我们感谢J. Vergara博士（加州大学洛杉矶分校）分享EGFP-CaV1.1（兔）野生型质粒。我们感谢耶鲁大学生理学系电子实验室，特别是Henrik Abildgaard设计和建造具有跟踪和保持电路的光电二极管。这项工作得到了美国国立卫生研究院拨款R01-AR075726和R01-NS103777的支持

天然骨骼肌细胞中的功能性定点荧光测定

作者报告没有利益冲突。

本文描述了在肌纤维中进行FSDF的分步协议，以研究来自CaV1.1通道的单个电压传感器运动。尽管该技术中组合的步骤数量和方法的多样性可能看起来很复杂，但大多数这些技术通常用于生物物理学家/细胞生物学家实验室。因此，明显的复杂性主要在于将所有各种方法组合在一个单一的集成技术中。通常，在执行多步骤方法时，开始时进行的微小修改的影响只能在后续步骤的后面检测到。通...

响应活细胞中已知电刺激而跟踪离子通道构象重排的能力是分子生理学有价值的信息来源1。电压门控离子通道是感知跨膜电压变化的膜蛋白，其功能也受电压变化的影响2。上个世纪电压钳技术的发展使生理学家能够实时研究电压门控离子通道响应膜去极化而携带的离子电流3。电压钳技术的使用对于理解可兴奋细胞（如神经元和肌肉）的电特性至关重要。在 1970 年代，电压钳优化允许检测电压门控钙 （Ca V） 和钠 （NaV） 通道4，5 中的门控电流（或电荷移动）。门控电流是非线性电容电流，由电压传感器的运动产生，以响应细胞膜6上的电场变化。门控电流被认为是分子重排的电表现形式，在离子通道打开之前或伴随7。虽然这些电流测量提供了有关通道功能的宝贵信息，但离子电流和门控电流都是电压门控通道7的分子间和分子内构象重排的间接读数。
功能性定点荧光测定法（FSDF;也称为电压钳式荧光测定法，VCF）是在1990年代初开发的8，并且首次提供了直接查看局部构象变化和通道蛋白功能的能力实时。使用通道诱变、电生理学和异源表达系统的组合，可以荧光标记和跟踪特定通道或受体的运动部分，以响应激活刺激9，10。该方法已被广泛用于研究电压门控离子通道8，10，11，12，13，14，15，16，17，18，19中的电压传感机制。权威评论见10，20，21，22，23。
Ca V和NaV通道对于电信号的启动和传播至关重要，由一个主α1亚基组成，该亚基具有一个中心孔和四个不相同的电压传感域2。除了它们独特的一级结构外，Ca V和NaV通道还表示为具有辅助亚基24的多亚基复合物。电压依赖性钾通道（K V）由四个亚基组成，看起来像NaV或CaV25的单个结构域。Ca V 和 NaV 通道的孔形成和电压感应 α1 亚基由单个多肽编码六个独特跨膜段的四个独立结构域（S1-S6;图1A）24，26.由S1至S4跨膜段组成的区域形成电压传感域（VSD），S5和S6跨膜段形成孔域26。在每个VSD中，S4 α螺旋含有带正电荷的精氨酸或赖氨酸（图1A，B），它们响应膜去极化而移动7。数十年的研究和高度多样化的实验方法的结果支持这样一个前提，即S4段向外移动，产生门控电流，以响应膜去极化6。
FSDF 测量与离子通道或其他蛋白质上的特定半胱氨酸残基（即 S4 α-螺旋）偶联的硫醇反应性染料的荧光变化，通过定点诱变工程，因为通道响应膜去极化或其他刺激起作用10。事实上，FSDF最初是为了研究KV通道中的S4段（被提议为通道的主要电压传感器）是否在门控电荷响应膜电位8，10的变化而移动时移动。在电压门控离子通道的情况下，FSDF可以解析四个VSD的独立构象重排（在任何给定时间跟踪一个VSD），同时进行通道函数测量。事实上，使用这种方法，已经表明单个VSD似乎在通道激活和失活的特定方面存在差异性参与12，27，28，29，30。确定每个VSD对通道功能的贡献具有高度相关性，可用于进一步阐明通道操作并潜在地确定药物开发的新靶点。
FSDF在异源表达系统中的使用对于我们从还原论的角度进一步理解通道功能非常有帮助10，23。像许多还原论方法一样，它有优点，但也有局限性。例如，一个主要的限制是异源系统中通道纳米环境的部分重构。通常，离子通道与许多辅助亚基和许多其他改变其功能的蛋白质相互作用31。原则上，使用多个蛋白质编码构建体或多顺反子质粒可以在异源系统中表达不同的通道及其附属亚基，但它们的天然环境不能完全重建30，32。
我们小组最近在天然解离的骨骼肌纤维中发表了FSDF的变体，用于研究激发 - 收缩耦合（ECC）的早期步骤33，34，肌肉纤维电去极化与肌肉收缩的激活相关的过程35，36。这种方法首次允许在成人分化肌纤维37的天然环境中对来自电压门控L型Ca2+通道（CaV1.1，也称为DHPR）的单个S4电压传感器进行运动跟踪。这是通过考虑这种细胞类型的多种特征来实现的，包括细胞的电活动允许快速刺激诱导的自我传播去极化，通过体内电穿孔表达cDNA质粒的能力，细胞内通道的自然高表达和区室组织，以及它与高速成像和电生理记录设备的兼容性。以前，我们使用高速线扫描共聚焦显微镜作为检测设备37。现在，使用光电二极管进行信号采集，介绍了该技术的变体。这种基于光电二极管的检测系统可以促进该技术在其他实验室中的实施。
本文描述了一种在天然电池中利用FSDF研究CaV1.1的单个电压传感器运动的分步协议。虽然CaV1.1通道在整个手稿中被用作一个例子，但该技术可以应用于其他离子通道，受体或表面蛋白的细胞外可访问结构域。

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			<td>895000</td>
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			<td>Fetal Bovine Serum - Premium, Heat Inactivated</td>
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			<td>acrodisk&#160; syringe filter 0.2um supor membrane low protein binding non pyrogenic</td>
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			<td>MEM</td>
			<td>Invitrogen</td>
			<td>INV-11380037</td>
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			<td>MTS-5-TAMRA</td>
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			<td>OriginLab Corporation</td>
			<td>OriginPro 2022 (64-bit) SR1</td>
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			<td>NA</td>
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			<td>Olympus</td>
			<td>BFPC2</td>
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			<td>Platinum wire 0.5 mm, 99.9 % metals basis</td>
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			<td>NA</td>
			<td></td>
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functional site-directed fluorometry in native cells to study skeletal muscle excitability

功能性定点荧光测定法一直是研究许多膜蛋白（包括电压门控离子通道）的结构-功能关系的首选技术。这种方法主要用于异源表达系统，以同时测量膜电流、通道活性的电表现和荧光测量，报告局部结构域重排。功能性定点荧光测量将电生理学、分子生物学、化学和荧光结合到一个广泛的技术中，允许分别通过荧光和电生理学研究实时结构重排和功能。通常，这种方法需要一个工程电压门控膜通道，其中包含可以通过硫醇反应性荧光染料测试的半胱氨酸。直到最近，用于蛋白质的定点荧光标记的硫醇反应化学仅在非洲爪蟾卵母细胞和细胞系中进行，将该方法的范围限制在原代不可兴奋细胞上。本报告描述了功能性定点荧光测定法在成人骨骼肌细胞中的适用性，以研究激发-收缩耦合的早期步骤，即肌纤维电去极化与肌肉收缩激活相关的过程。本协议描述了使用体内电穿孔将半胱氨酸工程的电压门控Ca2 +通道（CaV1.1）设计和转染到成年小鼠短指屈肌纤维中的方法，以及功能性定点荧光测量所需的后续步骤。这种方法可以适用于研究其他离子通道和蛋白质。使用哺乳动物肌肉的功能性定点荧光测定法与研究兴奋性的基本机制特别相关。

The ability to track ion channel conformational rearrangements in response to a known electrical stimulus in a living cell is a source of valuable information for molecular physiology1. Voltage-gated ion channels are membrane proteins that sense changes in transmembrane voltage, and their function is also affected by voltage changes2. The development of voltage clamp techniques in the last century allowed physiologists to study, in real-time, ionic currents carried by voltage-gated ion channels in response to membrane depolarization3. The use of voltage clamp technology has been crucial in understanding the electrical properties of excitable cells such as neurons and muscle. In the 1970s, voltage clamp refinement allowed for the detection of gating currents (or charge movement) in voltage-gated calcium (CaV) and sodium (NaV) channels4,5. Gating currents are non-linear capacitive currents that arise from the movement of voltage sensors in response to changes in the electric field across the cell membrane6. Gating currents are considered an electrical manifestation of molecular rearrangements that precede or accompany ion channel opening7. While these current measurements provide valuable information regarding the channel&#39;s function, both ionic currents and gating currents are indirect readouts of inter- and intra-molecular conformational rearrangements of voltage-gated channels7.
Functional site-directed fluorometry (FSDF; also referred to as voltage clamp fluorometry, VCF) was developed in the early 1990s8 and, for the first time, provided the ability to directly view local conformational changes and the function of a channel protein in real time. Using a combination of channel mutagenesis, electrophysiology, and heterologous expression systems, it is possible to fluorescently tag and track the moving parts of specific channels or receptors in response to the activating stimulus9,10. This approach has been extensively used to study the voltage-sensing mechanisms in voltage-gated ion channels8,10,11,12,13,14,15,16,17,18,19. For authoritative reviews, see10,20,21,22,23.
The CaV and NaV channels, critical for the initiation and propagation of electrical signals, are composed of a main &#945;1 subunit, which possesses a central pore and four non-identical voltage sensing domains2. In addition to their distinct primary structure, CaV and NaV channels are expressed as multisubunit complexes with auxiliary subunits24. Voltage-dependent potassium channels (KV) consist of four subunits that look like a single domain of NaV or CaV25. The pore-forming and voltage-sensing &#945;1 subunit of CaV and NaV channels is formed by a single polypeptide coding for four individual domains of six unique transmembrane segments (S1-S6; Figure 1A)24,26. The region comprised by S1 to S4 transmembrane segments form the voltage sensing domain (VSD) and S5 and S6 transmembrane segments form the pore domain26. In each VSD, the S4 &#945;-helix contains positively charged arginine or lysine (Figure 1A,B) that move in response to membrane depolarization7. Several decades of research and the results from highly diverse experimental approaches support the premise that S4 segments move outward, generating gating currents, in response to membrane depolarization6.
FSDF measures the fluorescence changes of a thiol-reactive dye conjugated to a specific cysteine residue (i.e., the S4 &#945;-helix) on an ion channel or other protein, engineered via site-directed mutagenesis, as the channel functions in response to membrane depolarization or other stimuli10. In fact, FSDF was originally developed to investigate whether the S4 segment in KV channels, proposed to be the main voltage sensor of the channel, moves when the gating charges move in response to changes in membrane potential8,10. In case of voltage-gated ion channels, FSDF can resolve independent conformational rearrangements of the four VSDs (tracking one VSD at any given time), concurrently with channel function measurements. Indeed, using this approach, it has been shown that individual VSDs appear to be differentially involved in specific aspects of channel activation and inactivation12,27,28,29,30. Identifying the contribution of each VSD to the channels&#39; function is of high relevance and can be used to further elucidate channel operation and potentially identify new targets for drug development.
The use of FSDF in heterologous expression systems has been extremely helpful in furthering our understanding of channel function from a reductionist perspective10,23. Like many reductionist approaches, it presents advantages but also has limitations. For instance, one major limitation is the partial reconstitution of the channel nano environment in the heterologous system. Often, ion channels interact with numerous accessory subunits and numerous other proteins that modify their function31. In principle, different channels and their accessory subunits can be expressed in heterologous systems with the use of multiple protein coding constructs or polycistronic plasmids, but their native environment cannot be fully reconstituted30,32.
Our group recently published a variant of FSDF in native dissociated skeletal muscle fibers for the study of early steps of excitation-contraction coupling (ECC)33,34, the process by which muscle fiber electrical depolarization is linked to the activation of muscle contraction35,36. For the first time, this approach allowed the motion tracking of individual S4 voltage-sensors from the voltage-gated L-type Ca2+ channel (CaV1.1, also known as DHPR) in the native environment of an adult differentiated muscle fiber37. This was accomplished by considering multiple characteristics of this cell type, including the electrical activity of the cell allowing fast stimulation-induced self-propagated depolarization, the ability to express cDNA plasmid through in vivo electroporation, the natural high expression and compartmental organization of the channels within the cell, and its compatibility with high-speed imaging and electrophysiological recording devices. Previously, we used a high-speed line scanning confocal microscope as a detecting device37. Now, a variation of the technique is presented using a photodiode for signal acquisition. This photodiode-based detection system could facilitate the implementation of this technique in other laboratories.
Here, a step-by-step protocol to utilize FSDF in native cells for the study of individual voltage-sensor movement from CaV1.1 is described. While the CaV1.1 channel has been used as an example throughout this manuscript, this technique could be applied to extracellularly accessible domains of other ion channels, receptors, or surface proteins.

作者聚焦：天然细胞中的功能性定点定向荧光测定法研究骨骼肌兴奋性  

天然细胞中的功能性定点荧光测定法，用于研究骨骼肌兴奋性

CaV1.1, a member of the voltage-gated ion channels, has four distinct voltage sensors. Evidence suggests that some voltage sensors contribute more to ryanodine receptor activation or calcium current. We aim to be able to identify the precise role of each voltage sensor in excitation-contraction coupling and calcium channel activation.Excitation-contraction coupling has been studied since the early 50s, yet the molecular detail of how this process occur are still unknown. Recent advance in cyro-electron microscopic structure of the channel, the characterization of novel CaV1.1 accessory protein, the discovery of channel alternative splicing variant, and the identification of disease-causing mutation have reignited the interest in this field. Many techniques are used in our field, from classical electrophysiology and molecular biology to more novel techniques such as cryo-electron microscopy, molecular dynamic simulation, targeted protein degradation, and functional site-directed fluorometry as well as engineered cells or animal models.Currently, the fuel faces several experimental challenges. In a skeletal muscle, proper trafficking and communication between CaV1.1 and RyR1, along with many regulatory proteins, is crucial to support excitation-contraction coupling. The methods to directly study these protein-protein interactions between CaV1.1 and RyR1 are missing or incomplete.The laboratory of Dr.Martin Schneider has been working on excitation-contraction coupling for decades, characterizing voltage-sensing mechanisms in CaV1.1, calcium release, and localized calcium-release events, known as calcium sparks. Recently, our laboratory has been implementing new optical techniques to investigate various steps of excitation-contraction coupling and voltage sensor motion in functioning adult muscle cells. While this has been done previously in a heterologous expression system, our protocol now allows tracking conformational changes in CaV1.1 voltage sensors during a propagated action potential in his native environment.Our new technique will allow us to investigate the precise movement of the voltage sensor needed for excitation-contraction coupling. We want to know which charge residue in each S4 are critical for its function, or exactly each S4 translocate, and all that's translocation is linked to CaV1.1 opening or ryanodine receptor activation.

当响应于重复场刺激而触发传播动作电位时，可以跟踪特定电压传感器的运动以响应特定的去极化频率。 如图6A所示，可以跟踪VSD-II标记螺旋的运动，以响应在10 Hz（即间隔100 ms）施加的两个连续去极化中的每一个。信号漂白可以通过减去迹线的基线来校正（图6B）。对第一和第二响应的进一步时间放大（图6C）可以精确观察这些?...

Watch this Scientific Journal Video about Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability at JoVE.com

功能性定点荧光测定法是一种实时研究蛋白质结构域运动的方法。该技术在天然细胞中的应用修改现在允许检测和跟踪来自小鼠分离骨骼肌纤维中电压门控Ca2+ 通道的单电压传感器运动。

Functional site-directed fluorometry has been the technique of choice to investigate the structure-function relationship of numerous membrane proteins, ...

CaV1.1是电压门控离子通道的成员，具有四个不同的电压传感器。有证据表明，一些电压传感器对Ryanodine受体激活或钙电流的贡献更大。我们的目标是能够识别每个电压传感器在激励-收缩耦合和钙通道激活中的精确作用。自50年代初以来，人们一直在研究激发-收缩耦合，但该过程如何发生的分子细节仍然未知。通道的圆柱电子微观结构的最新进展，新型CaV1.1辅助蛋白的表征，通道替代剪接变体的发现以及致病突变的鉴定重新点燃了对该领域的兴趣。我们的领域使用了许多技术，从经典的电生理学和分子生物学到更新颖的技术，如冷冻电子显微镜、分子动力学模拟、靶向蛋白质降解和功能性定点荧光测定以及工程细胞或动物模型。目前，这种燃料面临着几个实验挑战。在骨骼肌中，CaV1.1和RyR1之间的适当运输和通讯以及许多调节蛋白对于支持激发-收缩耦合至关重要。直接研究CaV1.1和RyR1之间这些蛋白质 - 蛋白质相互作用的方法缺失或不完整。Martin Schneider博士的实验室几十年来一直致力于激励-收缩耦合，表征CaV1.1中的电压传感机制，钙释放和局部钙释放事件，称为钙火花。最近，我们的实验室一直在实施新的光学技术，以研究功能性成人肌肉细胞中激发 - 收缩耦合和电压传感器运动的各个步骤。虽然这以前已经在异源表达系统中完成，但我们的协议现在允许在其原生环境中传播动作电位期间跟踪CaV1.1电压传感器的构象变化。我们的新技术将使我们能够研究激励-收缩耦合所需的电压传感器的精确运动。我们想知道每个S4中的哪些电荷残基对其功能至关重要，或者每个S4转位，并且所有易位都与CaV1.1开放或Ryanodine受体激活有关。

functional-site-directed-fluorometry-native-cells-to-study-skeletal

Research

JoVE Journal

Biology

Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability

657 次观看.  University of Maryland School of Medicine. CaV1.1是电压门控离子通道的成员，具有四个不同的电压传感器。有证据表明，一些电压传感器对Ryanodine受体激活或钙电流的贡献更大。我们的目标是能够识别每个电压传感器在激励-收缩耦合和钙通道激活中的精确作用。自50年代初以来，人们一直在研究激发-收缩耦合，但该过程如何发生的分子细节仍然未知。通道的圆柱电子微观结构的最新进展，新型CaV1.1辅助蛋白的表...

视频: 作者聚焦：天然细胞中的功能性定点定向荧光测定法研究骨骼肌兴奋性  

Functional site-directed fluorometry has been the technique of choice to investigate the structure-function relationship of numerous membrane proteins, including voltage-gated ion channels. This approach has been used primarily in heterologous expression systems to simultaneously measure membrane currents, the electrical manifestation of the channels&#39; activity, and fluorescence measurements, reporting local domain rearrangements. Functional site-directed fluorometry combines electrophysiology, molecular biology, chemistry, and fluorescence into a single wide-ranging technique that permits the study of real-time structural rearrangements and function through fluorescence and electrophysiology, respectively. Typically, this approach requires an engineered voltage-gated membrane channel that contains a cysteine that can be tested by a thiol-reactive fluorescent dye. Until recently, the thiol-reactive chemistry used for the site-directed fluorescent labeling of proteins was carried out exclusively in Xenopus oocytes and cell lines, restricting the scope of the approach to primary non-excitable cells. This report describes the applicability of functional site-directed fluorometry in adult skeletal muscle cells to study the early steps of excitation-contraction coupling, the process by which muscle fiber electrical depolarization is linked to the activation of muscle contraction. The present protocol describes the methodologies to design and transfect cysteine-engineered voltage-gated Ca2+ channels (CaV1.1) into muscle fibers of the flexor digitorum brevis of adult mice using in vivo electroporation and the subsequent steps required for functional site-directed fluorometry measurements. This approach can be adapted to study other ion channels and proteins. The use of functional site-directed fluorometry of mammalian muscle is particularly relevant to studying basic mechanisms of excitability.

Functional site-directed fluorometry is a method to study protein domain motions in real time. Modification of this technique for its application in native cells now allows the detection and tracking of single voltage-sensor motions from voltage-gated Ca2+ channels in murine isolated skeletal muscle fibers.

科研

生物学

Construct Design for Transfecting Cysteine-Engineered Voltage-Gated Ca2+ Channels in Murine Isolated Skeletal Muscle Fibers

Construct Design for Transfecting Cysteine-Engineered Voltage-Gated Ca2+ Channels in Murine Isolated Skeletal Muscle Fibers  

To begin, generate a wild-type, fluorescently-tagged CaV1.1 CDNA construct. Here, a plasmid coating for the enhanced green fluorescent protein or EGFP-tagged alpha subunit of rabbit Cav1.1 is used. The presence of the cytomegalovirus promoter allows strong transfection efficiency in muscle fibers.Introduce cystine substitution in the channel that would be tracked with fluorometry using a commercially available, site-directed mutagenesis kit. Here the cysteines are inserted at a position corresponding to the membrane extracellular interface of one of the four S4 of the channel. With the help of GFP observation and ionic current recording, confirm that neither the inserted cysteines nor the presence thiol-reactive dye impacts the voltage dependence and the conductance of the channel.