In excitation–contraction coupling (ECC), when the skeletal muscle action potential (AP) propagates into the transverse tubules, it modifies the conformational state of the voltage-gated calcium channels (CaV1.1). CaV1.1 serves as the voltage sensor for activation of calcium release from the sarcoplasmic reticulum (SR); however, many questions about this function persist. CaV1.1 α1 subunits contain four distinct homologous domains (I–IV). Each repeat includes six transmembranal helical segments; the voltage-sensing domain (VSD) is formed by S1–S4 segments, and the pore domain is formed by helices S5–S6. Because, in other voltage-gated channels, individual VSDs appear to be differentially involved in specific aspects of channel gating, here we thus hypothesized that not all the VSDs in CaV1.1 contribute equally to calcium-release activation. Yet, the voltage-sensor movements during an AP (the physiological stimulus for the muscle fiber) have not been previously measured in muscle. Reorientation of VSDs I–IV in CaV1.1 during an AP should generate a small but measurable electrical current. Still, neither the voltage-sensor charge movement during the AP nor the contribution of the individual VSDs to voltage-gated calcium release have been previously monitored. Here, we electrically monitor VSD movements using an AP voltage-clamp technique applied to muscle fibers. We introduce AP-fluorometry, a variant of the functional site-directed fluorescence, to track the movement of each VSD via a cysteine substitution on the extracellular region of S4 of each VSD and its labeling with a cysteine-reacting fluorescent probe, which served as an optical reporter of local rearrangements. Independent optical recordings of AP and calcium transients were performed to establish the temporal correlation between AP, AP-elicited charge movement, VSDs conformational changes, and calcium release flux. Our results support the hypothesis that not all VSDs in CaV1.1 contribute to ECC.

 National Institutes of Health
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This work is supported by National Institutes of Health grants R37AR055099, R01AR075726 (to M.F. Schneider), R01NS103777 (to R.A. Banniester), and T32AR007592 (to Q. Banks).

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