We thank Dr. J. Vergara (University of California, Los Angeles) for sharing the EGFP-CaV1.1 (rabbit) wild-type plasmid. We thank the Yale Department of Physiology Electronics Laboratory and especially Henrik Abildgaard for the design and construction of the photodiode with track and hold circuit. This work was supported by the National Institutes of Health grants R01-AR075726 and R01-NS103777

This protocol was approved by the University of Maryland Institutional Animal Care and Use Committee. The following protocol has been divided into multiple subsections, consisting of (1) molecular construct design and cysteine reacting dye selection, (2) in vivo electroporation, (3) muscle dissection and fiber isolation, (4) acquisition setup description, (5) assessment of enhanced green fluorescent protein (EGFP) positive fiber electrical activity and cysteine staining, and (6) signal acquisition and processing...

Functional Site-Directed Fluorometry in Native Skeletal Muscle Cells

Here, a step-by-step protocol to conduct FSDF in muscle fibers for the study of individual voltage sensor motions from the CaV1.1 channel is described. Even though the number of steps and the diversity of approaches that are combined in this technique may appear complex, most of these techniques are often routinely used in biophysicist/cell biologist laboratories. Thus, the apparent complexity resides mainly in the combination of all the various approaches in a single integrated technique. Often when carrying ...

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.

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functional site-directed fluorometry in native cells to study skeletal muscle excitability

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.

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

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

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.

When propagating action potentials are triggered in response to repetitive field stimulation, it is possible to track specific voltage sensor motion in response to a specific frequency of depolarization. As shown in Figure 6A, the motion of VSD-II-tagged helices can be tracked in response to each of two successive depolarizations applied at 10 Hz (i.e., spaced by 100 ms). Signal bleaching can be corrected by subtracting baseline to the trace (Figure 6B). Further...

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

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.

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

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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.

Assessment of EGFP-Positive Fiber Electrical Activity, Cysteine Staining, and Signal Acquisition

637 Views.  University of Maryland School of Medicine.  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.

Functional Site-Directed Fluorometry in Native Skeletal Muscle Cells

Summary