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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.


Related in: MedlinePlus

The polarity of the Trp265 indole nitrogen modulates retigabine sensitivity.(a) Chemical structures of Trp (with ring positions labelled) and F3-Trp, accompanied by colorimetric representations of electrostatic surface potentials. (b) Current magnitudes in oocytes injected with KCNQ3*[Trp265TAG] mRNA and either an unconjugated tRNA (pdCpA; n=5) or tRNA amino acylated with F3-Trp (n=12, *P<0.05, Student's t-test). (c) Exemplar currents from F3-Trp-rescued KCNQ3*[Trp265TAG] channels in the presence and absence of retigabine. (d) Conductance–voltage relationships illustrating the effects of 1 μM retigabine on Trp-rescued (n=12) and F3-Trp-rescued KCNQ3*[Trp265TAG] (n=12) channels. (e) Activation kinetics (−20 mV) for F3-Trp-rescued channels (n=3), in the presence and absence of 100 μM retigabine (*P<0.05, Student's t-test). (f) Concentration–response curves for retigabine effects on numerous fluoro-Trp analogues (n=9–12 per Trp analogue) substituted at position Trp265, illustrating enhanced retigabine potency with increased fluorination. (g) Conductance–voltage relationships with indicated retigabine concentrations on a KCNQ3 mutant (Asn220Cys, n=4) with an intrinsic hyperpolarizing shift in gating. Conductance–voltage relationships for F3-Trp substituted at Trp265TAG are shown for comparison. (h) Concentration–response curves for retigabine effects on KCNQ3* (n=5) and KCNQ3*[Asn220Cys] (n=4) channels. In all panels, error bars represent s.e.m.
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f4: The polarity of the Trp265 indole nitrogen modulates retigabine sensitivity.(a) Chemical structures of Trp (with ring positions labelled) and F3-Trp, accompanied by colorimetric representations of electrostatic surface potentials. (b) Current magnitudes in oocytes injected with KCNQ3*[Trp265TAG] mRNA and either an unconjugated tRNA (pdCpA; n=5) or tRNA amino acylated with F3-Trp (n=12, *P<0.05, Student's t-test). (c) Exemplar currents from F3-Trp-rescued KCNQ3*[Trp265TAG] channels in the presence and absence of retigabine. (d) Conductance–voltage relationships illustrating the effects of 1 μM retigabine on Trp-rescued (n=12) and F3-Trp-rescued KCNQ3*[Trp265TAG] (n=12) channels. (e) Activation kinetics (−20 mV) for F3-Trp-rescued channels (n=3), in the presence and absence of 100 μM retigabine (*P<0.05, Student's t-test). (f) Concentration–response curves for retigabine effects on numerous fluoro-Trp analogues (n=9–12 per Trp analogue) substituted at position Trp265, illustrating enhanced retigabine potency with increased fluorination. (g) Conductance–voltage relationships with indicated retigabine concentrations on a KCNQ3 mutant (Asn220Cys, n=4) with an intrinsic hyperpolarizing shift in gating. Conductance–voltage relationships for F3-Trp substituted at Trp265TAG are shown for comparison. (h) Concentration–response curves for retigabine effects on KCNQ3* (n=5) and KCNQ3*[Asn220Cys] (n=4) channels. In all panels, error bars represent s.e.m.

Mentions: To investigate the possible involvement of other modalities of Trp interactions with the drug, we also examined the consequences of fluorination of Trp265 on retigabine effects. Trp fluorination is typically used to modify the electrostatic surface potential and test for the effects of cation–pi interactions (Fig. 4a)32. We were unable to detect functional channels carrying the F4-Trp side chain (fluorines at ring positions 4,5,6 and 7, as numbered in Fig. 4a); however, robust current rescue was observed for less fluorinated derivatives such as F3-Trp (Fig. 4b,c). Retigabine potentiation of KCNQ3* channels was retained with F3-Trp substitution at Trp265 (Fig. 4c–e), a substitution that still potently diminishes the negative electrostatic potential on the face of the side chain (Fig. 4a), indicating that a cation–pi interaction of Trp265 with retigabine or some other entity (perhaps another channel residue) is not required for retigabine effects. It is noteworthy that progressive fluorination of Trp265 caused variable effects on KCNQ3*-gating properties (Table 1), with F1-Trp causing a modest depolarizing shift of the activation V1/2, while F3-Trp significantly shifted gating in the hyperpolarizing direction. This lack of an obvious trend also suggests that an endogenous cation–pi interaction involving Trp265 does not contribute significantly to the gating process.


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)

The polarity of the Trp265 indole nitrogen modulates retigabine sensitivity.(a) Chemical structures of Trp (with ring positions labelled) and F3-Trp, accompanied by colorimetric representations of electrostatic surface potentials. (b) Current magnitudes in oocytes injected with KCNQ3*[Trp265TAG] mRNA and either an unconjugated tRNA (pdCpA; n=5) or tRNA amino acylated with F3-Trp (n=12, *P<0.05, Student's t-test). (c) Exemplar currents from F3-Trp-rescued KCNQ3*[Trp265TAG] channels in the presence and absence of retigabine. (d) Conductance–voltage relationships illustrating the effects of 1 μM retigabine on Trp-rescued (n=12) and F3-Trp-rescued KCNQ3*[Trp265TAG] (n=12) channels. (e) Activation kinetics (−20 mV) for F3-Trp-rescued channels (n=3), in the presence and absence of 100 μM retigabine (*P<0.05, Student's t-test). (f) Concentration–response curves for retigabine effects on numerous fluoro-Trp analogues (n=9–12 per Trp analogue) substituted at position Trp265, illustrating enhanced retigabine potency with increased fluorination. (g) Conductance–voltage relationships with indicated retigabine concentrations on a KCNQ3 mutant (Asn220Cys, n=4) with an intrinsic hyperpolarizing shift in gating. Conductance–voltage relationships for F3-Trp substituted at Trp265TAG are shown for comparison. (h) Concentration–response curves for retigabine effects on KCNQ3* (n=5) and KCNQ3*[Asn220Cys] (n=4) channels. In all panels, error bars represent s.e.m.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: The polarity of the Trp265 indole nitrogen modulates retigabine sensitivity.(a) Chemical structures of Trp (with ring positions labelled) and F3-Trp, accompanied by colorimetric representations of electrostatic surface potentials. (b) Current magnitudes in oocytes injected with KCNQ3*[Trp265TAG] mRNA and either an unconjugated tRNA (pdCpA; n=5) or tRNA amino acylated with F3-Trp (n=12, *P<0.05, Student's t-test). (c) Exemplar currents from F3-Trp-rescued KCNQ3*[Trp265TAG] channels in the presence and absence of retigabine. (d) Conductance–voltage relationships illustrating the effects of 1 μM retigabine on Trp-rescued (n=12) and F3-Trp-rescued KCNQ3*[Trp265TAG] (n=12) channels. (e) Activation kinetics (−20 mV) for F3-Trp-rescued channels (n=3), in the presence and absence of 100 μM retigabine (*P<0.05, Student's t-test). (f) Concentration–response curves for retigabine effects on numerous fluoro-Trp analogues (n=9–12 per Trp analogue) substituted at position Trp265, illustrating enhanced retigabine potency with increased fluorination. (g) Conductance–voltage relationships with indicated retigabine concentrations on a KCNQ3 mutant (Asn220Cys, n=4) with an intrinsic hyperpolarizing shift in gating. Conductance–voltage relationships for F3-Trp substituted at Trp265TAG are shown for comparison. (h) Concentration–response curves for retigabine effects on KCNQ3* (n=5) and KCNQ3*[Asn220Cys] (n=4) channels. In all panels, error bars represent s.e.m.
Mentions: To investigate the possible involvement of other modalities of Trp interactions with the drug, we also examined the consequences of fluorination of Trp265 on retigabine effects. Trp fluorination is typically used to modify the electrostatic surface potential and test for the effects of cation–pi interactions (Fig. 4a)32. We were unable to detect functional channels carrying the F4-Trp side chain (fluorines at ring positions 4,5,6 and 7, as numbered in Fig. 4a); however, robust current rescue was observed for less fluorinated derivatives such as F3-Trp (Fig. 4b,c). Retigabine potentiation of KCNQ3* channels was retained with F3-Trp substitution at Trp265 (Fig. 4c–e), a substitution that still potently diminishes the negative electrostatic potential on the face of the side chain (Fig. 4a), indicating that a cation–pi interaction of Trp265 with retigabine or some other entity (perhaps another channel residue) is not required for retigabine effects. It is noteworthy that progressive fluorination of Trp265 caused variable effects on KCNQ3*-gating properties (Table 1), with F1-Trp causing a modest depolarizing shift of the activation V1/2, while F3-Trp significantly shifted gating in the hyperpolarizing direction. This lack of an obvious trend also suggests that an endogenous cation–pi interaction involving Trp265 does not contribute significantly to the gating process.

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.


Related in: MedlinePlus