Atomic basis for therapeutic activation of neuronal potassium channels

By Robin Y. Kim, Michael C. Yau, Jason D. Galpin, Guiscard Seebohm, Christopher A Ahern, Stephen Pless and Harley Kutara.

Published in Nature Communications 2915 Sep 3;6:8116.
PMID: 26333338. PMCID: PMC4561856. Link to Pubmed page.

Core Facility: Membrane Protein Expression/Purification.

Figure 5. Detailed characterization of secondary retigabine binding residues and alternative binding site orientations. (a) Conductance–voltage relationships were gathered for the indicated KCNQ3* mutant channels (n=4–6 per mutant), in 0, 100 or 300 μM retigabine. (b) Maximal ΔV1/2 in 300 μM retigabine measured in each mutant channel. Error bars in a,b represent s.e.m. (c) Retigabine was docked into a molecular model of the pore-forming domain of KCNQ3 (see ref. 19). Two orientations are shown with the carbamate group in either the vicinity of Leu314 (‘original’ model) or Trp265 (‘flip’ model). The two binding models are superimposed in the ‘overlay’, showing the similar space occupied by both drug orientations.


Retigabine is a recently approved anticonvulsant that acts by potentiating neuronal M-current generated by KCNQ2–5 channels, interacting with a conserved Trp residue in the channel pore domain. Using unnatural amino-acid mutagenesis, we subtly altered the properties of this Trp to reveal specific chemical interactions required for retigabine action. Introduction of a non-natural isosteric H-bond-deficient Trp analogue abolishes channel potentiation, indicating that retigabine effects rely strongly on formation of a H-bond with the conserved pore Trp. Supporting this model, substitution with fluorinated Trp analogues, with increased H-bonding propensity, strengthens retigabine potency. In addition, potency of numerous retigabine analogues correlates with the negative electrostatic surface potential of a carbonyl/carbamate oxygen atom present in most KCNQ activators. These findings functionally pinpoint an atomic-scale interaction essential for effects of retigabine and provide stringent constraints that may guide rational improvement of the emerging drug class of KCNQ channel activators.


Figure 1.Multiple retigabine molecules modulate KCNQ2 and KCNQ3 channel subunits via an S5 Trp side chain.

Figure 5.Detailed characterization of secondary retigabine binding residues and alternative binding site orientations.

Figure 8.Diverse structures of KCNQ openers. Multiple structures of KCNQ channel openers are presented to highlight the overall features of an amide group flanked by various ring structures. Our findings highlight the importance of the amide carbonyl for interaction with KCNQ3 Trp 265 and likely equivalent positions in KCNQ2, 4 and 5. Drugs depicted are (a) retigabine, (b) ztz-240 (described in ref. 24), (c) acrylamide (s)-1, (d) BMS-204352 and (e) an unnamed experimental drug described in ref. 43.