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Mechanistic insights into retigabine modulation of neuronal KCNQ channels Kim, Robin Yongkyu


Epilepsy is among the most prevalent neurological disorders, affecting approximately 1% of the human population. There are many types of epilepsies, with diverse aetiologies, and many therapies that target ion channels as a means to combat neuronal hyper-excitability. However, most anti-epileptic drugs today target a limited number of ion channel types, mainly voltage-gated sodium and calcium channels. Retigabine is a recently approved anti-epileptic drug that operates through a novel mechanism of activating voltage-gated potassium channels. Previous research has established neuronal KCNQ channels as the therapeutic target of retigabine. However, detailed insights regarding the molecular mechanisms of retigabine action are lacking, such as its mode of binding, the factors underlying its ability to stabilize channel opening, and the stoichiometry of its action. A lack of such knowledge hampers the development of more potent and specific channel openers devoid of side effects associated with this first-generation drug. The work presented in this thesis utilizes various research techniques to investigate retigabine pharmacology at a molecular level. In the first objective, retigabine binding to KCNQ3 channels is investigated using unnatural amino-acid mutagenesis. The data pinpoint an essential hydrogen-bonding interaction that likely occurs between an S5 tryptophan residue and a carbonyl oxygen moiety present in most KCNQ activating drugs, providing the highest resolution understanding of the retigabine pharmacophore to date. In the second objective, voltage-clamp fluorometry is used to track conformational changes of the voltage-sensing domain of KCNQ3 channels. The data illustrate a network of interactions between the voltage-sensing and ion conducting regions of the channel protein that is dependent on the anionic membrane phospholipid PIP2; these interactions are not only essential for channel function, but also for retigabine binding to modulate channel voltage sensing downstream of binding in the pore domain. Finally, using iii concatenated KCNQ3 constructs to express channels with variable stoichiometry of retigabine binding sites, we demonstrate that a minimum of one retigabine sensitive channel subunit is required for functional pharmacological effects. Overall, this work provides novel insights applicable to the development of retigabine derivatives with greater therapeutic impact, and improves our understanding of lipid and drug regulation of KCNQ channels.

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