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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

Nonsense suppression for amino-acid incorporation in KCNQ3* channels.(a) Schematic diagram of the nonsense suppression method, in which mRNA (with a stop codon at Trp265) and amino-acylated tRNA are co-injected into Xenopus oocytes. Incorporation of the unnatural amino acid enables readthrough of the stop codon and expression of functional channels. (b) Current magnitudes in oocytes injected with KCNQ3*[Trp265TAG] mRNA and either an unconjugated tRNA (pdCpA; n=5) or tRNA amino-acylated with Trp (n=8, *P<0.05, Student's t-test). (c) Activation kinetics of KCNQ3* (n=5) and KCNQ3*[Trp265TAG] (n=8) channels rescued with Trp, in the presence or absence of 100 μM retigabine (*P<0.05, Student's t-test). (d–f) Exemplar currents from oocytes injected with KCNQ3*[Trp265TAG] mRNA, and indicated synthetic tRNAs. (g) Conductance–voltage relationships for Trp-rescued KCNQ3*[Trp265TAG] (n=8), with retigabine response, illustrating faithful incorporation of the desired side chain at position 265. For KCNQ3* channels V1/2=−44±1 mV, k=7.5±0.5 mV; for Trp-rescued KCNQ3*[Trp265TAG] V1/2=−43±2 mV, k=7.9±0.5 mV (± indicates s.e.). Error bars throughout represent s.e.m.
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f2: Nonsense suppression for amino-acid incorporation in KCNQ3* channels.(a) Schematic diagram of the nonsense suppression method, in which mRNA (with a stop codon at Trp265) and amino-acylated tRNA are co-injected into Xenopus oocytes. Incorporation of the unnatural amino acid enables readthrough of the stop codon and expression of functional channels. (b) Current magnitudes in oocytes injected with KCNQ3*[Trp265TAG] mRNA and either an unconjugated tRNA (pdCpA; n=5) or tRNA amino-acylated with Trp (n=8, *P<0.05, Student's t-test). (c) Activation kinetics of KCNQ3* (n=5) and KCNQ3*[Trp265TAG] (n=8) channels rescued with Trp, in the presence or absence of 100 μM retigabine (*P<0.05, Student's t-test). (d–f) Exemplar currents from oocytes injected with KCNQ3*[Trp265TAG] mRNA, and indicated synthetic tRNAs. (g) Conductance–voltage relationships for Trp-rescued KCNQ3*[Trp265TAG] (n=8), with retigabine response, illustrating faithful incorporation of the desired side chain at position 265. For KCNQ3* channels V1/2=−44±1 mV, k=7.5±0.5 mV; for Trp-rescued KCNQ3*[Trp265TAG] V1/2=−43±2 mV, k=7.9±0.5 mV (± indicates s.e.). Error bars throughout represent s.e.m.

Mentions: To investigate the underlying mechanism of retigabine interactions with this essential Trp side chain, we employed unnatural amino-acid mutagenesis to introduce subtly altered Trp variants (Fig. 2a). With this method, a stop codon (TAG) is placed in the ion channel gene at a site of interest, and this mRNA is co-injected into Xenopus laevis oocytes along with a synthetic amino-acylated tRNA (carrying an unnatural amino acid) that is orthogonal to Xenopus tRNA synthetic pathways28. When the ribosomal translation machinery encounters the introduced TAG stop codon, the complementary synthetic tRNA (with a CUA anticodon) enables readthrough and incorporation of the appended amino acid into full-length ion channels (Fig. 2a)29.


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)

Nonsense suppression for amino-acid incorporation in KCNQ3* channels.(a) Schematic diagram of the nonsense suppression method, in which mRNA (with a stop codon at Trp265) and amino-acylated tRNA are co-injected into Xenopus oocytes. Incorporation of the unnatural amino acid enables readthrough of the stop codon and expression of functional channels. (b) Current magnitudes in oocytes injected with KCNQ3*[Trp265TAG] mRNA and either an unconjugated tRNA (pdCpA; n=5) or tRNA amino-acylated with Trp (n=8, *P<0.05, Student's t-test). (c) Activation kinetics of KCNQ3* (n=5) and KCNQ3*[Trp265TAG] (n=8) channels rescued with Trp, in the presence or absence of 100 μM retigabine (*P<0.05, Student's t-test). (d–f) Exemplar currents from oocytes injected with KCNQ3*[Trp265TAG] mRNA, and indicated synthetic tRNAs. (g) Conductance–voltage relationships for Trp-rescued KCNQ3*[Trp265TAG] (n=8), with retigabine response, illustrating faithful incorporation of the desired side chain at position 265. For KCNQ3* channels V1/2=−44±1 mV, k=7.5±0.5 mV; for Trp-rescued KCNQ3*[Trp265TAG] V1/2=−43±2 mV, k=7.9±0.5 mV (± indicates s.e.). Error bars throughout represent s.e.m.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4561856&req=5

f2: Nonsense suppression for amino-acid incorporation in KCNQ3* channels.(a) Schematic diagram of the nonsense suppression method, in which mRNA (with a stop codon at Trp265) and amino-acylated tRNA are co-injected into Xenopus oocytes. Incorporation of the unnatural amino acid enables readthrough of the stop codon and expression of functional channels. (b) Current magnitudes in oocytes injected with KCNQ3*[Trp265TAG] mRNA and either an unconjugated tRNA (pdCpA; n=5) or tRNA amino-acylated with Trp (n=8, *P<0.05, Student's t-test). (c) Activation kinetics of KCNQ3* (n=5) and KCNQ3*[Trp265TAG] (n=8) channels rescued with Trp, in the presence or absence of 100 μM retigabine (*P<0.05, Student's t-test). (d–f) Exemplar currents from oocytes injected with KCNQ3*[Trp265TAG] mRNA, and indicated synthetic tRNAs. (g) Conductance–voltage relationships for Trp-rescued KCNQ3*[Trp265TAG] (n=8), with retigabine response, illustrating faithful incorporation of the desired side chain at position 265. For KCNQ3* channels V1/2=−44±1 mV, k=7.5±0.5 mV; for Trp-rescued KCNQ3*[Trp265TAG] V1/2=−43±2 mV, k=7.9±0.5 mV (± indicates s.e.). Error bars throughout represent s.e.m.
Mentions: To investigate the underlying mechanism of retigabine interactions with this essential Trp side chain, we employed unnatural amino-acid mutagenesis to introduce subtly altered Trp variants (Fig. 2a). With this method, a stop codon (TAG) is placed in the ion channel gene at a site of interest, and this mRNA is co-injected into Xenopus laevis oocytes along with a synthetic amino-acylated tRNA (carrying an unnatural amino acid) that is orthogonal to Xenopus tRNA synthetic pathways28. When the ribosomal translation machinery encounters the introduced TAG stop codon, the complementary synthetic tRNA (with a CUA anticodon) enables readthrough and incorporation of the appended amino acid into full-length ion channels (Fig. 2a)29.

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