KCNQ Channels Research Articles

Evaluation of the Potential Targets of Shenxian–Shengmai Oral Liquid in Treating Sick Sinus Syndrome Based on Network Pharmacology and Molecular Docking

ABSTRACTShenxian–Shengmai (SXSM) is a Chinese patent medicine used in the treatment of sick sinus syndrome (SSS). However, its active chemical compounds and the underlying molecular mechanisms remain unclear. In this study, we researched the underlying mechanisms of SXSM in treating SSS. We conducted network analysis and molecular docking to identify the small molecules and core targets responsible for the therapeutic efficacy of SXSM on SSS. In vitro experiments were performed to verify the potential therapeutic mechanism. Network pharmacological analysis identified 17 core targets. Among these, BMP4, KCNH2, KCNMA1, and KCNQ1 were identified to be involved in various biological processes, such as the formation and regulation of the cardiac pacemaking system and potassium ion transmembrane transport. The experimental analysis revealed that SXSM could upregulate the expression of the Bmp4/Tbx3/Hcn4 pathway and the expression of Kcnh2, Kcnma1, and Kcnq1 channels, which protected and improved the pacemaking function of pacemaker cells (P cells) and increased the heart rate. These findings provide a scientific basis in the study of the mechanism of traditional Chinese medicine in the treatment of SSS.

Dual effects of mefenamic acid on the IKs molecular complex.

Mutations in both KCNQ1 and KCNE1, which together form the cardiac IKs current, are associated with inherited conditions such as long and short QT syndromes. Mefenamic acid, a non-steroidal anti-inflammatory drug, is an IKs potentiator and may be utilised as an archetype to design therapeutically useful IKs agonists. However, here we show that mefenamic acid can also act as an IKs inhibitor, and our data reveal its dual effects on KCNQ1/KCNE1 channels. Effects of mefenamic acid on wild type (WT) and mutant KCNQ1/KCNE1 channels expressed in tsA201 cells were studied using whole cell patch clamp. Molecular dynamics simulations were used to determine trajectory clustering. Mefenamic acid inhibits WT IKs at high concentrations while preserving some attributes of current potentiation. Inhibitory actions of mefenamic acid are unmasked at lower drug concentrations by KCNE1 and KCNQ1 mutations in the mefenamic acid binding pocket, at the extracellular end of KCNE1 and in the KCNQ1 S6 helix. Mefenamic acid does not inhibit KCNQ1 in the absence of KCNE1 but inhibits IKs current in a concentration-dependent manner in the mutant channels. Inhibition involves modulation of pore kinetics and/or voltage sensor domain-pore coupling in WT and in the KCNE1 E43C mutant. This work highlights the importance of structural motifs at the extracellular inter-subunit interface of KCNQ1 and KCNE1 channels, and their interactions, in determining the nature of drug effects on the IKs channel complex and has important implications for treating patients with specific long QT mutations.

PUFA stabilizes a conductive state of the selectivity filter in IKs channels.

In cardiomyocytes, the KCNQ1/KCNE1 channel complex mediates the slow delayed-rectifier current (IKs), pivotal during the repolarization phase of the ventricular action potential. Mutations in IKs cause long QT syndrome (LQTS), a syndrome with a prolonged QT interval on the ECG, which increases the risk of ventricular arrhythmia and sudden cardiac death. One potential therapeutical intervention for LQTS is based on targeting IKs channels to restore channel function and/or the physiological QT interval. Polyunsaturated fatty acids (PUFAs) are potent activators of KCNQ1 channels and activate IKs channels by binding to two different sites, one in the voltage sensor domain - which shifts the voltage dependence to more negative voltages - and the other in the pore domain - which increases the maximal conductance of the channels (Gmax). However, the mechanism by which PUFAs increase the Gmax of the IKs channels is still poorly understood. In addition, it is unclear why IKs channels have a very small single-channel conductance and a low open probability or whether PUFAs affect any of these properties of IKs channels. Our results suggest that the selectivity filter in KCNQ1 is normally unstable, contributing to the low open probability, and that the PUFA-induced increase in Gmax is caused by a stabilization of the selectivity filter in an open-conductive state.

PUFA stabilizes a conductive state of the selectivity filter in IKs channels

In cardiomyocytes, the KCNQ1/KCNE1 channel complex mediates the slow delayed-rectifier current (IKs), pivotal during the repolarization phase of the ventricular action potential. Mutations in IKs cause long QT syndrome (LQTS), a syndrome with a prolonged QT interval on the ECG, which increases the risk of ventricular arrhythmia and sudden cardiac death. One potential therapeutical intervention for LQTS is based on targeting IKs channels to restore channel function and/or the physiological QT interval. Polyunsaturated fatty acids (PUFAs) are potent activators of KCNQ1 channels and activate IKs channels by binding to two different sites, one in the voltage sensor domain – which shifts the voltage dependence to more negative voltages – and the other in the pore domain – which increases the maximal conductance of the channels (Gmax). However, the mechanism by which PUFAs increase the Gmax of the IKs channels is still poorly understood. In addition, it is unclear why IKs channels have a very small single-channel conductance and a low open probability or whether PUFAs affect any of these properties of IKs channels. Our results suggest that the selectivity filter in KCNQ1 is normally unstable, contributing to the low open probability, and that the PUFA-induced increase in Gmax is caused by a stabilization of the selectivity filter in an open-conductive state.

The fully activated open state of KCNQ1 controls the cardiac "fight-or-flight" response.

The cardiac KCNQ1 + KCNE1 (IKs) channel regulates heart rhythm under both normal and stress conditions. Under stress, the β-adrenergic stimulation elevates the intracellular cyclic adenosine monophosphate (cAMP) level, leading to KCNQ1 phosphorylation by protein kinase A and increased IKs, which shortens action potentials to adapt to accelerated heart rate. An impaired response to the β-adrenergic stimulation due to KCNQ1 mutations is associated with the occurrence of a lethal congenital long QT syndrome (type 1, also known as LQT1). However, the underlying mechanism of β-adrenergic stimulation of IKs remains unclear, impeding the development of new therapeutics. Here, we find that the unique properties of KCNQ1 channel gating with two distinct open states are key to this mechanism. KCNQ1's fully activated open (AO) state is more sensitive to cAMP than its intermediate open state. By enhancing the AO state occupancy, the small molecules ML277 and C28 are found to effectively enhance the cAMP sensitivity of the KCNQ1 channel, independent of KCNE1 association. This finding of enhancing AO state occupancy leads to a potential novel strategy to rescue the response of IKs to β-adrenergic stimulation in LQT1 mutants. The success of this approach is demonstrated in cardiac myocytes and also in a high-risk LQT1 mutation. In conclusion, the present study not only uncovers the key role of the AO state in IKs channel phosphorylation, but also provides a target for antiarrhythmic strategy.

Lipophilic compounds restore function to neurodevelopmental-associated KCNQ3 mutations

A major driver of neuronal hyperexcitability is dysfunction of K+ channels, including voltage-gated KCNQ2/3 channels. Their hyperpolarized midpoint of activation and slow activation and deactivation kinetics produce a current that regulates membrane potential and impedes repetitive firing. Inherited mutations in KCNQ2 and KCNQ3 are linked to a wide spectrum of neurodevelopmental disorders (NDDs), ranging from benign familial neonatal seizures to severe epileptic encephalopathies and autism spectrum disorders. However, the impact of these variants on the molecular mechanisms underlying KCNQ3 channel function remains poorly understood and existing treatments have significant side effects. Here, we use voltage clamp fluorometry, molecular dynamic simulations, and electrophysiology to investigate NDD-associated variants in KCNQ3 channels. We identified two distinctive mechanisms by which loss– and gain–of function NDD-associated mutations in KCNQ3 affect channel gating: one directly affects S4 movement while the other changes S4-to-pore coupling. MD simulations and electrophysiology revealed that polyunsaturated fatty acids (PUFAs) primarily target the voltage-sensing domain in its activated conformation and form a weaker interaction with the channel’s pore. Consistently, two such compounds yielded partial and complete functional restoration in R227Q- and R236C-containing channels, respectively. Our results reveal the potential of PUFAs to be developed into therapies for diverse KCNQ3-based channelopathies.

EXPLORING THE ROLE OF NGS IN GENETIC PROFILING FOR UNDERSTANDING NON-VIOLENT SUDDEN CARDIAC DEATH IN ASTANA

Sudden cardiac death is an unexpected and often unpredictable event that can have serious consequences for the health and lives of individuals not only in middle age but also in young individuals. Mutations, particularly in genes associated with cardiovascular disease, may play a main role in the development of such events, but their exact role and impact require detailed study and prevention of the development of new mutations by using the Next-generation sequencing (NGS). To evaluate the diagnostic importance of next-generation sequencing (NGS) by using our developed cardiogenetic panel in molecular autopsy in sudden non-violent deaths. A prospective study was conducted from 2018 to 2023 focusing of young individuals aged 18 to 45 years resigning in Astana who died of non-violent CVD. The data were analyzed from RSCE "Forensic Expertise Centre of the Ministry of Justice of the Republic of Kazakhstan" "Research Institute of Forensic Expertise" in Astana, blood samples were obtained from SCD victims for further DNA extraction. We implemented a next-generation sequencing (NGS) using the customized panel to target enrichment of coding sequences of 96 candidate genes in molecular autopsies of young adults who died of SCD. All identified genetic variants were classified according to ACMG guidelines in cases with non-diagnostic and diagnostic cardiac abnormalities (ischemic heart disease postmortem). Targeted sequencing and stepwise filtering of annotated variants identified 251 unique variants in 86 genes out of 96 in the entire group of cases studied. In patients with SCD with ischemic changes in the heart postmortem, the most frequent mutations were identified in the TTN, MYBPC3, LAMA2, MYH6 and GAA genes. Pathogenic variants in the genes of ion channels KCNQ1, KCNJ2, SCN5A, RYR2 were detected in individuals with non-diagnostic structural anomalies of the heart. Genetic screening of individuals who died of SCD identified variants with likely pathogenic functional effects with high frequency in 63% and 35% of SCD cases with non-diagnostic and diagnostic cardiac abnormalities, respectively, showing the high diagnostic value of genetic screening using a panel of genes for targeted NGS. Data from our prospective study have shown that genetic testing using NGS in young Astana victims with SCD results in a significant number of mutations, consequently, may allow genetic counselling of relatives to prevent SCD due to coronary heart disease and arrhythmias and may support differential diagnosis postmortem. Supported by Committee of Science of the of Science and Higher Education of Republic of Kazakhstan (AP1486990) and Nazarbayev University CRP(211123CRP1608).

Differential expression of ion channel coding genes in the endometrium of women experiencing recurrent implantation failures

Our study probed the differences in ion channel gene expression in the endometrium of women with Recurrent Implantation Failure (RIF) compared to fertile women. We analyzed the relative expression of genes coding for T-type Ca2+, ENaC, CFTR, and KCNQ1 channels in endometrial samples from 20 RIF-affected and 10 control women, aged 22–35, via microarray analysis and quantitative real-time PCR. Additionally, we examined DNA methylation in the regulatory region of KCNQ1 using ChIP real-time PCR. The bioinformatics component of our research included Gene Ontology analysis, protein–protein interaction networks, and signaling pathway mapping to identify key biological processes and pathways implicated in RIF. This led to the discovery of significant alterations in the expression of ion channel genes in RIF women’s endometrium, most notably an overexpression of CFTR and reduced expression of SCNN1A, SCNN1B, SCNN1G, CACNA1H, and KCNQ1. A higher DNA methylation level of KCNQ1’s regulatory region was also observed in RIF patients. Gene-set enrichment analysis highlighted a significant presence of genes involved with ion transport and membrane potential regulation, particularly in sodium and calcium channel complexes, which are vital for cation movement across cell membranes. Genes were also enriched in broader ion channel and transmembrane transporter complexes, underscoring their potential extensive role in cellular ion homeostasis and signaling. These findings suggest a potential involvement of ion channels in the pathology of implantation failure, offering new insights into the mechanisms behind RIF and possible therapeutic targets.

Sympathetic Motor Neuron Dysfunction is a Missing Link in Age-Associated Sympathetic Overactivity.

Overactivity of the sympathetic nervous system is a hallmark of aging. The cellular mechanisms behind this overactivity remain poorly understood, with most attention paid to likely central nervous system components. In this work, we hypothesized that aging also affects the function of motor neurons in the peripheral sympathetic ganglia. To test this hypothesis, we compared the electrophysiological responses and ion-channel activity of neurons isolated from the superior cervical ganglia of young (12 weeks), middle-aged (64 weeks), and old (115 weeks) mice. These approaches showed that aging does impact the intrinsic properties of sympathetic motor neurons, increasing spontaneous and evoked firing responses. A reduction of M current emerged as a major contributor to age-related hyperexcitability. Thus, it is essential to consider the effect of aging on motor components of the sympathetic reflex as a crucial part of the mechanism involved in sympathetic overactivity.

Abstract Mo063: Hacking the ubiquitin code to distinctively modulate ion channel functional expression

Ubiquitin is a powerful modulator of ion channel fate, yet the mechanistic basis and scope of its regulation has remained elusive due to the complexity of the ubiquitin code and an inability to achieve substrate-specific control of the ubiquitome. To circumvent this and to specifically interrogate the role of distinct ubiquitin linkages on the cardiac repolarizing I Ks channel KCNQ1 (Q1), we developed a toolkit of engineered deubiquitinases (enDUBs) featuring a GFP/YFP-targeted nanobody fused to catalytic domains of DUBs with preferences for hydrolyzing distinctive polyubiquitin linkage types: OTUD1 - K63; OTUD4 - K48; Cezanne - K11; TRABID - K27/K33; and USP21 - non-specific. Co-expressing each of the enDUBs with YFP-tagged Q1 channels in HEK293 cells led to decreased channel ubiquitination but produced quantitative and qualitative differences in Q1 expression. NanoCezanne and nanoTRABID significantly increased Q1 surface density and ionic currents, whereas nanoOTUD1 and nanoUSP21 yielded more moderate effects. In sharp contrast, nanoOTUD4 downregulated Q1 surface density and currents. The impact on steady-state Q1 surface density was achieved by divergent trafficking mechanisms – nanoTRABID and nanoCezanne increased channel forward trafficking to the surface, while nanoOTUD4 reduced channel forward trafficking. The E3 ligases NEDD4L and ITCH both eliminate Q1 functional expression but with different polyubiquitin signatures as assessed by mass spectrometry. The enDUBs nanoOTUD1, nanoCezanne and nanoUSP21 protected Q1 from NEDD4L; whereas nanoTRABID, nanoOTUD1, nanoCezanne and nanoUSP21 shielded Q1 from ITCH. In adult guinea ventricular cardiomyocytes, the enDUBs displayed context-specific regulatory effects, where nanoOTUD1, nanoCezanne and nanoUSP21 significantly enhanced Q1 surface expression and stability, with nanoTRABID having more moderate effects, while nanoOTUD4 sharply decreased Q1 trafficking to the sarcolemma. Finally, linkage-specific enDUBs demonstrated varying capabilities to rescue distinct trafficking-deficient mutant Q1 channels that cause long QT syndrome and predispose to sudden cardiac death. The results reveal a rich multi-faceted role for linkage-specific ubiquitination in regulating ion channel trafficking with implications to develop targeted therapies for trafficking-deficit ion channelopathies.

Plural molecular and cellular mechanisms of pore domain KCNQ2 encephalopathy.

KCNQ2 variants in children with neurodevelopmental impairment are difficult to assess due to their heterogeneity and unclear pathogenic mechanisms. We describe a child with neonatal-onset epilepsy, developmental impairment of intermediate severity, and KCNQ2 G256W heterozygosity. Analyzing prior KCNQ2 channel cryoelectron microscopy models revealed G256 as a node of an arch-shaped non-covalent bond network linking S5, the pore turret, and the ion path. Co-expression with G256W dominantly suppressed conduction by wild-type subunits in heterologous cells. Ezogabine partly reversed this suppression. G256W/+ mice have epilepsy leading to premature deaths. Hippocampal CA1 pyramidal cells from G256W/+ brain slices showed hyperexcitability. G256W/+ pyramidal cell KCNQ2 and KCNQ3 immunolabeling was significantly shifted from axon initial segments to neuronal somata. Despite normal mRNA levels, G256W/+ mouse KCNQ2 protein levels were reduced by about 50%. Our findings indicate that G256W pathogenicity results from multiplicative effects, including reductions in intrinsic conduction, subcellular targeting, and protein stability. These studies provide evidence for an unexpected and novel role for the KCNQ2 pore turret and introduce a valid animal model of KCNQ2 encephalopathy. Our results, spanning structure to behavior, may be broadly applicable because the majority of KCNQ2 encephalopathy patients share variants near the selectivity filter.

KCNQ1 is an essential mediator of the sex-dependent perception of moderate cold temperatures

Low temperatures and cooling agents like menthol induce cold sensation by activating the peripheral cold receptors TRPM8 and TRPA1, cation channels belonging to the TRP channel family, while the reduction of potassium currents provides an additional and/or synergistic mechanism of cold sensation. Despite extensive studies over the past decades to identify the molecular receptors that mediate thermosensation, cold sensation is still not fully understood and many cold-sensitive peripheral neurons do not express the well-established cold sensor TRPM8. We found that the voltage-gated potassium channel KCNQ1 (Kv7.1), which is defective in cardiac LQT1 syndrome, is, in addition to its known function in the heart, a highly relevant and sex-specific sensor of moderately cold temperatures. We found that KCNQ1 is expressed in skin and dorsal root ganglion neurons, is sensitive to menthol and cooling agents, and is highly sensitive to moderately cold temperatures, in a temperature range at which TRPM8 is not thermosensitive. C-fiber recordings from KCNQ1-/- mice displayed altered action potential firing properties. Strikingly, only male KCNQ1-/- mice showed substantial deficits in cold avoidance at moderately cold temperatures, with a strength of the phenotype similar to that observed in TRPM8-/- animals. While sex-dependent differences in thermal sensitivity have been well documented in humans and mice, KCNQ1 is the first gene reported to play a role in sex-specific temperature sensation. Moreover, we propose that KCNQ1, together with TRPM8, is a key instrumentalist that orchestrates the range and intensity of cold sensation.

The mitochondrial calcium uniporter inhibitor Ru265 increases neuronal excitability and reduces neurotransmission via off-target effects.

Excitotoxicity due to mitochondrial calcium (Ca2+) overloading can trigger neuronal cell death in a variety of pathologies. Inhibiting the mitochondrial calcium uniporter (MCU) has been proposed as a therapeutic avenue to prevent calcium overloading. Ru265 (ClRu(NH3)4(μ-N)Ru(NH3)4Cl]Cl3) is a cell-permeable inhibitor of the mitochondrial calcium uniporter (MCU) with nanomolar affinity. Ru265 reduces sensorimotor deficits and neuronal death in models of ischemic stroke. However, the therapeutic use of Ru265 is limited by the induction of seizure-like behaviours. We examined the effect of Ru265 on synaptic and neuronal function in acute brain slices and hippocampal neuron cultures derived from mice, in control and where MCU expression was genetically abrogated. Ru265 decreased evoked responses from calyx terminals and induced spontaneous action potential firing of both the terminal and postsynaptic principal cell. Recordings of presynaptic Ca2+ currents suggested that Ru265 blocks the P/Q type channel, confirmed by the inhibition of currents in cells exogenously expressing the P/Q type channel. Measurements of presynaptic K+ currents further revealed that Ru265 blocked a KCNQ current, leading to increased membrane excitability, underlying spontaneous spiking. Ca2+ imaging of hippocampal neurons showed that Ru265 increased synchronized, high-amplitude events, recapitulating seizure-like activity seen in vivo. Importantly, MCU ablation did not suppress Ru265-induced increases in neuronal activity and seizures. Our findings provide a mechanistic explanation for the pro-convulsant effects of Ru265 and suggest counter screening assays based on the measurement of P/Q and KCNQ channel currents to identify safe MCU inhibitors.

Selective inhibition of A-fiber-mediated excitatory transmission underlies the analgesic effects of KCNQ channel opening in the spinal dorsal horn

Neuronal voltage-gated KCNQ (Kv7) channels, expressed centrally and peripherally, mediate low-threshold and non-inactivating M-currents responsible for the control of tonic excitability of mammalian neurons. Pharmacological opening of KCNQ channels has been reported to generate analgesic effects in animal models of neuropathic pain. Here, we examined the possible involvement of central KCNQ channels in the analgesic effects of retigabine, a KCNQ channel opener. Behaviorally, intraperitoneally applied retigabine exerted analgesic effects on thermal and mechanical hypersensitivity in male mice developing neuropathic pain after partial sciatic nerve ligation, which was antagonized by the KCNQ channel blocker XE991 preadministered intraperitoneally and intrathecally. Intrathecally applied retigabine also exerted analgesic effects that were inhibited by intrathecally injected XE991. We then explored the synaptic mechanisms underlying the analgesic effects of retigabine in the spinal dorsal horn.Whole-cell recordings were made from dorsal horn neurons in spinal slices with attached dorsal roots from adult male mice developing neuropathic pain, and the effects of retigabine on miniature and afferent-evoked postsynaptic currents were examined. Retigabine reduced the amplitude of A-fiber-mediated EPSCs without affecting C-fiber-mediated excitatory synaptic transmission. A-fiber-mediated EPSCs remained unaltered by retigabine in the presence of XE991, consistently with the behavioral findings. The frequency and amplitude of mEPSCs were not affected by retigabine. Thus, opening of KCNQ channels in the central terminals of primary afferent A-fibers inhibits excitatory synaptic transmission in the spinal dorsal horn, most likely contributing to the analgesic effect of retigabine.

A mutation in the cardiac KV7.1 channel possibly disrupts interaction with Yotiao protein

Here, we characterized the p.Arg583His (R583H) Kv7.1 mutation, identified in two unrelated families suffered from LQT syndrome. This mutation is located in the HС-HD linker of the cytoplasmic portion of the Kv7.1 channel. This linker, together with HD helix are responsible for binding the A-kinase anchoring protein 9 (AKAP9), Yotiao. We studied the electrophysiological characteristics of the mutated channel expressed in CHO–K1 along with KCNE1 subunit and Yotiao protein, using the whole-cell patch-clamp technique. We found that R583H mutation, even at the heterozygous state, impedes IKs activation. Molecular modeling showed that HС and HD helixes of the C-terminal part of Kv7.1 channel are swapped along the C-terminus length of the channel and that R583 position is exposed to the outer surface of HC-HD tandem coiled-coil. Interestingly, the adenylate cyclase activator, forskolin had a smaller effect on the mutant channel comparing with the WT protein, suggesting that R583H mutation may disrupt the interaction of the channel with the adaptor protein Yotiao and, therefore, may impair phosphorylation of the KCNQ1 channel.

Glial KCNQ K+ channels control neuronal output by regulating GABA release from glia in C. elegans

KCNQs are voltage-gated K+ channels that control neuronal excitability and are mutated in epilepsy and autism spectrum disorder (ASD). KCNQs have been extensively studied in neurons, but their function in glia is unknown. Using voltage, calcium, and GABA imaging, optogenetics, and behavioral assays, we show here for the first time in Caenorhabditis elegans (C.elegans) that glial KCNQ channels control neuronal excitability by mediating GABA release from glia via regulation of the function of L-type voltage-gated Ca2+ channels. Further, we show that human KCNQ channels have the same role when expressed in nematode glia, underscoring conservation of function across species. Finally, we show that pathogenic loss-of-function and gain-of-function human KCNQ2 mutations alter glia-to-neuron GABA signaling in distinct ways and that the KCNQ channel opener retigabine exerts rescuing effects. This work identifies glial KCNQ channels as key regulators of neuronal excitability via control of GABA release from glia.

Potential therapeutic targets for hypotension in duchenne muscular dystrophy

Duchenne Muscular Dystrophy (DMD) is marked by genetic mutations occurring in the DMD gene, which is widely expressed in the cardiovascular system. In addition to developing cardiomyopathy, patients with DMD have been reported to be susceptible to the development of symptomatic hypotension, although the mechanisms are unclear. Analysis of single-cell RNA sequencing data has identified potassium voltage-gated channel subfamily Q member 5 (KCNQ5) and possibly ryanodine receptor 2 (RyR2) as potential candidate hypotension genes whose expression is significantly upregulated in the vascular smooth muscle cells of DMD mutant mice. We hypothesize that heightened KCNQ5 and RyR2 expression contributes to decreased arterial blood pressure in patients with DMD. Exploring pharmacological approaches to inhibit the KCNQ5 and RyR2 channels holds promise in managing the systemic hypotension observed in individuals with DMD. This avenue of investigation presents new prospects for improving clinical outcomes for these patients.

A small-molecule activation mechanism that directly opens the KCNQ2 channel.

Pharmacological activation of voltage-gated ion channels by ligands serves as the basis for therapy and mainly involves a classic gating mechanism that augments the native voltage-dependent open probability. Through structure-based virtual screening, we identified a new scaffold compound, Ebio1, serving as a potent and subtype-selective activator for the voltage-gated potassium channel KCNQ2 and featuring a new activation mechanism. Single-channel patch-clamp, cryogenic-electron microscopy and molecular dynamic simulations, along with chemical derivatives, reveal that Ebio1 engages the KCNQ2 activation by generating an extended channel gate with a larger conductance at the saturating voltage (+50 mV). This mechanism is different from the previously observed activation mechanism of ligands on voltage-gated ion channels. Ebio1 caused S6 helices from residues S303 and F305 to perform a twist-to-open movement, which was sufficient to open the KCNQ2 gate. Overall, our findings provide mechanistic insights into the activation of KCNQ2 channel by Ebio1 and lend support for KCNQ-related drug development.

Mid-Infrared Photons Alleviate Tinnitus by Activating the KCNQ2 Channel in the Auditory Cortex.

Tinnitus is a phantom auditory sensation often accompanied by hearing loss, cognitive impairments, and psychological disturbances in various populations. Dysfunction of KCNQ2 and KCNQ3 channels-voltage-dependent potassium ion channels-in the cochlear nucleus can cause tinnitus. Despite the recognized significance of KCNQ2 and KCNQ3 channels in the auditory cortex, their precise relationship and implications in the pathogenesis of tinnitus remain areas of scientific inquiry. This study aimed to elucidate the pathological roles of KCNQ2 and KCNQ3 channels within the auditory cortex in tinnitus development and examine the therapeutic potential of mid-infrared photons for tinnitus treatment. We utilized a noise-induced tinnitus model combined with immunofluorescence, electrophysiological recording, and molecular dynamic simulation to investigate the morphological and physiological alterations after inducing tinnitus. Moreover, invivo irradiation was administered to verify the treatment effects of infrared photons. Tinnitus was verified by deficits of the gap ratio with similar prepulse inhibition ratio and auditory brainstem response threshold. We observed an important enhancement in neuronal excitability in the auditory cortex using patch-clamp recordings, which correlated with KCNQ2 and KCNQ3 channel dysfunction. After irradiation with infrared photons, excitatory neuron firing was inhibited owing to increased KCNQ2 current resulting from structural alterations in the filter region. Meanwhile, deficits of the acoustic startle response in tinnitus animals were alleviated by infrared photons. Furthermore, infrared photons reversed the abnormal hyperexcitability of excitatory neurons in the tinnitus group. This study provided a novel method for modulating neuron excitability in the auditory cortex using KCNQ2 channels through a nonthermal effect. Infrared photons effectively mitigated tinnitus-related behaviors by suppressing abnormal neural excitability, potentially laying the groundwork for innovative therapeutic approaches for tinnitus treatment.

Phox2b-expressing neurons contribute to breathing problems in Kcnq2 loss- and gain-of-function encephalopathy models

Loss- and gain-of-function variants in the gene encoding KCNQ2 channels are a common cause of developmental and epileptic encephalopathy, a condition characterized by seizures, developmental delays, breathing problems, and early mortality. To understand how KCNQ2 dysfunction impacts behavior in a mouse model, we focus on the control of breathing by neurons expressing the transcription factor Phox2b which includes respiratory neurons in the ventral parafacial region. We find Phox2b-expressing ventral parafacial neurons express Kcnq2 in the absence of other Kcnq isoforms, thus clarifying why disruption of Kcnq2 but not other channel isoforms results in breathing problems. We also find that Kcnq2 deletion or expression of a recurrent gain-of-function variant R201C in Phox2b-expressing neurons increases baseline breathing or decreases the central chemoreflex, respectively, in mice during the light/inactive state. These results uncover mechanisms underlying breathing abnormalities in KCNQ2 encephalopathy and highlight an unappreciated vulnerability of Phox2b-expressing ventral parafacial neurons to KCNQ2 pathogenic variants.