这项工作是由国家卫生部授予R21 - HL094828 - 01授予博​​士Poelzing研究院支持。

1。准备噪声功能和输入信号<ol><li>创建噪音的功能，包含所需的频率成分。这可以通过在频域描述所需的频率成分，然后计算逆快速傅立叶变换。在这项研究中，1 - 15千赫。傅立叶变换和逆傅里叶变换在这项研究中中所描述的所有计算使用Matlab的FFT和IFFT功能。 </li><li>适当规模的噪音功能的振幅。在这项研究中，噪声功能，缩放等零到峰值噪声功能的幅度是50或100毫伏。 </li><li>适用于正在使用的数据采集软件的使用方法，创建一个激励文件。对于Clampex 8，首先要创建一个文本文件，用适当的头。标题下方，插入在第一列的单次扫描的时间增量。时间增量应在测量中使用的采样间隔相同的时间间隔。对于每个电压阶跃协议的扫描插入在每个时间步所需的精确电压。这应包括噪音的功能。 </li></ol> 2。执行电压钳测量<ol><li>内采集软件兼容与以前生成的刺激文件的测量协议。在Clampex，有一个菜单，允许用户与当前协议相关联的激励文件。 </li><li>将模型（或生物）细胞的测量设备。 </li><li>如期进行实验。控制的目的，确保包括定期测量，不包括任何噪音功能。 </li></ol> 3。发布实验分析<ol><li>计算个人录制的IV曲线。如果录音是在稳定状态时的噪音功能，IV曲线可以创建使用稳态部分的录音时间外的噪音功能的范围。如果录制处于稳定状态，噪音的功能可能会干扰IV曲线的计算，所以，第二个记录应无噪音的功能目前。 </li><li>对于中的每个记录的电压步骤，计算快速傅立叶变换的噪声功能插入记录的部分。结合傅立叶变换，每个电压阶跃到amxn矩阵，其中m是在FFT的频率数，而n是电压步骤。在此配置中，矩阵的每一行代表一个单频的振幅电压在实验中的所有步骤。 </li><li>针对每一个频率（即在上述矩阵的每一行）3.1产生的IV曲线相关联行和记录的相关系数。 </li><li>画出相关系数随频率的可视化高度相关IV曲线的频率。由于直流分量载内的FFT第一频率，这个频率的相关系数应始终&gt; 0.99。 </li></ol> 4。代表性的成果： 代表钳位电压测量显示为细胞模型（图1A）（图1B）插入电压阶跃协议噪音功能。 IV曲线计算模型细胞（图1C）。每次扫描，从图1的录音，FFT计算超过时限插入噪声功能（参见图1A，1B红色框）。图2a和2b显示分别显示在图1A和1B，录音计算的FFT。目视检查后，出现的直流分量（以红色突出显示），以模仿的IV曲线的形状。无噪音的功能，直流以上的所有频率似乎已经接近零振幅（图2A）。噪音的功能是当插入，1至15千赫的频率有明显的视觉振幅（图2B）。图3显示的关联比对IV曲线的电压步骤的范围内各个频率振幅的结果。图3A - C的显示时，实验控制的条件下（无噪声功能）和噪声幅度分别为50和100 mV时，相关系数。请注意在所有情况下，出现的直流成分几乎完全相关的IV曲线。事实上，所有的录音，这个频率的相关系数大于0.99（R = 0.9996 ± 1E - 5，平均±标准偏差）。当我们在图3A（控制条件），有没有频率除了直流分量，其幅度明显IV曲线相关。具体地说，这些频率的相关系数大于0.5。最低幅度噪声功能（50毫伏）插入后，这些相同的频率仍然相关系数小于0.5。为相关系数SE的频率也没有成为噪声的幅度大于0.5时提高到100 mV。 <img src="/files/ftp_upload/2361/2361fig1.jpg" alt="图1" /> 图1模型细胞录音：电压钳记录一个细胞模型（1A）及（1B）电压步进协议中包含一个噪音功能。对于第一个和最后一个20毫秒每次扫描，潜力保持在控股的潜力（0 mV时）。每个电压步长80毫秒，和噪音功能的步骤开始后插入40毫秒。噪声功能持续时间30毫秒，含有1至15千赫的频率。电压为加强回回，在-150至+50 mV的5 mV递增。还表现为细胞模型的IV曲线（1C）。为了使录音更容易阅读，只有五分之一扫描是包括在1A和1B，但所有的扫描，在1C。 <img src="/files/ftp_upload/2361/2361fig2.jpg" alt="图2" /> 图2 FFT录音：FFT的计算为30毫秒每次扫描部分噪音的功能是要插入（红色方块图1A，1B范围内）。图2a和2b显示的FFT计算分别与无噪音功能。再次，为清晰起见，，只有FFT五分之一的扫描图中，但所有的扫描，在未来的计算。 <img src="/files/ftp_upload/2361/2361fig3.jpg" alt="图3" /> 图3四频率的相关性：录制的IV曲线和对个人的电压步骤范围频率的振幅之间的相关性的结果显示。图1A - 1C显示相关系数分别控制的条件下从0到20 kHz的频率，并在50或100毫伏的噪声功能的存在。 <img src="/files/ftp_upload/2361/2361fig4.jpg" alt="图4" /> 图4再创造的IV曲线：细胞模型（图1C相同）和两个频率的幅度IV曲线叠加。第一频率的直流分量（R = 0.995），第二个是随机选择的频率低相关性（r = 0.3212）。频率振幅缩小约IV曲线的幅度相同。

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有技术障碍，目前防止研究人员同时测量后确定多大的电流应归功于每个通道类型的意图的多种离子通道类型。由于此限制，通常是研究离子通道是单独使用，如电压，电流和动作电位钳技术（1）为了研究异源表达系统通常使用的通道类型，（2）当工作从组织中分离出的细胞，如心肌细胞， ，必须使用其他手段来阻止各种离子通道。例如，钠通道，可以通过一个缓慢的去极化电压斜坡灭活，3内向整流钾通道可以阻止外氯化钡，第4和可阻断钙离子通道的使用维拉帕米5 。 使用的方法之一，部分克服了这个限制是通过两个渠道类型来衡量当前通量同时，选择性地阻断一个渠道类型与相应的代理后，再重复测量。两次测量的加减就可以被用来估计电流的通道被阻塞占的数量 。然而，这种技术有两个主要限制。首先，化学制剂没有被确定，可以选择性地阻断每个离子通道，和一些广泛使用的药物与其他类型的通道的非特异性相互作用 。5,7秒，它可以从这种技术不能确定是否一个渠道是由另一调制通道。例如，资产净值的1.5和2.1 KIR表达异构已被证明在豚鼠心室，并已建议，两个通道之间存在一种协同关系，例如，更高的KIR 2.1在右心室的表达抑制传导速度。8目前，这无法验证。 在这项研究中，我们建议，阻抗谱可能是一种有用的工具，用于研究离子通道同时测量多个类型。虽然提出的方法从来没有被用来辨别从两个通道同时测量类型的电流，阻抗谱已被用于研究离子通道的功能等方面。古德曼和艺术表现出使用龟听觉毛细胞电流钳协议可以修改，调整一个单元，以不同的频率，并在跨膜电位的振荡是由于一个内向整流钾离子通道和Ca 2 +通道之间的相互作用。 9韩和弗雷泽表明，阻抗，可在一个单元格来衡量一个宽的频率范围（100 Hz到5 MHz），并观察阻抗增加， 当 K + 或Ca 2 +通道被堵塞，可能是一个简单的方法来检测通道高通量药物屏幕块。10林和Fishman使用复杂的电导研究一个内向整流钾离子通道，11个其他团体已插入单一的频率和电压钳位到不同类型的通道协议的动力学特性，结果显示，观察到的频率响应，同意与预期的响应某些频率，而不是其他。12,13 Millonas和Hanck建议的原因，某些频率没有产生预期的反应是马尔可夫模型中存在的多速率常数12这类研究，以及其他表明，有实例，当同时使用阻抗谱测量从离子通道的离子电流与理论频率响应不同意。这是不是在这项研究中关注的问题，因为在这项研究中的方法的目的是要找出与膜的电路的基本假设独立的电流幅值相关的频率。 Furtheremore，电流的幅度计算，从没有任何噪音功能插入到他们的录音部分。其他一些研究也目前许多离子通道模型参展的所有用自己的速率常数众多国家进行非传导。14,15,16汤普森等人发现，KCSA通道的选择性过滤器有不同的结合位点的Na +李+和K +，和移动之一约束力的站点通过的选择性过滤器一个离子移动到另一个的充满活力的成本是什么使得通道优先进行通过其孔隙钾离子。本文17，我们插入一个范围成一个电压阶跃协议（噪音的功能）的频率和频率的振幅高度关联的整体电流幅值。由于强有力的证据已提出建议，多速率常数发挥离子传导通过分化的作用T频道，推出这些速率常数相关的频率可能会导致某些频率产生共鸣，或与电流的幅度，否则不会有高度关联。在这项研究中表现出的技术是执行上的细胞模型，这是一个并联的RC电路，通常是用来测试的电压钳位电路和数据采集设备。预计不会直流以外的任何频率与电流的大小相关联，这是在我们的数据显示。我们还表明，另外噪音功能没有造成任何频率高度相关的电流幅值。这两项调查结​​果是至关重要的，因为它们显示测量设备和噪声功能本身并不能造成任何频率电流幅值相关。当未来的研究中使用膜含有离子通道进行测量，它预计，取决于所使用的通道，选择性过滤器或可能的孔隙率常数对应的频率会影响通道的频率响应，影响频率高或关联度低，与当前的振幅。 由于这种方法是一种用于研究离子通道的新技术，也有未来的研究可以遵循的方向。首先，这项技术应该被用来描述特定的隔离通道的频率响应。其他工作也需要进行校准的频率振幅的电流幅度。一旦多渠道的特点是单独的，应同时测量多通道类型。该技术也可以适应动作电位钳，电流钳，并刺激研究使用。虽然这是一种新技术，它说明了什么，可能是一种强有力的方式，使以前不可能的电生理测量和离子通道的生理作用提供了有价值的新见解。

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Matlab</td>
<td>&nbsp;</td>
<td>Mathworks</td>
<td>n/a</td>
<td>Natick, MA</td>
</tr>
<tr>
<td>Clampex 8</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>Clampex 8</td>
<td>Sunnyvale, CA</td>
</tr>
<tr>
<td>Integrating Patch Clamp Amplifier</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>Axopatch 200</td>
<td>Sunnyvale, CA</td>
</tr>
<tr>
<td>Headstage</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>CV202</td>
<td>Sunnyvale, CA</td>
</tr>
<tr>
<td>16-Bit Data Acquisition System</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>Digidata 1322A</td>
<td>Sunnyvale, CA</td>
</tr>
<tr>
<td>Model Cell</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>Patch 1 Model Cell</td>
<td>Sunnyvale, CA</td>
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recapitulation of an ion channel iv curve using frequency components

简介：目前，有没有既定的方法来测量多种离子通道类型的同时，并分解到每个通道类型的部分占被测电流。这项研究表明，阻抗谱可用于识别与稳态电流在电压钳实验测量的幅度，高度相关的特定频率。该方法包括插入一个电压一步协议将包含特定频率的噪音功能。在工作​​中，一个细胞模型是用来证明没有很高的相关性，介绍了电压钳位电路，还噪音功能本身不引入任何高度相关时，没有离子通道是存在。前的技术可应用于含有离子通道，这种验证是必要的。提出的协议的目的是演示如何噪音功能特征的单一离子通道类型的频率响应。一旦特定的频率已确定在一个单独的通道类型，它们可以被用来重现的稳态电流电压（IV）曲线。高度相关的一个通道类型和微创与其他类型的通道相关的频率，然后可以用来估计同时测量多通道类型的电流贡献。 方法：使用标准电压一步协议（-150至+50 mV时，为5mV步骤）的细胞模型上进行钳位电压测量。噪音的功能，包含1-15千赫频率（零峰值幅度：50或100mV的）的平等程度，分别插入到每个电压的一步。计算每一步的潜力和无噪音的输出信号的快速傅立叶变换（FFT）的真正组成部分。每个频率的电压阶跃函数幅度与相应的电压电流的幅值。 结果与结论：在噪音的情况下（控制）的直流分量之外的所有频率的大小相关性较差（| R | &lt;0.5）的IV曲线，直流分量，而在所有的测量相关系数大于0.999。个别频率和IV曲线之间的相关性的质量并没有改变时的噪音功能被添加到电压阶跃协议。同样，增加幅度噪声功能的同时，也没有增加相关。控制测量表明，电压钳位电路本身不会引起0赫兹以上的频率，以高度关联与稳态的IV曲线。同样，在存在噪声功能测量表明，噪声功能不会导致0赫兹以上的频率与稳态的IV曲线没有离子通道是存在关联时。基于此验证，该方法可以适用于含有单一离子通道类型的识别频率，其振幅相关专门与该通道类型的意图。

Watch this Scientific Journal Video about Recapitulation of an Ion Channel IV Curve Using Frequency Components at JoVE.com

Recapitulation of an Ion Channel IV Curve Using Frequency Components

有技术上的障碍，通过多种离子通道同时测量电流磁通后雪亮的跨膜电流的部分是由于每个通道的类型。为了满足这一需要，这种方法提出了一种方法来生成个别通道，使用特定的频率成分类型的IV曲线。

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 ...

recapitulation-of-an-ion-channel-iv-curve-using-frequency-components

Research

JoVE Journal

Biology

13.4K 次观看.  University of Utah. 有技术上的障碍，通过多种离子通道同时测量电流磁通后雪亮的跨膜电流的部分是由于每个通道的类型。为了满足这一需要，这种方法提出了一种方法来生成个别通道，使用特定的频率成分类型的IV曲线。

视频: Recapitulation of an Ion Channel IV Curve Using Frequency Components

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI样品制备：超薄层的方法

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

科研

生物学

Major Components of the Light Microscope

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

光镜下的主要组成部分

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for ...

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

自制的网站定向诱变整个质粒

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as tumor-propagating cells, or tumor-initiating cells. It is thought that targeting these self-renewing cells for destruction will block tumor progression and stop relapse, greatly improving patient prognosis1. The most common way to determine the frequency of self-renewing cells within a tumor is a limiting dilution cell transplantation assay, in which tumor cells are transplanted into recipient animals at increasing doses; the proportion of animals that develop tumors is used the calculate the number of self-renewing cells within the original tumor sample2, 3. Ideally, a large number of animals would be used in each limiting dilution experiment to accurately determine the frequency of tumor-propagating cells. However, large scale experiments involving mice are costly, and most limiting dilution assays use only 10-15 mice per experiment. 
Zebrafish have gained prominence as a cancer model, in large part due to their ease of genetic manipulation and the economy by which large scale experiments can be performed. Additionally, the cancer types modeled in zebrafish have been found to closely mimic their counterpart human disease4. While it is possible to transplant tumor cells from one fish to another by sub-lethal irradiation of recipient animals, the regeneration of the immune system after 21 days often causes tumor regression5. The recent creation of syngeneic zebrafish has greatly facilitated tumor transplantation studies 6-8. Because these animals are genetically identical, transplanted tumor cells engraft robustly into recipient fish, and tumor growth can be monitored over long periods of time. Syngeneic zebrafish are ideal for limiting dilution transplantation assays in that tumor cells do not have to adapt to growth in a foreign microenvironment, which may underestimate self-renewing cell frequency9, 10. Additionally, one-cell transplants have been successfully completed using syngeneic zebrafish8 and several hundred animals can be easily and economically transplanted at one time, both of which serve to provide a more accurate estimate of self-renewing cell frequency.
Here, a method is presented for creating primary, fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) in syngeneic zebrafish, and transplanting these tumors at limiting dilution into adult fish to determine self-renewing cell frequency. While leukemia is provided as an example, this protocol is suitable to determine the frequency of tumor-propagating cells using any cancer model in the zebrafish.

Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.

量化传播肿瘤细胞的频率使用斑马鱼的同源有限稀释细胞移植

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as ...

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

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

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

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

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

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 ...

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and neurotransmitter loading in synaptic vesicles. This acidification is described to be mediated by proton ATPases, coupled to ClC chloride transporters. Highly-conserved electroneutral protons transporters, the Na+/H+ exchangers (NHE) 6, 7 and 9 are also expressed in these compartments. Mutations in their genes have been linked with human cognitive and neurodegenerative diseases. Paradoxically, their roles remain elusive, as their intracellular localization has prevented detailed functional characterization. This manuscript shows a method to solve this problem. This consists of the selection of mutant cell lines, capable of surviving acute cytosolic acidification by retaining intracellular NHEs at the plasma membrane. It then depicts two complementary protocols to measure the ion selectivity and activity of these exchangers: (i) one based on intracellular pH measurements using fluorescence video microscopy, and (ii) one based on the fast kinetics of lithium uptake. Such protocols can be extrapolated to measure other non-electrogenic transporters. Furthermore, the selection procedure presented here generates cells with an intracellular retention defective phenotype. Therefore these cells will also express other vesicular membrane proteins at the plasma membrane. The experimental strategy depicted here may therefore constitute a potentially powerful tool to study other intracellular proteins that will be then expressed at the plasma membrane together with the vesicular Na+/H+ exchangers used for the selection.

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

娜功能分析<sup&gt; +</sup&gt; / H<sup&gt; +</sup&gt;细胞内的隔间使用质子打死选择器来表达它们的质膜

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and ...

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers such as epithelia also form a barrier that separates the inside from the outside or different compartments of multicellular organisms. A key feature of these barriers is the differential distribution of ions across cell membranes or cell layers. Two properties allow this distribution: 1) membranes and epithelia display selective permeability to specific ions; 2) ions are transported through pumps across cell membranes and cell layers. These properties play crucial roles in maintaining tissue physiology and act as signaling cues after damage, during repair, or under pathological condition. The ion-selective self-referencing microelectrode allows measurements of specific fluxes of ions such as calcium, potassium or sodium at single cell and tissue levels. The microelectrode contains an ionophore cocktail which is selectively permeable to a specific ion. The internal filling solution contains a set concentration of the ion of interest. The electric potential of the microelectrode is determined by the outside concentration of the ion. As the ion concentration varies, the potential of the microelectrode changes as a function of the log of the ion activity. When moved back and forth near a source or sink of the ion (i.e. in a concentration gradient due to ion flux) the microelectrode potential fluctuates at an amplitude proportional to the ion flux/gradient. The amplifier amplifies the microelectrode signal and the output is recorded on computer. The ion flux can then be calculated by Fick&#8217;s law of diffusion using the electrode potential fluctuation, the excursion of microelectrode, and other parameters such as the specific ion mobility. In this paper, we describe in detail the methodology to measure extracellular ion fluxes using the ion-selective self-referencing microelectrode and present some representative results.

Transporters in cell membranes allow differential segregation of ions across cell membranes or cell layers and play crucial roles during tissue physiology, repair and pathology. We describe the ion-selective self-referencing microelectrode that allows the measurement of specific ion fluxes at single cells and tissues in vivo.

细胞外离子流的使用离子选择性自引用的微电极技术测量

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

可视化Stromule频率与荧光显微镜

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...