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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
该协议已获得马里兰大学机构动物护理和使用委员会的批准。以下协议分为多个子部分，包括（1）分子构建体设计和半胱氨酸反应染料选择，（2） 体内 电穿孔，（3）肌肉解剖和纤维分离，（4）采集设置说明，（5）评估增强绿色荧光蛋白（EGFP）阳性纤维电活性和半胱氨酸染色，以及（6）信号采集和处理。此外，在每节的开头，在骨骼肌纤维中应用FSDF时，会详细介绍一些相关的注意事项…
Representative Results
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?…
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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
Materials
| Hyaluronidase | SIGMA ALDRICH | H3884-50mg | |
| 0.5 mL Eppendorf tube | Millipore Sigma | EP022363719-500EA | |
| 1 mL syringe | Millipore Sigma | Z683531-100EA | tuberculine slip tip |
| 1/2” long 29-gauge sterile insulin needle and syringe | Becton Dikinson | 324702 | |
| 35 mm non coated plastic plate | Falcon, Corning | 353001 | |
| 60 mm non coated plastic plate | Falcon, Corning | 351007 | |
| Alcoholic whip | PDI | B60307 | |
| Alexa-533 cube LP | Chroma | 49907 | Ex: 530/30x; BS: 532; Em: 550lp |
| Arc lamp | Sutter Instrumets | LB-LS 672 | |
| Artificial tears cream | Akorn | NDC 59399-162-35 | |
| Borosilicate glass Pasteur pipet 5 3/4" | VWR | 14672-200 | |
| BTS (N-benzyl-p-toluene sulphonamide) | SIGMA ALDRICH | 203895 | |
| collagenase type I | SIGMA ALDRICH | C0130-1g | |
| Cotton tip | VWR | VWR-76048-960-BG | |
| Double electrode array (for electroporation) | BTX harvard apparatus | 45-0120 | 10mm 2 needle array tips |
| EGFP cube | Chroma | 39002AT | Ex: 480/30x; BS 505; Em: 535/40m |
| Electroporation apparatus device | BTX harvard apparatus | ECM 830 | |
| EPC10 | HEKA Elektronik GmbH (Harvard Bioscience) | 895000 | |
| FBS | Biotechne, R&D Systems | RND-S11150H | Fetal Bovine Serum – Premium, Heat Inactivated |
| glass coverslip 35 mm dish | MatTek Life Science | P35G-1.5-14-C | |
| Isoflurane | Fluriso (Isoflurane) Liquid for Inhalation | 502017-250ml | |
| Isothermal heating pad | Braintree scientific inc | 39DP | |
| Laminin | Thermo Fisher | INV-23017015 | Laminin Mouse Protein, Natural |
| Latex bulb | VWR | 82024-554 | |
| LED 530 nm | Sutter Instrumets | 5A-530 | |
| Low binding protein 0.2 μm sterile filter | Pall | FG4579 | acrodisk syringe filter 0.2um supor membrane low protein binding non pyrogenic |
| MEM | Invitrogen | INV-11380037 | |
| MTS-5-TAMRA | Biotium | 89410-784 | MTS-5-TAMRA |
| OriginPro Analysis Software | OriginLab Corporation | OriginPro 2022 (64-bit) SR1 | |
| Photodiode | Custom Made | NA | |
| PlanApo 60x oil 1.4 N.A/∞/0.17 | Olympus | BFPC2 | |
| Platinum wire 0.5 mm, 99.9 % metals basis | SIGMA | 267228-1G | To manufacyte field stimulation electrode |
| Pulse Generator | WPI | Pulsemaster A300 | |
| Shutter drive controller | Uniblitz | 100-2B | |
| Shuttter | Uniblitz | VS2582T0-100 | |
| S-MEM | Invitrogen | INV-11380037 | |
| Sterile bench pad | VWR | DSI-B1623 | |
| Sterile saline | SIGMA ALDRICH | S8776 | |
| Sylgard 184 Silicone Elastomer kit | Dow Corning | 1419447-1198 | |
| Vaporizer for Anesthesia | Parkland Scientific | V3000PK | |
| Voltage generator | Custom Made | NA |
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Cite This Article
Bibollet, H., Bennett, D. F., Schneider, M. F., Hernández-Ochoa, E. O. Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability. J. Vis. Exp. (196), e65311, doi:10.3791/65311 (2023).