Visualizing the divergent conformational dynamics of KCNH channels

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Project Summary The KCNH channel family includes both the Human ether á go-go related gene (hERG, KCNH2) potassium channel that is expressed in the heart and responsible for repolarizing the action potential and, the mammalian ether á go-go gene (EAG, KCNH1) potassium channel is expressed in neuronal tissue and contributes to electrical excitability. The role of hERG in cardiac health is well studied and mutations in hERG cause Long QT type 2 syndrome. Comparatively, the physiological role of EAG is relatively unstudied, yet human EAG is over expressed in many types of cancer and newly identified genetic mutations are linked to epileptogenic Temple- Baraitser and Zimmerman-Leband syndromes. Additionally, although EAG is inhibited by calcium sensor proteins CaM and S100B, the stoichiometry, calcium occupancy and cooperativity remain to be uncovered. While hERG and EAG channels share high sequence similarity, domain topology, and structural similarity they have highly divergent gating kinetics and regulation. We hypothesize that each KCNH channel has divergent and distinct gating dynamics that give rise to unique channel kinetics to tune individual channels for their precise physiological roles and these dynamics are altered by physiologically relevant effectors. In this proposal we measure and model the dynamics of the structurally solved KCNH channels hERG and EAG. We use non- canonical amino acids (ncAA) as small genetically encoded non-perturbing probes to study channel dynamics. We examine the characteristic slow deactivation of hERG that has been partially attributed to voltage dependent potentiation (VDP) and manifests as a hyperpolarizing shift in the voltage dependence of deactivation compared to activation. VDP is reduced in response to lowered extracellular pH which can occur during acute disease states and accelerates hERG deactivation. We incorporate the fluorescent ncAA 3-[(6-acetyl-2- naphthalenyl)amino]-L-alanine (L-ANAP) in hERG and use transition metal Förster resonance energy transfer (tmFRET) to measure dynamic motions at 10-20Å resolution to measure hERG VDP dynamics and examine how it is altered by pH. We will use distances obtained from tmFRET as constraints to visualize VDP in hERG with Rosetta modeling. We then examine the role of the highly conserved KCNH intrinsic ligand motif (IL) in EAG kinetics. In EAG, mutations in the IL alter channel kinetics to slow activation and abolish the Cole-Moore shift. We incorporate the photo-crosslinkable ncAA 4-benzoyl-L-phenylalanine (BZF) at the IL and use ultraviolet light to examine the loss of EAG IL dynamics on channel kinetics. Finally, with a traditional FRET approach we aim to determine the conserved nature of calcium sensor protein regulation of EAG and examine if mutations linked to TB/ZL syndromes alter EAG calcium regulation as it is unclear if calcium dependent channel inhibition is lost in disease states. Due to the roles of hERG in cardiac excitability and arrhythmia, and EAG in TB/ZL and cancer, determining the dynamic gating mechanisms of these channels directly impacts health and disease.