The skeletal muscle L-type Ca²⁺ channel (Ca_V1.1) works primarily as a voltage sensor for skeletal muscle action potential (AP)-evoked Ca²⁺ release. Ca_V1.1 contains four distinct voltage-sensing domains (VSDs), yet the contribution of each VSD to AP-evoked Ca²⁺ release remains unknown. To investigate the role of VSDs in excitation-contraction coupling (ECC), we encoded cysteine substitutions on each S4 voltage-sensing segment of Ca_V1.1, expressed each construct via in vivo gene transfer electroporation, and used in cellulo AP fluorometry to track the movement of each Ca_V1.1 VSD in skeletal muscle fibers. We first provide electrical measurements of Ca_V1.1 voltage sensor charge movement in response to an AP waveform. Then we characterize the fluorescently labeled channels' VSD fluorescence signal responses to an AP and compare them with the waveforms of the electrically measured charge movement, the optically measured free myoplasmic Ca²⁺, and the calculated rate of Ca²⁺ release from the sarcoplasmic reticulum for an AP, the physiological signal for skeletal muscle fiber activation. A considerable fraction of the fluorescence signal for each VSD occurred after the time of peak Ca²⁺ release, and even more occurred after the earlier peak of electrically measured charge movement during an AP, and thus could not directly reflect activation of Ca²⁺ release or charge movement, respectively. However, a sizable fraction of the fluorometric signals for VSDs I, II, and IV, but not VSDIII, overlap the rising phase of charge moved, and even more for Ca²⁺ release, and thus could be involved in voltage sensor rearrangements or Ca²⁺ release activation.

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