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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.


Multiple retigabine molecules modulate KCNQ2 and KCNQ3 channel subunits via an S5 Trp side chain.(a,b) Conductance–voltage relationships for (a) KCNQ2 (n=3) and KCNQ2[Trp236Phe] (n=6), and (b) KCNQ3* (n=5) and KCNQ3*[Trp265Phe] (n=3) homomeric channels along with indicated mutants (retigabine concentration of 100 μM). (c) Conductance–voltage relationships for heteromeric combinations of KCNQ2 and KCNQ3 (1:1 ratio of injected mRNA, with or without Trp→Phe mutations as indicated, n=5 for each combination), used to generate channels with reduced numbers of retigabine binding sites. (d) Summary of V1/2 shifts in saturating 100 μM retigabine for mutations of KCNQ2 Trp236 and KCNQ3 Trp265 as indicated (*P<0.05 in a paired Students t-test comparing control versus 100 μM retigabine in each experimental oocyte, n=3–6 per mutant). Only a Trp at either position is sufficient for retigabine sensitivity. (e) Exemplar currents of KCNQ3* and KCNQ3*[Trp265Phe] mutant coexpressed with CiVSP, illustrating that the Trp side chain responsible for retigabine sensitivity is not required for PIP2 sensitivity. (f) Summary data of tail current magnitude (−20 mV) after prepulses to a range of voltages, in oocytes expressing KCNQ3* (n=5) or KCNQ3*[Trp265Phe] (n=5) channels, along with CiVSP. In all panels, error bars represent s.e.m.
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f1: Multiple retigabine molecules modulate KCNQ2 and KCNQ3 channel subunits via an S5 Trp side chain.(a,b) Conductance–voltage relationships for (a) KCNQ2 (n=3) and KCNQ2[Trp236Phe] (n=6), and (b) KCNQ3* (n=5) and KCNQ3*[Trp265Phe] (n=3) homomeric channels along with indicated mutants (retigabine concentration of 100 μM). (c) Conductance–voltage relationships for heteromeric combinations of KCNQ2 and KCNQ3 (1:1 ratio of injected mRNA, with or without Trp→Phe mutations as indicated, n=5 for each combination), used to generate channels with reduced numbers of retigabine binding sites. (d) Summary of V1/2 shifts in saturating 100 μM retigabine for mutations of KCNQ2 Trp236 and KCNQ3 Trp265 as indicated (*P<0.05 in a paired Students t-test comparing control versus 100 μM retigabine in each experimental oocyte, n=3–6 per mutant). Only a Trp at either position is sufficient for retigabine sensitivity. (e) Exemplar currents of KCNQ3* and KCNQ3*[Trp265Phe] mutant coexpressed with CiVSP, illustrating that the Trp side chain responsible for retigabine sensitivity is not required for PIP2 sensitivity. (f) Summary data of tail current magnitude (−20 mV) after prepulses to a range of voltages, in oocytes expressing KCNQ3* (n=5) or KCNQ3*[Trp265Phe] (n=5) channels, along with CiVSP. In all panels, error bars represent s.e.m.

Mentions: Retigabine strongly affects voltage-dependent gating of KCNQ2 and KCNQ3* channels (Fig. 1a,b) by generating a substantial hyperpolarizing shift of the V1/2 of activation by ∼40–60 mV (KCNQ3* refers to KCNQ3[Ala315Thr]—see Methods). These effects have also been demonstrated in KCNQ4 and KCNQ5, but are absent in KCNQ1 because of the absence of an essential Trp residue (Trp236 in KCNQ2, Fig. 1a; Trp 265 in KCNQ3, Fig. 1b: Leu246 in KCNQ1)1426. This Trp side chain lies in the pore-forming S5 transmembrane segment, near the intracellular voltage-operated gate of the channel. As KCNQ2–5 subunits generally assemble as heteromers in the central nervous system1416, we tested the effects of retigabine in oocytes co-injected with KCNQ2 and KCNQ3 and observed large shifts of activation to more negative voltages, although not quite as large as with KCNQ2 or KCNQ3 alone (Fig. 1c). Some KCNQ channel activator molecules also cause a marked increase in peak current; however, the effects of retigabine on KCNQ3* channels are quite modest (rarely greater than a 15% increase in peak current), and throughout this study we have focused on the large gating shifts observed in these channels.


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)

Multiple retigabine molecules modulate KCNQ2 and KCNQ3 channel subunits via an S5 Trp side chain.(a,b) Conductance–voltage relationships for (a) KCNQ2 (n=3) and KCNQ2[Trp236Phe] (n=6), and (b) KCNQ3* (n=5) and KCNQ3*[Trp265Phe] (n=3) homomeric channels along with indicated mutants (retigabine concentration of 100 μM). (c) Conductance–voltage relationships for heteromeric combinations of KCNQ2 and KCNQ3 (1:1 ratio of injected mRNA, with or without Trp→Phe mutations as indicated, n=5 for each combination), used to generate channels with reduced numbers of retigabine binding sites. (d) Summary of V1/2 shifts in saturating 100 μM retigabine for mutations of KCNQ2 Trp236 and KCNQ3 Trp265 as indicated (*P<0.05 in a paired Students t-test comparing control versus 100 μM retigabine in each experimental oocyte, n=3–6 per mutant). Only a Trp at either position is sufficient for retigabine sensitivity. (e) Exemplar currents of KCNQ3* and KCNQ3*[Trp265Phe] mutant coexpressed with CiVSP, illustrating that the Trp side chain responsible for retigabine sensitivity is not required for PIP2 sensitivity. (f) Summary data of tail current magnitude (−20 mV) after prepulses to a range of voltages, in oocytes expressing KCNQ3* (n=5) or KCNQ3*[Trp265Phe] (n=5) channels, along with CiVSP. In all panels, error bars represent s.e.m.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Multiple retigabine molecules modulate KCNQ2 and KCNQ3 channel subunits via an S5 Trp side chain.(a,b) Conductance–voltage relationships for (a) KCNQ2 (n=3) and KCNQ2[Trp236Phe] (n=6), and (b) KCNQ3* (n=5) and KCNQ3*[Trp265Phe] (n=3) homomeric channels along with indicated mutants (retigabine concentration of 100 μM). (c) Conductance–voltage relationships for heteromeric combinations of KCNQ2 and KCNQ3 (1:1 ratio of injected mRNA, with or without Trp→Phe mutations as indicated, n=5 for each combination), used to generate channels with reduced numbers of retigabine binding sites. (d) Summary of V1/2 shifts in saturating 100 μM retigabine for mutations of KCNQ2 Trp236 and KCNQ3 Trp265 as indicated (*P<0.05 in a paired Students t-test comparing control versus 100 μM retigabine in each experimental oocyte, n=3–6 per mutant). Only a Trp at either position is sufficient for retigabine sensitivity. (e) Exemplar currents of KCNQ3* and KCNQ3*[Trp265Phe] mutant coexpressed with CiVSP, illustrating that the Trp side chain responsible for retigabine sensitivity is not required for PIP2 sensitivity. (f) Summary data of tail current magnitude (−20 mV) after prepulses to a range of voltages, in oocytes expressing KCNQ3* (n=5) or KCNQ3*[Trp265Phe] (n=5) channels, along with CiVSP. In all panels, error bars represent s.e.m.
Mentions: Retigabine strongly affects voltage-dependent gating of KCNQ2 and KCNQ3* channels (Fig. 1a,b) by generating a substantial hyperpolarizing shift of the V1/2 of activation by ∼40–60 mV (KCNQ3* refers to KCNQ3[Ala315Thr]—see Methods). These effects have also been demonstrated in KCNQ4 and KCNQ5, but are absent in KCNQ1 because of the absence of an essential Trp residue (Trp236 in KCNQ2, Fig. 1a; Trp 265 in KCNQ3, Fig. 1b: Leu246 in KCNQ1)1426. This Trp side chain lies in the pore-forming S5 transmembrane segment, near the intracellular voltage-operated gate of the channel. As KCNQ2–5 subunits generally assemble as heteromers in the central nervous system1416, we tested the effects of retigabine in oocytes co-injected with KCNQ2 and KCNQ3 and observed large shifts of activation to more negative voltages, although not quite as large as with KCNQ2 or KCNQ3 alone (Fig. 1c). Some KCNQ channel activator molecules also cause a marked increase in peak current; however, the effects of retigabine on KCNQ3* channels are quite modest (rarely greater than a 15% increase in peak current), and throughout this study we have focused on the large gating shifts observed in these channels.

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.