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

Kv1.1, 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 cryo-electron microscope structure of the channel, the characterization of novel Kv1.1 accessory protein, the discovery of channel alternative splicing variants, 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 side-directed fluorometry, as well as engineer cells or animal models.

Currently, the field faces several experimental challenges. In a skeletal muscle, proper trafficking and communication between Kv1.1 and RYR1, along with many regulatory proteins is crucial to support excitation contraction coupling. The methods to directly study these protein-protein interactions between Kv1.1 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 Kv1.1, calcium release, and localized at 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 heterologous expression system, our protocol now allows tracking confirmational changes in Kv1.1 voltage sensors during a propagated action potential in its 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 translocation is linked to Kv1.1 opening or reanogen receptor activation. 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 rabid Cav1.1 is used. The presence of the cytomegalovirus promoter allows strong transfection efficiency in muscle fibers. Introduce cysteine 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, confirmed that neither the inserted cysteines, nor the presence of thiol-reactive dye impacts the voltage dependence and the conductance of the channel. Turn on all the acquisition setup components for the fluorometry measurement.

Place the 35 millimeter glass-bottom dish containing the dissociated muscle fibers on the stage of the microscope. Carefully remove the culture media with a 1, 000 microliter pipette and replace it with two milliliters of room temperature Ringer's solution. Using a mechanical or motorized manipulator, place the two platinum wires perpendicular to the bottom of the dish.

Ensure the electrode terminals are aligned relative to the fibers longitudinal axis and a few millimeters away from the fibrous ends. Adjust the electrode positioning by rotating the dish or moving each electrode if mounted on an independent micromanipulator. Turn on the transmitted light and find the fibers in the field of view, using a 20X objective.

Move the EGFP filter cube into the light pathway. After turning off the transmitted light, activate the 488 nanometer excitation light using a remote controlled light shutter. To identify the EGFP positive fibers, center the fibers with the brightest EGFP signal in the middle of the field of view.

Use a diaphragm positioned in the excitation light path to focus the excitation light in a specific area of the fiber. This enables signal acquisition where the EGFP Cav1.1 signal is maximal. Adjust the fiber position with the mechanical stage to align with the diaphragm light path and store the fiber XY location.

After returning to the first saved localization, set the duration of two sequential stimulation pulses to one millisecond, amplitude to 20 volts, and the polarity of the pulses to alternating. Then use a manual trigger switch to deliver the two sequential pulses. During the stimulation, observe two concentric homogenous fiber contractions in response to the two pulses of opposite polarity.

If no contraction, or only one concentric contraction is observed with alternating polarity pulses, discard the fibers from the rest of the experiment. Add two microliters of 10 millimolar MTS-5-TAMRA solution directly into the dish and gently mix with a 1, 000 microliter pipette. Incubate for four to five minutes to allow the diffusion of the fluorescent thiol molecule into the transverse tubule system lumen.

Through field stimulation, apply bipolar repetitive stimulations to evoke successive action potential trains at a rate of 50 hertz for 300 milliseconds every one second for five minutes. Remove the staining solution from the dish with a 1, 000 microliter pipette and replace it with two milliliters of room temperature ringer solution. Let the stained fiber recover from the staining protocol for at least 10 minutes.

After reassessing the fiber health and electrical activity with two alternating pulses, move the MTS-5-TAMRA filter cube into the light pathway and activate the 533 nanometer excitation light with a remote controlled light shutter to visually confirm staining on the fibers. Next, using the stored location on the motorized stage, place the fiber in the middle of the field with the MTS-5-TAMRA filter cube, the diaphragm, and the 60X oil immersion objective. After reorienting the field stimulation platinum wires at each end of the fiber, align the wires on the main axis of the fibers in a straight line and space them five millimeters apart with the fiber in the center.

On the acquisition software, select the protocol that will be used for acquisition. This step is controlled only with the computer and triggers all the downstream devices for electrical stimulation and light path shutter control. For each protocol, minimize the total acquisition time as much as possible to avoid signal bleaching, but allow a few tens of milliseconds of light illumination and signal acquisition before the first electrical stimulation to record a baseline.

Run the protocol by hitting Execute. The recorded signal is automatically displayed on the screen. The small amplitude of the signal and the quick mechanical contraction of the fiber often hide most of the signal and display a movement artifact.

Add one micromolar of 100 millimolar and benzoyl per toluene sulfonimide to the recording solution to minimize the contractual responses and further distinguish the signal arising from S4 motions due to fiber activation. After a few minutes, run the same protocol a second time. The movement artifact is now removed, but a small fluorescence change corresponding to the S4 movement remains.

Move the dish slightly using the mechanical stage to acquire a signal from an area without any fibers or debris. Run the protocol a final time to record the background fluorescence, Export the traces and use a spreadsheet program to express the signal as Delta F over F zero as a function of time and apply baseline correction if required. The examples of transmitted and fluorescent images of the dissected and not dissociated muscle expressing an EGFP Kv1.1 construct are shown here.

The representative images of a muscle fiber expressing an EGFP Kv1.1 VSD3 construct before and after MTS-5-TAMRA staining are shown in this figure. This dye also stains endogenous cysteines of non-transfected fibers. The confocal images of an EGFP Kv1.1 VSD3 construct and MTS-5-TAMRA staining show a double band pattern, characteristic of Kv1.1 localization on the transverse tubule system of the muscle fiber.

The representative fluorometric recording in response to two stimuli and measured with a photodiode is shown here. Both signals are normalized by their minimum value and plotted as a function of time, relative to their respective depolarization. These recordings show that the rising phase and the time to peak of the signal are similar for both depolarizations imposed on the fiber.