Project Summary/Abstract The long‐term goals of this project are to develop a high‐resolution understanding of ion channel function and regulation. Our studies focus on uncovering the architectural foundations that underlie the modulation of exemplar classes from the voltage‐gated ion channel (VGIC) superfamily and seek to address the fundamental question of how conformational changes in channel intracellular domains control and shape VGIC function. Many VGIC superfamily members, including Kv7 voltage‐gated potassium channels and BacNaV bacterial voltage‐gated sodium channels, share a common cytoplasmic domain architecture in which the pore domain and a four‐stranded coiled‐coil frame a metastable membrane proximal domain that acts as a receiver for modulatory signals. We aim to understand how such metastable domains sense inputs from the calcium sensor calmodulin in Kv7s and from temperature in BacNaVs and transmit signals to the channel pore. The prevalence of similar intracellular elements among diverse VGICs suggests that the principles derived from these studies will have broad impact in defining how such intracellular modules shape channel responses. A second effort is directed at defining the architecture of a class of intracellular endolysosomal VGICs known as Two‐Pore‐Channels (TPCs) and that have limited structural characterization. These channels possess a unique tandem transmembrane architecture and respond to a variety of intracellular signals, including calcium. Elaboration of the underlying structural framework of exemplar VGICs is essential for understanding how these and other VGICs are integrated into intracellular signaling pathways and for developing novel ways to intervene to control channel function. Our efforts encompass a multidisciplinary approach that includes biochemical, biophysical, X‐ray crystallographic, and cryo‐electronmicroscopy studies to probe structure and electrophysiological measurements to dissect function. Because of their important role in human physiology, VGICs are the targets for drugs with great utility for the treatment of cardiac arrhythmias, hypertension, congestive heart failure, epilepsy, and chronic pain. Thus, understanding their structures and mechanisms of action at atomic level detail should greatly assist the development of valuable therapeutic agents for a wide range of human ailments.