The role of KCNQ channel activators in management of major depressive disorder.

Depression, a complex disorder with significant treatment challenges, necessitates innovative therapeutic approaches to address its multifaceted nature and enhance treatment outcomes. The modulation of KCNQ potassium (K+) channels, pivotal regulators of neuronal excitability and neurotransmitter release, is a promising innovative therapeutic target in psychiatry. Widely expressed across various tissues, including the nervous and cardiovascular systems, KCNQ channels play a crucial role in modulating membrane potential and regulating neuronal activity. Recent preclinical evidence suggests that KCNQ channels, particularly KCNQ3, contribute to the regulation of neuronal excitability within the reward circuitry, offering a potential target for alleviating depressive symptoms, notably anhedonia. Studies using animal models demonstrate that interventions targeting KCNQ channels can restore dopaminergic firing balance and mitigate depressive symptoms. Human studies investigating the effects of KCNQ channel activators, such as ezogabine, have shown promising results in alleviating depressive symptoms and anhedonia. The aforementioned observations underscore the therapeutic potential of KCNQ channel modulation in depression management and highlight the need and justification for phase 2 and phase 3 dose-finding studies as well as studies prespecifying symptomatic targets in depression including anhedonia.

KCNQ potassium channels modulate Wnt activity in gastro-oesophageal adenocarcinomas.

Voltage-sensitive potassium channels play an important role in controlling membrane potential and ionic homeostasis in the gut and have been implicated in gastrointestinal (GI) cancers. Through large-scale analysis of 897 patients with gastro-oesophageal adenocarcinomas (GOAs) coupled with in vitro models, we find KCNQ family genes are mutated in ∼30% of patients, and play therapeutically targetable roles in GOA cancer growth. KCNQ1 and KCNQ3 mediate the WNT pathway and MYC to increase proliferation through resultant effects on cadherin junctions. This also highlights novel roles of KCNQ3 in non-excitable tissues. We also discover that activity of KCNQ3 sensitises cancer cells to existing potassium channel inhibitors and that inhibition of KCNQ activity reduces proliferation of GOA cancer cells. These findings reveal a novel and exploitable role of potassium channels in the advancement of human cancer, and highlight that supplemental treatments for GOAs may exist through KCNQ inhibitors.

Dehydroepiandrosterone Sulfate (DHEAS) Is an Endogenous Kv7 Channel Modulator That Reduces Kv7/M-Current Suppression and Inflammatory Pain.

Neuronal Kv7 voltage-gated potassium channels generate the M-current and regulate neuronal excitability. Here, we report that dehydroepiandrosterone sulfate (DHEAS) is an endogenous Kv7 channel modulator that attenuates Gq-coupled receptor-induced M-current suppression. DHEAS reduced muscarinic agonist-induced Kv7-current suppression of Kv7.1, Kv7.2, Kv7.4, or Kv7.5 homomeric currents and endogenous M-currents in rat sympathetic ganglion neurons. However, DHEAS per se did not alter the voltage dependence of these Kv7 homomeric channels or the m1 receptor-induced activation of phospholipase C or protein kinase C. DHEAS-treated Kv7.2 homomeric currents became resistant to depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) induced by voltage-activated phosphatase, Ci-VSP or eVSP. Our computational models predicted a novel binding site for DHEAS in the cytoplasmic domain of Kv7 subunits. A single-point mutation of the predicted key histidine into cysteine in the rat Kv7.2 subunit, rKv7.2(H558C), resulted in a loss of effects of DHEAS on muscarinic Kv7 current suppression. Furthermore, in vivo administration of DHEAS in mice of both sexes reduced late phase pain responses in the formalin paw test. However, it did not have effects on early phase responses in the formalin paw test or responses in the hot plate test. Coadministration of a selective Kv7 inhibitor, XE991, and DHEAS eliminated analgesic effects of DHEAS in late phase responses in the formalin paw test. Collectively, these results suggest that DHEAS attenuates M-current suppression by stabilizing PIP2-Kv7 subunit interaction and can mitigate inflammatory pain.SIGNIFICANCE STATEMENT M-current suppression induced by stimulation of Gq-coupled receptors is a form of Kv7 current modulation that can reversibly increase neuronal excitability. This study demonstrates that DHEAS, an endogenous steroid hormone, is a novel Kv7 channel modulator that can attenuate M-current suppression without affecting basal Kv7 channel kinetics. Administration of DHEAS in vivo alleviated inflammatory pain in rodents. These results suggest that the degree of M-current suppression can be dynamically regulated by small molecules. Therefore, this novel form of Kv7 channel regulation holds promising potential as a therapeutic target for sensitized nervous activities, such as inflammatory pain.

KCNQ2/3 Gain-of-Function Variants and Cell Excitability: Differential Effects in CA1 versus L2/3 Pyramidal Neurons.

Gain-of-function (GOF) pathogenic variants in the potassium channels KCNQ2 and KCNQ3 lead to hyperexcitability disorders such as epilepsy and autism spectrum disorders. However, the underlying cellular mechanisms of how these variants impair forebrain function are unclear. Here, we show that the R201C variant in KCNQ2 has opposite effects on the excitability of two types of mouse pyramidal neurons of either sex, causing hyperexcitability in layer 2/3 (L2/3) pyramidal neurons and hypoexcitability in CA1 pyramidal neurons. Similarly, the homologous R231C variant in KCNQ3 leads to hyperexcitability in L2/3 pyramidal neurons and hypoexcitability in CA1 pyramidal neurons. However, the effects of KCNQ3 gain-of-function on excitability are specific to superficial CA1 pyramidal neurons. These findings reveal a new level of complexity in the function of KCNQ2 and KCNQ3 channels in the forebrain and provide a framework for understanding the effects of gain-of-function variants and potassium channels in the brain.SIGNIFICANCE STATEMENT KCNQ2/3 gain-of-function (GOF) variants lead to severe forms of neurodevelopmental disorders, but the mechanisms by which these channels affect neuronal activity are poorly understood. In this study, using a series of transgenic mice we demonstrate that the same KCNQ2/3 GOF variants can lead to either hyperexcitability or hypoexcitability in different types of pyramidal neurons [CA1 vs layer (L)2/3]. Additionally, we show that expression of the recurrent KCNQ2 GOF variant R201C in forebrain pyramidal neurons could lead to seizures and SUDEP. Our data suggest that the effects of KCNQ2/3 GOF variants depend on specific cell types and brain regions, possibly accounting for the diverse range of phenotypes observed in individuals with KCNQ2/3 GOF variants.

Functional characterization and in vitro pharmacological rescue of KCNQ2 pore mutations associated with epileptic encephalopathy.

Mutations in the KCNQ2 gene encoding KV7.2 subunit that mediates neuronal M-current cause a severe form of developmental and epileptic encephalopathy (DEE). Electrophysiological evaluation of KCNQ2 mutations has been proved clinically useful in improving outcome prediction and choosing rational anti-seizure medications (ASMs). In this study we described the clinical characteristics, electrophysiological phenotypes and the in vitro response to KCNQ openers of five KCNQ2 pore mutations (V250A, N258Y, H260P, A265T and G290S) from seven patients diagnosed with KCNQ2-DEE. The KCNQ2 variants were transfected into Chinese hamster ovary (CHO) cells alone, in combination with KCNQ3 (1:1) or with wild-type KCNQ2 (KCNQ2-WT) and KCNQ3 in a ratio of 1:1:2, respectively. Their expression and electrophysiological function were assessed. When transfected alone or in combination with KCNQ3, none of these mutations affected the membrane expression of KCNQ2, but most failed to induce a potassium current except A265T, in which trace currents were observed when co-transfected with KCNQ3. When co-expressed with KCNQ2-WT and KCNQ3 (1:1:2), the currents at 0 mV of these mutations were decreased by 30%-70% compared to the KCNQ2/3 channel, which could be significantly rescued by applying KCNQ openers including the approved antiepileptic drug retigabine (RTG, 10 µM), as well as two candidates subjected to clinical trials, pynegabine (HN37, 1 µM) and XEN1101 (1 µM). These newly identified pathologic variants enrich the KCNQ2-DEE mutation hotspots in the pore-forming domain. This electrophysiological study provides a rational basis for personalized therapy with KCNQ openers in DEE patients carrying loss-of-function (LOF) mutations in KCNQ2.

Intracellular zinc protects Kv7 K+ channels from Ca2+/calmodulin-mediated inhibition.

Zinc (Zn) is an essential trace element; it serves as a cofactor for a great number of enzymes, transcription factors, receptors, and other proteins. Zinc is also an important signaling molecule, which can be released from intracellular stores into the cytosol or extracellular space, for example, during synaptic transmission. Amongst cellular effects of zinc is activation of Kv7 (KCNQ, M-type) voltage-gated potassium channels. Here, we investigated relationships between Kv7 channel inhibition by Ca2+/calmodulin (CaM) and zinc-mediated potentiation. We show that Zn2+ ionophore, zinc pyrithione (ZnPy), can prevent or reverse Ca2+/CaM-mediated inhibition of Kv7.2. In the presence of both Ca2+ and Zn2+, the Kv7.2 channels lose most of their voltage dependence and lock in an open state. In addition, we demonstrate that mutations that interfere with CaM binding to Kv7.2 and Kv7.3 reduced channel membrane abundance and activity, but these mutants retained zinc sensitivity. Moreover, the relative efficacy of ZnPy to activate these mutants was generally greater, compared with the WT channels. Finally, we show that zinc sensitivity was retained in Kv7.2 channels assembled with mutant CaM with all four EF hands disabled, suggesting that it is unlikely to be mediated by CaM. Taken together, our findings indicate that zinc is a potent Kv7 stabilizer, which may protect these channels from physiological inhibitory effects of neurotransmitters and neuromodulators, protecting neurons from overactivity.

Potassium channels and epilepsy.

With the development and application of next-generation sequencing technology, the aetiological diagnosis of genetic epilepsy is rapidly becoming easier and less expensive. Additionally, there is a growing body of research into precision therapy based on genetic diagnosis. The numerous genes in the potassium ion channel family constitute the largest family of ion channels: this family is divided into different subtypes. Potassium ion channels play a crucial role in the electrical activity of neurons and are directly involved in the mechanism of epileptic seizures. In China, scientific research on genetic diagnosis and studies of precision therapy for genetic epilepsy are progressing rapidly. Many cases of epilepsy caused by mutation of potassium channel genes have been identified, and several potassium channel gene targets and drug candidates have been discovered. The purpose of this review is to briefly summarize the progress of research on the precise diagnosis and treatment of potassium ion channel-related genetic epilepsy, especially the research conducted in China. Here in, we review several large cohort studies on the genetic diagnosis of epilepsy in China in recent years, summarized the proportion of potassium channel genes. We focus on the progress of precison therapy on some hot epilepsy related potassium channel genes: KCNA1, KCNA2, KCNB1, KCNC1, KCND2, KCNQ2, KCNQ3, KCNMA1, and KCNT1.

Triclosan is a KCNQ3 potassium channel activator.

KCNQ channels participate in the physiology of several cell types. In neurons of the central nervous system, the primary subunits are KCNQ2, 3, and 5. Activation of these channels silence the neurons, limiting action potential duration and preventing high-frequency action potential burst. Loss-of-function mutations of the KCNQ channels are associated with a wide spectrum of phenotypes characterized by hyperexcitability. Hence, pharmacological activation of these channels is an attractive strategy to treat epilepsy and other hyperexcitability conditions as are the evolution of stroke and traumatic brain injury. In this work we show that triclosan, a bactericide widely used in personal care products, activates the KCNQ3 channels but not the KCNQ2. Triclosan induces a voltage shift in the activation, increases the conductance, and slows the closing of the channel. The response is independent of PIP2. Molecular docking simulations together with site-directed mutagenesis suggest that the putative binding site is in the voltage sensor domain. Our results indicate that triclosan is a new activator for KCNQ channels.

Phenotypic Spectrum in a Family Sharing a Heterozygous KCNQ3 Variant.

BACKGROUND AND PURPOSE: Mutations in KCNQ3 have classically been associated with benign familial neonatal and infantile seizures and more recently identified in patients with neurodevelopmental disorders and abnormal electroencephalogram (EEG) findings. We present 4 affected patients from a family with a pathogenic mutation in KCNQ3 with a unique constellation of clinical findings. METHODS: A family of 3 affected siblings and mother sharing a KCNQ3 pathogenic variant are described, including clinical history, genetic results, and EEG and magnetic resonance imaging (MRI) findings. RESULTS: This family shows a variety of clinical manifestations, including neonatal seizures, developmental delays, autism spectrum disorder, and anxiety. One child developed absence epilepsy, 2 children have infrequent convulsive seizures that have persisted into childhood, and their parent developed adult-onset epilepsy. An underlying c.1091G>A (R364H) variant in KCNQ3 was found in all affected individuals. CONCLUSIONS: The phenotypic variability of KCNQ3 channelopathies continues to expand as more individuals and families are described, and the variant identified in this family adds to the understanding of the manifestations of KCNQ3-related disorders.

KCNQ2 and KCNQ5 form heteromeric channels independent of KCNQ3.

KCNQ2 and KCNQ3 channels are associated with multiple neurodevelopmental disorders and are also therapeutic targets for neurological and neuropsychiatric diseases. For more than two decades, it has been thought that most KCNQ channels in the brain are either KCNQ2/3 or KCNQ3/5 heteromers. Here, we investigated the potential heteromeric compositions of KCNQ2-containing channels. We applied split-intein protein trans-splicing to form KCNQ2/5 tandems and coexpressed these with and without KCNQ3. Unexpectedly, we found that KCNQ2/5 tandems form functional channels independent of KCNQ3 in heterologous cells. Using mass spectrometry, we went on to demonstrate that KCNQ2 associates with KCNQ5 in native channels in the brain, even in the absence of KCNQ3. Additionally, our functional heterologous expression data are consistent with the formation of KCNQ2/3/5 heteromers. Thus, the composition of KCNQ channels is more diverse than has been previously recognized, necessitating a re-examination of the genotype/phenotype relationship of KCNQ2 pathogenic variants.

Cannabidiol activates neuronal Kv7 channels.

Cannabidiol (CBD), a chemical found in the Cannabis sativa plant, is a clinically effective antiepileptic drug whose mechanism of action is unknown. Using a fluorescence-based thallium flux assay, we performed a large-scale screen and found enhancement of flux through heterologously expressed human Kv7.2/7.3 channels by CBD. Patch-clamp recordings showed that CBD acts at submicromolar concentrations to shift the voltage dependence of Kv7.2/7.3 channels in the hyperpolarizing direction, producing a dramatic enhancement of current at voltages near -50 mV. CBD enhanced native M-current in mouse superior cervical ganglion starting at concentrations of 30 nM and also enhanced M-current in rat hippocampal neurons. The potent enhancement of Kv2/7.3 channels by CBD may contribute to its effectiveness as an antiepileptic drug by reducing neuronal hyperexcitability.

Hyperexcitable arousal circuits drive sleep instability during aging.

Sleep quality declines with age; however, the underlying mechanisms remain elusive. We found that hyperexcitable hypocretin/orexin (Hcrt/OX) neurons drive sleep fragmentation during aging. In aged mice, Hcrt neurons exhibited more frequent neuronal activity epochs driving wake bouts, and optogenetic activation of Hcrt neurons elicited more prolonged wakefulness. Aged Hcrt neurons showed hyperexcitability with lower KCNQ2 expression and impaired M-current, mediated by KCNQ2/3 channels. Single-nucleus RNA-sequencing revealed adaptive changes to Hcrt neuron loss in the aging brain. Disruption of Kcnq2/3 genes in Hcrt neurons of young mice destabilized sleep, mimicking aging-associated sleep fragmentation, whereas the KCNQ-selective activator flupirtine hyperpolarized Hcrt neurons and rejuvenated sleep architecture in aged mice. Our findings demonstrate a mechanism underlying sleep instability during aging and a strategy to improve sleep continuity.

Impaired Kv7 channel activity in the central amygdala contributes to elevated sympathetic outflow in hypertension.

AIMS: Elevated sympathetic outflow is associated with primary hypertension. However, the mechanisms involved in heightened sympathetic outflow in hypertension are unclear. The central amygdala (CeA) regulates autonomic components of emotions through projections to the brainstem. The neuronal Kv7 channel is a non-inactivating voltage-dependent K+ channel encoded by KCNQ2/3 genes involved in stabilizing the neuronal membrane potential and regulating neuronal excitability. In this study, we investigated if altered Kv7 channel activity in the CeA contributes to heightened sympathetic outflow in hypertension. METHODS AND RESULTS: The mRNA and protein expression levels of Kv7.2/Kv7.3 in the CeA were significantly reduced in spontaneously hypertensive rats (SHRs) compared with Wistar-Kyoto (WKY) rats. Lowering blood pressure with coeliac ganglionectomy in SHRs did not alter Kv7.2 and Kv7.3 channel expression levels in the CeA. Fluospheres were injected into the rostral ventrolateral medulla (RVLM) to retrogradely label CeA neurons projecting to the RVLM (CeA-RVLM neurons). Kv7 channel currents recorded from CeA-RVLM neurons in brain slices were much smaller in SHRs than in WKY rats. Furthermore, the basal firing activity of CeA-RVLM neurons was significantly greater in SHRs than in WKY rats. Bath application of specific Kv7 channel blocker 10, 10-bis (4-pyridinylmethyl)-9(10H)-anthracnose (XE-991) increased the excitability of CeA-RVLM neurons in WKY rats, but not in SHRs. Microinjection of XE-991 into the CeA increased arterial blood pressure (ABP) and renal sympathetic nerve activity (RSNA), while microinjection of Kv7 channel opener QO-58 decreased ABP and RSNA, in anaesthetized WKY rats but not SHRs. CONCLUSIONS: Our findings suggest that diminished Kv7 channel activity in the CeA contributes to elevated sympathetic outflow in primary hypertension. This novel information provides new mechanistic insight into the pathogenesis of neurogenic hypertension.

Autism-associated mutations in KV7 channels induce gating pore current.

Autism spectrum disorder (ASD) adversely impacts >1% of children in the United States, causing social interaction deficits, repetitive behaviors, and communication disorders. Genetic analysis of ASD has advanced dramatically through genome sequencing, which has identified >500 genes with mutations in ASD. Mutations that alter arginine gating charges in the voltage sensor of the voltage-gated potassium (KV) channel KV7 (KCNQ) are among those frequently associated with ASD. We hypothesized that these gating charge mutations would induce gating pore current (also termed ω-current) by causing an ionic leak through the mutant voltage sensor. Unexpectedly, we found that wild-type KV7 conducts outward gating pore current through its native voltage sensor at positive membrane potentials, owing to a glutamine in the third gating charge position. In bacterial and human KV7 channels, gating charge mutations at the R1 and R2 positions cause inward gating pore current through the resting voltage sensor at negative membrane potentials, whereas mutation at R4 causes outward gating pore current through the activated voltage sensor at positive potentials. Remarkably, expression of the KV7.3/R2C ASD-associated mutation in vivo in midbrain dopamine neurons of mice disrupts action potential generation and repetitive firing. Overall, our results reveal native and mutant gating pore current in KV7 channels and implicate altered control of action potential generation by gating pore current through mutant KV7 channels as a potential pathogenic mechanism in autism.

The SUMO-specific protease SENP2 plays an essential role in the regulation of Kv7.2 and Kv7.3 potassium channels.

Sentrin/small ubiquitin-like modifier (SUMO)-specific protease 2 (SENP2)-deficient mice develop spontaneous seizures in early life because of a marked reduction in M currents, which regulate neuronal membrane excitability. We have previously shown that hyper-SUMOylation of the Kv7.2 and Kv7.3 channels is critically involved in the regulation of the M currents conducted by these potassium voltage-gated channels. Here, we show that hyper-SUMOylation of the Kv7.2 and Kv7.3 proteins reduced binding to the lipid secondary messenger PIP2. CaM1 has been shown to be tethered to the Kv7 subunits via hydrophobic motifs in its C termini and implicated in the channel assembly. Mutation of the SUMOylation sites on Kv7.2 and Kv7.3 specifically resulted in decreased binding to CaM1 and enhanced CaM1-mediated assembly of Kv7.2 and Kv7.3, whereas hyper-SUMOylation of Kv7.2 and Kv7.3 inhibited channel assembly. SENP2-deficient mice exhibited increased acetylcholine levels in the brain and the heart tissue because of increases in the vagal tone induced by recurrent seizures. The SENP2-deficient mice develop seizures followed by a period of sinus pauses or atrioventricular conduction blocks. Chronic administration of the parasympathetic blocker atropine or unilateral vagotomy significantly prolonged the life of the SENP2-deficient mice. Furthermore, we showed that retigabine, an M-current opener, reduced the transcription of SUMO-activating enzyme SAE1 and inhibited SUMOylation of the Kv7.2 and Kv7.3 channels, and also prolonged the life of SENP2-deficient mice. Taken together, the previously demonstrated roles of PIP2, CaM1, and retigabine on the regulation of Kv7.2 and Kv7.3 channel function can be explained by their roles in regulating SUMOylation of this critical potassium channel.

Place fields of single spikes in hippocampus involve Kcnq3 channel-dependent entrainment of complex spike bursts.

Hippocampal pyramidal cells encode an animal's location by single action potentials and complex spike bursts. These elementary signals are believed to play distinct roles in memory consolidation. The timing of single spikes and bursts is determined by intrinsic excitability and theta oscillations (5-10 Hz). Yet contributions of these dynamics to place fields remain elusive due to the lack of methods for specific modification of burst discharge. In mice lacking Kcnq3-containing M-type K+ channels, we find that pyramidal cell bursts are less coordinated by the theta rhythm than in controls during spatial navigation, but not alert immobility. Less modulated bursts are followed by an intact post-burst pause of single spike firing, resulting in a temporal discoordination of network oscillatory and intrinsic excitability. Place fields of single spikes in one- and two-dimensional environments are smaller in the mutant. Optogenetic manipulations of upstream signals reveal that neither medial septal GABA-ergic nor cholinergic inputs alone, but rather their joint activity, is required for entrainment of bursts. Our results suggest that altered representations by bursts and single spikes may contribute to deficits underlying cognitive disabilities associated with KCNQ3-mutations in humans.

The Amyloid Precursor Protein C99 Fragment Modulates Voltage-Gated Potassium Channels.

BACKGROUND/AIMS: The Amyloid Precursor Protein (APP) is involved in the regulation of multiple cellular functions via protein-protein interactions and has been most studied with respect to Alzheimer's disease (AD). Abnormal processing of the single transmembrane-spanning C99 fragment of APP contributes to the formation of amyloid plaques, which are causally related to AD. Pathological C99 accumulation is thought to associate with early cognitive defects in AD. Here, unexpectedly, sequence analysis revealed that C99 exhibits 24% sequence identity with the KCNE1 voltage-gated potassium (Kv) channel ß subunit, comparable to the identity between KCNE1 and KCNE2-5 (21-30%). This suggested the possibility of C99 regulating Kv channels. METHODS: We quantified the effects of C99 on Kv channel function, using electrophysiological analysis of subunits expressed in Xenopus laevis oocytes, biochemical and immunofluorescence techniques. RESULTS: C99 isoform-selectively inhibited (by 30-80%) activity of a range of Kv channels. Among the KCNQ (Kv7) family, C99 isoform-selectively inhibited, shifted the voltage dependence and/or slowed activation of KCNQ2, KCNQ3, KCNQ2/3 and KCNQ5, with no effects on KCNQ1, KCNQ1-KCNE1 or KCNQ4. C99/APP co-localized with KCNQ2 and KCNQ3 in adult rat sciatic nerve nodes of Ranvier. Both C99 and full-length APP co-immunoprecipitated with KCNQ2 in vitro, yet unlike C99, APP only weakly affected KCNQ2/3 activity. Finally, C99 altered the effects on KCNQ2/3 function of inhibitors tetraethylammounium and XE991, but not openers retigabine and ICA27243. CONCLUSION: Our findings raise the possibility of C99 accumulation early in AD altering cellular excitability by modulating Kv channel activity.

Loss of KCNQ2 or KCNQ3 Leads to Multifocal Time-Varying Activity in the Neonatal Forebrain Ex Vivo.

Epileptic encephalopathies represent a group of disorders often characterized by refractory seizures, regression in cognitive development, and typically poor prognosis. Dysfunction of KCNQ2 and KCNQ3 channels has emerged as a major cause of neonatal epilepsy. However, our understanding of the cellular mechanisms that may both explain the origins of epilepsy and inform treatment strategies for KCNQ2 and KCNQ3 dysfunction is still lacking. Here, using mesoscale calcium imaging and pharmacology, we demonstrate that in mouse neonatal brain slices, conditional loss of Kcnq2 from forebrain excitatory neurons (Pyr:Kcnq2 mice) or constitutive deletion of Kcnq3 leads to sprawling hyperactivity across the neocortex. Surprisingly, the generation of time-varying hypersynchrony in slices from Pyr:Kcnq2 mice does not require fast synaptic transmission. This is in contrast to control littermates and constitutive Kcnq3 knock-out mice where activity is primarily driven by fast synaptic transmission in the neocortex. Unlike in the neocortex, hypersynchronous activity in the hippocampal formation from Kcnq2 conditional and Kcnq3 constitutive knock-out mice persists in the presence of synaptic transmission blockers. Thus, we propose that loss of KCNQ2 or KCNQ3 function differentially leads to network hyperactivity across the forebrain in a region-specific and macro-circuit-specific manner.

CRISPR knockdown of Kcnq3 attenuates the M-current and increases excitability of NPY/AgRP neurons to alter energy balance.

OBJECTIVE: Arcuate nucleus neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons drive ingestive behavior. The M-current, a subthreshold non-inactivating potassium current, plays a critical role in regulating NPY/AgRP neuronal excitability. Fasting decreases while 17ß-estradiol increases the M-current by regulating the mRNA expression of Kcnq2, 3, and 5 (Kv7.2, 3, and 5) channel subunits. Incorporating KCNQ3 into heteromeric channels has been considered essential to generate a robust M-current. Therefore, we investigated the behavioral and physiological effects of selective Kcnq3 deletion from NPY/AgRP neurons. METHODS: We used a single adeno-associated viral vector containing a recombinase-dependent Staphylococcus aureus Cas9 with a single-guide RNA to selectively delete Kcnq3 in NPY/AgRP neurons. Single-cell quantitative measurements of mRNA expression and whole-cell patch clamp experiments were conducted to validate the selective knockdown. Body weight, food intake, and locomotor activity were measured in male mice to assess disruptions in energy balance. RESULTS: The virus reduced the expression of Kcnq3 mRNA without affecting Kcnq2 or Kcnq5. The M-current was attenuated, causing NPY/AgRP neurons to be more depolarized, exhibit a higher input resistance, and require less depolarizing current to fire action potentials, indicative of increased excitability. Although the resulting decrease in the M-current did not overtly alter ingestive behavior, it significantly reduced the locomotor activity as measured by open-field testing. Control mice on a high-fat diet exhibited an enhanced M-current and increased Kcnq2 and Kcnq3 expression, but the M-current remained significantly attenuated in KCNQ3 knockdown animals. CONCLUSIONS: The M-current plays a critical role in modulating the intrinsic excitability of NPY/AgRP neurons that is essential for maintaining energy homeostasis.

In Silico Predictions of KCNQ Variant Pathogenicity in Epilepsy.

BACKGROUND: Variants in KCNQ2 and KCNQ3 may cause benign neonatal familial seizures and early infantile epileptic encephalopathy. Previous reports suggest that in silico models cannot predict pathogenicity accurately enough for clinical use. Here we sought to establish a model to accurately predict the pathogenicity of KCNQ2 and KCNQ3 missense variants based on available in silico prediction models. METHODS: ClinVar and gnomAD databases of reported KCNQ2 and KCNQ3 missense variants in patients with neonatal epilepsy were accessed and classified as benign, pathogenic, or of uncertain significance. Sensitivity, specificity, and classification accuracy for prediction of pathogenicity were determined and compared for 10 widely used prediction algorithms program. A mathematical model of the variants (KCNQ Index) was created using their amino acid location and prediction algorithm scores to improve prediction accuracy. RESULTS: Using clinically characterized variants, the free online tool PROVEAN accurately predicted pathogenicity 92% of the time and the KCNQ Index had an accuracy of 96%. However, when including the gnomAD database as benign variants, only the KCNQ Index was able to predict pathogenicity with an accuracy greater than 90% (sensitivity = 93% and specificity = 98%). No model could accurately predict the phenotype of variants. CONCLUSION: We show that KCNQ channel variant pathogenicity can be predicted by a novel KCNQ Index in neonatal epilepsy. However, more work is needed to accurately predict the patient's epilepsy phenotype from in silico algorithms.