这项工作是由国家心脏，肺和血液研究所（NIH R01-HL089564）的资助KLB和博士后奖学金由美国心脏协会（09PRE2260209）和Arthur J.施密特基金会BKM。

1。手术切除的小肠肠系膜血管商场<ol><li>麻醉300-400克SD大鼠（4％）与异氟醚吸入给药。 </li><li>执行中线剖腹手术暴露的小肠系膜。 Exteriorize通过腹部切口非常小心，以避免创伤暴露的肠管及肠系膜小肠和大肠。轻轻地扇了的肠系膜超过无菌纱布。 </li><li>外科手术切除小肠和大肠的一部分，包括盲肠（沿利润率和肠系膜上动脉/静脉）从主分支的起源仔细剪裁血管。 </li><li>切除的组织转移到烧杯中含有冰冷解剖溶液（ 表1）。 </li><li>切除的小肠，大鼠安乐死，而在麻醉状态下。 </li></ol> 2。解剖一个ð清洁动脉<ol><li>转移切除小肠和肠系膜街机冷却的解剖室内（4-5°C）含有夹层的解决方案。该室包括一个100毫米的玻璃陪替氏培养皿用SYLGARD 184弹性体基容纳解剖标签;维持在4-5℃的循环水浴冷却的碱是被放置在陪替氏培养皿。 </li><li>使用的整个肠道手术剪刀剪下约10-12厘米的段。处切断的肠段保持其脉管连接的每一端，然后移除未使用的从腔室离开一个段及其连接的血管（肠系膜街机）孤立肠。 </li><li>牵制的小肠段，在近前侧的腔室，使得主分支肠系膜上动脉/静脉是在从中心（ 图1）的中心，并在随后的订单的分支辐射扩频肠系膜。 PL<html>王牌引脚上的肠段（而不是血管）。 </li><li>区分其相对明亮的红色，厚厚的墙壁和特性的V形分支，而不是U形见于静脉的分支动脉静脉。 </li><li>可视化通过解剖显微镜的照明，仔细清除脂肪组织和结缔组织的周围的第二 -四阶动脉接近（在图1中的虚线圆），使用微型钳和剪刀肠子。控股邻近的静脉，有利于清除脂肪组织和结缔组织。避免不必要的动脉延伸。 </li><li>肠系膜动脉血管的结缔组织清除就可以被分离出，并用于细胞分离（单细胞膜片钳技术研究，第3-4）或插管的压力myography（第5章）。剪切分支点之间的线段，通常最长为1厘米的长度。 </li></ol>jove_title"&gt; 3。细胞分离膜片钳研究<ol><li>准备0.5 L的隔离解决方案（ 见表1），并把它分成2份，然后再调整pH值。的第一部分（通常为100毫升）应被加热至37℃下，pH值应调节到7.2，在37℃下（37°C隔离解决方案）。的第二部分的分离解决方案应被冷却到4℃，pH值应调整至7.2，在4℃（冰镇的分离解决方案）。在pH调节后，这两种解决方案的渗透压必须被调整，通过添加适当量的D-葡萄糖或H 2 O至298毫渗</li><li>转让2毫升37°C隔离解决方案，以小玻璃瓶中，在37°C孵育步骤3.4使用。加入20毫克的牛血清白蛋白（BSA），10毫升37°C隔离解决方案，解散，重新加热到37°C，并调整pH值至7.2。 2毫升上述溶液中添加4毫克型胶原酶VIII（Sigma公司，批号089K8612:633ü尼特/毫克胶原酶活性，285单位/ mg Caseinase的活动，1.6单位/ mg梭菌活动），0.64毫克弹性蛋白酶IV型（Sigma公司，批号110M7025V 5.9单位/毫克，加入40μl的16毫克/毫升的股票，准备在37°。 C隔离解决方案）1和2μl中号DL-二硫苏糖醇（DTT Sigma公司）预暖的酶液37°C，在准备步骤3.5。 </li><li> 2-3段的肠系膜动脉转移到含2毫升冰冷的隔离解决方案和地方的冰盘上的35毫米组织培养皿中。切断肠系膜上动脉段为3-4毫米长的碎片。 </li><li>使用巴斯德吸管与火抛光头（防止动脉血管的损害）转移到一个玻璃小瓶（15×45毫米，1 DRAM），含2毫升预热的37°C的动脉段隔离解决方案，并培育30分钟，37 °C。 </li><li> 30分钟后，小心吸37°C的隔离解决方案使用巴斯德吸管，加2毫升温水酶溶液，室温放置35分钟在37℃下</li><li>酶溶液中孵育35分钟后，立即在冰面上，将小瓶吸酶溶液和添加2毫升冰冷的隔离解决方案（重复的愿望和新鲜的冰镇隔离解决方案增加了4倍）。然后，在一个体积为1毫升的冰镇隔离解决方案， 轻轻捣碎动脉段与抛光的巴斯德吸移管释放出单个的肠系膜动脉平滑肌的细胞（MASMCs）的10-20倍。 </li><li>定期验证心肌细胞外观上的35毫米的菜，将一滴细胞悬液，在显微镜下观看。健康的MASMCs应该有一个平滑细长的外观。不是所有的在动脉壁的细胞将被释放。将细胞悬浮液应保存在冰中。 </li><li> MASMC健康的另一项有用的指标是生理的Ca 2 +浓度的耐受性。浴小号填写录音室（生物科学工具，CSC-25L）辨率含有2mM CaCl 2的 （组合物在室温（RT）的pH值为7.3， 见表1）和位置琼脂桥（用2％琼脂的2 M氯化钾弯曲玻璃毛细管填充，参比电极插入（华纳仪器，REF -3L））箱内。使用抛光的巴斯德吸移管，约100-200微升细胞悬浮液转移到一个记录室，使细胞粘附15分钟。完好无损的细胞不应该在2mM的显着收缩的CaCl 2的溶液（的细胞应该出现是细长的或棒状成球，而不是向上舍入）。 </li><li>第一等分的细胞悬浮液后加入到记录室，浴溶液中的0.25毫升含有2mM CaCl 2的应该被添加到细胞悬浮液的剩余量。在此解决方案中，这种其它的细胞悬浮液可保持在冰上，并用于膜片钳记录长达6小时。 </li></ol> 4。使用全细胞膜片钳测量的KV7钾电流或膜电位<ol><li>后心肌细胞坚持的记录室（25毫米编号1轮盖玻片，华纳仪器）的玻璃基体，启动的浴溶液（ 表1）的连续灌注。记录的KV7电流的KV1和KV2家庭的延迟整流钾电流的隔离，沐浴液，应补充与100μMGDCL。 </li><li>制作从:硼硅酸盐玻璃（Kimax-51，金布尔大通）使用吸移管牵拉和microforge的一个补丁移液器;MΩ内部溶液（ 表1）充满时其电阻应是4-5。外套的下1/3的吸移管（〜1-2毫米以上的前端），SYLGARD 184。 </li><li>对于记录的KV7电流MASMCs，应采用穿孔膜片钳全​​细胞电压钳技术。使用的穿孔修补程序配置有助于防止depletio的n的膜磷脂酰肌醇4,5 -二磷酸（PIP 2）和随后的快（在5分钟内）破旧的KV7的电流，也就是通常观察到使用常规的破裂补丁配置。补编内部溶液（ 表1）与120微克/毫升两性霉素B:3μl的2毫克/毫升两性霉素B库存（在DMSO中制备并保持冻结在小等分试样和避光）添加到0.5毫升的内部溶液。填充的补丁移液管的前端与两性霉素B无内部溶液通过移液管尖中的两性霉素B无内部溶液浸渍，然后在另一端进行抽吸，使用10毫升注射器附着通过一个管件（的Tygonŕ 3603）。加入两性霉素B含吸管的解决方案使用的Eppendorf Microloader移液器吸头的顶部，直到移液器，约有一半的充满溶​​液。删除任何气泡，轻轻拍打移液管。立即用移液管。 </li><li>将滴管到所述电极保持器，并使用显微操作降低到一个细长的心肌细胞的表面上（接近，但不是在细胞核中， 图2的顶部）的吸移管。当移液管阻力增加6〜10MΩ，适用吸力，实现了千兆密封（&gt; 10GΩ）。 </li><li>设置保持电压为0毫伏，而等待膜穿孔。应用100毫秒的电压的步骤至+10 mV，然后到-10 mV，使用放大器来补偿快速移液器电容瞬变。的外观，全细胞电容的瞬变，小持续正电流（通常为2-10 PA）的同时，将指示存在的功能KV7渠道。 </li><li>成功与两性霉素B穿孔会降低接电阻为35MΩ或更少。当前记录，然后可以开始使用5秒-4 mV的保持电压的电压阶跃协议。我们使用-4 mV的保持电压，以实现接近最大延迟同属的其他类型的失活tifier钾电流（主要由KV1和KV2通道介导的），否则将污染的记录的相对小的非灭活KV7电流。长（5秒）的电压阶跃协议是要实现稳态激活KV7的渠道，这很慢，电压依赖性激活和失活。 KV7电流的持续电压步骤期间不灭活。 </li><li>要记录KV7电流 - 电压（IV）关系，适用于5秒的电压阶跃到+16 mV的电压范围从-84毫伏，在哪些的KV7通道的开放概率最小，在哪些KV7通道的开放概率达到高原，在每个测试电压，5秒后退一步，以保持电压（-4毫伏），持续10秒。这将需要约350分钟的全系列的测试脉冲（ 图3A）。执行2-3控制IV录音，以确保记录的电流随时间是稳定的。情节平均端的脉冲电流（平均电流记录的最后1秒）对电压创建的IV曲线（ 图3D）。如果电流是稳定的，控制的IV曲线应约叠加。 </li><li>在应用任何药物开始连续记录电流在-20 mV的一个时间过程设计的协议。至少5分钟的药物应用之前，确保稳定的记录的电流。应用药物为5-15分钟，或直至达到一个稳定状态（ 图3C），然后终止该时间过程协议和记录两个IV曲线，使用的电压步协议。冲蚀的药物，并记录药物清洗过程中的时间，然后通过记录两个额外的IV曲线。 </li><li>在实验结束后，10的μMlinopirdine或10μM的XE991，不可逆抑制剂KV7通道的应用，这些药物应充分抑制记录在负-20毫伏（ 图3D）的电压的电流。 </li><li&gt;每个实验后用洗涤剂，乙醇和热水清洗室和灌注系统，以除去所用的药物的任何痕迹。 </li></ol> 5。使用压力Myography完整的动脉段<ol><li>购自丹麦的，MYO技术（DMT A / S，丹麦，型号110P）的压力肌动描记系统用于的压力myography实验。安装在不锈钢腔动脉段插入到一个端部和可动到另一端的合适的玻璃套管的水平固定的左玻璃套管。两个压力换能器（权的P1和P2上的左侧）是建立在以监测动脉管腔内的压力。将被用于正确的玻璃套管的流体从动脉内的压力调整到其近似的生理水平。首先，预填充开放10ml注射器（用作一个重力进料压力柱），与管腔溶液（ 表1）。提高注射器推解决方案TION，通过聚乙烯管材照顾到右边的玻璃套管，油管或套管，以避免空气中的气泡。左边的套管应预先填写流明解决方案，通过一个1毫升注射器。填充压力肌动描记室容器浴溶液（ 表1）用10ml。松散暂停前的每一侧的玻璃套管上的细的尼龙缝合线。 </li><li>小心转移解剖肠系膜动脉段（通常为3 次或4 阶段，约200 - 350微米的直径;从步骤2.6）从解剖腔肌动描记的压力室，通过保持到该段的一端部上，使用微镊子。 </li><li>可视化通过照明解剖显微镜，按住动脉段的一端，并轻轻将它滑到的左玻璃套管和使用的尼龙缝合线固定结束。轻轻冲洗管腔液通过安装的容器，在容器内，以清除血液和碎片。左边的注射器的阀，然后应关闭，使得这一边提出反对从右侧被调节的压力流体静态列。 </li><li>使用显微操作器，位置更接近可移动的合适的玻璃套管的动脉段和拉段在它的右端，最后固定​​它与尼龙缝合线。剪掉多余的缝合线。 </li><li>在舞台上安装的压力肌动描记单位的压力肌动描记显微镜。将盖在压力肌动描记室。盖的腔室中包含的的灌流入口和连接到它的吸入管。连接灌流进气歧管，使灌流室温浴缸或60毫米KCl溶液通过重力的压力肌动描记室。试验物质，如被发​​现在膜片钳测量影响KV7通道功能的药物或激素，一般是直接施加浴溶液中，不经由浴支持erfusion或在管腔溶液。将使用沐浴液灌流，洗出的KCl溶液。的吸入管连接到真空系统，以除去过量的灌流。所述温度传感器插入到浴溶液中，通过该开口设置在肌动描记器的腔室盖。 </li><li>连接的压力肌动描记单元的肌醇接口系统，硬件的myoview软件（DMT），使通信的肌动描记单元。 </li><li>打开的myoview的软件在电脑上。在"参数"窗口中，设置目标温度为35°C的温度公差为1°C，80毫米汞柱和80毫米汞柱的目标流出压力的目标流入压力。加载校准文件和校准的容器外径，使动脉直径的变化，准确地记录在微米。调整对比度必要的安装部分的映衬下，使一个很好的观点。如何使用软件的详细说明设置在用户手册从DMT。 </li><li>逐步提高用10ml注射器（作为一个重力进料压力柱），时间为15分钟以上一个，直到P1压力传感器读数为80毫米汞柱。虽然提高的压力列，检查是否有泄漏的容器。如果有任何泄漏，丢弃段，装入另一个动脉段（重复步骤从5.2）。调整套管必要时之间的距离。应调整的距离，在这样一种方式，加压动脉段大约是直的（但是未拉伸），并形成了一个肩类似的关系（ 图4A）的图案。 </li><li>开启压力肌动描记室的加热通过在的肌接口系统的控制。允许浴溶液逐渐升温，直到它达到36-37℃。 </li><li>的使用的XYZ调整的目标中的DMT显微镜必要时保持容器在焦点和方便准确地跟踪的ch法兰外血管直径。 </li><li>短暂灌流（约30秒），60 mM KCl中，检查安装的容器的可行性。一个可行的血管收缩加入KCl和迅速扩张后，立即冲洗60毫米氯化钾（superfuse约60毫升的沐浴液洗出的氯化钾）。在本试验中，调整的浴溶液，以确切地是10毫升的体积。浴的温度降低的KCl的测试和冲洗过程中，因为这些解决方案是在室温下，但腔室的加热器将还原温度为36-37℃下，在几分钟之内。 </li><li>允许动脉平衡至少30分钟（直径应该是稳定的，至少在最后10分钟在此期间）。添加浓缩的试验物质，以直接的浴溶液，轻轻的来回移液混合浴溶液中的试验物质，得到适当的最终浓度在10毫升的腔室容积的腔室的边缘附近。陈另外的测试物质（s）中的血管直径以下的水电站可以监视在实况视频图像，并在图像分析窗口，同时也绘制实验正在进行中。 </li><li>实验完成后，所存储的数据文件（*。肌）可以打开作为一个电子表格文件，作进一步的分析。 </li></ol> 6。有代表性的结果 KV7电流从肠系膜动脉肌细胞，使用前一个电压阶跃协议（ 图3A，B黑色痕迹）和在治疗过程中与KV7通道活化剂锌吡啶硫酮（ZnPyr，100nM的， 图3A，B所示的结果在图3中示出典型的录音红色痕迹）。 3细胞相似的电压的步骤的数据进行平均，以制备的电流-电压（IV）曲线， 如图3D所示。一位代表过程中提高电流幅值测量连续的电压钳制在-20 mV的白枕克治疗与100nM的锌吡啶硫酮在图3C中所示， 图4B中示出的时间过程的效果的ZnPyr上肠系膜动脉的外径使用压力myography测量容器100 PM精 ​​氨酸加压素（AVP）是预缩之前除了100纳米ZnPyr。在图4C中示出的平均剂量-反应数据ZnPyr诱导肠系膜动脉扩张。 <img src="/files/ftp_upload/4263/4263fig1.jpg" alt="图1"/> 图1。的肠系膜商场，在解剖室内的画面。 <img src="/files/ftp_upload/4263/4263fig2.jpg" alt="图2"/> 图照片的心肌细胞在其表面上与补丁移液器。 <img src="/files/ftp_upload/4263/4263fig3.jpg" alt="图3"/> 图3。原始的痕迹KV7电流，时间疗程的药<html>应用程序，IV曲线。 A.家庭响应于电压的步骤（前面板）治疗前（控制，黑色）和以下稳定在100nM的ZnPyr（红色）的存在下，在一个单一的MASMC中记录在连续电流的痕迹（下面板）。 B.代表电流的痕迹A（用箭头表示），控制（黑色）和100:NM ZnPyr（红色）。灰色条表示的时间间隔平均电流幅值测量电流 - 电压（IV）曲线。在右边的箭头表示跨步电压。 C. KV7 100nM的ZnPyr电流增强的时间过程中测量具有连续的电压钳位在-20毫伏（巴）。 D.平均IV曲线组（n = 3）来自KV7电流密度之前记录的（对照组，实心圆），在治疗过程中与100nM的ZnPyr（空心圆），并用10μMXE991（实心三角形）的治疗过程中。减去非特异性泄漏电流帕斯莫尔等人所描述的:1。 ENT"&gt; <img src="/files/ftp_upload/4263/4263fig4.jpg" alt="图4"/> 图4。:ZnPyr，诱导放宽预缩与100 PM AVP的肠系膜动脉。 A.安装在照片的肠系膜上动脉的压力肌动描记的设置。 B.该船只的外径变动的时间过程中响应于应用100 PM AVP（开放式酒吧）随后应用的100nM的ZnPyr的（填充巴）。 C.条形图总结的100纳米ZnPyr引起的vasodilaion的结果。

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没有利益冲突的声明。

这里所描述的方法和实验方法相当强劲，并且能够产生明确的和可重复的结果时一丝不苟的细节。良好的电生理记录和收缩/扩张动脉段是依赖于细胞和动脉血管的健康。细胞制剂可以改变每一天，甚至使用相同的协议。隔离解决方案，可用于长达2周，但如果细胞制备的质量低，随之而来的两个天应准备新的隔离解决方案。我们已经发现，一批一批，因此隔离条件（时间的培养和酶的浓度）酶活?...

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 目录编号 </td><td>评论</td></tr><tr><td>氯化钠</td><td>西格玛</td><td> S5886 </td><td>剖析解决方案:145 沐浴液电:140 电:10内部解决方案 为心肌细胞的隔离解决方案:140 浴的压力myography解决方案:145 流明的压力myography解决方案:145 </td></tr><tr><td>氯化钾</td><td>西格玛</td><td> P5405 </td><td>剖析解决方案:4.7 沐浴液电:5.36 电:135内部解决方案 隔离解决方案肌:5.36 浴的压力myography解决方案:4.7 流明的压力myography解决方案:4.7 </td></tr><tr><td>钾EGTA </td><td>西格玛</td><td> E4378 </td><td>电:0.05内部解决方案</td></tr><tr><td> HEPES </td><td>西格玛</td><td> H9136 </td><td>沐浴液电:10 电:10内部解决方案 隔离解决方案，为心肌细胞*:10 </td></tr><tr><td>磷酸氢二钠</td><td>西格玛</td><td> S5136 </td><td>隔离解决方案，为心肌细胞*:0.34 </td></tr><tr><td>磷酸氢二钾</td><td>西格玛</td><td> P5655 </td><td>隔离解决方案，为心肌细胞*:0.44 </td></tr><tr><td>氯化镁</td><td>西格玛</td><td> M2393 </td><td>沐浴液电*:1.2 内部电:1解决方案 隔离解决方案肌:1.2 </td></tr><tr><td>氯化钙</td><td>西格玛</td><tD&gt; C7902 </td><td>沐浴液电:2 为心肌细胞的隔离解决方案:0.05 </td></tr><tr><td>磷酸钠</td><td> Fisher Scientific则</td><td> BP331-1 </td><td>剖析解决方案:1.2 浴的压力myography解决方案:1.2 流明的压力myography解决方案:1.2 </td></tr><tr><td>硫酸镁</td><td>西格玛</td><td> M2643 </td><td>剖析解决方案:1.17 浴的压力myography解决方案:1.17 流明的压力myography解决方案:1.17 </td></tr><tr><td> MOPS </td><td> Fisher Scientific则</td><td> BP308 </td><td>剖析解决方案: 浴的压力myography解决方案:3 流明的压力myography解决方案:3 </td></tr><tr><td>丙酮酸</td><td>西格玛</td><td> P4562 </td><td>剖析解决方案: 沐浴解决方案压力myography:2 流明的压力myography解决方案:2 </td></tr><tr><td> EDTA二水</td><td>研究有机物</td><td> 9572E </td><td>剖析解决方案:0.02 浴的压力myography解决方案:0.02 流明的压力myography解决方案:0.02 </td></tr><tr><td> D-葡萄糖</td><td>西格玛</td><td> G7021 </td><td>剖析解决方案: 沐浴液电:10 电:20内部解决方案 隔离解决方案，为心肌细胞*:10 浴的压力myography解决方案:5 流明的压力myography解决方案:5 </td></tr><tr><td>牛血清白蛋白</td><td>西格玛</td><td> A3912 </td><td>剖析解决方案:1％ 流明压力myography溶液:1％ </td></tr><tr><td> pH值</td><td></td><td></td><td>剖析搜索解决方案N:7.4 沐浴液电*:7.3 电:7.2的内部解决方案 隔离解决方案肌:7.2 浴的压力myography解决方案:7.4 流明的压力myography解决方案:7.4 </td></tr><tr><td>渗透压</td><td></td><td></td><td>剖析解决方案:300 沐浴液电:298 电:298内部解决方案 隔离解决方案肌:298 浴的压力myography解决方案:300 流明的压力myography解决方案:300 </td></tr><tr><td></td><td></td><td></td><td> * 11 </td></tr><tr><td></td><td></td><td></td><td> 表1中，在实验中使用的解决方案的组件。 </td></tr></tbody></table>

exploring arterial smooth muscle kv7 potassium channel function using patch clamp electrophysiology and pressure myography

电阻动脉的壁内的平滑肌细胞的收缩或松弛确定动脉直径并由此控制的血液流至容器内，并有助于全身血压。收缩过程中被限制主要是由胞内游离钙浓度（〔Ca 2 +] cyt的），它是依次由多种离子转运和频道控制。离子通道是常见的中间体，在信号转导通路激活血管活性激素对血管收缩或舒张功能。离子通道往往是有针对性的治疗药物，无论是有意（ 如钙离子通道阻断剂诱导的血管舒张和降低血压）或无意的（ 如引起不必要的心血管副作用）。 KV7（KCNQ）电压激活的钾离子通道最近被牵连的生理和治疗的重要塔尔格ETS调节平滑肌收缩。为了阐明KV7渠道在生理信号转导和治疗药物的行动中的具体作用，我们需要研究他们的活动是如何在细胞水平上调节，以及评估他们的贡献的背景下，完整的动脉。 大鼠肠系膜动脉提供了一个有用的模型系统。动脉可以很容易地解剖，结缔组织清除，并用孤立的动脉肌细胞膜片钳电生理准备，或为血管收缩剂/血管舒张反应的在相对生理条件下测量插管，加压。在这里，我们描述了这两种类型的测量方法，并提供一些例子，实验一体化设计可提供更清楚地了解这些离子通道在调节血管张力的作用。

Watch this Scientific Journal Video about Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography at JoVE.com

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

测量KV7（KCNQ）钾离子通道活性的孤立动脉细胞（中与测量大蟒/扩张的反应（使用压力myography），同时采用膜片钳电生理技术）可以揭示有关的重要信息的的KV7渠道作用在血管平滑肌生理和药理学。

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 ...

exploring-arterial-smooth-muscle-kv7-potassium-channel-function-using

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

15.1K 次观看.  Loyola University Chicago. 测量KV7（KCNQ）钾离子通道活性的孤立动脉细胞（中与测量大蟒/扩张的反应（使用压力myography），同时采用膜片钳电生理技术）可以揭示有关的重要信息的的KV7渠道作用在血管平滑肌生理和药理学。

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

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

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 ...

Focal Ca2+ Transient Detection in Smooth Muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Focal Ca2+ Transient Detection in Smooth Muscle

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

焦距钙 2 +</sup瞬态检测平滑肌

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.

人脐动脉平滑肌细胞的分离（HUASMC）

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

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 ...

Assessing Murine Resistance Artery Function Using Pressure Myography

Pressure myograph systems are exquisitely useful in the functional assessment of small arteries, pressurized to a suitable transmural pressure. The near physiological condition achieved in pressure myography permits in-depth characterization of intrinsic responses to pharmacological and physiological stimuli, which can be extrapolated to the in vivo behavior of the vascular bed. Pressure myograph has several advantages over conventional wire myographs. For example, smaller resistance vessels can be studied at tightly controlled and physiologically relevant intraluminal pressures. Here, we study the ability of 3rd order mesenteric arteries (3-4 mm long), preconstricted with phenylephrine, to vaso-relax in response to acetylcholine. Mesenteric arteries are mounted on two cannulas connected to a pressurized and sealed system that is maintained at constant pressure of 60 mmHg. The lumen and outer diameter of the vessel are continuously recorded using a video camera, allowing real time quantification of the vasoconstriction and vasorelaxation in response to phenylephrine and acetylcholine, respectively.
	To demonstrate the applicability of pressure myography to study the etiology of cardiovascular disease, we assessed endothelium-dependent vascular function in a murine model of systemic hypertension. Mice deficient in the &alpha;1 subunit of soluble guanylate cyclase (sGC&alpha;1-/-) are hypertensive when on a 129S6 (S6) background (sGC&alpha;1-/-S6) but not when on a C57BL/6 (B6) background (sGC&alpha;1-/-B6). Using pressure myography, we demonstrate that sGC&alpha;1-deficiency results in impaired endothelium-dependent vasorelaxation. The vascular dysfunction is more pronounced in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice, likely contributing to the higher blood pressure in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice. 
	Pressure myography is a relatively simple, but sensitive and mechanistically useful technique that can be used to assess the effect of various stimuli on vascular contraction and relaxation, thereby augmenting our insight into the mechanisms underlying cardiovascular disease.

In pressure myography, an intact small segment of a vessel is mounted onto two small cannulas and pressurized to a suitable luminal pressure. Here, we describe the method to measure vasorelaxation response of the mouse 3rd order mesenteric arteries in c57 and sGC&alpha;1-/- mice using pressure myography.

评估抗小鼠动脉功能使用压力Myography

Pressure myograph systems are  exquisitely useful in the functional assessment of small arteries, pressurized  to a suitable transmural pressure. The  ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

单通道细胞贴附膜片钳记录

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

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

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

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 ...

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum &#8211; if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.

平滑肌功能中分离出来的组织浴，应用药理学研究测量

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...

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.

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

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