Corbin-Leftwich A , Mossadeq SM , Ha J , Ruchala I , Le AH , Villalba-Galea CA .

KL2 TR000057 NCATS NIH HHS , KL2TR000057 NCATS NIH HHS , UL1 TR000058 NCATS NIH HHS , UL1TR000058 NCATS NIH HHS

	Figure 1. K+ deactivation rate decreases as a function of activation. (A) K+ currents recorded from Xenopus oocytes coexpressing human KV7.2 and KV7.3 channels using the cut-open voltage-clamp technique. Test pulses ranging from −120 mV to +60 mV were applied from an HP of −90 mV. Deactivation of the current was driven at −105 mV (arrow). (B) Normalized deactivating currents from activating pulses from −30 to +60 mV. Deactivation slowed down after higher activating potentials. (C) Deactivation time constant (τDEACT) versus test pulse potential (τDEACT–VPULSE) plot. Error bars represent standard deviation. (D) Kinetics scheme describing alternative pathways for the deactivation of the heteromeric KV7.2/KV7.3 channel. Voltage-dependent transitions are noted with V.
	Figure 2. Effect of Retigabine on the activity of the heteromeric KV7.2/KV7.3 channel. (A) K+ currents recorded in the absence or presence of 1–5-µM Retigabine using the same protocol shown in Fig. 1. (B) Average normalized K+ current amplitude measured at the beginning of the deactivating currents (ITAIL; arrows in A). Normalized ITAIL versus test pulse potential (ITAIL–VPULSE) plots incrementally shift toward negative potentials as the concentration of Retigabine (RTG) was increased. Individual ITAIL–VPULSE plots were fitted to a double Boltzmann distribution, and the weighted average half-maximum potential (weighted V1/2) was plotted as a function of the concentration of Retigabine (inset). Although the weighted V1/2 values were not statistically different, this parameter changed to more negative potentials as a function of the concentration of Retigabine in all our recordings. Error bars represent standard deviation. n = 5–8.
	Figure 3. Deactivation time constant increase as a function of the duration of the activating pulse. (A and B) K+ currents were activated by 40-mV pulses with a duration varying from 10 to 4,175 ms in the absence (A) and presence (B) of 1-µM Retigabine. The duration of the pulse increased 1.3-fold for each trace. After activation, the K+ current was deactivated at −90 mV. (C) τDEACT–tPULSE plot shows that τDEACT increased as the activating pulse was longer (black squares). In the presence of Retigabine, τDEACT increased further. (D) The same plot as in C, but the tPULSE is displayed in a logarithmic scale to highlight the τDEACT–tPULSE relationship for shorter activating pulses and showing that τDEACT was unaltered by Retigabine for pulses shorter than 500 ms. Error bars represent standard deviation. Control, n = 7; Retigabine, n = 6.
	Figure 4. Deactivation from the depolarized and resting potential open states. (A) Deactivation at −90 mV of the K+ current elicited by a 100-ms depolarization to 40 mV in the absence (black trace) and presence of 1-µM Retigabine (red trace). (B) Deactivation at −90 mV of the K+ current elicited by holding the membrane potential at −50 mV also in the absence (black trace) and presence of 1-µM Retigabine (red trace). (C–-F) Deactivation time constant (τDEACT) versus deactivating potential (τDEACT–VDEACT) plot of channels activated by a 100-ms depolarization to 40 mV (C) or by holding the membrane potential at −60 mV, −50 mV, and −40 mV (D, E, and F, respectively) in the absence (black squares) and presence of 1-µM Retigabine (RTG; red circles). Error bars represent standard deviation. t test; n = 5–8. *, P < 0.05, t test.
	Figure 5. The resting potential, but not the threshold potential for triggering AP, was affected by Retigabine. (A) APs were recorded from Xenopus oocytes coexpressing both the α and β subunits of NaV1.4, ShakerIR, KV7.2, and KV7.3. The APs were recorded using the loose two-electrode voltage-clamp technique (see Materials and methods for details) in the absence (black trace) or presence of 1-µM (red trace) or 10-µM (green trace) Retigabine. The gray trace shows the response of VM when a subthreshold was applied. The arrow indicates the moment at which AP threshold (VTHR) is reached. (B) Effect of Retigabine (RTG) on the resting membrane potential (VREST) and AP threshold (VTHR). (C) Minimum current injection needed to evoke an AP (ITHR). The values of ITHR were normalized by ITHR in the absence of Retigabine (n = 4). (D) Example of a phase plot calculated from the APs shown on A. VREST was strongly affected by Retigabine (blue star), whereas VTHR remained fairly unaltered. Although the depolarization phase was affected by Retigabine (blue arrow), the late phase of repolarization (yellow arrow) remained unaltered. Arrows indicate the progress of the AP in time during the stimulation phase (purple arrow), depolarization phase (green arrow), and repolarization phase (orange arrow). The open blue arrow points at the times when APs reached their maximum voltage. The open yellow arrow points at the late repolarization phase. Error bars represent standard deviation. t test; n = 5. *, P < 0.002 with respect to control; **, P < 0.07 with respect to 1-µM Retigabine; ***, P < 0.0025 with respect to control; #, P < 0.072 with respect to control.
	Figure 6. Proposed kinetic scheme for the activity of KV7.2/KV7.3 and the effect of Retigabine. The scheme contains five global states, which are different with respect to each other depending on the activation status of the VSD and the pore domain (P). In the scheme, VSD represents the four voltage sensors of the channel. At negative potentials, the VSDs are in the deactivated state (VSDD) and the pore is, therefore, closed (PC0). Upon depolarization, the VSDs are activated (VSDA), allowing the pore to sojourn between a conductive and a nonconductive state (PO1 and PC1, respectively). Holding the membrane depolarized potentials keeps the VSDs in the VSDA state while allowing a second transition in the pore. As a result, the pore sojourns between a second pair of conductive and nonconductive states (PO2 and PC2, respectively) and the channel becomes Retigabine sensitive. Deactivation from these states is slower than from the pair PC1–PO1. Retigabine further decreases the rate of deactivation from the pair PC2–PO2 while leaving deactivation unaffected from the other pair.

Abidi, A recurrent KCNQ2 pore mutation causing early onset epileptic encephalopathy has a moderate effect on M current but alters subcellular localization of Kv7 channels. 2015, Pubmed

Abidi, A recurrent KCNQ2 pore mutation causing early onset epileptic encephalopathy has a moderate effect on M current but alters subcellular localization of Kv7 channels. 2015, Pubmed
Akemann, Effect of voltage sensitive fluorescent proteins on neuronal excitability. 2009, Pubmed
Bean, The action potential in mammalian central neurons. 2007, Pubmed
Benndorf, Probability fluxes and transition paths in a Markovian model describing complex subunit cooperativity in HCN2 channels. 2012, Pubmed
Bezanilla, How membrane proteins sense voltage. 2008, Pubmed
Bezanilla, Distribution and kinetics of membrane dielectric polarization. 1. Long-term inactivation of gating currents. 1982, Pubmed
Brown, Neural KCNQ (Kv7) channels. 2009, Pubmed
Brown, Muscarinic suppression of a novel voltage-sensitive K+ current in a vertebrate neurone. 1980, Pubmed
Charlier, A pore mutation in a novel KQT-like potassium channel gene in an idiopathic epilepsy family. 1998, Pubmed
Colquhoun, Relaxation and fluctuations of membrane currents that flow through drug-operated channels. 1977, Pubmed
Dedek, Myokymia and neonatal epilepsy caused by a mutation in the voltage sensor of the KCNQ2 K+ channel. 2001, Pubmed , Xenbase
Gunthorpe, The mechanism of action of retigabine (ezogabine), a first-in-class K+ channel opener for the treatment of epilepsy. 2012, Pubmed
Jentsch, Neuronal KCNQ potassium channels: physiology and role in disease. 2000, Pubmed
Kim, Atomic basis for therapeutic activation of neuronal potassium channels. 2015, Pubmed , Xenbase
Kuzmenkin, Gating of the bacterial sodium channel, NaChBac: voltage-dependent charge movement and gating currents. 2004, Pubmed
Labro, Molecular mechanism for depolarization-induced modulation of Kv channel closure. 2012, Pubmed , Xenbase
Lacroix, Properties of deactivation gating currents in Shaker channels. 2011, Pubmed
Lange, Refinement of the binding site and mode of action of the anticonvulsant Retigabine on KCNQ K+ channels. 2009, Pubmed , Xenbase
Li, Bimodal regulation of an Elk subfamily K+ channel by phosphatidylinositol 4,5-bisphosphate. 2015, Pubmed
Linley, M channel enhancers and physiological M channel block. 2012, Pubmed
Long, Atomic structure of a voltage-dependent K+ channel in a lipid membrane-like environment. 2007, Pubmed
Long, Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. 2005, Pubmed
Main, Modulation of KCNQ2/3 potassium channels by the novel anticonvulsant retigabine. 2000, Pubmed , Xenbase
Maljevic, KV7 channelopathies. 2010, Pubmed
Mastrangelo, Novel Genes of Early-Onset Epileptic Encephalopathies: From Genotype to Phenotypes. 2015, Pubmed
Miceli, The Voltage-Sensing Domain of K(v)7.2 Channels as a Molecular Target for Epilepsy-Causing Mutations and Anticonvulsants. 2011, Pubmed
Miceli, A novel KCNQ3 mutation in familial epilepsy with focal seizures and intellectual disability. 2015, Pubmed
Miceli, Early-onset epileptic encephalopathy caused by gain-of-function mutations in the voltage sensor of Kv7.2 and Kv7.3 potassium channel subunits. 2015, Pubmed
Miceli, Molecular pharmacology and therapeutic potential of neuronal Kv7-modulating drugs. 2008, Pubmed
Männikkö, Hysteresis in the voltage dependence of HCN channels: conversion between two modes affects pacemaker properties. 2005, Pubmed , Xenbase
Noebels, Potassium Channels (including KCNQ) and Epilepsy 2012, Pubmed
Olcese, Correlation between charge movement and ionic current during slow inactivation in Shaker K+ channels. 1997, Pubmed , Xenbase
Orhan, Retigabine/Ezogabine, a KCNQ/K(V)7 channel opener: pharmacological and clinical data. 2012, Pubmed
Pennefather, Idiosyncratic gating of HERG-like K+ channels in microglia. 1998, Pubmed
Peretz, Meclofenamic acid and diclofenac, novel templates of KCNQ2/Q3 potassium channel openers, depress cortical neuron activity and exhibit anticonvulsant properties. 2005, Pubmed
Peters, Conditional transgenic suppression of M channels in mouse brain reveals functions in neuronal excitability, resonance and behavior. 2005, Pubmed , Xenbase
Piper, Gating currents associated with intramembrane charge displacement in HERG potassium channels. 2003, Pubmed , Xenbase
Priest, S3-S4 linker length modulates the relaxed state of a voltage-gated potassium channel. 2013, Pubmed
Rundfeldt, Investigations into the mechanism of action of the new anticonvulsant retigabine. Interaction with GABAergic and glutamatergic neurotransmission and with voltage gated ion channels. 2000, Pubmed
Rundfeldt, The novel anticonvulsant retigabine activates M-currents in Chinese hamster ovary-cells tranfected with human KCNQ2/3 subunits. 2000, Pubmed
Schenzer, Molecular determinants of KCNQ (Kv7) K+ channel sensitivity to the anticonvulsant retigabine. 2005, Pubmed , Xenbase
Shapiro, Infrared light excites cells by changing their electrical capacitance. 2012, Pubmed , Xenbase
Shirokov, Two classes of gating current from L-type Ca channels in guinea pig ventricular myocytes. 1992, Pubmed
Singh, A novel potassium channel gene, KCNQ2, is mutated in an inherited epilepsy of newborns. 1998, Pubmed
Soh, Conditional deletions of epilepsy-associated KCNQ2 and KCNQ3 channels from cerebral cortex cause differential effects on neuronal excitability. 2014, Pubmed
Syeda, The Sensorless Pore Module of Voltage-gated K+ Channel Family 7 Embodies the Target Site for the Anticonvulsant Retigabine. 2016, Pubmed
Tatulian, Activation of expressed KCNQ potassium currents and native neuronal M-type potassium currents by the anti-convulsant drug retigabine. 2001, Pubmed
Vargas, An emerging consensus on voltage-dependent gating from computational modeling and molecular dynamics simulations. 2012, Pubmed
Villalba-Galea, S4-based voltage sensors have three major conformations. 2008, Pubmed
Villalba-Galea, Voltage-Controlled Enzymes: The New JanusBifrons. 2012, Pubmed
Villalba-Galea, Hv1 proton channel opening is preceded by a voltage-independent transition. 2014, Pubmed
Villalba-Galea, Charge movement of a voltage-sensitive fluorescent protein. 2009, Pubmed , Xenbase
Villalba-Galea, Sensing charges of the Ciona intestinalis voltage-sensing phosphatase. 2013, Pubmed
Wainger, Intrinsic membrane hyperexcitability of amyotrophic lateral sclerosis patient-derived motor neurons. 2014, Pubmed
Wang, KCNQ2 and KCNQ3 potassium channel subunits: molecular correlates of the M-channel. 1998, Pubmed , Xenbase
Wickenden, N-(6-chloro-pyridin-3-yl)-3,4-difluoro-benzamide (ICA-27243): a novel, selective KCNQ2/Q3 potassium channel activator. 2008, Pubmed
Wickenden, Retigabine, a novel anti-convulsant, enhances activation of KCNQ2/Q3 potassium channels. 2000, Pubmed
Wuttke, Neutralization of a negative charge in the S1-S2 region of the KV7.2 (KCNQ2) channel affects voltage-dependent activation in neonatal epilepsy. 2008, Pubmed , Xenbase
Wuttke, The new anticonvulsant retigabine favors voltage-dependent opening of the Kv7.2 (KCNQ2) channel by binding to its activation gate. 2005, Pubmed , Xenbase
Wuttke, Peripheral nerve hyperexcitability due to dominant-negative KCNQ2 mutations. 2007, Pubmed , Xenbase
Xiao, Hysteresis in human HCN4 channels: a crucial feature potentially affecting sinoatrial node pacemaking. 2010, Pubmed
Xiong, Zinc pyrithione-mediated activation of voltage-gated KCNQ potassium channels rescues epileptogenic mutants. 2007, Pubmed