Name: application of electrophysiology measurement to study the activity of electro-neutral transporters
Uploaded: 2018-02-03T14:00:00.000+00:00
Duration: 11 min 51 s
Description: Watch this Scientific Journal Video about Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters at JoVE.com

作者要感谢埃里克 Fishback (得梅因大学, 得梅因, 爱荷华州, 美国) 为他的协助拍摄和编辑视频。Dr. 标记多诺维茨 (约翰霍普金斯大学医学院, 巴尔的摩, 美国马里兰州) 提供了稳定表达 NHE3-wt 或 NHE3-YRK 的 PS120 成纤维样细胞。

注: 需要两个放大器来记录 electroneutral 转运体的活动, 与膜片钳夹, 膜片钳放大器, 以维持细胞在整个细胞配置和高阻抗放大 (静电计) 通过伊势记录传送器活动 (见材料表)。膜片钳放大器直接连接到采集板端子。静电计连接到用于过滤和放大信号的差分放大器。差分放大器连接到采集板端子。Capmeter, 用于监视单元容量的补丁钳软件, 已用于以前的研究20 (参见材料表)。记录室和灌注解决方案保持在37° c。
1. 太阳能管的制备
<ol><li>将管从硼硅酸盐玻璃毛细管拉到外径为 1.2 mm (参见材料表), 使用吸管拉出器2 。</li>
	<li>用 microforge 抛光吸管。吸管尖端的直径应为2-3 µm。</li>
	<li>将管放在管中, 将吸管端朝上 (请参阅材料表)。 注意: 在通风良好的化学通风柜中执行步骤 1.4-1.6。</li>
	<li>混合由1滴 (150 µL) 双 (二甲氨基) 二甲基硅烷和10滴四氯化碳组成的硅混合物 (参见材料表)。</li>
	<li>把硅的混合物倒入罐子里, 把盖子紧紧地关上。</li>
	<li>在室温下用硅混合物在化学油烟罩中孵育24-48 小时, 直到混合物完全蒸发。</li>
	<li>用适当的离子选择树脂回填吸管以创建大约2-4 毫米的柱。 注: 氢载体 I 鸡尾酒 B 可以用于记录他活动 (请参阅材料表)。</li>
	<li>轻叩吸管的尖端, 将离子选择的树脂分布到针尖上, 并将吸管垂直放置在罐内, 并将吸管顶端向下。</li>
	<li>Checkunder 的解剖显微镜, 树脂鸡尾酒已经达到了末端的尖端。如果需要, 施加积极的压力, 以推动树脂到年底。</li>
	<li>用适当的溶液 (〜100μ l) 回填半吸管。在这里, 使用100毫米氯化钾和10毫米 HEPES 在 pH 7.0 如果使用载体 I 鸡尾酒 B。</li>
	<li>将伊势连接到静电计, 并将伊势的尖端放入用于记录的沐浴液中。零静电计。</li>
	<li>通过改变浴液的 pH 值来测试 H+-选择性势响应。平均 H+-选择性势响应为每单位 pH 值 58 mV。</li>
	<li>定期评估对校准的伊势响应。</li>
</ol>2. 用于记录的膜片钳管的制备
注意: 推荐了唐纳德 Hilgemann 在他关于单通道录制21的文章中描述的方法。
<ol><li>在吸管拉拔器的2、22、23中, 从硼硅酸盐玻璃毛细管 (2.0 mmOD/1.5 mm ID) 上拉出管。</li>
	<li>使用 microforge 安装在倒置显微镜的舞台上, 使宽吸管尖端 (10-22 μ m)23。将软玻璃珠 (0.5 毫升) 融化在 microforge 的厚铂线上。 注: 需要较厚的铂线 (〜 0.5 mm) 制造巨型补丁管。</li>
	<li>位置的补丁吸管接近软玻璃珠, 并应用全热, 直到小费开始消退。铂金丝会微微向 centerduring 加热。</li>
	<li>关闭热量, 并推动吸管尖端进入软化玻璃珠。冷却线将收回和打破吸管尖端。</li>
	<li>重新应用热量来平滑吸管尖端, 并创建一个所需直径的吸管尖端。尖端的直径取决于要修补的细胞的大小 (本研究中 8-10 μ m)。</li>
</ol>3. 细胞的制备
<ol><li>加热磷酸盐缓冲盐水 (PBS) 没有 Ca2 +和镁2 +, 胰蛋白酶-EDTA 溶液和生长培养基到在37° c。</li>
	<li>用 PBS 清洗两次细胞。使用1毫升的缓冲区为35毫米直径细胞培养板。</li>
	<li>在35毫米直径细胞培养板中加入0.5 毫升的胰蛋白酶。 注意: 当细胞开始旋转时, 摇动盘子以消除附着细胞。</li>
	<li>通过添加5毫升 (35 毫米直径细胞培养板) 的完全生长培养基补充10% 胎牛血清停止胰蛋白酶作用。 注: 胰蛋白酶的反应时间为细胞型依赖性。</li>
	<li>把细胞放在一个摇动的振动器上, 以防形成结块。</li>
	<li>在 trypsinization 后使用2小时的单元格。 注意: 时间可能会因单元格线而异。</li>
</ol>4. 吸管内灌注系统的制备
<ol><li>切割适当大小的石英导管 (150 µm 外径/75 µm ID), 以准备吸管内灌注线。</li>
	<li>将石英管的一侧插入一个200µL 吸管尖端。把石英管粘在尖端上, 用锋利的刀片把尖端的末端切下来。</li>
	<li>将灌注线 (石英导管粘到尖端) 连接到一小块硅管, 作为吸管内灌注液的蓄水池。</li>
	<li>将硅管连接至注射器, 以产生正压。</li>
	<li>通过灌注口的吸管支架的聚乙烯管插入石英导管的自由端 (请参阅材料表)。</li>
	<li>用吸管溶液对灌注线进行评估。</li>
</ol>5. 解决方案的编写
<ol><li>为记录他 (115 毫米的天门冬氨酸钾 (KAsp), 1 毫米 EGTA, 0.5 毫米氯化镁2, 10 毫米镁 ATP, 12.5 毫米 HEPES, 12.5 毫米管, 12.5 毫米拖把, 和 12.5 mm MES, pH 6.0 调整与天门冬氨酸) 准备一个沉重缓冲的吸管解决方案。 注: 准备库存吸管溶液和过滤器。在录音前加入 Mg ATP。</li>
	<li>每次准备一个新的沐浴液 (140 毫米氯化钠, 2 毫米 CaCl2, 2 毫米氯化镁2, 和0.1 毫米三盐酸 pH 8.2)。</li>
</ol>6. 传送者活动记录
<ol><li>保持记录室和所有解决方案在37° c, 因为运输者活动是温度依赖。</li>
	<li>将细胞放入记录室。等待, 直到他们下降到会议厅底部, 但不让他们附加到会议厅。</li>
	<li>选择一个合适的单元格。用眼千分尺测量细胞的直径。选择具有相似直径的单元格。 注意: 一般来说, 一个大的细胞会有更多的细胞表面, 可能更多的转运蛋白在其细胞膜上表达, 从而也有更多的运输活动。然而, 这不一定是正确的, 因为运输活动取决于特定的运输者和细胞类型的研究。</li>
	<li>将离子选择性微电极吸管安装到微上, 将膜片夹钳与胞内溶液一起回填, 确保溶液到达吸管的尖端2。 注意: 它可能需要几个温和的水龙头, 以消除在吸管尖端的气泡。</li>
	<li>将膜片夹钳移到电生理学的头部阶段设置为2,22,23。 注意: 重要的是保持头部在大约45度角的水平轴, 因为这确保了一个入口的角度, 适合形成一个高耐药密封的吸管。</li>
	<li>将小的正压 (~ 5 厘米 H2O) 应用到膜片吸管, 以减少堵塞笔尖的机会, 并将其放入沐浴液中。</li>
	<li>一旦尖端靠近细胞, 停止移动膜片钳吸管。</li>
	<li>释放正压, 而吸管触及细胞, 并施加轻微的负压 (〜5厘米 H2O), 以获得 > 1000 ω (一个千兆欧姆) 密封在细胞膜上的补丁吸管的23。</li>
	<li>将贴片吸管与细胞放在同一个焦平面上, 和移动的补丁吸管与细胞附近的伊势 (〜 5-10 µm)2,5 , 但避免让该细胞联系伊势 (图 1A)。</li>
	<li>确保保持电位为 0 mV。</li>
	<li>用短吸力系列断裂密封, 获得全细胞构型。</li>
	<li>移动的伊势 (或膜片钳吸管) 缓慢左-右或向上-记录运输者的活动 (图 1B)。</li>
	<li>记录至少5-8 峰值 (图 1C, 从 A 到 B)。</li>
	<li>在记录过程中应用方波扰动来监测密封和细胞膜电容的质量 (图 1)。</li>
	<li>从单元格表面和大容量解决方案 (h<img alt="Equation 1" src="/files/ftp_upload/56630/56630eq1.jpg">+) 之间的自由 h+浓度差估计 h+通量 () <img alt="Equation 2" src="/files/ftp_upload/56630/56630eq2.jpg"> <img alt="Equation 3" src="/files/ftp_upload/56630/56630eq3v2.jpg"> 注意: 和分别为 H <img alt="Equation 4" src="/files/ftp_upload/56630/56630eq4.jpg"> <img alt="Equation 5" src="/files/ftp_upload/56630/56630eq5.jpg"> +和缓冲区扩散系数, BT 是总缓冲区集中, KD 是该缓冲区的离解常数, r是单元格半径, 并且确定在自由 h <img alt="Equation 6" src="/files/ftp_upload/56630/56630eq6.jpg"> +中对小的更改的绑定 h+的集中状态的稳态变化。H+表示解中的自由质子。为了与先前的研究相一致, H+通量通过计算 pA<img alt="Equation 1" src="/files/ftp_upload/56630/56630eq1.jpg">3、5中的流量 (/法拉第) 的当前等效性来转换为电生理磁通单元。</li>
	<li>将质子通量正常化到可以从细胞大小或细胞膜电容计算出来的细胞表面。</li>
</ol>

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作者没有利益冲突透露。

尽管运输者具有重要的作用, 但用于研究其活动的方法是无效和不充分的。一个限制是, 可用的方法测量离子运动介导的运输活动, 而不考虑波动的细胞内离子组成在实验中4。该方法确保了细胞外和胞内离子组成的精确控制, 并为修饰细胞内离子、蛋白质和脂质组成提供了有力的工具3,4,5。
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离子和溶质在细胞膜上的传输对于细胞的存活和有机体的生存是必不可少的1。离子和溶质的选择性运输是通过一系列专门的通道和转运蛋白来实现的。这些蛋白质的突变往往导致各种临床条件, 呈现通道和转运蛋白潜在靶点的药理治疗1。遗憾的是, 了解通道和传送器功能和调节的机制通常受到可用来研究其活动的方法的限制2,3,4。
具体地说, 转运体可以大致分为两大类, 这取决于它们在溶质转运过程中是否改变了细胞跨膜电位: 改变 electrogenic 离子转运蛋白 [如,磷酸磷酸钠共运输 2a (NaPi2a), 钠钙交换器 (NCX), etc。或非改变的 electroneutral 离子转运体 [例如,钠-质子交换器 (他), 氯化钠 co 转运体, NaPi2c,等。利用放射性同位素和荧光膜-permeant 染料的吸收, 对两类转运体的活性进行了广泛的研究2。这两种方法都通过测量特定细胞质离子的体积浓度的变化来估计转运体的活动, 这两种方法都有局限性, 如灵敏度和时间分辨率, 以及对细胞内的控制不足。环境.事实上, 许多运输者的活动取决于运载的离子的细胞质集中 (例如, NHE3, NCX), 并且这些离子集中的变动在调控运输者活动将扮演一个重要角色2,3,5. 用经典方法对这些调节机构进行精确的测量是有限的。
为了克服这些限制, 膜片钳方法用于研究传送器活动2,6。具体地说, 自引用离子选择性电极 (太阳能)7,8与补丁夹紧系统相结合, 最近允许测量 electroneutral 传送器活动3,4,5. 太阳能根据事实运输者活动创造离子梯度在接近的接近度细胞膜。在一个重复的、振荡的时尚中, 移动到离细胞膜的一个势值记录了电压差 (µV)。电压差可以转换成离子通量的值, 使用的校准方法, 适用菲克的第一定律的扩散2,9。当太阳能被用来检测离子从细胞中移出的通量时, 采用膜片钳的方法来控制细胞膜电位和胞内离子组成。此外, 该巨型膜片钳技术的应用, 不仅可以修改的细胞内组成的离子, 但也脂质和蛋白质3,5。
总之, 膜片钳方法的通用性与其他方法相比, 研究运输活动, 使补丁夹紧适合克服这些其他方法的共同限制。自参照太阳能和膜片钳技术的结合提供了在严格控制的实验环境中测量 electroneutral 转运体活动的独特可能性, 并发现新的生物物理和分子细胞膜传输特性3,4,5。该方法已成功地用于研究他的活动。哺乳动物他蛋白家族催化胞外钠的 electroneutral 净交换 (na+) 的细胞内质子 (H+)10,11使用内向 Na+渐变。在哺乳动物中, 他蛋白家族包括11相关的蛋白质 (NHE1-9 和 NHA1-2) 和精子特异的他10,12,13。
NHEs (SLC9a 家族) 被发现无所不在在大多数生物体从简单的原到更高的真核生物和参与了各种重要的细胞功能10,11, 包括控制细胞盐防御原, 维持酸碱的稳态和细胞体积, 并调节各种专门上皮的盐和水的吸收10,12,14,15。通过几项研究, 确定了 NHEs 的关键生物学作用及其功能的意义;然而, 由于方法论上的限制, 很少有研究对哺乳动物 NHEs 的生物物理和分子性质进行调查.4。近年来, 自引用太阳能在全细胞贴片钳夹中的应用, 揭示了由离子、蛋白质和磷脂的细胞内浓度变化所调控的他异构体的新分子机制3, 4。
具体地说, 本手稿中提供的协议概述了研究他型 3 (NHE3) 的活动和规则的方法和方法, 这是一个在 Na+, Cl-, 小贩3的吸收中的主要参与者.刷膜的肾脏和肠道上皮细胞14,16。报告了 NHE3 活性对胞内 phosphoinositides (phosphatidylinositide 45--1,6-[pi (45) P2] 和 phosphatidylinositide 34, 5-三磷酸 [pi (34, 5] P3]) 灵敏度的新认识。细胞传输蛋白, 如通道和转运体, 由 phosphoinositides17调节, NHE3 直接绑定 pi (45) P2 和 pi (34, 5) P318。根据目前的文献, 无论是肌可能是相关的生理学或病理生理调节的 NHE35,18,19。我们的研究结果支持 pi (45) P2 和 pi (34, 5) P3 在调节 NHE3 活动中的单独角色。这种区别是可能的, 因为应用的伊势技术与全细胞膜片钳记录结合。这项技术还允许控制肌细胞的内容通过细胞内灌注不同的 phosphoinositides 在测量 NHE3 活动。

<table><tbody><tr><td>Patch clamp Amplifier</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Dual Channel Differential Electrometer (HiZ-223)</td><td>Warner Instruments</td><td>64-1650</td><td></td></tr><tr><td>Differential Amplifier (DP-301)</td><td>Warner Instruments</td><td>64-0044</td><td></td></tr><tr><td>Patch Clamp Software Based on MatLab</td><td>MatLab with acquisition toolbox</td><td></td><td>The capmeter software is recommended</td></tr><tr><td>ThermoClamp-1 Temperature Control System</td><td>&amp;nbsp;AutoMate Scientific</td><td>03-11-LL</td><td>In-line heater</td></tr><tr><td>Single-Channel Temperature Controller (TC-324C)</td><td>Warner Instruments</td><td>64-2400</td><td></td></tr><tr><td>Single-Barrel Standard Borosilicate Glass Tubing</td><td>World Precision Instruments</td><td>1B120F-3&amp;nbsp;&amp;nbsp;</td><td>Used for ion selective electrodes</td></tr><tr><td>Micropipette Storage Jar</td><td>World Precision Instruments</td><td>E212</td><td></td></tr><tr><td>Bis(dimethylamino)dimethylsilane</td><td>Sigma-Aldrich</td><td>14755-100ML</td><td></td></tr><tr><td>Carbon tetrachloride</td><td>Sigma-Aldrich</td><td>319961-500ML</td><td></td></tr><tr><td>Hydrogen ionophore I - cocktail B</td><td>Sigma-Aldrich</td><td>95293</td><td></td></tr><tr><td>Thin Wall Borosilicate Tubing</td><td>&amp;nbsp;Sutter Instrument</td><td>B200-156-15</td><td>Used for patch clamp pipette&amp;nbsp;</td></tr><tr><td>Soft glass (Corning 8161 Patch Glass)</td><td>Warner Instruments</td><td>64-0815</td><td></td></tr><tr><td>Silica Capillary Tubing (150um OD/75um ID)</td><td>Molex (Polymicro Technologies)</td><td>106815-0018</td><td>Used for intra-pipette perfusion system</td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich</td><td>P9333</td><td></td></tr><tr><td>Potassium aspartate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A6558</td><td></td></tr><tr><td>EGTA</td><td>Sigma-Aldrich</td><td>E3889</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>M4880</td><td></td></tr><tr><td>Mg-ATP</td><td>Sigma-Aldrich</td><td>A9187</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>PIPES</td><td>Sigma-Aldrich</td><td>P6757</td><td></td></tr><tr><td>MOPS</td><td>Sigma-Aldrich</td><td>M1254</td><td></td></tr><tr><td>MES</td><td>Sigma-Aldrich</td><td>M3671</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma-Aldrich</td><td>C5670</td><td></td></tr><tr><td>Tris</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Sigma-Aldrich</td><td>H1758</td><td></td></tr><tr><td>Apyrase</td><td>Sigma-Aldrich</td><td>A6535</td><td></td></tr><tr><td>Phosphatidylinositol(4,5) bisphosphate diC8</td><td>Echelon Biosciences</td><td>P-4508</td><td></td></tr><tr><td>Phosphatidylinositol 3,4,5-trisphosphate diC8</td><td>Echelon Biosciences</td><td>P-3908</td><td></td></tr></tbody></table>

application of electrophysiology measurement to study the activity of electro-neutral transporters

离子通过细胞膜的传输确保了细胞内和外部的离子含量的精细控制, 这对于细胞存活是必不可少的。这些运输机制由专业运输者蛋白质的活动斡旋。具体来说, pH 动态是精细控制的等离子膜质子 (H+) 挤出系统, 如 Na+/H+交换器 (他) 蛋白家族。尽管广泛努力研究他规则的机制, 但我们目前对他家族的生物物理和分子性质的理解是不充分的, 因为有效地测量他活动的方法的有效性有限。.在这份手稿中, 我们使用了 h+选择性电极在全细胞膜膜片钳记录, 以测量他诱导的 h+通量。我们提出这种方法, 以克服一些典型的使用方法来测量他活动, 如放射性吸收和荧光膜 permeants 的一些限制。使用所述方法测量他活动, 可以提高灵敏度和时间分辨率, 更有效地控制胞内 H+浓度。H+-选择性电极基于这样一个事实, 即运输者活动在靠近细胞膜的附近产生离子梯度。一个 h+-选择性电极在一个重复的、振荡的方式上移动到和远离细胞膜, 记录一个依赖于 h+通量的电压差。当 h+-选择性电极用于检测从电池中移出的 h+磁通时, 采用全细胞构型的膜片钳法来控制细胞内离子的组成。此外, 应用巨型膜片钳技术, 不仅可以改变细胞内的组成, 不仅离子, 但也脂质。用该技术方法测定了他型 3 (NHE3) 的转运体活性, 研究了 phosphoinositides NHE3 调节的分子基础。

应用全细胞膜膜片钳记录中的自参考势, 研究了 phosphoinositides 对 NHE3 活性的调节作用。PS120 成纤维样细胞24, 缺乏内源性细胞膜 NHEs 的表达。NHE3 野生型 (NHE3-wt) 或 NHE3 突变体不束缚 phosphoinositides [酪氨酸 501, 精氨酸503和赖氨酸505用丙氨酸取代, Y501A/R503A/K505A (NHE3-YRK)] 稳定表达在 PS120 成纤维样细胞18。
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Watch this Scientific Journal Video about Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters at JoVE.com

Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

本文介绍了质子选择性电极和膜片夹紧方法在质子传输系统中的应用。这些方法克服了一般用于研究质子转运活动的一些技术限制, 如灵敏度适中、时间分辨力和细胞内环境控制不足。

电生理测量在中性转运体活性研究中的应用

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. ...

application-electrophysiology-measurement-to-study-activity-electro

Research

JoVE Journal

Biology

6.9K 次观看.  Des Moines University. 本文介绍了质子选择性电极和膜片夹紧方法在质子传输系统中的应用。这些方法克服了一般用于研究质子转运活动的一些技术限制, 如灵敏度适中、时间分辨力和细胞内环境控制不足。

视频: Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

Measuring the Induced Membrane Voltage with Di-8-ANEPPS

Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell membrane, resulting in a voltage across the membrane. This voltage, termed the induced membrane voltage (also induced transmembrane voltage, or induced transmembrane potential difference) and denoted by &Delta;&Phi;, exists only as long as the external field is present. If the resting voltage is present on the membrane, the induced voltage superimposes (adds) onto it. By using one of the potentiometric fluorescent dyes, such as di-8-ANEPPS, it is possible to observe the variations of &Delta;&Phi; on the cell membrane and to measure its value noninvasively. di-8-ANEPPS becomes strongly fluorescent when bound to the lipid bilayer of the cell membrane, with the change of the fluorescence intensity proportional to the change of &Delta;&Phi;. This video shows the protocol for measuring &Delta;&Phi; using di-8-ANEPPS and also demonstrates the influence of cell shape on the amplitude and spatial distribution of &Delta;&Phi;.

External electric field induces a voltage on the membrane of a cell, termed the induced membrane voltage (&Delta;&Phi;). By using the potentiometric dye di-8-ANEPPS, it is possible to measure the &Delta;&Phi; noninvasively. This video shows the protocol for measuring &Delta;&Phi; using di-8-ANEPPS.

诱导膜电压测量与DI - 8 - ANEPPS

Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell ...

科研

生物学

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 ...

生物化学

Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting

Potassium chloride cotransporters 2 (KCC2) is a member of the solute carrier family 12 (SLC12) of cation-chloride-cotransporters (CCCs), found exclusively in the neuron and is essential for the proper functioning of Cl- homeostasis and consequently functional GABAergic inhibition. Failure in proper regulation of KCC2 is deleterious and has been associated with the prevalence of several neurological diseases, including epilepsy. There has been considerable progress with regard to understanding the mechanisms involved in the regulation of KCC2, accredited to the development of techniques that enable researchers to study its functions and activities; either via direct (assessing kinase regulatory sites phosphorylation) or indirect (observing and monitoring GABA activity) investigations. Here, the protocol highlights how to investigate KCC2 phosphorylation at kinase regulatory sites - Thr906 and Thr1007- using western blotting technique. There are other classic methods used to directly measure KCC2 activity, such as rubidium ion and thallium ion uptake assay. Further techniques such as patch-clamp-electrophysiology are used to measure GABA activity; hence, indirectly reflecting activated and/or inactivated KCC2 as informed by the assessment of intracellular chloride ion homeostasis. A few of these additional techniques will be briefly discussed in this manuscript.

The present protocol highlights the application of western blotting technique to study the functions and activities of neuronal K-Cl co-transporter KCC2. The protocol describes the investigation of KCC2 phosphorylation at kinase regulatory sites Thr906/1007 via western blotting. Also, additional methods to confirm KCC2 activity are briefly highlighted in this text.

使用蛋白质印迹法研究神经元K-Cl共转运蛋白KCC2的功能和活性

Potassium chloride cotransporters 2 (KCC2) is a member of the solute carrier family 12 (SLC12) of cation-chloride-cotransporters (CCCs), found exclusively ...

Recapitulation of an Ion Channel IV Curve Using Frequency Components

INTRODUCTION: Presently, there are no established methods to measure multiple ion channel types simultaneously and decompose the measured current into portions attributable to each channel type. This study demonstrates how impedance spectroscopy may be used to identify specific frequencies that highly correlate with the steady state current amplitude measured during voltage clamp experiments. The method involves inserting a noise function containing specific frequencies into the voltage step protocol. In the work presented, a model cell is used to demonstrate that no high correlations are introduced by the voltage clamp circuitry, and also that the noise function itself does not introduce any high correlations when no ion channels are present. This validation is necessary before the technique can be applied to preparations containing ion channels. The purpose of the protocol presented is to demonstrate how to characterize the frequency response of a single ion channel type to a noise function. Once specific frequencies have been identified in an individual channel type, they can be used to reproduce the steady state current voltage (IV) curve. Frequencies that highly correlate with one channel type and minimally correlate with other channel types may then be used to estimate the current contribution of multiple channel types measured simultaneously.
METHODS: Voltage clamp measurements were performed on a model cell using a standard voltage step protocol (-150 to +50 mV, 5mV steps). Noise functions containing equal magnitudes of 1-15 kHz frequencies (zero to peak amplitudes: 50 or 100mV) were inserted into each voltage step. The real component of the Fast Fourier transform (FFT) of the output signal was calculated with and without noise for each step potential. The magnitude of each frequency as a function of voltage step was correlated with the current amplitude at the corresponding voltages.
RESULTS AND CONCLUSIONS: In the absence of noise (control), magnitudes of all frequencies except the DC component correlated poorly (|R|&lt;0.5) with the IV curve, whereas the DC component had a correlation coefficient greater than 0.999 in all measurements. The quality of correlation between individual frequencies and the IV curve did not change when a noise function was added to the voltage step protocol. Likewise, increasing the amplitude of the noise function also did not increase the correlation. Control measurements demonstrate that the voltage clamp circuitry by itself does not cause any frequencies above 0 Hz to highly correlate with the steady-state IV curve. Likewise, measurements in the presence of the noise function demonstrate that the noise function does not cause any frequencies above 0 Hz to correlate with the steady-state IV curve when no ion channels are present. Based on this verification, the method can now be applied to preparations containing a single ion channel type with the intent of identifying frequencies whose amplitudes correlate specifically with that channel type.

There are technical obstacles to measuring current flux through multiple ion channels simultaneously, and later discerning what portion of the transmembrane current is due to each channel type. To address this need, this method presents a way to generate the IV curve of individual channel types using specific frequency components.

重演的离子通道IV曲线的使用频率分量

INTRODUCTION: Presently, there are no established methods to measure multiple ion channel types simultaneously and decompose the measured current into ...

A Micro-agar Salt Bridge Electrode for Analyzing the Proton Turnover Rate of Recombinant Membrane Proteins

To date, more than 50% of all pharmacological drugs target the transport kinetics of membrane proteins. The electrophysiological characterization of membrane carrier proteins reconstituted in lipid bilayer membranes is a powerful but delicate method for the assessment of their physicochemical and pharmacological properties. The substrate turnover number is a unique parameter that allows the comparison of the activity of different membrane proteins. In an electrogenic transport, the gradient of the translocated substrate creates a membrane potential that directly correlates to the substrate turnover rate of the protein. By using silver chloride electrodes, a diffusion potential, also called liquid junction potential, is induced, which alters electrode potential and significantly disturbs precise membrane potential measurements. Diffusion potential can be minimized by a salt bridge, which balances electrode potential. In this article, a micro-agar salt bridge is designed to improve the electrophysiological set-up, which uses micropipettes for the membrane formation. The salt solution is filled into a microcapillary pipette tip, stabilized by the addition of agarose, and can be easily mounted to a standard electrode. The electrode potential of a micro-salt bridge electrode is more stable compared to a standard electrode. The implementation of this system stabilizes electrode potential and allows more precise measurements of membrane potential generated by a pH gradient. Using this system, the proton turnover rates of the mitochondrial carriers UCP1 and UCP3 are reinvestigated and compared to earlier measurements.

In electrophysiological measurements, the presence of a diffusion potential disturbs the precise measurement of the reverse potential by altering the electrode potential. Using a micro-agar salt bridge, the impact of the diffusion potential is minimized, which allows a more precise measurement of substrate turnover numbers of reconstituted recombinant membrane proteins.

一种微琼脂盐桥电极分析重组膜蛋白质子周转率

To date, more than 50% of all pharmacological drugs target the transport kinetics of membrane proteins. The electrophysiological characterization of ...

神经科学

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+,K+-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+ or Li+ transport by Na+,K+- or gastric H+,K+-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb+ (Li+) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na+/2K+ transport stoichiometry of the Na+,K+-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+,K+-ATPase. In principle, the assay is not limited to K+-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

测量离子转运的Na，K，H，K-ATP酶活性的<em&gt;爪蟾</em卵母细胞的原子吸收分光光度法：另一种放射性同位素检测

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating ...

Introduction to Solid Supported Membrane Based Electrophysiology

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated sensor chip and a phosphatidylcholine monolayer on top. This assembly is mounted into a cuvette system containing the reference electrode, a chlorinated silver wire.
After adsorption of membrane fragments or proteoliposomes containing the membrane protein of interest, a fast solution exchange is used to induce the transport activity of the membrane protein. In the single solution exchange protocol two solutions, one non-activating and one activating solution, are needed. The flow is controlled by pressurized air and a valve and tubing system within a faraday cage.
The kinetics of the electrogenic transport activity is obtained via capacitive coupling between the SSM and the proteoliposomes or membrane fragments. The method, therefore, yields only transient currents. The peak current represents the stationary transport activity. The time dependent transporter currents can be reconstructed by circuit analysis.
This method is especially suited for prokaryotic transporters or eukaryotic transporters from intracellular membranes, which cannot be investigated by patch clamp or voltage clamp methods.

Here we present an electrophysiological method based on solid supported membranes with focus on its applications for the characterization of electrogenic membrane transporters.

固体支持基于膜的电

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated ...

<meta charset="utf8"></head><body>评估电穿孔对基因工程加标 HEK 细胞的影响

Monitoring Electroporation-Induced Changes in Action Potential Generation in Genetically Engineered Tet-On Spiking HEK cells

Excitable cells such as&#160;neuronal and muscle cells can be primary targets in rapidly emerging electroporation-based treatments. However, they can be affected by electric pulses even in therapies where they are not the primary targets, and this can cause adverse side effects. Therefore, to optimize the electroporation-based treatments of excitable and non-excitable tissues, there is a need to study the effects of electric pulses on excitable cells, their ion channels, and excitability in vitro. For this purpose, a protocol was developed for optical monitoring of changes in action potential generation due to electroporation on a simple excitable cell model of genetically engineered tet-on spiking HEK cells. With the use of a fluorescent potentiometric dye, the changes in transmembrane voltage were monitored under a fluorescence microscope, and relevant parameters of cell responses were extracted automatically with a MATLAB application. This way, the excitable cell responses to different electric pulses and the interplay between excitation and electroporation could&#160;be efficiently evaluated.

<meta charset="utf8"></head><body>监测电穿孔诱导的基因工程 Tet-On 加标 HEK 细胞中动作电位产生的变化

For studying responses of excitable cells in vitro, the protocol describes optical monitoring of changes in action potential generation due to electroporation on a simple excitable cell model of genetically engineered tet-on spiking HEK cells as well as changes in transmembrane voltage with automated extraction of relevant parameters.

监测电穿孔诱导的基因工程 Tet-On 加标 HEK 细胞中动作电位产生的变化

Excitable cells such as&#160;neuronal and muscle cells can be primary targets in rapidly emerging electroporation-based treatments. However, they can be ...

电生理测量在中性转运体活性研究中的应用

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诱导膜电压测量与DI - 8 - ANEPPS

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

使用蛋白质印迹法研究神经元K-Cl共转运蛋白KCC2的功能和活性

重演的离子通道IV曲线的使用频率分量

一种微琼脂盐桥电极分析重组膜蛋白质子周转率

测量离子转运的Na，K，H，K-ATP酶活性的

固体支持基于膜的电

监测电穿孔诱导的基因工程 Tet-On 加标 HEK 细胞中动作电位产生的变化

监测电穿孔诱导的基因工程 Tet-On 加标 HEK 细胞中动作电位产生的变化

监测电穿孔诱导的基因工程 Tet-On 加标 HEK 细胞中动作电位产生的变化