KCNH channels such as EAG and hERG serve important physiological roles in the nervous system and are targets for disease such as epilepsy and cardiac arrhythmia. They are emerging biomarkers for malignancy and proliferation in a wide range of blood cancers and tumors. Unique to this family of channels are highly conserved intracellular domains that have evolved over the millennia to serve unique physiological roles. In the previous project period, the PI and Co-I resolved details about the role of the C-terminal cyclic nucleotide- binding homology domain (CNBhD) in gating, and provided first insights into dynamic behavior of the Per-Arnt- Sim (PAS) domain. We generated new reagents in the form of single-chain fragment (scFv) antibodies, which we showed exerted therapeutic potential with a beneficial ceiling effect that could confer protection against arrhythmia. Here we have developed a new model for dynamic modulation of gating via the interactions of the PAS domain, CNBhD and the C-linker based on recent cryo-EM structures of closed and open channels. We will test hypotheses emerging from the EAG1 cryo-EM structure with calmodulin (CaM) to elucidate the mechanism by which CaM inhibits EAG1 channel function. We will test a new hypothesis for how the PAS-cap modulates channel gating scFv antibodies as tools to monitor state-dependent changes in accessibility and by immobilizing the domain by crosslinking substituted unnatural amino acids. We will count the number of PAS domains in heteromeric hERG channels comprising PAS-containing (1a) and PAS-less (1b) subunits in both heterologous systems and native tissues. To answer this long-standing question of hERG stoichiometry, we will use isoform-specific scFvs in combination with a novel single-molecule technology that can detect individual binding events of antibodies with modest affinities to untagged channel subunits and is equally applicable to native tissues. The broad range of biochemical, biophysical and functional approaches reflect highly complementary strengths of the participating laboratories. Given the importance of the KCNH family of channels to so many physiological and disease processes, the advances expected from the work here, made possible by recent cutting-edge conceptual and technical developments, will have broad implications.