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Defining the molecular mechanisms of subtype-specific KCNQ2/3 potassium channel activators

University of British Columbia

Retigabine is the first approved anti-epileptic drug that acts via activation of voltage-gated potassium channels, targeting KCNQ channels that underlie the neuronal M-current. Retigabine exhibits little specificity between KCNQ2-5, which all contain a Trp residue in the pore domain that is essential for retigabine actions. The retigabine analog ICA 069673 (‘ICA73’) exhibits much stronger effects than retigabine on KCNQ2 channels, including a large hyperpolarizing shift of the voltage-dependence of activation, and roughly two-fold enhancement of peak current. Unlike retigabine, ICA73 exhibits strong subtype specificity for KCNQ2 over KCNQ3, and appears to have a unique mechanism of action, because pore mutations that abolish retigabine action (KCNQ2 Trp236Phe) do not affect ICA73 sensitivity. Based on ICA73 sensitivity of chimeric constructs of the transmembrane segments of KCNQ2 and KCNQ3, this drug appears to interact with the KCNQ2 voltage sensor (S1-S4) rather than the pore region targeted by retigabine. KCNQ2 point mutants in the voltage sensor were generated based on KCNQ2/KCNQ3 sequence differences, and screened for ICA73 sensitivity. These experiments reveal that KCNQ2 residues Phe168 and Ala181 in the S3 segment are essential determinants of ICA73 subtype specificity. Mutations at either position in KCNQ2 abolish the ICA73-mediated gating shift, while retaining retigabine sensitivity. Interestingly, KCNQ2[A181P] mutant channels show little ICA73-mediated gating shift, but retain current potentiation by the drug. When Phe168 and Ala181 are substituted into KCNQ3 ([L198F] and [P211A]), ICA73-sensitivity can be partially rescued. These results demonstrate that retigabine and ICA73 act via distinct mechanisms, and provide the first insights into channel residues that underlie subtype specificity of KCNQ channel openers. Further mutagenic scanning of the voltage sensor, and screening for potential ICA73 binding residues in solvent accessible pockets have also generated new insights into KCNQ channel function, despite not identifying additional residues essential for ICA73 sensitivity. Taken together, findings presented in this thesis have laid a foundation for further understanding of diverse mechanisms of action of KCNQ potassium channel openers, which may lead to more targeted and rational approaches for drug design.

Text

2016-10-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/57894

Pharmacology and Therapeutics

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/