The overall objectives are to apply nuclear magnetic resonance (NMR) techniques in concert with experimental functional approaches to elucidate the molecular structure and regulatory mechanisms of neuronal L-type voltage-gated Ca2+ channel (CaV1.2) and its atomic-level structural and functional association with calcium binding protein-1 (CaBP1), -actinin1 (ACTN1) and calmodulin (CaM). During the next five years, we will use NMR, fluorescence, microcalorimetry, cryoEM, x-ray crystallography, and computational analysis to delineate the structure and dynamics of CaM and CaBP1 each bound to CaV1.2. Site-directed mutagenesis and electrophysiology functional analysis will be integrated with atomic– level structural information to understand how CaM, ACTN1 and CaBP1 each reciprocally control the Ca2+- dependent channel activity of CaV1.2 in neuronal functions. By pursuing these studies, we hope to gain an atomic-level understanding of how CaM, ACTN1 and CaBP1 each regulate CaV1.2 involved in controlling neuronal excitability and gene expression. In particular, we want to understand how protein target binding sites work in concert with calcium-binding sites to confer Ca2+-dependent channel activity of CaV1.2 at the postsynaptic membrane. This structural information will probe how L-type channel regulation may be connected to neurological disorders, including epilepsy, Alzheimer’s disease, and Timothy Syndrome. The Specific Aims are three-fold: (1) Determine the structural basis of CaV1.2 channel activation promoted by CaM and ACTN1; (2) Elucidate the structure and functional role of a CaM intermediate with 2 Ca2+ bound and determine its role in regulating Ca2+-dependent inactivation (CDI); (3) Determine the structural basis of how CaBP1 suppresses Ca2+-dependent inactivation (CDI) of CaV1.2.