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

Area of Science:

• Membrane biophysics
• Molecular physiology
• Cellular electrophysiology

Background:

• Functional site-directed fluorometry (fSDF) investigates membrane protein structure-function relationships using electrophysiology and fluorescence.
• Previous applications were limited to non-excitable cells (e.g., Xenopus oocytes, cell lines).
• Studying excitation-contraction coupling in muscle requires methods applicable to excitable cells.

Purpose of the Study:

• To adapt and apply functional site-directed fluorometry in adult mammalian skeletal muscle cells.
• To investigate the early steps of excitation-contraction coupling.
• To enable real-time measurements of voltage-gated calcium channel (CaV1.1) function and structural rearrangements in muscle.

Main Methods:

• Design and transfection of cysteine-engineered CaV1.1 channels into adult mouse flexor digitorum brevis muscle fibers via in vivo electroporation.
• Application of thiol-reactive fluorescent dyes for site-specific labeling.
• Simultaneous measurement of membrane currents (electrophysiology) and fluorescence (structural rearrangements).

Main Results:

• Demonstrated the successful applicability of fSDF in adult skeletal muscle fibers.
• Established a protocol for studying CaV1.1 channel function in vivo.
• Provided a method to link electrical signals to mechanical output in muscle contraction.

Conclusions:

• Functional site-directed fluorometry is a viable technique for studying ion channels and protein function in adult mammalian skeletal muscle.
• This approach is crucial for understanding the basic mechanisms of muscle excitability and excitation-contraction coupling.
• The methodology can be adapted for diverse ion channels and proteins in excitable tissues.