Activation of skeletal muscle fibers, which is a prerequisite for all bodily movements, is initiated by the muscle fiber action potential (AP). This wave of electrical depolarization spreads along the fiber away from the neuromuscular junction and radially into the transverse tubules (TTs), causing positively charged membrane voltage sensor domains (VSDs) in the TT membrane Ca2+ channel (Cav1.1) to trigger Ca2+ release via the abutting skeletal muscle ryanodine receptor (RyR1) Ca2+ release channels in the adjacent sarcoplasmic reticulum membrane. However, the molecular mechanisms coupling TT VSD movements to SR RyR1 release channel activation are poorly understood, and the roles of the four individual VSDs within each Cav1.1 are not established. Furthermore, there are no previous studies of VSD movement in response to an AP in any cell type. Here in Aim 1 we first determine the time course (Q(t)) of total VSD charge movement during the AP waveform, and compare it to the time course of Ca2+ release (Aim 1) in adult muscle fibers. In Aims 2 and 3 we examine the time course of the individual VSD movements during an AP. We compare the VSD time courses to the time course of Q(t) and of activation of SR Ca2+ release via RyR1. VSD components that are obviously slow or less voltage dependent compared to the measured Ca2+ release would not be capable of activating the RyR1 Ca2+ release channel. We will characterize the VSD components that do occur prior to and coincident with RyR1 channel opening in response to an AP, and are thus candidates for regulatory effectors of channel activation. In Aim 2 we track VSD movements using cys residues introduced individually in Cav1.1 near the extracellular end of each of the S4 transmembrane helices and fluorescently reacted. In Aim 3 we use artificial fluorescent amino acids introduced near the cytoplasmic end or within the transmembrane S4 segment itself or in the Cav1.1 alpha I-II and II-III cytoplasmic loops considered critical for Cav1.1-RyR1 coupling. In Aim 4 we experimentally determine the effects of charge-eliminating mutations of the VSDs which cause human diseases (either hypokalemic periodic paralysis or malignant hyperthermia). We use high speed (<50 µs/line) line-scan confocal imaging of fibers containing fluorescently stained or fluorescent residues near or in each VSD. We will also use Ca2+ indicators to monitor Ca2+ signals and calculate the underlying Ca2+ release flux from the SR during a single AP in intact voltage clamped fibers. Our studies will elucidate basic molecular mechanisms regulating Ca2+ release in skeletal muscle and the roles of Cav1.1 voltage sensor charges that are mutated in hypokalemic periodic paralysis and malignant hyperthermia. This project has immediate high impact for basic membrane biophysics of muscle and channel activation, for multiple disciplines and in the long-term the potential to further our understanding of the pathophysiology of problems of both locomotion and br...