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。
本协议中最重要的一步是准备一个长期稳定的伊势25。在我们的经验中, 不稳定的伊势不是很好的硅 (例如,离子选择树脂在伊势吸管侧上重新分布), 它可以通过势信号中的常数和不可控漂移来检测。此外, 在实验过程中, 应密切监测伊势中树脂的分布。沐浴液可以将离子选择性树脂推进到伊势吸管中, 在树脂25之前创建一列沐浴液。在这种情况下, 伊势将记录离子浓度从这一列, 成为响应变化的离子梯度在实验期间。在100毫秒9的范围内, 调整势的几何是获得势响应的关键, 并克服了与离子梯度的变化相比较的伊势时间响应的可能延迟。
所提出的方法的主要局限性是从膜片钳测量的运输活动在上皮细胞。当分离的支持和维持在悬浮, 极化上皮细胞可能会失去在顶端或侧膜上的转运体极化定位。
在研究极化上皮中的通道和转运体时, 应采用改进的膜片钳位测量方法。考虑到在通过提供的公式将 H+渐变转换为通量时, 单元格可能近似于圆柱体而不是球体是很重要的。在这种情况下, 公式应更改为, 其中λ是圆柱的长度, r 是圆柱半径, ad x 是从圆柱中点的径向距离, 在该位置上, 渐变是被测量的<img alt="Equation 7" src="/files/ftp_upload/56630/56630eq7.jpg"> 5。这一强大的技术研究 electroneutral 转运体的未来发展, 应侧重于修改的方法, 以优化记录细胞生长在单层。

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 border="0" cellpadding="0" cellspacing="0" width="961">
	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:367px;">Patch clamp Amplifier</td>
			<td style="width:219px;">Molecular Devices</td>
			<td style="width:101px;"></td>
			<td style="width:276px;"></td>
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		<tr height="34">
			<td height="34" style="height:34px;">Dual Channel Differential Electrometer (HiZ-223)</td>
			<td style="width:219px;">Warner Instruments</td>
			<td style="width:101px;">64-1650</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Differential Amplifier (DP-301)</td>
			<td style="width:219px;">Warner Instruments</td>
			<td style="width:101px;">64-0044</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Patch Clamp Software Based on MatLab</td>
			<td style="width:219px;">MatLab with acquisition toolbox</td>
			<td style="width:101px;"></td>
			<td>The capmeter software is recommended</td>
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		<tr height="34">
			<td height="34" style="height:34px;width:367px;">ThermoClamp-1 Temperature Control System</td>
			<td>&#160;AutoMate Scientific</td>
			<td style="width:101px;">03-11-LL</td>
			<td>In-line heater</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:367px;">Single-Channel Temperature Controller (TC-324C)</td>
			<td style="width:219px;">Warner Instruments</td>
			<td style="width:101px;">64-2400</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:367px;">Single-Barrel Standard Borosilicate Glass Tubing</td>
			<td>World Precision Instruments</td>
			<td style="width:101px;">1B120F-3&#160;&#160;</td>
			<td style="width:276px;">Used for ion selective electrodes</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:367px;">Micropipette Storage Jar</td>
			<td>World Precision Instruments</td>
			<td>E212</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:367px;">Bis(dimethylamino)dimethylsilane</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td>14755-100ML</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:367px;">Carbon tetrachloride</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td colspan="2">319961-500ML</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:367px;">Hydrogen ionophore I - cocktail B</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:101px;">95293</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:367px;">Thin Wall Borosilicate Tubing</td>
			<td style="width:219px;">&#160;Sutter Instrument</td>
			<td style="width:101px;">B200-156-15</td>
			<td style="width:276px;">Used for patch clamp pipette&#160;</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:367px;">Soft glass (Corning 8161 Patch Glass)</td>
			<td style="width:219px;">Warner Instruments</td>
			<td style="width:101px;">64-0815</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Silica Capillary Tubing (150um OD/75um ID)</td>
			<td style="width:219px;">Molex (Polymicro Technologies)</td>
			<td>106815-0018</td>
			<td style="width:276px;">Used for intra-pipette perfusion system</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Potassium chloride</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:367px;">Potassium aspartate&#160;</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td>A6558</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:367px;">EGTA</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:367px;">Magnesium chloride</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:101px;">M4880</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:367px;">Mg-ATP</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:101px;">A9187</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:367px;">HEPES</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:367px;">PIPES</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td>P6757</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:367px;">MOPS</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td>M1254</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:367px;">MES</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td>M3671</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:367px;">Calcium chloride</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td>C5670</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:367px;">Tris</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:101px;">T1503</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:367px;">Hydrochloric acid</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:367px;">Apyrase</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td>A6535</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Phosphatidylinositol(4,5) bisphosphate diC8</td>
			<td>Echelon Biosciences</td>
			<td>P-4508</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">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。
这些跟踪显示在图 2中。细胞被 heldin 为一个完整的细胞结构, 由一个吸管进行膜片夹紧, 而伊势被放置在细胞前面。在这个位置, 伊势记录了游离 H+接近细胞膜的浓度 (图 1A);然后, µm (或膜片钳吸管) 被手动移动到侧向 50, 并记录了解决方案中的自由 H+的浓度 (图 1B)。记录的电压差异, 对应的两个位置的补丁吸管与细胞在前面或远处的伊势, 是代表 NHE3 活动 (图 1C)。apyrase (atp-diphosphatase) 内吸管灌注降低 atp 的细胞内浓度, 随后降低肌含量5。与先前报告的研究结果一致5,1819, 由 apyrase 处理介导的减少的肌含量减少了 NHE3 活性 (~ 50%), 这被记录为电压下降振荡振幅 (图 2)。apyrase (5 单位/毫升) 对 NHE3 活性的影响是完全逆转的吸管内灌注 apyrase 结合2µM PI (34, 5) P3 (图 2和 3A)。这些发现得到了 PS120 细胞中记录的 NHE3 活动的支持, 稳定表达 NHE3 突变体 (NHE3-YRK), 无法绑定 phosphatidylinositides18。apyrase 单独或与 PIP3 的联合灌注对 NHE3-YRK 突变体的 NHE3 活性没有影响 (图 3B)。apyrase 治疗负鼠肾脏 (OK) 细胞内源性 NHE3 的活性也受到抑制 (3。PI (34, 5) P3 的内吸管灌注逆转了 apyrase 在 OK 细胞中诱导的 NHE3 活性的抑制作用 (图 3C)。这些结果表明, PI (34, 5) P3 对 NHE3 活性的影响不是细胞类型特异的。
接下来, 我们的目的是测试 PIP3 的特异性, 调节 apyrase 介导的 NHE3 活动的变化。我们通过分析其他 phosphoinositides (如 PI (45) P2) 灌注后 apyrase 依赖性还原对 NHE3 活性的影响。PI (45) P2 (10 µM) 不影响由 apyrase (图 4A) 介导的减少的 NHE3 活动。最后, 非水解 ATP 模拟的 AMP-PNP 的灌注并没有阻断 theapyrase 依赖性的 NHE3 活动抑制 (图 4B)。这些发现表明 pi (34, 5) P3 的作用, 但不是 pi (45) P2, 在 ATP 耗尽后 NHE3 的调节。NHE3 调节中 pi (45) P2 和 pi (34、5) P3 的分离作用是可能的, 这是因为在伊势测量和全细胞膜膜片钳记录5的同时, 细胞间的灌注 phosphoinositides。pi (34、5) P3 而非 pi (45) P2 在 NHE3 活动的调节中的具体作用对于确定不同的胞内 phosphoinositides 调节 NHE3 的功能是否有价值。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56630/56630fig1.jpg"> 图 1.H+磁通测量的示意性表示及他活性与 pH 微电极记录的电位差之间的相关性. 记录设置的示意图表示。初始实验设置的A.图。该细胞是由膜片钳吸管在整个细胞的配置, 并放置在前面的伊势。在这个位置上, 伊势记录了离细胞膜近的自由 H+的浓度。B.在该位置的解决方案中, 手动移动侧移50µm 并记录自由 H+的浓度。C.使用伊势记录的电压差异的图形表示。振荡从位置 A (细胞在伊势的前面) 到 B (细胞远离伊势) 是代表他活动。该软件使用登记的电压差异记录的伊势和产生方波扰动 (20 mV, 0.2 赫), 以监测细胞膜电容和质量的膜片钳密封。<a href="//cloudflare.jove.com/files/ftp_upload/56630/56630fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56630/56630fig2.jpg"> 图 2.一个记录集的例子, NHE3 运输活动在一个单一的 PS120 细胞稳定表达 NHE3-wt 前后灌注与 apyrase 和 PI (34, 5) P3 (PIP3)。 在最初的记录, 细胞被耗尽的 ATP 的内部吸管灌注 apyrase。NHE3 活动逐渐减少, 并通过 PIP3 内灌流进行恢复。Apyrase 加法由红色酒吧指示, 并且 PIP3 加法由绿色酒吧指示。<a href="//cloudflare.jove.com/files/ftp_upload/56630/56630fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56630/56630fig3.jpg"> 图 3.ATP 损耗和 PIP3 灌注对 PS120 和 OK 细胞 NHE3 转运体活动的影响。 apyrase 介导的 ATP 耗竭对 NHE3 活性的影响及保留 apyrase 对 PIP3 的 NHE3 活性的影响: A. PS120 细胞表达 NHE3-wt 或B.PS120 细胞表达 NHE3-YRK, NHE3 突变体无法绑定 phosphoinositides。C. OK 细胞表达内生 NHE3。实心圆圈显示单个实验的平均数据 (四在相同条件下进行的实验)。误差线表示的是标准误差 (SE)。* P < 0.05 与对照, 方差分析。$p < 0.5, $p < 0.01 apyrase vs. apyrase + PIP3, 方差分析。<a href="//cloudflare.jove.com/files/ftp_upload/56630/56630fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56630/56630fig4.jpg"> 图 4.PI (45) P2 (PIP2) 和 ANP-PNP 灌注对 ATP 耗尽后 NHE3 活性的影响。 A.的效果PIP2 或B.ANP-PNP 内流灌注对 apyrase 依赖性减少 PS120 细胞 NHE3 活性的影响表达 NHE3-wt. 实心圆圈显示单个实验的平均数据 (四在相同条件下进行的实验)。误差线代表的手段± < 0.05, ** < 0.01 与对照, 方差分析。<a href="//cloudflare.jove.com/files/ftp_upload/56630/56630fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

Universidad San Sebastian

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

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

计算机生成的动物模型的刺激

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Applying Microfluidics to Electrophysiology

Microfluidics can be integrated with standard electrophysiology techniques to allow new experimental modalities.  Specifically, the motivation for the microfluidic brain slice device is discussed including how the device docks to standard perfusion chambers and the technique of passive pumping which is used to deliver boluses of neuromodulators to the brain slice.  By simplifying the device design, we are able to achieve a practical solution to the current unmet electrophysiology need of applying multiple neuromodulators across multiple regions of the brain slice.  This is achieved by substituting the standard coverglass substrate of the perfusion chamber with a thin microfluidic device bonded to the coverglass substrate.  This was then attached to the perfusion chamber and small holes connect the open-well of the perfusion chamber to the microfluidic channels buried within the microfluidic substrate.  These microfluidic channels are interfaced with ports drilled into the edge of the perfusion chamber to access and deliver stimulants.  This project represents how the field of microfluidics is transitioning away from proof-of concept device demonstrations and into practical solutions for unmet experimental and clinical needs.

应用微流体电

Microfluidics can be integrated with standard electrophysiology techniques to allow new experimental modalities.  Specifically, the motivation for the ...

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

半自动小红桃光学心跳分析

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

脊髓电

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small molecule studies and drug development 4. Many epigenetic regulators have only recently been discovered and are still in the process of being classified. Among these enzymes are lysine demethylases which remove methyl groups from lysines on histones and other proteins. Due to the novel nature of this class of enzymes, few assays have been developed to study their activity. This has been a road block to both the classification and high throughput study of histone demethylases. Currently, very few demethylase assays exist. Those that do exist tend to be qualitative in nature and cannot simultaneously discern between the different lysine methylation states (un-, mono-, di- and tri-). Mass spectrometry is commonly used to determine demethylase activity but current mass spectrometric assays do not address whether differentially methylated peptides ionize differently. Differential ionization of methylated peptides makes comparing methylation states difficult and certainly not quantitative (Figure 1A). Thus available assays are not optimized for the comprehensive analysis of demethylase activity.
Here we describe a method called MassSQUIRM (mass spectrometric quantitation using isotopic reductive methylation) that is based on reductive methylation of amine groups with deuterated formaldehyde to force all lysines to be di-methylated, thus making them essentially the same chemical species and therefore ionize the same (Figure 1B). The only chemical difference following the reductive methylation is hydrogen and deuterium, which does not affect MALDI ionization efficiencies. The MassSQUIRM assay is specific for demethylase reaction products with un-, mono- or di-methylated lysines. The assay is also applicable to lysine methyltransferases giving the same reaction products. Here, we use a combination of reductive methylation chemistry and MALDI mass spectrometry to measure the activity of LSD1, a lysine demethylase capable of removing di- and mono-methyl groups, on a synthetic peptide substrate 5. This assay is simple and easily amenable to any lab with access to a MALDI mass spectrometer in lab or through a proteomics facility. The assay has ~8-fold dynamic range and is readily scalable to plate format 5.

We present a method for using MALDI mass spectrometry and reductive methylation chemistry to quantify changes in lysine methylation.

赖氨酸去甲基化酶活性的定量测量中的应用的MassSQUIRM

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small ...

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 ...

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can also be used to investigate and validate novel techniques and algorithms in volumetric medical imaging and therapies. Creating 3D high-resolution atlases of different organs1,2,3 is another application of histology volume reconstruction. This provides a resource for investigating tissue structures and the spatial relationship between various cellular features. We present an image registration approach for histology volume reconstruction, which uses a set of optical blockface images. The reconstructed histology volume represents a reliable shape of the processed specimen with no propagated post-processing registration error. The Hematoxylin and Eosin (H&#38;E) stained sections of two mouse mammary glands were registered to their corresponding blockface images using boundary points extracted from the edges of the specimen in histology and blockface images. The accuracy of the registration was visually evaluated. The alignment of the macrostructures of the mammary glands was also visually assessed at high resolution.
This study delineates the different steps of this image registration pipeline, ranging from excision of the mammary gland through to 3D histology volume reconstruction. While 2D histology images reveal the structural differences between pairs of sections, 3D histology volume provides the ability to visualize the differences in shape and volume of the mammary glands.

We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.

3维组织学音量和其研究小鼠乳腺腺体的应用改造

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

肿瘤细胞球体的高通量图像分析:一个用户友好的软件应用来测量球体的大小自动准确

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

幼虫活动的测量<em&gt;果蝇</em&gt;活动监视器

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...