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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 position of the Trp 265 indole nitrogen is essential for retigabine sensitivity.(a) Chemical structures of Trp and Ind side chains, illustrating the subtle change in the position of the indole nitrogen atom. (b) Exemplar currents elicited from a Xenopus oocyte with Ind-rescued KCNQ3*[Trp265TAG] channels illustrating retigabine insensitivity. (c) Current magnitudes in oocytes injected with KCNQ3*[Trp265TAG] mRNA and either an unconjugated tRNA (pdCpA; n=5) or tRNA amino-acylated with Ind (n=7, *P<0.05, Student's t-test). (d) Conductance–voltage relationships for Ind-rescued KCNQ3*[Trp265TAG], in the presence and absence of retigabine, illustrating the importance of the correct positioning of the N–H group. For KCNQ3*[Trp265Trp], V1/2=−43±2 mV, k=7.9±0.5 mV; for KCNQ3*[Trp265Ind], V1/2=−48±2 mV, k=7.3±0.6 mV (no statistical significance, ±indicates s.e.). (e) Activation kinetics for KCNQ3*[Trp265Ind] measured at −20 mV in the presence and absence of 100 μM retigabine (n=7, no statistical significance). In all panels, error bars represent s.e.m.
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f3: The position of the Trp 265 indole nitrogen is essential for retigabine sensitivity.(a) Chemical structures of Trp and Ind side chains, illustrating the subtle change in the position of the indole nitrogen atom. (b) Exemplar currents elicited from a Xenopus oocyte with Ind-rescued KCNQ3*[Trp265TAG] channels illustrating retigabine insensitivity. (c) Current magnitudes in oocytes injected with KCNQ3*[Trp265TAG] mRNA and either an unconjugated tRNA (pdCpA; n=5) or tRNA amino-acylated with Ind (n=7, *P<0.05, Student's t-test). (d) Conductance–voltage relationships for Ind-rescued KCNQ3*[Trp265TAG], in the presence and absence of retigabine, illustrating the importance of the correct positioning of the N–H group. For KCNQ3*[Trp265Trp], V1/2=−43±2 mV, k=7.9±0.5 mV; for KCNQ3*[Trp265Ind], V1/2=−48±2 mV, k=7.3±0.6 mV (no statistical significance, ±indicates s.e.). (e) Activation kinetics for KCNQ3*[Trp265Ind] measured at −20 mV in the presence and absence of 100 μM retigabine (n=7, no statistical significance). In all panels, error bars represent s.e.m.

Mentions: Next, we tested retigabine sensitivity of KCNQ3* channels synthesized with the ‘Ind' amino acid at position 265. This unnatural amino-acid side chain ablates the hydrogen-bonding capability of Trp by shifting the position of the indole nitrogen atom (Fig. 3a)31, but retains the steric and chemical compositions of Trp. KCNQ3*[Trp265TAG] currents were efficiently rescued with Ind relative to the pdCpA control, yielding relatively large voltage-activated K+ currents that closely resembled KCNQ3* (Fig. 3b,c). Remarkably, however, this single atom alteration in each of the KCNQ3* subunits entirely abolishes retigabine activation of KCNQ3* channels (Fig. 3d,e). Notably, Trp265Ind channels displayed WT-like gating, indicating that this residue does not play a significant role in conformational stabilization of channel states in the absence of drug. However, given the potent impact of such a minor manipulation, we examined the effects of the Trp265Ind substitution on channel gating more closely, to rule out the possibility of significant perturbation of channel function. We observed no statistically significant difference in the V1/2 or slope of the voltage dependence of activation of KCNQ3* and KCNQ3*[Trp265Ind] (see Table 1 and Fig. 3d,e). Last, as described earlier, the more disruptive Trp265Phe mutation did not alter the inhibition of KCNQ3* currents by PIP2 depletion (mediated by CiVSP). Taken together, these observations demonstrate a highly specific effect of the Trp265Ind substitution on drug interactions (with little disruption of intrinsic voltage-dependent gating or lipid regulation), indicating that H-bond formation with Trp265 is a crucial step for retigabine action.


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 position of the Trp 265 indole nitrogen is essential for retigabine sensitivity.(a) Chemical structures of Trp and Ind side chains, illustrating the subtle change in the position of the indole nitrogen atom. (b) Exemplar currents elicited from a Xenopus oocyte with Ind-rescued KCNQ3*[Trp265TAG] channels illustrating retigabine insensitivity. (c) Current magnitudes in oocytes injected with KCNQ3*[Trp265TAG] mRNA and either an unconjugated tRNA (pdCpA; n=5) or tRNA amino-acylated with Ind (n=7, *P<0.05, Student's t-test). (d) Conductance–voltage relationships for Ind-rescued KCNQ3*[Trp265TAG], in the presence and absence of retigabine, illustrating the importance of the correct positioning of the N–H group. For KCNQ3*[Trp265Trp], V1/2=−43±2 mV, k=7.9±0.5 mV; for KCNQ3*[Trp265Ind], V1/2=−48±2 mV, k=7.3±0.6 mV (no statistical significance, ±indicates s.e.). (e) Activation kinetics for KCNQ3*[Trp265Ind] measured at −20 mV in the presence and absence of 100 μM retigabine (n=7, no statistical significance). In all panels, error bars represent s.e.m.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4561856&req=5

f3: The position of the Trp 265 indole nitrogen is essential for retigabine sensitivity.(a) Chemical structures of Trp and Ind side chains, illustrating the subtle change in the position of the indole nitrogen atom. (b) Exemplar currents elicited from a Xenopus oocyte with Ind-rescued KCNQ3*[Trp265TAG] channels illustrating retigabine insensitivity. (c) Current magnitudes in oocytes injected with KCNQ3*[Trp265TAG] mRNA and either an unconjugated tRNA (pdCpA; n=5) or tRNA amino-acylated with Ind (n=7, *P<0.05, Student's t-test). (d) Conductance–voltage relationships for Ind-rescued KCNQ3*[Trp265TAG], in the presence and absence of retigabine, illustrating the importance of the correct positioning of the N–H group. For KCNQ3*[Trp265Trp], V1/2=−43±2 mV, k=7.9±0.5 mV; for KCNQ3*[Trp265Ind], V1/2=−48±2 mV, k=7.3±0.6 mV (no statistical significance, ±indicates s.e.). (e) Activation kinetics for KCNQ3*[Trp265Ind] measured at −20 mV in the presence and absence of 100 μM retigabine (n=7, no statistical significance). In all panels, error bars represent s.e.m.
Mentions: Next, we tested retigabine sensitivity of KCNQ3* channels synthesized with the ‘Ind' amino acid at position 265. This unnatural amino-acid side chain ablates the hydrogen-bonding capability of Trp by shifting the position of the indole nitrogen atom (Fig. 3a)31, but retains the steric and chemical compositions of Trp. KCNQ3*[Trp265TAG] currents were efficiently rescued with Ind relative to the pdCpA control, yielding relatively large voltage-activated K+ currents that closely resembled KCNQ3* (Fig. 3b,c). Remarkably, however, this single atom alteration in each of the KCNQ3* subunits entirely abolishes retigabine activation of KCNQ3* channels (Fig. 3d,e). Notably, Trp265Ind channels displayed WT-like gating, indicating that this residue does not play a significant role in conformational stabilization of channel states in the absence of drug. However, given the potent impact of such a minor manipulation, we examined the effects of the Trp265Ind substitution on channel gating more closely, to rule out the possibility of significant perturbation of channel function. We observed no statistically significant difference in the V1/2 or slope of the voltage dependence of activation of KCNQ3* and KCNQ3*[Trp265Ind] (see Table 1 and Fig. 3d,e). Last, as described earlier, the more disruptive Trp265Phe mutation did not alter the inhibition of KCNQ3* currents by PIP2 depletion (mediated by CiVSP). Taken together, these observations demonstrate a highly specific effect of the Trp265Ind substitution on drug interactions (with little disruption of intrinsic voltage-dependent gating or lipid regulation), indicating that H-bond formation with Trp265 is a crucial step for retigabine action.

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