Cole, Bethan Aimee ORCID: 0000-0002-6214-016X (2021) Function and Pharmacological Modulation of the Epilepsy-Associated KNa1.1 (KCNT1) Potassium Channel. PhD thesis, University of Leeds.
Abstract
Gain-of-function missense pathogenic variants of KCNT1, the gene encoding the sodium-activated potassium channel subunit KNa1.1, are associated with intractable early-onset epilepsies. Variants in diverse regions of the channel are hypothesised to interfere with channel gating, although the mechanisms of gating are poorly defined. Patients are heterozygous and there is a lack of information about how pathogenic variants increase channel activity and also the behaviour of heterotetrametric channels comprised of wildtype and variant subunits. Inhibition of hyperactive KNa1.1 channels with class I antiarrhythmic quinidine has had variable success in patients due to low potency, non-selectivity and poor blood-brain-barrier penetration. Seven disease-causing variants were selected from across the spectrum of disorders and involving different protein domains. Whole cell electrophysiology was used to characterise homomeric and heteromeric KNa1.1 channel assemblies carrying epilepsy-causing variants expressed in CHO cells. We found that all disease-causing KNa1.1 variants lowered the energetic barrier associated with the sodium-dependent activation gate by destabilising the inactivated channel conformation. Some variants also had effects on selectivity filter gating. Using the previously determined cryo-EM structure of KNa1.1 and mutational analysis, we identified how quinidine binds within the intracellular pore vestibule. Using in silico methods and whole cell electrophysiology, six small molecule compounds and four FDA-approved drugs were identified that inhibit KNa1.1 channels with low- and sub-micromolar potency. Finally, using mutagenesis and whole cell electrophysiology, six residues involved in Na+-activation were identified, located on the cytoplasmic RCK (regulators of conductance of K+) domains. The results provide a common mechanism for how disease-causing variants cause gain-of-function. In addition to providing more information about the gating of KNa1.1, identification of the Na+-activation mechanisms will aid development of alternative drug modalities in future. We have identified potential therapeutic drugs that could be repurposed for treating KCNT1-related epilepsies, or possible pharmacophores that could be starting points for developing potent and selective inhibitors.
Metadata
Supervisors: | Lippiat, Jonathan and Clapcote, Steven |
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Related URLs: | |
Keywords: | Epilepsy, ion channels, EIMFS, (AD)SHE, KCNT1, KNa1.1, potassium channel |
Awarding institution: | University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > Institute of Membrane and Systems Biology (Leeds) |
Depositing User: | Miss Bethan Aimee Cole |
Date Deposited: | 06 Jun 2022 10:49 |
Last Modified: | 31 Jan 2024 09:50 |
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