Neuronal Kv7 Channels Research Articles

Epileptic channelopathies caused by neuronal Kv7 (KCNQ) channel dysfunction.

Seizures are the most common neurological manifestation in the newborn period, with an estimated incidence of 1.8-3.5 per 1000 live births. Prolonged or intractable seizures have a detrimental effect on cognition and brain function in experimental animals and are associated with adverse long-term neurodevelopmental sequelae and an increased risk of post-neonatal epilepsy in humans. The developing brain is particularly susceptible to the potentially severe effects of epilepsy, and epilepsy, especially when refractory to medications, often results in a developmental and epileptic encephalopathy (DEE) with developmental arrest or regression. DEEs can be primarily attributed to genetic causes. Given the critical role of potassium (K+) currents with distinct subcellular localization, biophysical properties, modulation, and pharmacological profile in regulating intrinsic electrical properties of neurons and their responsiveness to synaptic inputs, it is not too surprising that genetic research in the past two decades has identified several K+ channel genes as responsible for a large fraction of DEE. In the present article, we review the genetically determined epileptic channelopathies affecting three members of the Kv7 family, namely Kv7.2 (KCNQ2), Kv7.3 (KCNQ3), and Kv7.5 (KCNQ5); we review the phenotypic spectrum of Kv7-related epileptic channelopathies, the different genetic and pathogenetic mechanisms, and the emerging genotype-phenotype correlations which may prove crucial for prognostic predictions, disease management, parental counseling, and individually tailored therapeutic attempts.

M-Current Suppression, Seizures and Lipid Metabolism: A Potential Link Between Neuronal Kv7 Channel Regulation and Dietary Therapies for Epilepsy.

Neuronal Kv7 channel generates a low voltage-activated potassium current known as the M-current. The M-current can be suppressed by various neurotransmitters that activate Gq-coupled receptors. Because the M-current stabilizes membrane potential at the resting membrane potential, its suppression transiently increase neuronal excitability. However, its physiological and pathological roles in vivo is not well understood to date. This review summarizes the molecular mechanism underlying M-current suppression, and why it remained elusive for many years. I also summarize how regulation of neuronal Kv7 channel contributes to anti-seizure action of valproic acid through inhibition of palmitoylation of a Kv7 channel binding protein, and discuss about a potential link with anti-seizure mechanisms of medium chain triglyceride ketogenic diet.

A novel and potent neuronal Kv7 channel opener SCR2682 alleviates chronic pain in rats

Activation of neuronal Kv7/KCNQ/M‐current emerges as an effective intervention for treatment of hyperexcitability‐related neurological diseases including chronic pain that is prevalent and unmet medical need. The aim of this study was to evaluate the anti‐nociceptive effects of a novel and potent Kv7/KCNQ/M channel opener SCR2682 on chronic pain in rats. The pharmacological effects of SCR2682 on native M current and firing of dorsal root ganglia (DRG) neurons were examined using electrophysiological recordings. Two in vivo rat models of complete Freund’s adjuvant (CFA)‐induced inflammatory pain and spared nerve injury (SNI)‐induced neuropathic pain were used to evaluate anti‐nociceptive effects of SCR2682 by measuring the paw withdrawal mechanical threshold (PWMT) and/or thermal latency (PWTL). Locomotor activity in normal rats was tested for safety evaluation. The expression of KCNQ2 in L4 and L5 DRGs of rat SNI model was detected by quantitative RT‐PCR and western blot.We found that SCR2682 (0.1 μM) or an FDA approved anti‐epileptic drug retigabine (RTG) (10 μM) increased M current in rat DRG neurons approximately 30%, which was blocked by Kv7 channel inhibitor XE991 (3 μM). SCR2682 caused the resting membrane potential (RMP) hyperpolarization to −66.4 ± 3.7 mV from −56.7 ± 2.8 mV. Current clamp recordings revealed that perfusion of SCR2682 abolished the firing spikes of DRG neurons. In rat model of CFA‐induced inflammatory pain, intraperitoneal (i.p.) administration of SCR2682 at doses of 0.5, 1 and 2 mg/kg increased the PWMT and PWTL in a dose‐dependent manner, as compared with RTG (7 mg/kg) that also increased the PWMT and PWTL. In the model of SNI‐induced neuropathic pain, SCR2682 (0.5, 1 and 2 mg/kg) or RTG (7 mg/kg) increased the PWMT, and the anti‐nociceptive effect of SCR2682 (2 mg/kg) could be reversed by the channel specific blocker XE991 (3 mg/kg, i.p.). Furthermore, SCR2682 (2 mg/kg.) or RTG (7 mg/kg) significantly increased the expression of KCNQ2 mRNA and protein in DRG of SNI rats, and co‐injection of XE991 (3 mg/kg) with SCR2682 reduced the expression levels of KCNQ2. In the open field test, there was no significant difference in the travel total distance and mean speed of rats treated with RTG (7 mg/kg) or SCR2682 (0.5, 1 and 2 mg/kg). Taken together, our data show that the novel compound SCR2682 reduces the excitability of DRG sensory neurons by activating Kv7 channel, and intraperitoneal administration of SCR2682 alleviates chronic pain in rats.Support or Funding InformationThis project was supported by grants awarded to K.W. from National Natural Sciences Foundation of China (81573410) and the Ministry of Science and Technology of China (2018ZX09711001‐004‐006) and Science and Technology Program of Guangdong (2018B030334001).

Voltage Sensor Movement of Neuronal Kv7 Channels

Voltage Sensor Movement of Neuronal Kv7 Channels

Synthesis and Pharmacological Characterization of Conformationally Restricted Retigabine Analogues as Novel Neuronal Kv7 Channel Activators.

Kv7 K+ channels represent attractive pharmacological targets for the treatment of different neurological disorders, including epilepsy. In this paper, 42 conformationally restricted analogues of the prototypical Kv7 activator retigabine have been synthesized and tested by electrophysiological patch-clamp experiments as Kv7 agonists. When compared to retigabine (0.93 ± 0.43 μM), the EC50s for Kv7.2 current enhancements by compound 23a (0.08 ± 0.04 μM) were lower, whereas no change in potency was observed for 24a (0.63 ± 0.07 μM). In addition, compared to retigabine, 23a and 24a showed also higher potency in activating heteromeric Kv7.2/Kv7.3 and homomeric Kv7.4 channels. Molecular modeling studies provided new insights into the chemical features required for optimal interaction at the binding site. Stability studies evidenced improved chemical stability of 23a and 24a in comparison with retigabine. Overall, the present results highlight that the N5-alkylamidoindole moiety provides a suitable pharmacophoric scaffold for the design of chemically stable, highly potent and selective Kv7 agonists.

Electrophysiological and pharmacological characterization of a novel and potent neuronal Kv7 channel opener SCR2682 for antiepilepsy.

Voltage-gated Kv7/KCNQ/M potassium channels play an essential role in the control of membrane potential and neuronal excitability. Activation of the neuronal Kv7/KCNQ/M-current represents an attractive therapeutic strategy for treatment of hyperexcitability-related neuropsychiatric disorders such as epilepsy, pain, and depression, which is an unmet medical need. In this study, we synthesized and characterized a novel compound, N-(4-(2-bromo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)-2,6-dimethylphenyl)-3,3-dimethylbutanamide (SCR2682) 2,6-dimethyl-4-(piperidin-yl) phenyl)-amide derivative, that exhibits selective and potent activation of neuronal Kv7/KCNQ/M-channels. Whole-cell patch-clamp recordings of human embryonic kidney 293 cells expressing Kv7.2/Kv7.3 channels show that SCR2682 selectively activates the channel current in a dose-dependent manner with an EC50 of 9.8 ± 0.4 nM, which is ∼100-fold more potent than a U.S. Food and Drug Administration-approved antiepileptic drug (retigabine) for treatment of partial epilepsy. SCR2682 shifts voltage-dependent activation of the Kv7.2/7.3 current toward more negative membrane potential, to about -37 mV (V1/2). SCR2682 also activates the native M-current in rat hippocampal or cortical neurons, causing marked hyperpolarization and potent inhibition of neuronal firings. Mechanistically, mutating the tryptophan residue 236 located at the fifth transmembrane segment of Kv7.2 abolishes the chemical activation of the channel by SCR2682. Furthermore, intraperitoneal or intragastric administration of SCR2682 results in a dose-dependent inhibition of seizures by maximal electroshock. Taken together, our findings demonstrate that a novel small molecule, SCR2682, selectively and potently activates neuronal Kv7 channels and reverses epileptic seizures in rodents. Thus, SCR2682 may warrant further evaluation for clinical development of antiepileptic therapy.-Zhang, F., Liu, Y., Tang, F., Liang, B., Chen, H., Zhang, H., Wang, K. Electrophysiological and pharmacological characterization of a novel and potent neuronal Kv7 channel opener SCR2682 for antiepilepsy.

Sa1123 – Activation of Kv7 Channels in Enteric Neurons Inhibits Epithelial Chloride Secretion: Potential Target in the Treatment of Diarrhea

Sa1123 – Activation of Kv7 Channels in Enteric Neurons Inhibits Epithelial Chloride Secretion: Potential Target in the Treatment of Diarrhea

Electrophysiological and Pharmacological Characterization of a Novel and Potent Neuronal Kv7 Opener SCR2682 for Anti-Epilepsy

Voltage-gated Kv7/KCNQ/M-potassium channels play an essential role in control of membrane potential and neuronal excitability. Activation of neuronal Kv7/KCNQ/M-current represents an attractive therapeutic strategy for treatment of hyperexcitability-related neuropsychiatric disorders such as epilepsy, pain and schizophrenia. In this study, we synthesized and characterized a novel series of 2,6-dimethyl-4-(piperidin-yl)phenyl)-amide derivatives that exhibit selective and potent activation of neuronal Kv7/KCNQ/M channels. Whole-cell patch clamp recordings of HEK293 cells expressing Kv7.2/Kv7.3 channels show that a representative compound SCR2682 selectively activates the channel current in dose-dependent manner with an EC50 of 9.8 ± 0.4 nM, which is about 100-fold potent than antiepileptic drug retigabine approved by FDA for partial epilepsy. Compound SCR2682 shifts the voltage-dependent activation of Kv7.2/7.3 current towards more negative membrane potential about −37 mV (V1/2). SCR2682 also activates native M current of rat hippocampal or cortical neurons, causing a marked hyperpolarization and potent inhibition of evoked action potentials. In rat epileptic models, intraperitoneal and intragastric administrations of SCR2682 results in a dose-dependent inhibition of seizures. Taken together, our findings demonstrate that a novel SCR2682 selectively and potently activates neuronal Kv7 channels and reverses epileptic activity in rats. Thus, SCR2682 may warrant further evaluation for clinical development of antiepileptic therapy.

The paracetamol metabolite N-acetylp-benzoquinone imine reduces excitability in first- and second-order neurons of the pain pathway through actions on KV7 channels.

Paracetamol (acetaminophen, APAP) is one of the most frequently used analgesic agents worldwide. It is generally preferred over nonsteroidal anti-inflammatory drugs because it does not cause typical adverse effects resulting from the inhibition of cyclooxygenases, such as gastric ulcers. Nevertheless, inhibitory impact on these enzymes is claimed to contribute to paracetamols mechanisms of action which, therefore, remained controversial. Recently, the APAP metabolites N-arachidonoylaminophenol (AM404) and N-acetyl-p-benzoquinone imine (NAPQI) have been detected in the central nervous system after systemic APAP administration and were reported to mediate paracetamol effects. In contrast to nonsteroidal anti-inflammatory drugs that rather support seizure activity, paracetamol provides anticonvulsant actions, and this dampening of neuronal activity may also form the basis for analgesic effects. Here, we reveal that the APAP metabolite NAPQI, but neither the parent compound nor the metabolite AM404, reduces membrane excitability in rat dorsal root ganglion (DRG) and spinal dorsal horn (SDH) neurons. The observed reduction of spike frequencies is accompanied by hyperpolarization in both sets of neurons. In parallel, NAPQI, but neither APAP nor AM404, increases currents through KV7 channels in DRG and SDH neurons, and the impact on neuronal excitability is absent if KV7 channels are blocked. Furthermore, NAPQI can revert the inhibitory action of the inflammatory mediator bradykinin on KV7 channels but does not affect synaptic transmission between DRG and SDH neurons. These results show that the paracetamol metabolite NAPQI dampens excitability of first- and second-order neurons of the pain pathway through an action on KV7 channels.

Pharmacological Targeting of Neuronal Kv7.2/3 Channels: A Focus on Chemotypes and Receptor Sites.

The Kv7 (KCNQ) subfamily of voltage-gated potassium channels consists of 5 members (Kv7.1-5) each showing characteristic tissue distribution and physiological roles. Given their functional heterogeneity, Kv7 channels represent important pharmacological targets for the development of new drugs for neuronal, cardiovascular and metabolic diseases. In the present manuscript, we focus on describing the pharmacological relevance and potential therapeutic applications of drugs acting on neuronally-expressed Kv7.2/3 channels, placing particular emphasis on the different chemotypes, and highlighting their pharmacodynamic and, whenever possible, pharmacokinetic peculiarities. The present work is based on an in-depth search of the currently available scientific literature, and on our own experience and knowledge in the field of neuronal Kv7 channel pharmacology. Space limitations impeded to describe the full pharmacological potential of Kv7 channels; thus, we have chosen to focus on neuronal channels composed of Kv7.2 and Kv7.3 subunits, and to mainly concentrate on their involvement in epilepsy. An astonishing heterogeneity in the molecular scaffolds exploitable to develop Kv7.2/3 modulators is evident, with important structural/functional peculiarities of distinct compound classes. In the present work we have attempted to show the current status and growing potential of the Kv7 pharmacology field. We anticipate a bright future for the field, and express our hopes that the efforts herein reviewed will result in an improved treatment of hyperexcitability (or any other) diseases.

Modulation of Kv7 channels and excitability in the brain.

Neuronal Kv7 channels underlie a voltage-gated non-inactivating potassium current known as the M-current. Due to its particular characteristics, Kv7 channels show pronounced control over the excitability of neurons. We will discuss various factors that have been shown to drastically alter the activity of this channel such as protein and phospholipid interactions, phosphorylation, calcium, and numerous neurotransmitters. Kv7 channels locate to key areas for the control of action potential initiation and propagation. Moreover, we will explore the dynamic surface expression of the channel modulated by neurotransmitters and neural activity. We will also focus on known principle functions of neural Kv7 channels: control of resting membrane potential and spiking threshold, setting the firing frequency, afterhyperpolarization after burst firing, theta resonance, and transient hyperexcitability from neurotransmitter-induced suppression of the M-current. Finally, we will discuss the contribution of altered Kv7 activity to pathologies such as epilepsy and cognitive deficits.

A Kv7.2 mutation associated with early onset epileptic encephalopathy with suppression-burst enhances Kv7/M channel activity.

Mutations in the KCNQ2 gene encoding the voltage-gated potassium channel subunit Kv7.2 cause early onset epileptic encephalopathy (EOEE). Most mutations have been shown to induce a loss of function or to affect the subcellular distribution of Kv7 channels in neurons. Herein, we investigated functional consequences and subcellular distribution of the p.V175L mutation of Kv7.2 (Kv7.2(V175L) ) found in a patient presenting EOEE. We observed that the mutation produced a 25-40 mV hyperpolarizing shift of the conductance-voltage relationship of both the homomeric Kv7.2(V175L) and heteromeric Kv7.2(V175L) /Kv7.3 channels compared to wild-type channels and a 10 mV hyperpolarizing shift of Kv7.2(V175L) /Kv7.2/Kv7.3 channels in a 1:1:2 ratio mimicking the patient situation. Mutant channels also displayed faster activation kinetics and an increased current density that was prevented by 1 μm linopirdine. The p.V175L mutation did not affect the protein expression of Kv7 channels and its localization at the axon initial segment. We conclude that p.V175L is a gain of function mutation. This confirms previous observations showing that mutations having opposite consequences on M channels can produce EOEE. These findings alert us that drugs aiming to increase Kv7 channel activity might have adverse effects in EOEE in the case of gain-of-function variants.

The Role of KV7.3 in Regulating Osteoblast Maturation and Mineralization.

KCNQ (KV7) channels are voltage-gated potassium (KV) channels, and the function of KV7 channels in muscles, neurons, and sensory cells is well established. We confirmed that overall blockade of KV channels with tetraethylammonium augmented the mineralization of bone-marrow-derived human mesenchymal stem cells during osteogenic differentiation, and we determined that KV7.3 was expressed in MG-63 and Saos-2 cells at the mRNA and protein levels. In addition, functional KV7 currents were detected in MG-63 cells. Inhibition of KV7.3 by linopirdine or XE991 increased the matrix mineralization during osteoblast differentiation. This was confirmed by alkaline phosphatase, osteocalcin, and osterix in MG-63 cells, whereas the expression of Runx2 showed no significant change. The extracellular glutamate secreted by osteoblasts was also measured to investigate its effect on MG-63 osteoblast differentiation. Blockade of KV7.3 promoted the release of glutamate via the phosphorylation of extracellular signal-regulated kinase 1/2-mediated upregulation of synapsin, and induced the deposition of type 1 collagen. However, activation of KV7.3 by flupirtine did not produce notable changes in matrix mineralization during osteoblast differentiation. These results suggest that KV7.3 could be a novel regulator in osteoblast differentiation.

Role of calmodulin in neuronal Kv7/KCNQ potassium channels and epilepsy

Neuronal Kv7/KCNQ channels are critical regulators of neuronal excitability since they potently suppress repetitive firing of action potentials. These voltage-dependent potassium channels are composed mostly of Kv7.2 / KCNQ2 and Kv7.3 / KCNQ3 subunits that show overlapping distribution throughout the brain and in the peripheral nervous system. They are also called ‘M-channels’ since their inhibition by muscarinic agonists leads to a profound increase in action potential firing. Consistent with their ability to suppress seizures and attenuate chronic inflammatory and neuropathic pain, mutations in the KCNQ2 and KCNQ3 genes are associated with benign familial neonatal convulsions, a dominantly-inherited epilepsy in infancy. Recently, de novo mutations in the KCNQ2 gene have been linked to early onset epileptic encephalopathy. Notably, some of these mutations are clustered in a region of the intracellular cytoplasmic tail of Kv7.2 that interacts with a ubiquitous calcium sensor, calmodulin. In this review, we highlight the recent advances in understanding the role of calmodulin in modulating physiological function of neuronal Kv7 channels including their biophysical properties, assembly, and trafficking. We also summarize recent studies that have investigated functional impact of epilepsy-associated mutations localized to the calmodulin binding domains of Kv7.2.

Chapter 2 - Potassium channel genes and benign familial neonatal epilepsy

Several potassium channel genes have been implicated in different neurological disorders including genetic and acquired epilepsy. Among them, KCNQ2 and KCNQ3, coding for KV7.2 and KV7.3 voltage-gated potassium channels, present an example how genetic dissection of an epileptic disorder can lead not only to a better understanding of disease mechanisms but also broaden our knowledge about the physiological function of the affected proteins and enable novel approaches in the antiepileptic therapy design. In this chapter, we focus on the neuronal KV7 channels and associated genetic disorders-channelopathies, in particular benign familial neonatal seizures, epileptic encephalopathy, and peripheral nerve hyperexcitability (neuromyotonia, myokymia) caused by KCNQ2 or KCNQ3 mutations. Furthermore, strategies using KV7 channels as targets or tools for the treatment of epileptic diseases caused by neuronal hyperexcitability are being addressed.

Characterization of a novel high-potency positive modulator of Kv7 channels

Kv7 channel activators decrease neuronal excitability and might potentially treat neuronal hyperexcitability disorders like epilepsy and mania. Here we introduce NS15370 ((2-(3,5-difluorophenyl)-N-[6-[(4-fluorophenyl)methylamino]-2-morpholino-3-pyridyl]acetamide)hydrochloride, an in vitro high-potency chemical analogue of retigabine, without effects on GABAA receptors. NS15370 activates recombinant homo- and heteromeric Kv7.2–Kv7.5 channels in HEK293 cells at sub-micromolar concentrations (EC50∼100nM, as quantified by a fluorescence based Tl+-influx assay). In voltage clamp experiments NS15370 exhibits a complex, concentration-dependent mode-of-action: At low concentrations it accelerates voltage-dependent activation rates, slows deactivations, and increases steady-state current amplitudes. Quantified by the peak-tail current method, the V½ value of the steady-state activation curve is shifted towards hyperpolarized potentials at concentrations ∼100 times lower than retigabine. However, in contrast to retigabine, NS15370 also introduces a distinct time-dependent current decrease, which eventually, at higher concentrations, causes suppression of the current at depolarized potentials, and an apparent “cross-over“ of the voltage-activation curve. In brain slices, NS15370 hyperpolarizes and increases spike frequency adaptation of hippocampal CA1 neurons and the compound reduces the autonomous firing of dopaminergic neurons in the substantia-nigra pars compacta. NS15370 is effective in rodent models of hyperexcitability: (i) it yields full protection against mouse 6Hz seizures and rat amygdala kindling discharges, two models of partial epilepsia; (ii) it reduces (+)-MK-801 hydrogen maleate (MK-801)-induced hyperactivity as well as chlordiazepoxide (CDP)+d-amphetamine (AMP)-induced hyperactivity, models sensitive to classic anti-psychotic and anti-manic treatments, respectively. Our findings with NS15370 consolidate neuronal Kv7 channels as targets for anti-epileptic and psychiatric drug development.

Structure of a Ca2+/CaM:Kv7.4 (KCNQ4) B-Helix Complex Provides Insight into M Current Modulation

Calmodulin (CaM) is an important regulator of Kv7.x (KCNQx) voltage-gated potassium channels. Channels from this family produce neuronal M currents and cardiac and auditory I(KS) currents and harbor mutations that cause arrhythmias, epilepsy, and deafness. Despite extensive functional characterization, biochemical and structural details of the interaction between CaM and the channel have remained elusive. Here, we show that both apo-CaM and Ca(2+)/CaM bind to the C-terminal tail of the neuronal channel Kv7.4 (KCNQ4), which is involved in both hearing and mechanosensation. Interactions between apo-CaM and the Kv7.4 tail involve two C-terminal tail segments, known as the A and B segments, whereas the interaction between Ca(2+)/CaM and the Kv7.4 C-terminal tail requires only the B segment. Biochemical studies show that the calcium dependence of the CaM:B segment interaction is conserved in all Kv7 subtypes. X-ray crystallographic determination of the structure of the Ca(2+)/CaM:Kv7.4 B segment complex shows that Ca(2+)/CaM wraps around the Kv7.4 B segment, which forms an α-helix, in an antiparallel orientation that embodies a variation of the classic 1-14 Ca(2+)/CaM interaction motif. Taken together with the context of prior studies, our data suggest a model for modulation of neuronal Kv7 channels involving a calcium-dependent conformational switch from an apo-CaM form that bridges the A and B segments to a Ca(2+)/CaM form bound to the B-helix. The structure presented here also provides a context for a number of disease-causing mutations and for further dissection of the mechanisms by which CaM controls Kv7 function.

Osmosensitivity through the PIP2availability: just add water

Osmosensitivity through the PIP₂availability: just add water

Isoform-specific Prolongation of Kv7 (KCNQ) Potassium Channel Opening Mediated by New Molecular Determinants for Drug-Channel Interactions

Kv7 channels, especially Kv7.2 (KCNQ2) and Kv7.3 (KCNQ3), are key determinants for membrane excitability in the brain. Some chemical modulators of KCNQ channels are in development for use as anti-epileptic drugs, such as retigabine (D-23129, N-(2-amino-4-(4-fluorobenzylamino)-phenyl)), which was recently approved for clinical use. In addition, several other compounds were also reported to potentiate activity of the Kv7 channels. It is therefore of interest to investigate compound-channel interactions, so that more insights may be gained to aid future development of therapeutics. We have conducted a screen of 20,000 compounds for KCNQ2 potentiators using rubidium flux combined with atomic absorption spectrometry. Here, we report the characterization of a series of new structures that display isoform specificity and induce a marked reduction of deactivation distinct from that of retigabine. Furthermore, KCNQ2(W236L), a previously reported mutation that abolishes sensitivity to retigabine, remains fully sensitive to these compounds. This result, together with mutagenesis and other studies, suggests that the reported compounds confer a unique mode of action and involve new molecular determinants on the channel protein, consistent with the idea of recognizing a new site on channel protein.

Gating currents from neuronal KV7.4 Channels: General features and correlation with the ionic conductance

The KV7 (KCNQ) subfamily of voltage-gated K+ channels consists of five members (KV7.1- KV7.5) giving rise to non-inactivating, and slowly activating/deactivating currents mainly expressed in cardiac (KV7.1) and neuronal (KV7.2- KV7.5) tissue. In the present study, using the cut-open oocyte voltage clamp, we studied the relation of the ionic currents from homomeric neuronal Kv7 channels (KV7.2-KV7.5) with the gating currents recorded after K+ conductance blockade from the same channels. Increasing the recording temperature from 18{degree sign}C to 28{degree sign}C accelerated activation/deactivation kinetics of the ionic currents in all homomeric KV7 channels (activation Q10s at 0 mV were 3.8, 4.1, 8.3, and 2.8 for Kv7.2, Kv7.3, Kv7.4 and Kv7.5 channels, respectively), without large changes in currents voltage-dependence; moreover, at 28{degree sign}C, ionic currents carried by KV7.4 channels also showed a significant increase in their maximal value. Gating currents were only resolved in KV7.4 and KV7.5 channels; the size of the ON gating charges at +40 mV was 1.34 ± 0.34 nC for KV7.4, and 0.79 ± 0.20 nC for KV7.5. At 28{degree sign}C, KV7.4 gating currents had the following salient properties: 1) similar time integral of QON and QOFF, indicating no charge immobilization; 2) a left-shift in the V1/2 of the QON/V when compared to the G/V (≈ 50 mV in the presence of 2 mM extracellular Ba2+); 3) a QON decay faster than ionic current activation; and 4) a rising phase in the OFF gating charge after depolarizations larger than 0 mV. These observations suggest that, in KV7.4 channels, VSD movement is followed by a slow and/or low bearing charge step linking to pore opening, a result which may help to clarify the molecular consequence of disease-causing mutations and drugs affecting channel gating.