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.

Public Health Relevance

KCNH channels and their interacting proteins are major components of the electrical machinery of the brain and are critical to learning and memory. KCNH channels also have the potential to serve as indicators of invasiveness for many types of cancer. The work in this proposal will advance our understanding of how these molecular machines contribute to such important processes and how, when they go awry, disease can result. Such insights are the first step toward finding new cures.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Silberberg, Shai D
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Wisconsin Madison
Schools of Medicine
United States
Zip Code
Pointer, Kelli B; Clark, Paul A; Eliceiri, Kevin W et al. (2017) Administration of Non-Torsadogenic human Ether-à-go-go-Related Gene Inhibitors Is Associated with Better Survival for High hERG-Expressing Glioblastoma Patients. Clin Cancer Res 23:73-80
Zhao, Yaxian; Goldschen-Ohm, Marcel P; Morais-Cabral, João H et al. (2017) The intrinsically liganded cyclic nucleotide-binding homology domain promotes KCNH channel activation. J Gen Physiol 149:249-260
Marques-Carvalho, Maria João; Oppermann, Johannes; Muñoz, Eva et al. (2016) Molecular Insights into the Mechanism of Calmodulin Inhibition of the EAG1 Potassium Channel. Structure 24:1742-1754
Harley, Carol A; Starek, Greg; Jones, David K et al. (2016) Enhancement of hERG channel activity by scFv antibody fragments targeted to the PAS domain. Proc Natl Acad Sci U S A 113:9916-21
Liu, Fang; Jones, David K; de Lange, Willem J et al. (2016) Cotranslational association of mRNA encoding subunits of heteromeric ion channels. Proc Natl Acad Sci U S A 113:4859-64
Jones, David K; Liu, Fang; Dombrowski, Natasha et al. (2016) Dominant negative consequences of a hERG 1b-specific mutation associated with intrauterine fetal death. Prog Biophys Mol Biol 120:67-76
Fernandes, Andreia S; Morais-Cabral, João H; Harley, Carol A (2016) Screening for Non-Pore-Binding Modulators of EAG K+ Channels. J Biomol Screen 21:758-65
Morais-Cabral, João H; Robertson, Gail A (2015) The enigmatic cytoplasmic regions of KCNH channels. J Mol Biol 427:67-76
Jones, David K; Liu, Fang; Vaidyanathan, Ravi et al. (2014) hERG 1b is critical for human cardiac repolarization. Proc Natl Acad Sci U S A 111:18073-7
Adaixo, Ricardo; Harley, Carol A; Castro-Rodrigues, Artur F et al. (2013) Structural properties of PAS domains from the KCNH potassium channels. PLoS One 8:e59265