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 ...

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><tbody><tr><td>Liposomicator, Avanti Polar Lipids Inc.</td><td>&amp;nbsp;Avanti Polar Lipids Inc.</td><td>610200</td><td></td></tr><tr><td>IEC Centra CL2 Benchtop</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Orion Research Model 701A Digital pH-mV meter</td><td></td><td></td><td>These can be found on Ebay.</td></tr><tr><td>Non-functional pH probe</td><td></td><td></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><td>DI-710 Data Logger</td><td>DATAQ instruments</td><td></td><td></td></tr><tr><td>WinDAQ acquisition software</td><td>DATAQ instruments</td><td></td><td></td></tr><tr><td>Pierce Disposable Plastic Columns, Gravity-flow, 2ml</td><td>Pierce (Thermo Scientific)</td><td>29922</td><td></td></tr><tr><td>KIMAX Culture Tubes, Disposable, Borosilicate Glass</td><td>Kimble Chase</td><td>73500-13100</td><td></td></tr><tr><td>Extruder Set With Holder/Heating Block</td><td>Avanti Polar Lipids Inc.</td><td>610000</td><td></td></tr><tr><td>Computer</td><td></td><td></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 ...

a-proteoliposome-based-efflux-assay-to-determine-single-molecule

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

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

9.8K 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.

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

Cholesterol Efflux Assay

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of cellular membranes, apoptosis and necrosis 1. Cells can source cholesterol from intracellular synthesis and from plasma lipoproteins, both sources are sufficient to fully satisfy cells' requirements for cholesterol. The processes of cholesterol synthesis and uptake are tightly regulated and deficiencies of cholesterol are rare 2. Excessive cholesterol is more common problem 3. With the exception of hepatocytes and to some degree adrenocortical cells, cells are unable to degrade cholesterol. Cells have two options to reduce their cholesterol content: to convert cholesterol into cholesteryl esters, an option with limited capacity as overloading cells with cholesteryl esters is also toxic, and cholesterol efflux, an option with potentially unlimited capacity. Cholesterol efflux is a specific process that is regulated by a number of intracellular transporters, such as ATP binding cassette transporter proteins A1 (ABCA1) and G1 (ABCG1) and scavenger receptor type B1. The natural acceptor of cholesterol in plasma is high density lipoprotein (HDL) and apolipoprotein A-I. 
The cholesterol efflux assay is designed to quantitate the rate of cholesterol efflux from cultured cells. It measures the capacity of cells to maintain cholesterol efflux and/or the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of the following steps. Step 1: labelling cellular cholesterol by adding labelled cholesterol to serum-containing medium and incubating with cells for 24-48 h. This step may be combined with loading of cells with cholesterol. Step 2: incubation of cells in serum-free medium to equilibrate labelled cholesterol among all intracellular cholesterol pools. This stage may be combined with activation of cellular cholesterol transporters. Step 3: incubation of cells with extracellular acceptor and quantitation of movement of labelled cholesterol from cells to the acceptor. If cholesterol precursors were used to label newly synthesized cholesterol, a fourth step, purification of cholesterol, may be required.
The assay delivers the following information: (i) how a particular treatment (a mutation, a knock-down, an overexpression or a treatment) affects the capacity of cell to efflux cholesterol and (ii) how the capacity of plasma acceptors to accept cholesterol is affected by a disease or a treatment. This method is often used in context of cardiovascular research, metabolic and neurodegenerative disorders, infectious and reproductive diseases.

The cholesterol assay is designed to quantitate the rate of cholesterol efflux from cultured cells and the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of labelling cells with cholesterol, equilibration of cholesterol among intracellular pools and release of cholesterol to an extracellular acceptor.

胆固醇外流检测

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of ...

科研

医学

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200&#160;nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

模型和方法来评价药物输送系统在整个细胞屏障的运输

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

生物工程

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used for ATP production. The amphiphilic nature of membrane proteins requires particular attention to their handling, and reconstitution into the natural lipid environment is indispensable when studying membrane transport processes like proton translocation. Here, we detail a method that has been used for the investigation of the proton-pumping mechanism of membrane redox enzymes, taking cytochrome bo3 from Escherichia coli as an example. A combination of electrochemistry and fluorescence microscopy is used to control the redox state of the quinone pool and monitor pH changes in the lumen. Due to the spatial resolution of fluorescent microscopy, hundreds of liposomes can be measured simultaneously while the enzyme content can be scaled down to a single enzyme or transporter per liposome. The respective single enzyme analysis can reveal patterns in the enzyme functional dynamics that might be otherwise hidden by the behavior of the whole population. We include a description of a script for automated image analysis.

Here, we present a protocol to study the molecular mechanism of proton translocation across lipid membranes of single liposomes, using cytochrome bo3 as an example. Combining electrochemistry and fluorescence microscopy, pH changes in the lumen of single vesicles, containing single or multiple enzyme, can be detected and analyzed individually.

电化学和荧光显微镜在质子泵膜酶研究中的单脂质体测定

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used ...

生物化学

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 ...

Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides an indirect way for amphiphiles to modulate protein function and a possible mechanism for "off-target" drug effects. We have previously developed an electrophysiological assay for detecting changes in lipid bilayer properties using linear gramicidin channels as probes 3,12. Gramicidin channels are mini-proteins formed by the transbilayer dimerization of two non-conducting subunits. They are sensitive to changes in their membrane environment, which makes them powerful probes for monitoring changes in lipid bilayer properties as sensed by bilayer spanning proteins. We now demonstrate a fluorescence assay for detecting changes in bilayer properties using the same channels as probes. The assay is based on measuring the time-course of fluorescence quenching from fluorophore-loaded large unilamellar vesicles due to the entry of a quencher through the gramicidin channels. We use the fluorescence indicator/quencher pair 8-aminonaphthalene-1,3,6-trisulfonate (ANTS)/Tl+ that has been successfully used in other fluorescence quenching assays 5,13. Tl+ permeates the lipid bilayer slowly 8 but passes readily through conducting gramicidin channels 1,14. The method is scalable and suitable for both mechanistic studies and high-throughput screening of small molecules for bilayer-perturbing, and potential "off-target", effects. We find that results using this method are in good agreement with previous electrophysiological results 12.

We introduce a fast fluorescence-based assay that monitors the rate of fluorescence quenching as a measure of gramicidin channel activity. The gramicidin channels are used as molecular force transducers to monitor changes in lipid bilayer properties as sensed by bilayer spanning proteins.

短杆菌肽为基础的荧光分析法，确定小分子的修改脂质双层属性的潜在

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter ...

生物学

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes all liposomes of the ensemble to be identical. However, new experimental platforms able to observe liposomes at the single-particle level have made it possible to perform highly sophisticated and quantitative studies on protein-membrane interactions or drug carrier properties on individual liposomes, thus avoiding errors from ensemble averaging. Here we present a protocol for preparing, detecting, and analyzing single liposomes using a fluorescence-based microscopy assay, facilitating such single-particle measurements. The setup allows for imaging individual liposomes in a massive parallel manner and is employed to reveal intra-sample size and compositional inhomogeneities. Additionally, the protocol describes the advantages of studying liposomes at the single liposome level, the limitations of the assay, and the important features to be considered when modifying it to study other research questions.

This protocol describes the fabrication of liposomes and how these can be immobilized on a surface and imaged individually in a massive parallel manner using fluorescence microscopy. This allows for the quantification of the size and compositional inhomogeneity between single liposomes of the population.

定量荧光显微镜为基础的单脂体测定，用于检测单个脂质体之间的成分不均匀性

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

内向整流钾通道的小分子调节剂的高通量筛选

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa

There is an emerging scientific need for reliable tools for monitoring membrane protein transport. We present a methodology leading to the reconstitution of efflux pumps from the Gram-negative bacteria Pseudomonas aeruginosa in a biomimetic environment that allows for an accurate investigation of their activity of transport. Three prerequisites are fulfilled: compartmentation in a lipidic environment, use of a relevant index for transport, and generation of a proton gradient. The membrane protein transporter is reconstituted into liposomes together with bacteriorhodopsin, a light-activated proton pump that generates a proton gradient that is robust as well as reversible and tunable. The activity of the protein is deduced from the pH variations occurring within the liposome, using pyranin, a pH-dependent fluorescent probe. We describe a step-by-step procedure where membrane protein purification, liposome formation, protein reconstitution, and transport analysis are addressed. Although they were specifically designed for an RND transporter, the described methods could potentially be adapted for use with any other membrane protein transporter energized by a proton gradient.

In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa

We present a protocol for the in vitro investigation of efflux pumps from Pseudomonas aeruginosa. This protocol allows for the generation of a robust, reversible, and tunable proton gradient within the liposome membrane and hence should be adaptable to any membrane protein energized by the protomotive force.

在 MexAB外排泵从铜绿假单胞菌的体外研究

There is an emerging scientific need for reliable tools for monitoring membrane protein transport. We present a methodology leading to the reconstitution ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

纯化野生型和突变CFTR蛋白的功能重建和通道活性测定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

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 ...

一个脂蛋白体为基础的外排检测，以确定氯的单分子性质

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