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Atomic basis for therapeutic activation of neuronal potassium channels.

Kim RY, Yau MC, Galpin JD, Seebohm G, Ahern CA, Pless SA, Kurata HT - Nat Commun (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, 2176 Health Sciences Mall, Vancouver, British Columbia, Canada V6T 1Z3.

ABSTRACT
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.

No MeSH data available.


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.
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f5: 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.

Mentions: Given the dramatic effects of the Trp265Ind substitution, we sought to clarify the contributions of other residues reported to play a role in retigabine binding. Using a chimeric approach between KCNQ1 and KCNQ3, previous work identified KCNQ3 residues Thr271 (S5 helix), Leu272 (S5 helix), Leu314 (pore helix) and Leu338 (S6 helix) as important contributors to a putative retigabine binding pocket19. We generated several mutations at each of these positions in KCNQ3* and tested retigabine effects over a broad concentration range (Fig. 5a,b). We observed that all functional mutants at these positions retained a large retigabine-mediated gating shift (Fig. 5b), although reduced potency was reflected by a shift to higher drug concentrations in many cases. Moreover, many of these mutations caused intrinsic shifts in channel gating in the absence of retigabine (Fig. 5a). Previous reports have suggested that many of these mutations strongly diminish retigabine action; however, our application of higher concentrations illustrates that, while potency of the drug is weakened, large gating shifts still occur. These data indicate that Thr271, Leu272, Leu314 and L338 make measurable contributions to retigabine binding affinity. However, the Trp265Ind substitution abolishes any response to retigabine at experimentally achievable concentrations. Thus, Trp265Ind remains the most structurally subtle, yet most disruptive, mutation we have identified in terms of retigabine sensitivity, highlighting the potential importance of H-bonding with the Trp265 side chain.


Atomic basis for therapeutic activation of neuronal potassium channels.

Kim RY, Yau MC, Galpin JD, Seebohm G, Ahern CA, Pless SA, Kurata HT - Nat Commun (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4561856&req=5

f5: 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.
Mentions: Given the dramatic effects of the Trp265Ind substitution, we sought to clarify the contributions of other residues reported to play a role in retigabine binding. Using a chimeric approach between KCNQ1 and KCNQ3, previous work identified KCNQ3 residues Thr271 (S5 helix), Leu272 (S5 helix), Leu314 (pore helix) and Leu338 (S6 helix) as important contributors to a putative retigabine binding pocket19. We generated several mutations at each of these positions in KCNQ3* and tested retigabine effects over a broad concentration range (Fig. 5a,b). We observed that all functional mutants at these positions retained a large retigabine-mediated gating shift (Fig. 5b), although reduced potency was reflected by a shift to higher drug concentrations in many cases. Moreover, many of these mutations caused intrinsic shifts in channel gating in the absence of retigabine (Fig. 5a). Previous reports have suggested that many of these mutations strongly diminish retigabine action; however, our application of higher concentrations illustrates that, while potency of the drug is weakened, large gating shifts still occur. These data indicate that Thr271, Leu272, Leu314 and L338 make measurable contributions to retigabine binding affinity. However, the Trp265Ind substitution abolishes any response to retigabine at experimentally achievable concentrations. Thus, Trp265Ind remains the most structurally subtle, yet most disruptive, mutation we have identified in terms of retigabine sensitivity, highlighting the potential importance of H-bonding with the Trp265 side chain.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, 2176 Health Sciences Mall, Vancouver, British Columbia, Canada V6T 1Z3.

ABSTRACT
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.

No MeSH data available.