Name: one-channel cell-attached patch-clamp recording
Uploaded: 2014-06-09T14:45:00
Description: Watch this Scientific Journal Video about One-channel Cell-attached Patch-clamp Recording at JoVE.com

这项工作是由F31NS086765（KAC）的支持，F31NS076235（MAP）和R01 NS052669（GKP）和EIA9100012。作者感谢艾琳Kasperek的专业知识，并与分子生物学和组织培养的援助;和杰森迈尔斯分享从早期的前额叶皮层神经元获得的数据。

1，细胞培养和蛋白表达<ol><li>保持HEK293细胞通道22和40之间（ATCC编号CRL-1573），它包括由ATCC执行的，在DMEM中单层培养在补充有10％胎牛血清（FBS）的通路，并在5％CO 2的1％青霉素/链霉素的混合物和37°C。实验之间，传代细胞进T25烧瓶在10毫升的最终体积5-20倍稀释液。注意：在这些通道使用的细胞可以保证良好的健康细胞，这将使以获得最佳的密封形成和补丁的稳定性。 </li><li>对于转染?...

<ol>
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在离子通道字段，一个重要的研究领域致力于了解的事件，导致信道的开口或通道的门控机制的序列。对于大多数信道，该过程是复杂的，涉及不能从宏观的多声道信号推导出几个反应步骤。相比之下，实验可设计其中观察在单通道记录打开/闭合事件的顺序可以产生约门控机制的更详细的信息。在这里所描述的方法，由NMDA受体所具有的位于外部的配体结合域允许将在恒定条件下执行微创的膜片?...

过生物膜的快速通信几乎完全依赖于低聚孔形成的膜蛋白，通常被称为信道。这些蛋白质在活化信号，门控机制，传导性能有很大的出入。通道蛋白的毛孔是有选择性的离子被列为离子通道;它们的活化产生的离子电流穿过细胞膜，并且它们的响应可以使用电生理技术来记录具有高分辨率的实时性。激活信号跨越的化学和物理输入，包括浓度梯度，机械和电气的力量和温度浩如烟海;因此，进一步的分类离子通道进入配体门控，机械敏感性，电压门控，或热敏感的类型。在这篇文章中，协议被描述为从一个配体门控通道，NMDA受体和机械敏感性离子通道，PIEZO1记录单声道活动，用膜片钳技术。0; 膜片钳电是最早，最广泛使用的实验方法足够灵敏，以允许观察单个分子1，2。除了这个精致的灵敏度，它已大大扩展了生物制剂适合于电生理记录，并允许离子通道在细胞膜完整的观察。首先，因为这两个电压钳位与当前记录被完成与同一电极，它可以用来通过小的细胞或膜的补丁记录的信号。该技术表明，离子通道并不局限于蛙肌肉，鳗鱼electroplaques或乌贼巨轴突3，4，而是它们所代表的跨膜信号传导机制无处不装置及有固有的单向或所有细胞膜类型的可兴奋膜多细胞有机体，并且还细胞内膜。进口antly，通过简单的连接玻璃吸管，以完整的细胞，记录跨膜电流的能力提供了前所未有的机会，从离子通道在家乡不间断的膜录制活动。因此，细胞贴附式膜片钳技术，这是在本协议中所述，允许连续监测离子通道的活性数十分钟或在他们的母语环境的时间。 在正常温度波动，所有的蛋白质，包括离子通道蛋白，经过了广泛的时间尺度上的结构变化，与代表最有可能通过侧链运动和整个的定位表示要慢得多，那么频繁变化最快和最频繁的重排结构域或亚基，或在某些情况下，通过翻译后修饰或蛋白质-蛋白质相互作用5,6。观察活动由一个分子产生长时间可以帮助了解功能离子通道的完好生理膜际动力学，并提供关于所观察到的分子的运行机制的有价值的信息。 与此相反的离子通道穿过细胞类型和发育阶段，知识的离子通道在原生膜的分子组成的多样性，人们越来越认识仍然有限。所有离子通道是多聚体蛋白，大部分天然离子通道的从几种类型的亚基产生的宽分子多样性，这是经常伴随着不同的电导和门控性质的蛋白质的组装。出于这个原因，定义分子组成的离子通道表达时在异源系统的研究。特别地，HEK293细胞，这是永生化人胚胎肾细胞7的品系，获得广泛接受，作为重组的离子通道的异源表达的优选系统。其中男子该升高的HEK293细胞的选择系统用于离子通道电Ÿ优点是易于进行培养的承受能力和保持长期稳定的培养，其进行翻译后折叠，加工和贩卖的哺乳动物蛋白质的能力，并且在许多情况下，其低的水平，甚至不存在内源性表达的感兴趣7,8的通道。表达重组的离子通道，并研究在HEK293细胞中其功能特性仍然是一个有价值的方法，以获得关于离子通道的结构 - 功能特性以及离子通道亚型和它们在天然组织角色的特定属性的信息。本文中所描述的协议同样可以很好地应用于表达在HEK293细胞重组离子通道和原生的离子通道。 综上所述，膜片钳技术，通过其前所未有的能力来解决信号从1分子遗体，迄今为止，最直接的方法，观察单个分子的行为。在其细胞贴附模式下，膜片钳记录允许长期观察期间，当一个分子中完成的，可以提供出色的洞察离子通道的操作。下面给出一个协议，用于获得高分辨率当前记录从含有一种离子通道蛋白的细胞贴附式膜片。

<table border="0" cellpadding="0" cellspacing="0" width="695">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:189px;">Chemicals</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:128px;">Various</td>
			<td style="width:205px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Borosillicate Glass</td>
			<td>Sutter</td>
			<td>BF-150-86-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bright field inverted microscope</td>
			<td>Olympus</td>
			<td>1x51</td>
			<td>Nikon also has similar microscopes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluroescent box</td>
			<td>X-cite</td>
			<td>Series 120</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Liquid Light Guide</td>
			<td>X-cite</td>
			<td>OEX-LG15</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micromanipulator</td>
			<td>Sutter Instruments</td>
			<td>MP-225</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oscilloscope</td>
			<td>Tektronix</td>
			<td>TDS1001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Amplifier</td>
			<td>Molecular Devices</td>
			<td colspan="2">Axon Axopatch 200B&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Table</td>
			<td>TMC</td>
			<td align="right">63561</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NIDAQ card</td>
			<td>National Instruments</td>
			<td>776844-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Puller</td>
			<td>Narishige</td>
			<td>PC-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polisher</td>
			<td>Narishige</td>
			<td>Microforge MF-830</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Faraday Cage</td>
			<td>TMC</td>
			<td align="right">8133306</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High Speed Pressure Clamp</td>
			<td>ALA Scientific Instruments</td>
			<td>ALA HSPC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pressue/Vaccuum Pump</td>
			<td>ALA Scientific Instruments</td>
			<td>ALA PV-PUMP</td>
			<td>For HSPC-1</td>
		</tr>
	</tbody>
</table>

one-channel cell-attached patch-clamp recording

离子通道蛋白是通过生物膜快速通信的通用设备。离子通量它们产生的瞬时签名取决于固有的每个通道蛋白，以及由其所产生和控制的机制的属性和表示当前研究的重要领域。关于离子通道蛋白的经营动态信息，可通过观察当前很长一段由单个分子制造来获得。这里描述的是一种协议，用于获取一条通道细胞贴附膜片钳记录电流为配体门控离子通道，NMDA受体，异源表达在HEK293细胞中或在本地皮层神经元。本发明还提供关于如何通过呈现在机械性敏感通道PIEZO1的例子中，方法适应其他感兴趣的离子通道的指示。这种方法可以对于该通道的电导特性和氧化聚乙烯的时间序列数据提供正封闭构象组成通道的激活机制，从而有助于了解他们在健康和疾病的功能。

重组NMDA受体 NMDA受体结合并以两个共激动剂随之而来的行动作出回应：谷氨酸和甘氨酸。他们组装成两个甘氨酸异源结合GluN1亚基和两个谷氨酸结合GluN2亚基。 GluN2亚基是由这四个基因（AD）和编码在大脑的最广泛形式的转录是GluN2A在成年和GluN2B在幼龄动物。由于NMDA受体亚型在原生的准备，表达受体在HEK293细胞（ 图1A）允许控制的亚基组成的，以及?...

Watch this Scientific Journal Video about One-channel Cell-attached Patch-clamp Recording at JoVE.com

One-channel Cell-attached Patch-clamp Recording

这里描述的是获得当前记录的很长一段距离与细胞贴附膜片钳技术一种离子通道的过程。这种方法允许观察，实时的背后的生物信号开闭通道构象的图案。这些数据的通知关于在不受干扰的生物膜通道属性。

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 ...

one-channel-cell-attached-patch-clamp-recording

Research

JoVE Journal

Biology

Methods for Patch Clamp Capacitance Recordings from the Calyx

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

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

膜片钳从花萼电容录音方法

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

科研

生物学

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

补丁钳爪蟾卵母细胞中表达的离子通道

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Pressure-polishing Pipettes for Improved Patch-clamp Recording

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

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

改进的膜片钳记录的压力抛光移液器

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

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

线虫神经肌肉交界处的解剖和录制

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

在电生理记录果蝇胚胎

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Ambulatory ECG Recording in Mice

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although the surface ECG is utilized for short-term measurements of waveform intervals, it is not practical for long-term studies of heart rate variability or the capture of rare episodes of arrhythmias. Implantable ECG telemeters offer the advantages of simple surgical implantation, long-term recording of electrograms in ambulatory mice, and scalability with simultaneous recordings of multiple animals. Here, we present a step-by-step guide to the implantation of telemeters for ambulatory ECG recording in mice. Careful adherence to aseptic technique is required for favorable survival results with the possibility of implantation and recording from weeks to months. Thus, implantable ECG telemetry is a valuable tool for detection of critical information on cardiac electrophysiology in ambulatory animal models such as the mouse. 

Telemetric ECG has emerged as an essential tool in evaluating animal models for cardiac arrhythmias and sudden cardiac death. Here, we present a stepwise guide to telemetric ECG recordings for application in long-term ambulatory ECG monitoring in mice.

在小鼠体内的动态心电图记录

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

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

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

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

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

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

Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

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

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

巨脂质体制剂成像和膜片钳电

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

Reconstitution of a 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 ...