A depolarization-initiated influx of Ca through voltage-gated Ca (CaV) channels gives rise to a plethora of physiological responses such as neurotransmitter release, muscle contraction and gene expression. Membrane depolarization is sensed by four transmembrane structures, the voltage sensor domains (VSDs), which surround and control the activation, deactivation and inactivation properties of a central Ca-selective pore governing the amount and timing of Ca influx. In contrast to homotetrameric KV channels, the CaV pore and four VSDs are encoded by a single long polypeptide chain (alpha1). Thus, each VSD has a unique primary amino acid sequence, suggesting distinct voltage-sensing properties. Critically, the voltage-sensing processes coupling membrane depolarization to Ca influx are still poorly understood and the molecular mechanisms by which auxiliary subunits, such as beta and alpha2delta, alter the voltage dependence of the channel, still need to be elucidated. This lack of knowledge persists in part because ionic and gating current measurements have not thus far captured the properties of individual VSDs in CaV channels. Using Voltage Clamp Fluorometry (VCF), we have resolved that the time- and voltage-dependent properties of each of the four VSDs of human CaV1.2 revealing their highly distinct functional properties. We now have the experimental tools and theoretical formulation to answer key unresolved questions on the operation of CaV1.2 channels, as delineated in four specific aims: (1) To establish the contribution of individual voltage sensing domains to CaV1.2 channel activation. (2) To establish the molecular mechanism by which accessory subunits regulate voltage-dependent activation of CaV1.2 channels. (2a) regulation by alpha2delta subunits (2b) regulation by beta subunits (3) To determine the role of each VSD in Voltage- and Ca-dependent Inactivation and (4) To develop a CaV1.2 model accounting for the operation and role of the four distinct VSDs. CaV1.2 channels specifically labeled at each VSD with small, environment-sensitive fluorophores will be voltage-clamped using the cut-open oocyte technique, so that voltage-evoked fluorescence changes will reflect local conformational rearrangements. A series of physically-relevant models consistent with the CaV1.2 structure and accounting for all experimentally- resolved aspects of CaV1.2 voltage-dependent operation, including the interactions governing excitation- evoked Ca influx. The innovative aspects of this proposal include (1) the experimental approach, unprecedented for the CaV superfamily; (2) the hypothesis that VSDs are the targets of regulation by modulatory subunits; (3) the premise, supported by striking preliminary results, that CaV1.2 VSDs are drivers and regulators for inactivation; (4) the theoretical approach proposes the first model consistent with the molecular architecture and asymmetry of CaV channels. Finally, this study will contribute to the understanding of the molecular mechanisms of pathological states caused by altered CaV1.2 voltage dependence, such as Timothy Syndrome.