We thank Susanne Fenz for discussing the possibility of reconstituting proteins by agarose swelling, Feng Ching Tsai for current measurements, and present and former members of the Bassereau group for support and assistance. The project was funded by the Agence Nationale de la Recherche (grant BLAN-0057-01), by the European Commission (NoE SoftComp), by the Universit&#233; Pierre et Marie Curie (grant from the FED21, Dynamique des Syst&#232;mes Complexes). M.G. was supported by an Institut Curie International PhD Fellowship, S.A. by a fellowship from the Fondation pour la Recherche M&#233;dicale, G.E.S.T. by a Marie Curie Incoming International Fellowship from the European Commission and a grant from the Universit&#233; Pierre et Marie Curie. The publication fees were covered by the Labex &#8216;CelTisPhyBio&#8217; (ANR-11-LABX0038).

1.溶液制备<ol><li>制备5ml含5mM的氯化钾，1mM的HEPES（pH 7.4）中或TRIS（pH为7.5），和2mM海藻糖&#39;的SUV缓冲器“的。过滤用0.2微米的针筒式过滤器的缓冲器和分成可存储在-20℃1ml等分。 注：其他详细信息的试剂和仪器的材料清单给出。 </li><li>准备40毫升GUV“成长缓冲”，这将填补室内GUV在电影补液。对于一个“低盐”的增长，结合的5mM的KCl，1mM的HEPES（pH 7.4）中或1mM的TRIS（pH为7.5）和〜400毫蔗糖。对于一个“生理食盐&#39;成长，结合的100mM氯化钾，5mM的HEPES（pH 7.4）中或5毫摩尔Tris（pH7.5）中，并〜200mM的蔗糖。 </li><li>通过组合的100mM氯化钾，5mM的HEPES（pH 7.4）中或5毫摩尔Tris（pH7.5）中，并〜200mM的葡萄糖制备40毫升&#39;观察缓冲器&#39;为在实验室中的外部溶液。 注意：这些缓冲区只EXAmples。见讨论，以适应缓冲区的其他实验。 </li><li>测量生长并观察缓冲器用渗透压计的渗透压摩尔浓度。添加蔗糖或葡萄糖的颗粒将它们匹配到1％以内，这样GUVs不会溶解或从生长室传送到观察室时塌陷。 注：在此浓度范围内，加入蔗糖1毫米（每40毫升13.7毫克）或葡萄糖（每40毫升7.2毫克）由〜1毫渗量增加渗透压。 </li><li>过滤生长和观测缓冲器用0.2微米的过滤器，并将其存储在4℃，以抑制细菌生长。 </li><li>溶解50毫克β-酪蛋白的10毫升的20mM TRIS（pH为7.5）缓冲液，以形成钝化的试验室的表面，使得GUVs不沾，散布和突发需要5毫克/毫升β-酪蛋白溶液。一旦β-酪蛋白完全溶解（最多几个小时，在4℃），过滤器（0.2微米）成0.5毫升等份可快速冷冻并储存在-20℃，供以后使用（储存在4℃下解冻等分试样通常可用于多达1周）。 </li></ol> 2. SUV准备<ol><li>准备并冻结继先前公布详细的协议28 PROTEO-的SUV等分。使用KvAP荧光在蛋白质标记与Alexa-488马来酰亚胺，还原成DPhPC越野车（10毫克/毫升）至1:10（质量比）的脂质比率。 注：野生型KvAP包含每个单体靠近细胞内C端（氨基酸247）一个半胱氨酸。 </li><li>荧光，脂质只有越野车： 注：在通风橱戴丁腈手套和防护眼镜处理氯仿。避免使用任何塑料如氯仿能够溶解它们。氯仿溶液可以储存在琥珀色玻璃小瓶用聚四氟乙烯帽和使用玻璃注射器转移。照顾之前和吹打脂质后，以冲洗所有玻璃器皿至少5至10倍，用氯仿。 <ol><li>制备100微升10含有0.5％（摩尔）的红色荧光脂质，德克萨斯红-DHPE中，通过将100微升DPhPC溶液（10毫克/毫升的氯仿溶液）用8.2微升德克萨斯红 - DHPE溶液（1毫克/毫升毫克/毫升DPhPC越野车在甲醇中）在1.5ml琥珀色玻璃管形瓶中。 </li><li>在化学罩干燥脂类向下在氮气流中，同时旋转小瓶。当膜看来是干燥，放置脂质的真空下3小时，以除去任何残余的溶剂。 </li><li>加入100微升的SUV缓冲器向脂质和涡大力直到没有脂质仍粘在小瓶的壁，该溶液是均匀的乳白色。 </li><li>超声处理脂质溶液中以形成越野车。调整小瓶的位置，直到超声波使大多数流动和流动瓶内，并注意不要进行不必要的加热解决方案。继续超声处理直到溶液变为半透明，或可能时，透明的（2-5分钟为尖超声处理，〜20分钟，浴超声处理）。 </li><li> Aliquo吨的SUV（ 例如，10微升或20微升），并冷冻（-20℃）以备后用。 注：血脂，特别是不饱和脂肪，可以轻松击穿。在20℃（或80℃）在氩气下商店脂质溶液和6个月内使用。脂质分解产物可与薄层色谱法来检测。 </li></ol></li></ol> 3. GUV生长的Electroformation <ol><li>准备electroformation室。 <ol><li>如果室内没有被清理，删除Windows，擦去所有密封胶和油脂，提取的电线，冲洗和擦洗使用水和乙醇（≥70％）交替组织室。 </li><li>擦电线井，淹没电线和腔室中的丙酮，和超声处理5分钟。消灭一切用纸巾再次使用丙酮。放入乙醇和超声腔室5分钟。 </li><li>组装室通过孔插入线，然后旋转擦拭线，以确保他们的ARË干净。放腔室在蒸馏水，超声处理5分钟，并用氮气干燥或空气流腔室。 </li></ol></li><li>制备30微升3毫克/毫升的SUV悬浮在SUV缓冲器。形成含有蛋白质 - GUVs，结合8微升PROTEO-的SUV（DPhPC 10毫克/毫升KvAP 1:10），2微升荧光的SUV（10毫克/毫升DPhPC，0.5摩尔％TexasRed-DHPE）和20μl的SUV缓冲器在1.5ml微量离心管中的最终蛋白质为1脂质（质量）比：12.5和0.1％（摩尔）TexasRed-DHPE。大力混合溶液。 <ol><li>可替换地，实行与脂质仅越野车的协议，简单地结合10微升荧光的SUV（10毫克/毫升DPhPC和0.5摩尔％TexasRed-DHPE）与20μl的SUV缓冲器。 </li></ol></li><li>存款SUV解决方案。 <ol><li>使用2微升吸管或5微升玻璃注射器沉积在导线在SUV溶液的小（&lt;0.2微升）液滴。大约1微升溶液是需要的沿着形成一系列滴1厘米线。确保墨滴足够小，间隔足够远，他们不碰或保险丝。 </li><li>让沉积的SUV干燥〜在露天30分钟。当所有的水滴纷纷落户，旋转钢丝等脂质沉积更容易观察的显微镜。 注意：如果SUV的不充分干燥，他们可以洗掉的电线时增长缓冲液加入，而干燥过多会损伤蛋白质。因为空气湿度影响干燥速率，干燥时间和/或空气湿度可以获得最佳的结果30进行调整。上的电线的脂质膜应该是在显微镜下可见。 </li></ol></li><li>装配该室。 <ol><li>密封该室底部：用注射器施加真空润滑脂的三个井周围的腔室的底部，并按下40毫米×22毫米盖玻片轻轻反对以密封腔室底部，使得它粘附没有间隙。密封腔室的（其中的电线出口）与侧面密封膏。在室内，概述了三口井的顶部应用真空润滑脂。 </li><li>慢慢加入生长缓冲，直到每个井被填充到顶部。避免在孔中的溶液中的任何快速运动，因为这可以去除脂质膜脱落的电极。 </li><li>轻轻按下顶盖滑动到油脂，注意不要打跑底部盖玻片关闭室。使用的组织去除缓冲器的任何滴在顶盖滑动的边缘。 注：这是一个很好的时间来检查显微镜下腔，以确认脂质薄膜一直保持在电线。 </li></ol></li><li>连接的信号发生器，以使用两个鳄鱼夹的导线。设置频率（10赫兹/ 500赫兹的正弦波低/高盐缓冲液），并用万用表测量并调整整个电线的电压0.7 / 0.35 V均方根（Vrms）的电压为低/高盐缓冲液。用铝箔覆盖的腔室，以保护荧光团从光。离开吨他GUVs中生长2至3小时的低盐缓冲液中，12小时或O / N的高盐缓冲液。 </li><li>断开发电机室，并小心地将它放在倒置显微镜来评价GUV增长。使用井缓慢，稳定的运动或流体可能会过早地从电线分离GUVs。 注：GUVs在电线上的边缘通常是在相衬（40X长工作距离物镜）可见，而GUVs上的导线的下半部的任何地方，可以看出用落射荧光。如果没有GUVs是可见的，尝试旋转电线看的上表面。 GUVs可以储存于4℃在生长室中数天。 </li></ol> 4. GUV增长凝胶辅助肿胀<ol><li>通过混合100毫克琼脂糖的10毫升纯水中制备出10毫升1％琼脂糖溶液。其加热至沸腾通过将其在微波中在480瓦〜20秒。搅拌以确保琼脂糖完全溶解。 注：该解决方案可以储存于4℃并在需要时加热。 </li><li>等离子清洗（空气等离子）掩盖幻灯1分钟，使琼脂糖解决方案将很好地传播它。用接下来的15分钟内盖滑梯等离子清洗的效果迅速消退。 </li><li>应用200微升暖琼脂糖溶液到每个22×22 mm 2的滑动，以便润湿整个表面上的溶液。倾斜滑动垂直触摸下缘组织，去除多余的液体，并留下琼脂糖只是薄薄的一层光滑的幻灯片上。 </li><li>放置在热板上或烘箱滑动，在60℃，并让它干燥至少30分钟。琼脂糖膜是通过眼几乎不可见。后的幻灯片冷却至室温，立即使用它们，或者在一个封闭的容器将它们存储为长达一周，在4℃。 </li><li>将琼脂糖涂覆的盖玻片在标准3.5厘米培养皿。 </li><li>准备SUV的解决方案，如第3.2和运用〜15微升的SUV溶液（3毫克/毫升的脂肪）〜30非常小水珠轻轻地放在琼脂糖表面。小心不要扭曲琼脂糖层太多。 </li><li>放置在氮气流平缓滑动约10-15分钟，并按照缓冲的蒸发通过肉眼作为液滴干燥。 </li><li>只要越野车已干燥，添加生长缓冲，以覆盖滑动面。对于一个小3.5厘米培养皿中使用约1 ml缓冲液。 </li><li>允许肿胀进行为约30分钟，然后使用倒置显微镜相衬或微分干涉对比（DIC）的审查GUVs的在腔室中的生长。 注：落射荧光观察是困难的，因为在凝胶和自发荧光琼脂糖的荧光团的强背景。 </li></ol> 5.采收和观测GUVs <ol><li>钝化观察室（ 如小培养皿或玻璃盖玻片），以便GUVs不沾，传播和突发的室底部。覆盖湛BER底部与β-酪蛋白溶液，孵育5分钟，冲洗用纯水的酪蛋白溶液，用空气或氮气流干燥，最后加观测缓冲液（ 例如，约5毫米深度要小培养皿）。 </li><li>收获GUVs。切的100微升移液管头的端部，以便所述开口较大（〜2毫米直径），并慢慢地向往如移液可以很容易地破坏GUVs的剪切应力。 <ol><li>对于电形成GUVs，轻轻除去顶部盖玻片打开生长室。直接放置在移液管尖的每个导线和抽吸以上〜50微升，同时移动枪头沿导线拆下GUVs。 注意：它可能有助于旋转电线收集GUVs的“另一面”的电线。 </li><li>对于“凝胶辅助消肿”GUVs，先点击培养皿的一侧几次帮助GUVs从盖玻片表面分离。定位枪头刚盖玻片和吸出上述50微升，而Pulling尖端背面上的表面上。直接收获GUVs在1.5ml微量离心管中，在4℃下长达1周转移到观察室中，或储存。 </li></ol></li><li>将观察室的倒置显微镜，加上GUVs到观察室，并等待几分钟，让GUVs，收于腔底部。 </li><li>调查相衬或DIC室快速定位光滑，球形（“无缺陷”）“GUV考生”。检查萤光每个“GUV候选人”，以排除内部嵌套任何含有较小的脂质体。最后，检查血脂荧光强度均匀，用一个单一的膜（ 即单层）兼容。 注意：在某些bilamellar（或多层状）囊泡，所述膜过于靠近在一起以得到解决，这样他们出现在单层相衬或DIC图像。然而，这些对象可以从实际unilame区分LLAR GUVs通过脂质荧光，这是更亮的两倍（或更多）。 </li></ol> 6.膜片钳GUVs <ol><li>使补丁移液器与使用推荐的吸管拉马程序规范硼硅玻璃毛细管1-2微米的尖端直径。 注：特殊处理如火抛光是没有必要的，而且吸液管可用于它们已被拉出后，如果它们被保持在封闭的盒子数天。 </li><li>通过用β-酪蛋白溶液（5毫克/毫升）培养，以确保GUVs不粘附，扩散和破裂的室表面钝化室。 5分钟后冲洗酪关闭。 </li><li>将接地电极，填充观察缓冲室，转移10微升的GUV暂停如步骤5.2和5.3描述，并等待几分钟，让GUVs，收于底部。 </li><li>填补了新鲜的补丁吸管解决方案（观察缓冲区或其他等渗溶液）和它安装在膜片钳放大器探头。 </li><li>通过腔搜索找到了“无缺陷”GUV在5.4节中描述，并检查它是否含有荧光蛋白。 </li><li>施加恒定正压力（&gt; 100帕，或大致1厘米H 2 o在一个压力计），以保持所述膜片吸管内部清洁，并插入膜片吸管进入腔室。使膜片吸管进入视场，施加测试脉冲来测量/补偿移液器电压偏移和电阻，并检查在荧光灯照明的吸管，以确认该尖端是干净的。 </li><li>使膜片吸管朝向GUV，并在必要时，同时降低了正压力，从而从膜片吸管的向外流动不会使GUV“跑掉”。当膜片吸管靠近GUV，施加负压（长达5厘米的H 2 O）与拉靠在膜片吸管的GUV。监视电阻作为“; GUV膜舌“进入补丁吸管和千兆欧密封形式。 </li><li>如果一个千兆欧密封没有形成，从腔室取出膜片吸管，并返回到步骤6.4。如果膜贴片形成的千兆欧密封，但GUV保持附着到所述移液管，通过从GUV拉远，爆破的GUV靠在腔室底部，或简单地移动所述吸移管从溶液中切除补丁。 </li><li>当内向外膜补丁已经切除从GUV和千兆欧密封是稳定的，关闭测试脉冲和施加电压的协议，如在图13中所示的那个。 注： 图13所示为一个由内而外的补丁中，电流流入补丁电极是“积极”的标准电惯例，V = V 浴 - V 吸管 。保持贴片在负电位（ 例如，V = -100毫伏）为〜30秒的地方KvAP在静止状态，而步骤（100毫秒至5秒），以更积极的潜力（ 例如，V = 100毫伏）就可以驾驶它到导通（ 即开放）活动状态。 </li><li>在膜补片测量结束后，打破了补丁以ZAP或压力脉冲，检查电压电极贴片偏移没有漂移。取出膜片吸管从室，并返回到步骤6.4。 </li></ol>

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The authors have nothing to disclose.

仿生模型系统用于研究蛋白质和膜的性能和相互作用的一个重要工具。相比像类脂膜或支持的脂质膜的其他重组系统，GUV基础的系统本数的机会，包括相当大的控制的膜组合物，紧张和几何形状，以及作为真正的无油。然而，掺入跨膜蛋白，如KvAP成GUVs需要常规的协议为脂质仅GUVs显著适应。这里介绍的electroformation协议是以前的特点，并用于研究在弯曲膜2,4,35膜蛋白的分布和动态变化的生?...

当研究支配生命系统的物理原理，自下而上途径允许一个实验，以控制系统的组成和不容易在基于细胞的系统1操作的其他参数。为基于膜的工艺，巨单层囊泡（GUVs，直径〜1-100微米）已被证明是一种非常有用的仿生系统2 - 7，因为它们非常适合于显微镜研究和显微8 - 10。虽然有许多不同的协议来产生GUVs，最分为两类-基乳液方法的基础上再水化脂质膜13 11,12和技术- 16。在乳化液为基础的方法中，GUV膜的内，外小叶被依次从脂质单层在水/油界面组装。这种方法适用于封装可溶性蛋白与在GUVs，和用于形成GUVs不对称小叶脂质组合物。然而，从乳液形成GUVs可以保留的痕量溶剂是改变膜的机械特性17，并且该方法不特别适合于跨膜蛋白重建。 膜补液方法依赖于以下事实干燥（脱水）导致许多脂类混合物以形成膜的多片层堆栈。如果此堆栈，然后放置在接触含水缓冲液，在叠层膜会移动它们之间，并在该叠层的表面分开为溶剂流动，个别膜可以分离，以形成GUVs 13,18（以及一个真正的动物园其它脂质对象）。然而，即使是对于最佳缓冲液和脂质组合物，这种典型的“自发溶胀”的方法具有相对低的无缺陷GUVs收率。一种广泛使用的方法，以提高无缺陷GUVs收率“electroformation221 ;,在其中交变电流（AC）场期间膜补液施加。而该机制仍然知之甚少，“electroformation”可以给予壮观GUV产率（&gt;在有利的情况下，90％）为低盐浓度缓冲液（&lt;5毫米）14,19，并且甚至可以在生理缓冲液的工作（约100毫米）使用较高的频率（500赫兹与10赫兹）的交流场和铂电极15。另一种方法，以促进无缺陷GUVs收率是“凝胶辅助肿胀”，其中的脂质溶液沉积在聚合物凝胶基质，而不是被动用于古典“自发溶胀（ 例如 ，玻璃，聚四氟乙烯）基底“。当所得到的脂质/凝胶膜再水化，GUVs能迅速形成，即使对于生理缓冲器16,20。 所有这些方法都可以产生可用于研究膜相关的现象，如脂质仅GUVs可溶性蛋白和膜之间的相互作用。然而，掺入跨膜蛋白进入GUVs，需要显著修改，以确保该蛋白质保持在功能状态在整个重构过程。而在有机溶剂（ 例如，氯仿，环己烷）脂质的解决方案非常适合用于制造脂质膜，跨膜蛋白是典型地仅当稳定它们的疏水跨膜结构域被嵌入脂质双层，或由洗涤剂胶束包围（ 例如，在蛋白质纯化）。因此，对于一重建的起始材料通常是由洗涤剂去除在形成的天然的膜，在洗涤剂溶液中纯化的蛋白质，或小单层含蛋白囊泡（PROTEO-越野车）和/或多层状囊泡（PROTEO-MLV的）存在脂质。大多数将这些膜蛋白进入GUVs方法分为三类。 直接InsertioN：跨膜蛋白悬浮在洗涤剂与预成形的，脂质-只，轻度清洁剂溶解的GUVs混合，并且将洗涤剂然后使用biobeads 21除去。而概念简单，这种方法需要的洗涤剂浓度的精确控制，因为太高的洗涤剂浓度可以溶解而过低的浓度可导致蛋白质折叠或聚集的GUVs。 GUV / PROTEO-SUV的融合：蛋白质在PROTEO-SUV的组合与预成形的，脂质-仅GUVs和融合促进了与特殊膜融合肽22或21的洗涤剂。典型地融合的程度有限，导致GUVs具有低蛋白质浓度。 脱水/再水化：包含蛋白质 - 类脂膜通过PROTEO-SUV的部分脱水（或PROTEO-MLV）溶液和GUVs形成然后生长作为用于纯脂质膜。明显的挑战是局部dehydrati期间保护蛋白质关于步骤23中 ，但该方法已被成功地用于重组跨膜蛋白，如细 ​​菌视紫红质，钙ATP酶，整合和VDAC成GUVs 7,23 - 25。 本文介绍的脱水/再水化协议，使含有该电压-门控钾通道，KvAP，从超嗜热古菌， 嗜pernix GUVs。KvAP具有高度的同源性，以真核电压依赖性钾通道26和一个公知的晶体结构27 ，使其成为一个很好的模型来研究电压门控的机制。生产PROTEO-SUV的已详细描述以前，不是本教程26,28,29的一部分。重要的是，KvAP PROTEO-SUV的不必要产生的每个GUV制剂，因为它们可以被存储在小（ 例如，10微升）等分在-80℃下延长的时间段（&gt; 1年）。 Electroformation或凝胶辅助溶胀然后可用于从KvAP PROTEO-SUV的生长GUVs（或PROTEO-MLV的）。 为electroformation协议的关键步骤被示于图1。含有蛋白质的SUV的溶液的液滴沉积在铂丝（ 图2中示出）。在SUV的悬挂部分脱水导致通过的SUV的融合，形成脂质蛋白膜。期间补液，一个交流电场施加到电极上，以协助脂质层脱层并形成GUVs。 10赫兹场效果很好时，使用“低盐”（&lt;5毫米）补液缓冲区28和GUVs需要几个小时才能成长。与此相反，生理缓冲液（含有〜100mM的盐）与一低电压，500赫兹AC场工作良好，但需要一个长期（〜12小时）肿胀周期15。此方法是基于使用ITO滑动24较早的协议，但是使用自定义腔室续癌宁两个铂丝， 如图2（见用于更简单的设计细节和建议的讨论中，即兴室）。 图3示出了凝胶辅助膨胀方法。该协议可以很好地缓冲生理食盐浓度，迅速，并且产生GUVs具有更均匀的蛋白质分布。然而，分离的，显然无缺陷GUVs收率（ 即 GUV膜是均匀的，在光学长度尺度，并且不括任何对象）较低，但它提供了足够数量的用于膜片钳和显微操作实验。此方法是基于使用琼脂糖凝胶，以产生脂质仅GUVs 16和需要更少的专门的设备比electroformation方法的协议。 GUVs用荧光显微镜的表征被描述，以及使用标准膜片钳设置到程序衡量KvAP活动“由内而外”切膜的补丁。 生长含蛋白GUVs可以比脂质仅GUVs更加困难。尤其是，最后的GUV收率上究竟如何在SUV溶液沉积和脱水以形成薄膜叠层灵敏依赖。有人未经任何以往的经验与GUVs，它可以是下面的其中隔膜是通过从有机溶剂沉积类脂形成的常规协议15,16到第一生长脂质仅GUVs很有帮助。一旦现有协议工作良好，SUV沉积和部分脱水然后可以使用脂质仅越野车，这也是非常有益的调整协议的新的脂质组合物时掌握。当GUVs脂质只SUV的可靠增长，它是那么只是一小步，产生含蛋白质GUVs从PROTEO-的SUV。

<table border="0" cellpadding="0" cellspacing="0" width="599">
	<tbody>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Name of the 
			Material/Equipment</td>
			<td style="width:132px;">Company</td>
			<td style="width:155px;">Catalog Number</td>
			<td style="width:149px;">Comments/ Description</td>
		</tr>
		<tr height="60">
			<td dir="LTR" height="60" style="height:60px;width:163px;">DPhPC (1,2-diphytanoyl-sn-glycero-3-phosphocholine)</td>
			<td style="width:132px;">Avanti Polar Lipids&#160;</td>
			<td>850356P</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="60">
			<td dir="LTR" height="60" style="height:60px;width:163px;">Egg PC L-&#945;-phosphatidylcholine (Egg, Chicken)&#160;</td>
			<td style="width:132px;">Avanti Polar Lipids&#160;</td>
			<td>840051P</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Egg PA L-&#945;-phosphatidic acid (Egg, Chicken)</td>
			<td style="width:132px;">Avanti Polar Lipids&#160;</td>
			<td>840101P</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="60">
			<td dir="LTR" height="60" style="height:60px;width:163px;">18:1 (&#916;9-Cis) PC (DOPC) 1,2-dioleoyl-sn-glycero-3-phosphocholine</td>
			<td style="width:132px;">Avanti Polar Lipids&#160;</td>
			<td>850375P</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">cholesterol (ovine wool, &#62;98%)&#160;</td>
			<td style="width:132px;">Avanti Polar Lipids&#160;</td>
			<td>700000P</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">TRed-DHPE</td>
			<td style="width:132px;">Invirtogen</td>
			<td>T-1395MP</td>
			<td style="width:149px;">labeled lipid</td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">BPTR-Ceramide</td>
			<td style="width:132px;">Invirtogen</td>
			<td>D-7540&#160;</td>
			<td style="width:149px;">labeled lipid</td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Choloroform</td>
			<td style="width:132px;">VWR</td>
			<td>22711.290</td>
			<td style="width:149px;">AnalaR Normapur</td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Acetone</td>
			<td style="width:132px;">VWR</td>
			<td>20066.296</td>
			<td style="width:149px;">AnalaR Normapur</td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Ethanol</td>
			<td style="width:132px;">VWR</td>
			<td>20821.330</td>
			<td style="width:149px;">AnalaR Normapur</td>
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		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Kimwipe</td>
			<td style="width:132px;">Kimtech</td>
			<td>7552</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Hamilton syringes&#160;</td>
			<td style="width:132px;">Hamilton Bonaduz AG</td>
			<td></td>
			<td style="width:149px;">diverse</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Amber vials and teflon cups</td>
			<td style="width:132px;">Sigma</td>
			<td>SU860083 and SU860076</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Parafilm</td>
			<td style="width:132px;">VWR</td>
			<td>291-1214</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">microcentrifuge tube</td>
			<td style="width:132px;">eppendorf</td>
			<td></td>
			<td style="width:149px;">diverse</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Agarose</td>
			<td style="width:132px;">Euromex</td>
			<td style="width:155px;">LM3 (1670-B, Tg 25.7C, Tm 64C)</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Sucrose</td>
			<td style="width:132px;">Sigma</td>
			<td>84097-1kg</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Glucose&#160;</td>
			<td style="width:132px;">Sigma</td>
			<td>G8270-1kg</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Trehalose</td>
			<td style="width:132px;">Sigma</td>
			<td>T9531-25G</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">KCl</td>
			<td style="width:132px;">Sigma</td>
			<td>P9333-1kg</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Hepes</td>
			<td style="width:132px;">Sigma</td>
			<td>H3375-100G</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">TRIS</td>
			<td style="width:132px;">Euromex</td>
			<td>EU0011-A</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Osmometer</td>
			<td style="width:132px;">Wescor</td>
			<td>Vapro</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">pH meter</td>
			<td style="width:132px;">Schott instruments</td>
			<td>Lab850</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Sonicator</td>
			<td style="width:132px;">Elmasonic</td>
			<td>S 180 H</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Syringe filter 0.2&#181;m</td>
			<td style="width:132px;">Sartorius steim</td>
			<td>Minisart 16532</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Function generator</td>
			<td style="width:132px;">TTi</td>
			<td>TG315</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Platinum wires</td>
			<td style="width:132px;">Goodfellow</td>
			<td>LS413074</td>
			<td style="width:149px;">99.99+%, d=0.5mm</td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Polytetrafluoroethylene</td>
			<td style="width:132px;">Goodfellow</td>
			<td></td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Dow Corning &#39;high vacuum grease&#39;</td>
			<td style="width:132px;">VWR</td>
			<td>1597418</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">sealing paste</td>
			<td style="width:132px;">Vitrex medical A/S, Denmark</td>
			<td>REF 140014</td>
			<td style="width:149px;">Sigillum Wax</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Cover slides 22x40 mm No1,5</td>
			<td style="width:132px;">VWR&#160;</td>
			<td>631-0136</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Cover slides 22x22 mm No1,5</td>
			<td style="width:132px;">VWR&#160;</td>
			<td>631-0125</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">plasma cleaner</td>
			<td style="width:132px;">Harrick</td>
			<td>PDC-32G</td>
			<td style="width:149px;">airplasma, setting &#39;high&#39;</td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">petri dishes</td>
			<td style="width:132px;">Falcon BD</td>
			<td>REF 353001</td>
			<td style="width:149px;">3.5 cmx1cm</td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">patch clamp amplifier</td>
			<td style="width:132px;">Axon instruments</td>
			<td>Multiclamp700B</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">DAQ Card</td>
			<td style="width:132px;">National Instruments</td>
			<td>PCI-6221</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Labview</td>
			<td style="width:132px;">National Instruments</td>
			<td>version 8.6</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="60">
			<td dir="LTR" height="60" style="height:60px;width:163px;">Glass pipettes boro silicate OD 1mm ID 0.58mm</td>
			<td style="width:132px;">Harvard Apparatus</td>
			<td>GC100-15</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Electrode holder</td>
			<td style="width:132px;">Warner Instruments&#160;</td>
			<td>Q45W-T10P</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Micromanipulator</td>
			<td style="width:132px;">Sutter&#160;</td>
			<td>MP-285</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">pipette puller</td>
			<td style="width:132px;">Sutter&#160;</td>
			<td>P-2000&#160;</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Camera</td>
			<td style="width:132px;">ProSilica&#160;</td>
			<td>GC1380</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Zeiss microscope</td>
			<td style="width:132px;">Zeiss</td>
			<td>Axiovert 135</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="60">
			<td dir="LTR" height="60" style="height:60px;width:163px;">Objective</td>
			<td style="width:132px;">Zeiss</td>
			<td></td>
			<td style="width:149px;">40x long working distance, Phase contrast</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Objective&#160;</td>
			<td style="width:132px;">Zeiss</td>
			<td></td>
			<td style="width:149px;">100x Plan-Apochromat NA 1.3</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Filterset GFP (for Alexa-488)</td>
			<td style="width:132px;">Horiba</td>
			<td>XF100-3</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Filterset Cy3 (for TexasRed)</td>
			<td style="width:132px;">Horiba</td>
			<td>XF101-2</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">beta-casein</td>
			<td style="width:132px;">Sigma&#160;</td>
			<td>C6905-1G</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">confocal microscope</td>
			<td style="width:132px;">Nikon Eclipse TE 2000-E</td>
			<td></td>
			<td style="width:149px;">D-Eclipse C1 confocal head</td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Objective</td>
			<td style="width:132px;">Nikon</td>
			<td></td>
			<td style="width:149px;">Plan Fluor 100&#215; NA1.3</td>
		</tr>
		<tr height="60">
			<td dir="LTR" height="60" style="height:60px;width:163px;">matlab</td>
			<td style="width:132px;">Mathworks</td>
			<td></td>
			<td style="width:149px;">for image processing and analysis of the current traces</td>
		</tr>
	</tbody>
</table>

reconstitution of a transmembrane protein, the voltage-gated ion channel, kvap, into giant unilamellar vesicles for microscopy and patch clamp studies

巨人单层囊泡（GUVs）是一种流行的仿生系统研究膜相关现象。然而，通常使用的协议来生长GUVs必须为了形成含有官能跨膜蛋白GUVs进行修改。本文介绍了两种脱水 - 再水化方法 - electroformation和凝胶辅助肿胀 - 以形成含有所述电压 - 门控钾通道，KvAP GUVs。在两种方法中，对含有蛋白质的小单层囊泡的溶液是部分脱水，形成一个叠层膜，然后使其在再水化缓冲液溶胀。为electroformation方法，所述膜沉积在铂电极以使得AC场可以在膜补液施加。与此相反，凝胶溶胀辅助方法使用琼脂糖凝胶基质，以提高膜补液。这两种方法都可以产生在低（ 例如，5毫米）和生理（ 例如，100毫摩尔）的盐浓度GUVs。所得GUVs的特征在于通过荧光显微镜，和使用该内面向外膜片钳构造重构信道的函数进行测量。而以交替的电场（electroformation）的存在下溶胀得到高产率的无缺陷GUVs，凝胶辅助溶胀方法产生更均匀的蛋白质的分布，不需要特殊的设备。

GUVs的生长可通过检查生长室，在显微镜下进行快速评估。对于electroformation，所述GUVs倾向于增长沿铂导线束， 如图4。在凝胶辅助肿胀，GUVs显示为球形的结构，可以快速成长，并融合在一起（ 图5）。 无缺陷GUVs更容易识别和传送到一个​​观察室之后进行评价。需要校准测量来严格评价GUV质量，并且一个系统的量化先前已公布的28。然而，?...

Watch this Scientific Journal Video about Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies at JoVE

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

跨膜蛋白，KvAP，变成巨大的单层囊泡（GUVs）的重组被证明两脱水补液方法 - electroformation和凝胶辅助肿胀。在两种方法中，将含有蛋白的小单层囊泡融合在一起，以形成可以随后通过荧光显微镜和膜片钳电生理学来研究GUVs。

跨膜蛋白的重组，电压门控离子通道，KvAP，进入巨人单层囊的显微镜和膜片钳研究

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

reconstitution-transmembrane-protein-voltage-gated-ion-channel-kvap

Research

JoVE Journal

Biology

18.9K 次观看.  Université Pierre et Marie Curie. 跨膜蛋白，KvAP，变成巨大的单层囊泡（GUVs）的重组被证明两脱水补液方法 - electroformation和凝胶辅助肿胀。在两种方法中，将含有蛋白的小单层囊泡融合在一起，以形成可以随后通过荧光显微镜和膜片钳电生理学来研究GUVs。 

视频: Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Methods for Patch Clamp Capacitance Recordings from the Calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal.

膜片钳从花萼电容录音方法

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. ...

科研

生物学

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 ...

Pressure-polishing Pipettes for Improved Patch-clamp Recording

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording. We first developed this method for fabricating pipettes suitable for recording from small (&lt;3 m) neuronal cell bodies. The basic principal is similar to glass-blowing and combines air pressure and heat to modify the shape of patch pipettes prepared by a conventional micropipette puller. It can be applied to so-called soft (soda lime) and hard (borosilicate) glasses. Generally speaking, pressure polishing can reduce pipette resistance by 25% without decreasing the diameter of the tip opening (Goodman and Lockery, 2000). It can be applied to virtually any type of glass and requires only the addition of a high-pressure valve and fitting to a microforge. This technique is essential for recording from ultrasmall cells (&lt;5 m) and can also improve single-channel recording by minimizing pipette resistance. The blunt shape is also useful for perforated-patch clamp recording since this tip shape results in a larger membrane bleb available for perforation.

This is a guide to modifying the shape of glass micropipettes. Specifically, by using heat and air pressure the taper is widened without increasing the tip opening, leading to lower pipette resistance. This is critical to obtain low noise recordings of small cells but is useful in many applications.

改进的膜片钳记录的压力抛光移液器

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording.  We first developed this method for fabricating pipettes suitable for ...

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 ...

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) ...

Measuring Peptide Translocation into Large Unilamellar Vesicles

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1. Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors1-3. Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms4,5, particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes6,7. In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides1,8. A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs)9-13. LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies14,15. Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II16,17. In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties18, to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell-penetrating peptides are cleaved as they encounter uninhibited trypsin encapsulated with the LUVs, leading to disassociation from the LUV membrane and a drop in FRET signal. The drop in FRET signal observed for a translocating peptide is significantly greater than that observed for the same peptide when the LUVs contain both trypsin and trypsin inhibitor, or when a peptide that does not spontaneously cross lipid membranes is exposed to trypsin-containing LUVs. This change in fluorescence provides a direct quantification of peptide translocation over time.

This protocol details a method for the quantitative measure of peptide translocation into large unilamellar lipid vesicles. This method also provides information about the rate of membrane translocation and can be used to identify peptides that efficiently and spontaneously cross lipid bilayers.

测量肽转运到大Unilamellar囊泡

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1.  Many of these are referred ...

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca2+]cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).
Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.
The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.

Measurements of Kv7 (KCNQ) potassium channel activity in isolated arterial myocytes (using patch clamp electrophysiological techniques) in parallel with measurements of constrictor/dilator responses (using pressure myography) can reveal important information about the roles of Kv7 channels in vascular smooth muscle physiology and pharmacology.

探讨动脉平滑肌的KV7钾离子通道功能使用膜片钳电生理和压力Myography

Contraction  or relaxation of smooth muscle cells within the walls of resistance arteries  determines the artery diameter and thereby controls flow of ...

Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify and explore the function of these important proteins. However, classical preparations, such as planar bilayers, limit the manipulations and experiments that can be performed on the reconstituted channel and its membrane environment. The more cell-like structure of giant liposomes permits traditional patch-clamp experiments without sacrificing control of the lipid environment.
Electroformation is an efficient mean to produce giant liposomes &gt;10 &mu;m in diameter which relies on the application of alternating voltage to a thin, ordered lipid film deposited on an electrode surface. However, since the classical protocol calls for the lipids to be deposited from organic solvents, it is not compatible with less robust membrane proteins like ion channels and must be modified. Recently, protocols have been developed to electroform giant liposomes from partially dehydrated small liposomes, which we have adapted to protein-containing liposomes in our laboratory.
We present here the background, equipment, techniques, and pitfalls of electroformation of giant liposomes from small liposome dispersions. We begin with the classic protocol, which should be mastered first before attempting the more challenging protocols that follow. We demonstrate the process of controlled partial dehydration of small liposomes using vapor equilibrium with saturated salt solutions. Finally, we demonstrate the process of electroformation itself. We will describe simple, inexpensive equipment that can be made in-house to produce high-quality liposomes, and describe visual inspection of the preparation at each stage to ensure the best results.

Reconstituting functional membrane proteins into giant liposomes of defined composition is a powerful approach when combined with patch-clamp electrophysiology. However, conventional giant liposome production may be incompatible with protein stability. We describe protocols for producing giant liposomes from pure lipids or small liposomes containing ion channels.

巨脂质体制剂成像和膜片钳电

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify ...

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 ...