Uncovering Druggable Allosteric Sites in HCN Channels - ABSTRACT Infantile epileptic encephalopathy (IEE) is a severe form of early-onset epilepsy and has been associated with de novo and inherited mutations in HCN1 (hyperpolarization-activated and cyclic-nucleotide gated) channels. Current drugs available to treat IEE are nonspecific. To create specific pharmaceuticals for IEE, disease-related polymorphisms have been cataloged and pathology has been attributed to aberrant trafficking to the cellular membrane or alteration of gating properties of HCN1 channels. The overall goal of this application is to develop a novel targeted approach to uncover druggable HCN1 allosteric sites using genomic, functional, and structural approaches. We propose to utilize both an allosteric inhibitor and an allosteric activator of HCN1 to uncover their mechanism of action by structural determination of complexes and investigation of their functional effects on WT and mutant channels. We will then determine the association of allosteric trajectories predicted by coevolution models with the mechanisms of action of these small molecules as well as pathogenic mutations in HCN1. Together with determining structures of other gating states, this will allow generation of a model of ion channel allostery and will provide a testable framework for structure-function relationships. Allosteric modulation of HCN channels will thus be predicted and exploited for rational design of candidate therapeutics. Our first and second specific aims are to determine the structures by single-particle cryo-EM of HCN1 in complex with an allosteric inhibitor, propofol, and in complex with an allosteric activator, PIP2, respectively. The candidate binding sites will be validated by mutagenesis followed by electrophysiology. Clues into the molecular reasons for the HCN1 selectivity for propofol despite strong sequence conservation across all HCN family members, will be provided by a bioinformatic co-evolution analysis. Statistical coupling analysis will identify and predict HCN1 allosteric pathways and we will determine their association with pathogenic missense mutations and propofol binding. Candidate positions will be tested by mutagenesis and electrophysiology. Our third specific aim is to obtain structures of distinct conformers of HCN1 channels by adjusting the sample conditions (e.g. liposomes with an established electrochemical gradient) as well as by targeting disease-associated HCN1 polymorphisms. This aim will not only aid in understanding the basics of channel gating by determining physiologically-relevant channel conformations, but also will facilitate structure-based drug design to modulate aberrant, disease-specific ion channel activity.
