Voltage sensor movements of Ca _V 1.1 during an action potential in skeletal muscle fibers

Abstract

The skeletal muscle L-type Ca2+ channel (CaV1.1) works primarily as a voltage sensor for skeletal muscle action potential (AP)-evoked Ca2+ release. CaV1.1 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 CaV1.1, expressed each construct via in vivo gene transfer electroporation, and used in cellulo AP fluorometry to track the movement of each CaV1.1 VSD in skeletal muscle fibers. We first provide electrical measurements of CaV1.1 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.

The role of action potential changes in depolarization-induced failure of excitation contraction coupling in mouse skeletal muscle

“…Release of Ca 2+ from the SR is governed by gating of Cav1.1 channels in the t-tubules ( Kovács et al, 1979 ; Rios and Brum, 1987 ; García et al, 1994 ). Although movement of the Cav1.1 gating charges responsible for triggering opening of RyR1 and Ca 2+ release from the SR is faster than opening of Cav1.1 channels, the movement of the gating charges in Ca V 1.1 is relatively slow compared to the upstroke of the AP ( Schneider and Chandler, 1973 ; Banks et al, 2021 ; Savalli et al, 2021 ; Banks et al, 2022 ) such that the wider the AP, the greater the charge movement until saturation is reached. Indeed, only ~65% of the total intramembrane charge is moved during an AP ( Banks et al, 2021 ; Banks et al, 2022 ).…”

Excitation-contraction coupling in mammalian skeletal muscle: Blending old and last-decade research

“…Since the DHPR carries a Ca 2+ current under voltage-clamp protocols in intact fibres (Skoglund et al, 2014;Dayal et al, 2017;Banks et al, 2021), it is expected to function the same as a response to an AP, highlighting its nature as a voltage-gated channel. The activated Ca 2+ inward current is slower, and with a slightly lower amplitude compared to the Ca v 1.2 present in the heart, however, the influx of Ca 2+ through this channel is not necessary for the skeletal muscle ECC and contraction (Caputo and Gimenez, 1967;Armstrong et al, 1972;Dayal et al, 2017;Idoux et al, 2020).…”

“…The voltage sensing function depends on the S4 enrichment in the positively charged aminoacids arginine and lysine. Their movement during the VSD operation produces a small, yet measurable, voltage-dependent intramembrane charge movement, i.e., a current, which precedes the activation of the Ca 2+ release from the SR (Schneider and Chandler, 1973;Rios and Brum, 1987;Banks et al, 2021). The peak of the charge movement time course follows the peak of the AP by 1.5 ms (Banks et al, 2021).…”

“…Their movement during the VSD operation produces a small, yet measurable, voltage-dependent intramembrane charge movement, i.e., a current, which precedes the activation of the Ca 2+ release from the SR (Schneider and Chandler, 1973;Rios and Brum, 1987;Banks et al, 2021). The peak of the charge movement time course follows the peak of the AP by 1.5 ms (Banks et al, 2021). It is intriguing why there are sizeable differences in the amplitudes, voltagedependence and time courses of the VSDI-IV movements (Banks et al, 2021;Savalli et al, 2021), and whether they actually tune in any way the Ca 2+ release from the SR. For instance, the VSDII and VSDIV seem to be the first ones activated, but the VSDI is so slowly activated that it seems not to be directly involved in the Ca 2+ release activation; contradictory results have been reported regarding the activation kinetics of VSDIII (Banks et al, 2021;Savalli et al, 2021).…”

“…The peak of the charge movement time course follows the peak of the AP by 1.5 ms (Banks et al, 2021). It is intriguing why there are sizeable differences in the amplitudes, voltagedependence and time courses of the VSDI-IV movements (Banks et al, 2021;Savalli et al, 2021), and whether they actually tune in any way the Ca 2+ release from the SR. For instance, the VSDII and VSDIV seem to be the first ones activated, but the VSDI is so slowly activated that it seems not to be directly involved in the Ca 2+ release activation; contradictory results have been reported regarding the activation kinetics of VSDIII (Banks et al, 2021;Savalli et al, 2021). In any case, the activation of either one or several of the DHPR´s VSD likely leads to a conformational change that gates the opening of the RyR1 in a cooperative way (Schneider and Chandler, 1973;Rios and Brum, 1987;Ríos et al, 1993).…”