Sequence determinants of subtype-specific actions of KCNQ channel openers

Abstract

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, due to conservation of a Trp residue in the pore domain that binds to the drug. The retigabine analog ICA-069673 ('ICA73') exhibits much stronger effects on KCNQ2 channels, including a large hyperpolarizing shift of the voltage-dependence of activation, roughly two-fold enhancement of peak current, and pronounced subtype specificity for KCNQ2 over KCNQ3. 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 F168 and A181 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, A181P mutant channels show little ICA73-mediated gating shift, but retain current potentiation by the drug. Mutations (L198F and P211A) that introduce these critical KCNQ2 residues at corresponding positions in KCNQ3 transplant partial ICA73 sensitivity. These findings demonstrate that retigabine and ICA73 act via distinct mechanisms, and reveal specific residues that underlie subtype specificity of KCNQ channel openers.

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