O和乙醇的丰富金额的电线。 </li><li>发生的FeCl 3（或漂白剂），直到导线的饱和溶液覆盖有均匀的深色涂层氯化银的。 </li></ol> 5.准备单层囊中<ol><li>夜的实验之前，膨胀将1g的Sephadex G-50珠在15ml外部缓冲液（1mM的氯化钾，150mM的K 2 SO 4，25mM的柠檬酸，pH4.5）中，轻轻摇动（不搅拌），用于至少3个小时，在室温后再使用。每个实验需要〜3个毫升肿珠。保持溶胀的珠粒于4℃几天至多。 </li><li>而脂质体解冻在室温，倒在每列〜3米升肿G-50珠。让他们干通过在RT重力流;这通常需要1-2分钟。 </li><li>通过使用0.4米聚四氟乙烯截止通过Mini-挤出机挤出的脂蛋白11次准备单层囊。 </li><li>放置在一个塑料圆底管色谱柱。旋柱以除去过量的溶液。在临床离心机离心20-30秒在1400 XG。 </li><li>丢弃流过，地点列在一个13×100毫米的玻璃管中，并添加100微升挤出囊泡到柱上。在临床离心机离心柱1分钟，在500×g离心。 </li><li>收集〜200微升流动通过的，将被添加到记录室，在步骤6.5。 </li></ol> 6.流出测量<ol><li>放置加入2ml的100mM氯化钾在基准（接地）室和1.8毫升外部缓冲液（1mM的氯化钾，150mM的K 2 SO 4，25mM的柠檬酸，pH4.5）中，以记录室中。内部的细微渗透压失衡和外部的解决方案不影响</li><li>启动收购程序。让基本平衡，这可能需要几分钟的时间。 </li><li>一旦信号达到稳定的基线（脂质体是准备和列干），开始记录（ 图3A）。 </li><li>加入15微升10mM的KCl溶液校准系统（ 图2A）。 </li><li>添加脂质体。阿细跳可能是可见的，由于不完全拆除外的Cl -从脂蛋白（ 图3A）。等到基线稳定。 </li><li>加入缬氨霉素（1微升以1mg / ml，在EtOH中）来启动流出（ 图3A）。 </li><li>让流出的运行时间过程，直到它高原（ 图3A）。 </li><li>加入40微升在外部缓冲器以溶解所有的脂质体（ 图3A）中制备1.5M的β-辛基葡糖苷（β-OG）。结束录制。 </li></ol> 7.数据nalysis <ol><li>从ASCII或文本格式导出跟踪文件，并将其导入到所选择的分析程序。 </li><li>测量的电压在以下各点（ 图3A）： 在记录开始时（V 0）; 此外校准脉冲（V CAL）之后; 在加入脂质体（V 脂 ）的; 在流出（Ⅴ 翅片 ）的端部; 在加入洗衣粉（V TOT）的; 基线的电压使V 0 = 0。 </li><li>使用能斯特 - 普朗克公式确定的实验值<img alt="公式3" fo:content-width=".3in" src="/files/ftp_upload/52369/52369eq3.jpg" />通过测量 <img alt="公式4" fo:content-width="3in" src="/files/ftp_upload/52369/52369eq4.jpg" /> 其中，卷校准是在校准脉冲在微升的体积，1800是腔室容积在开始的实验中表达1，L，[CL] 卡/中是氯-在毫米外部缓冲器的校准脉冲和浓度ΔVCAL的校准脉冲后测得的跳毫伏。 注：此经验确定α的有三个目的：它保证了系统正常响应，提供实验之间的仪器的响应的一致性的措施，以及检查是否有错误是在溶液中做出决策。 </li><li>每一步之后计算氯浓度如下： <img alt="公式5" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq5.jpg" /> <img alt="方程6" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq6.jpg" /> <img alt="公式7" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq7.jpg" /> 该因子四百分之三来自稀释15微升校准脉冲的进入2000微升最终体积。 </li><li>演算吃在每一步，ΔCL 脂/鳍/ TOT的变化[氯]。 </li><li>转换V（T）的氯（T）与 <img alt="方程8" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq8.jpg" /> 在临界点直接决定（CL）值，并将其进行比较，以计算出的值作为内部对照。 </li><li>标准化的跟踪，这样的氯相对 脂 = 0和Cl 相对 托特 = 1 <img alt="公式9" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq9.jpg" /></li><li>适合的流出时间过程，以下面的等式： <img alt="10式" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq10.jpg" /> 其中L是Cl的速率-漏是通过实验，通过测量确定的氯-从在相同条件下，τ制备无蛋白质的脂质体流出是过程的时间常数。 </li><li>计算V，初始速度： 其中，ΔCL 翅片表达于毫摩尔，V是在微升室的容积。 </li><li>计算运输率/电导率 <img alt="12式" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq12.jpg" /> 其中，p是在P / L时，MW为活性复合物的分子量以kDa表示</li></ol>

<ol>
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The authors declare no competing financial interests.

我们已经描述了详细的协议来测量Cl -的运输由纯化的阴离子选择性通道或转运蛋白重构于脂质体介导的。所使用的例子是原核H + / Cl -的换热器CLC-EC1。然而，该方法可以容易地适合于研究通过配体12,13,15，11,12的电压，或者通过更换银运动不同的阴离子选择性15,16门控通道：与一种适合于所考虑的离子氯化银电极。电极选择性离子比其它氯- ，如H <su...

在过去的二十年中，以过表达和纯化膜转运蛋白的能力显着增加：离子通道，初级和次级转运目前一般均从异源表达系统，以及天然来源纯化。新的方法来监测表达，改善和促进提取和提高这些蛋白质的稳定性正在不断开发1-5。这些技术的突破已经有助于触发的原子级的结构信息上的膜蛋白，这反过来，提高了我们自己的功能的结构基础的理解的爆炸。相比之下，我们的能力来探测纯化的蛋白质的功能性质没有增加以同样的速度，这样，在某些情况下，高分辨率的结构信息是伴随着定性功能数据，从而限制了我们的定量测试结构为基础的预测能力。因此，发展定量和可普及功能测定t是朝着膜蛋白功能的机械基础的澄清的关键步骤。 在这里，我们描述了可用于定量测定纯化和重组的Cl键的功能性质外排测定-通道和转运。该测定法的基本原则可以推广到各种运输系统，以及非离子转运蛋白。脂质体重组，纯化的Cl -的信道/转运在一个大的氯的存在下-梯度（ 图1A，B）。 Cl -的流出是通过加入一种离子载体的启动，以允许对离子通量，在我们的情况下，第K +离子载体缬氨霉素，其分流由氯建立的电压-梯度和设置初始膜电位至K的平衡电位+ 6,7。没有T他离子载体没有显著净Cl -的流出发生的，因为它防止了由一个跨膜电位的产生。 -流出，并且f 0，不含有活性蛋白的脂质体的分数τ，C1的时间常数：所述数据是由两个可测量参数（ 图1C）定量描述。从τ和f 0酉Cl -的传输速率，活性蛋白质的级分和活性复合体的分子质量可以衍生8。已知结构和功能的热交换器-该技术是利用脂蛋白重构与CLC-EC1，一个良好表征CLC型H + /氯这里示出。该测定是很容易推广到通道或转运与不同的离子选择性或其活性依赖于电压和/或配体的存在。另外，该测定可用于确定是否小分子直接影响重组蛋白，以定量这些化合物和如何膜组合物或脂质修饰试剂影响重构的通道和转运功能的影响。

<table border="0" cellpadding="0" cellspacing="0" width="995">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Name of Reagent/ Equipment</td>
			<td style="width:337px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liposomicator, Avanti Polar Lipids Inc.</td>
			<td style="width:337px;">&#160;Avanti Polar Lipids Inc.</td>
			<td>610200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IEC Centra CL2 Benchtop</td>
			<td style="width:337px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Orion Research Model 701A Digital pH-mV meter</td>
			<td style="width:337px;"></td>
			<td style="width:119px;"></td>
			<td>These can be found on Ebay.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Non-functional pH probe</td>
			<td style="width:337px;"></td>
			<td style="width:119px;"></td>
			<td>Any pH meter probe with silver wires will work. The glass/plastic coating needs to be removed and the wires cleaned.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160; DI-710 Data Logger</td>
			<td style="width:337px;">DATAQ instruments</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">WinDAQ acquisition software</td>
			<td style="width:337px;">DATAQ instruments</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pierce Disposable Plastic Columns, Gravity-flow, 2ml</td>
			<td style="width:337px;">Pierce (Thermo Scientific)</td>
			<td>29922</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KIMAX Culture Tubes, Disposable, Borosilicate Glass</td>
			<td style="width:337px;">Kimble Chase</td>
			<td>73500-13100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Extruder Set With Holder/Heating Block</td>
			<td style="width:337px;">Avanti Polar Lipids Inc.</td>
			<td align="right" style="width:119px;">610000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Computer</td>
			<td style="width:337px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

我们描述了一个详细而强大的协议来衡量Cl -的运输由纯化CLC-EC1，原核CLC-H型+ /氯介导-交换，重组的脂质体。该实验的示意图示于图3脂蛋白体重组，纯化的CLC-EC1和含有高内氯-浸渍在含低氯浴溶液- 。在这些条件下的净Cl -的流出防止通过在脂质体（ 图3A）正电荷的累积。此外缬氨霉素允许K +而穿过膜从而分流的电?...

Watch this Scientific Journal Video about A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters at JoVE.com

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 ...

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

9.7K 次观看.  Weill Cornell Medical College. 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.