Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201.
Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201.
Proc Natl Acad Sci U S A. 2021 Oct 5;118(40). doi: 10.1073/pnas.2026116118.
The skeletal muscle L-type Ca channel (Ca1.1) works primarily as a voltage sensor for skeletal muscle action potential (AP)-evoked Ca release. Ca1.1 contains four distinct voltage-sensing domains (VSDs), yet the contribution of each VSD to AP-evoked Ca 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 Ca1.1, expressed each construct via in vivo gene transfer electroporation, and used AP fluorometry to track the movement of each Ca1.1 VSD in skeletal muscle fibers. We first provide electrical measurements of Ca1.1 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 Ca, and the calculated rate of Ca 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 Ca 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 Ca 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 Ca release, and thus could be involved in voltage sensor rearrangements or Ca release activation.
骨骼肌 L 型钙通道 (Ca1.1) 主要作为骨骼肌动作电位 (AP) 引发的钙释放的电压传感器。Ca1.1 包含四个不同的电压感应域 (VSD)，但每个 VSD 对 AP 引发的钙释放的贡献仍不清楚。为了研究 VSD 在兴奋-收缩偶联 (ECC) 中的作用，我们在 Ca1.1 的每个 S4 电压感应段上编码了半胱氨酸取代，通过体内基因转移电穿孔表达每个构建体，并使用 AP 荧光法跟踪每个 Ca1.1 VSD 在骨骼肌纤维中的运动。我们首先提供 Ca1.1 电压传感器电荷运动对 AP 波形的电测量。然后，我们描述了荧光标记通道的 VSD 荧光信号对 AP 的响应，并将其与电测量的电荷运动、光学测量的游离细胞质 Ca 和从肌浆网计算的 Ca 释放速率以及骨骼肌纤维激活的生理信号进行比较。每个 VSD 的荧光信号的相当一部分发生在钙释放峰值之后，甚至更多的是在 AP 期间电测量的电荷运动的更早峰值之后，因此不能直接反映钙释放或电荷运动的激活。然而，VSDs I、II 和 IV 的荧光信号的相当一部分，但不是 VSDIII 的荧光信号，与电荷移动的上升相重叠，甚至更多的是钙释放，因此可能参与电压传感器重排或钙释放激活。
