Published: June 2nd, 2023
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, 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' 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.
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 underst....
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.......
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.......
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 .......
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....
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