Unnatural Amino Acid Mutagenesis Reveals the Critical Role of Hydrogen Bonding for Binding of Retigabine in the Pore of KCNQ Channels

Abstract

Retigabine is the prototypical member of a novel class of anti-epileptic drugs that act by potentiating neuronal KCNQ2/3 channels. Retigabine interacts directly with the pore of KCNQ2/3 channels, requiring a conserved Trp, present in both KCNQ2 and KCNQ3, but not KCNQ1. Application of 100 µM retigabine to KCNQ3 channels causes a leftward 40 mV shift of the G-V curve that can be strongly ameliorated with either Phe or Leu substitution of Trp 265, with minor effects on channel gating. We have used nonsense suppression to subtly alter the properties of this Trp residue (265) in homomeric KCNQ3[A315T] channels to probe the chemical features of retigabine binding in more detail. A tri-fluorinated Trp derivative (3,4,5-F-Trp), which reduces cation-pi binding ability of this side chain caused a 20 mV left-shift in the gating properties of KCNQ3, but these channels were still highly sensitive to retigabine, suggesting that a cation-pi interaction is not essential for retigabine interactions with KCNQ3. In contrast, Ind (an isosteric Trp derivative that lacks the indole nitrogen atom required for hydrogen bonding ability of Trp) substitution completely abolished he effects of retigabine with minimal effects on KCNQ3 gating. These observations demonstrate that retigabine binds KCNQ3 channels through an energetically significant H-bond interaction via the indole nitrogen of Trp 265. These findings stringently constrain models of retigabine binding to KCNQ channels, and will guide the rational development of improved retigabine derivatives.