Project Summary/Abstract The long-term goals of this project are to develop a high-resolution understanding of ion channel function, assembly, and regulation. Our studies focus on uncovering the architectural foundations that underlie the modulation and assembly of channels from the voltage-gated ion channel (VGIC) superfamily and seek to address the fundamental question of how these multi-subunit protein complexes are assembled into properly functioning forms. There are exemplar structures of representatives of most major VGIC classes that reveal a shared, intricately assembled, multi-domain architecture. Although it has been appreciated for decades that assembly of such proteins is critical for their proper function, there has been no direct structural information to inform how such complicated multi-unit assembles are put together or whether they interact with chaperone proteins that aid in their assembly. Among VGICs, the high-voltage activated class of voltage-gated calcium channels (CaV1s and CaV2s) represent a paradigmatic case whose function and trafficking is powerfully shaped by interactions between pore-forming CaV1 or CaV2 CaVa1 and auxiliary CaVb and CaVa2d subunits. Our studies focus on the first known structure of an ion channel:chaperone complex, recently determined by our lab, comprising the brain and heart CaV1.2 channel, CaVb3, and a nine subunit membrane protein chaperone assembly known as the Endoplasmic reticulum Membrane protein Complex (EMC). Binding to the EMC chaperone appears to prepare the CaVa1/CaVb pair for handoff to the CaVa2d to complete channel assembly. We aim to understand which EMC:channel interactions are important for channel biogenesis, how disease mutations affect EMC:CaV interactions, and the factors that drive the channel assembly handoff mechanism. The EMC interaction sites are conserved throughout the CaV1 and CaV2 families and may be shared by other VGIC superfamily members. A second effort is directed at defining other VGIC EMC clients and determining if they use common elements to interact with the EMC. Elaboration of the underlying structural framework of VGIC biogenesis is essential for understanding how VGICs are made and integrated into intracellular signaling pathways and for developing new ways to control channel function. Our efforts encompass a multidisciplinary approach that includes biochemical, biophysical, mass spectrometry, and cryo-electronmicroscopy studies to probe structure and cell biological and electrophysiological measurements to dissect function. Because of their important roles in human physiology, VGICs are targets for drugs with great utility for treating cardiac arrhythmias, hypertension, congestive heart failure, epilepsy, and chronic pain. Thus, understanding their structures mechanisms of assembly at atomic level detail should greatly assist development of valuable therapeutic agents for a wide range of human ailments.