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Complex Mechanisms of KCNQ Channel Activation by State-dependent Modulators

  • Author / Creator
    Wang, Caroline K
  • Epilepsy is among the most common neurological diseases, but approximately 30% of epileptics are unresponsive to available drugs, and many more suffer significant side effects. One reason for the persisting large fraction of drug-resistant or drug-intolerant patients may be that most drug development in this field has been limited towards just a few molecular targets. I investigated a new class of anti-epileptic drugs that targets voltage-gated potassium channels of the KCNQ family, mutations of which have been implicated in neonatal forms of epilepsy. The KCNQ channel activator retigabine (RTG) was only recently approved (~2011) for use as an antiepileptic in humans. However, RTG has little subtype specificity between KCNQ2-5, and efforts are underway to develop improved retigabine derivatives with better specificity. To do this, a deeper understanding of the mechanism behind KCNQ channel openers is needed. My research compares the mechanisms of RTG and a 'second-generation' KCNQ channel opener, ICA-069673 (ICA73), which is suggested to have a distinct binding site and mechanism from RTG. In Chapter 3, we investigated the state- and use-dependent properties of ICA73 and RTG on KCNQ2 channel activation. Using whole-cell patch clamp electrophysiology and fast-solution switching, we characterized the relationship between drug binding and channel activation, and found that ICA73 preferentially binds to open channels at more depolarized voltages, while RTG can readily access both open and closed channels, and is thus available to bind across a broader range of voltages. Additionally, we found that an alanine to proline mutation in KCNQ2, previously shown to alter ICA73 sensitivity, results in faster ICA73 unbinding despite the drug retaining state dependent binding. Both ICA73 and RTG bound in a use-dependent manner during rapid, successive depolarizations. In Chapter 4, we found that a mixture of ICA73 and RTG synergistically enhanced KCNQ channel activation in Xenopus oocytes using two-electrode voltage clamp. In an in vivo model of zebrafish larvae, RTG showed a clear, dose-dependent inhibition of seizure activity; ICA73 did not. Instead, another KCNQ opener, ML-213, was an exceptionally powerful anticonvulsant drug in larvae, cells, and oocytes. Further investigation of ML-213 actions on KCNQ2 W236F and KCNQ2 F168L, mutations known to disrupt RTG or ICA73 sensitivity respectively, showed that ML-213 sensitivity was most severely disrupted in W236F. Finally, in Chapter 5, we aligned sequences of KCNQ2 with that of KCNQ5, an ICA73-insensitive subtype, and identified several mutations that had altered ICA73 sensitivity. The KCNQ2 mutants K116P, I173T, and G186K exhibited attenuated ICA73-induced gating shifts. Collectively, the results from this thesis highlight fundamental differences in the mechanism of two KCNQ2 openers, ICA73 and RTG, which may be exploited for clinical therapy.

  • Subjects / Keywords
  • Graduation date
    Fall 2018
  • Type of Item
    Thesis
  • Degree
    Master of Science
  • DOI
    https://doi.org/10.7939/R35T3GG0W
  • License
    Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.