Clinical and genetic analysis of 23 Chinese children with epilepsy associated with KCNQ2 gene mutations.

OBJECTIVE: To summarize the clinical features and genetic mutation characteristics of Chinese children with KCNQ2-related epilepsy. METHODS: A cohort of children with genetically caused epilepsy was evaluated at Linyi People's Hospital from January 2017 to December 2023. After next-generation sequencing and pathogenicity analysis, we summarized the medical records and genetic testing data of the children who had KCNQ2 gene mutations. RESULTS: We identified 23 KCNQ2 gene mutations. 73.9% (n = 17) of the mutation sites were located in S5-S6 segments and the C-terminal region. In addition to the common phenotypes, 2 new phenotypes were identified: infantile convulsion with paroxysmal choreoathetosis (ICCA) and febrile seizure plus (FS+). Of all the cases with abnormal video-electro-encephalography, three cases with self-limited familial infantile epilepsy (SeLNE) exhibited a small number of multifocal discharges. Of the patients who have taken a particular antiepileptic drug, the statistics on the number of patients who have responded to the drug are as follows: oxcarbazepine (8/9, 88.9%), levetiracetam (5/7, 71.4%), phenobarbital (9/16, 56.3%), and topiramate (2/5, 40.0%). However, the efficacy of phenobarbital varied widely in treating SeLNE and KCNQ2-DEE. At the final follow-up, 1 case with SeLNE had a transient developmental regression and 7 cases with KCNQ2-DEE had mild to severe developmental backwardness. SIGNIFICANCE: Although clinically rare, we report 10 new KCNQ2 mutations and two new phenotypes: ICCA and FS+. This further expands genetic and phenotypic spectrum of KCNQ2-related epilepsy. The gene mutation sites are mostly located in S5-S6 segments and the C-terminal region, and the former is usually associated with KCNQ2-DEE. Sodium channel blockers (including oxcarbazepine and topiramate) and levetiracetam should be prioritized over phenobarbital for KCNQ2-DEE. Some cases with KCNQ2-related epilepsy may have transient developmental regression during periods of frequent seizures. Early treatment and early seizure control may be beneficial for willing outcomes in children with KCNQ2-DEE. PLAIN LANGUAGE SUMMARY: This article reports 23 cases of children with KCNQ2-related epilepsy, including 10 new mutation sites and 2 new phenotypes. It further expands the genetic and phenotypic spectrum of KCNQ2-related epilepsy. In addition, the article summarizes the gene mutation characteristics and clinical manifestations of children with KCNQ2-related epilepsy, with the expectation of providing a certain theoretical basis for the diagnosis and treatment of such patients.

GRT-X Stimulates Dorsal Root Ganglia Axonal Growth in Culture via TSPO and Kv7.2/3 Potassium Channel Activation.

GRT-X, which targets both the mitochondrial translocator protein (TSPO) and the Kv7.2/3 (KCNQ2/3) potassium channels, has been shown to efficiently promote recovery from cervical spine injury. In the present work, we investigate the role of GRT-X and its two targets in the axonal growth of dorsal root ganglion (DRG) neurons. Neurite outgrowth was quantified in DRG explant cultures prepared from wild-type C57BL6/J and TSPO-KO mice. TSPO was pharmacologically targeted with the agonist XBD173 and the Kv7 channels with the activator ICA-27243 and the inhibitor XE991. GRT-X efficiently stimulated DRG axonal growth at 4 and 8 days after its single administration. XBD173 also promoted axonal elongation, but only after 8 days and its repeated administration. In contrast, both ICA27243 and XE991 tended to decrease axonal elongation. In dissociated DRG neuron/Schwann cell co-cultures, GRT-X upregulated the expression of genes associated with axonal growth and myelination. In the TSPO-KO DRG cultures, the stimulatory effect of GRT-X on axonal growth was completely lost. However, GRT-X and XBD173 activated neuronal and Schwann cell gene expression after TSPO knockout, indicating the presence of additional targets warranting further investigation. These findings uncover a key role of the dual mode of action of GRT-X in the axonal elongation of DRG neurons.

Calcium-sensing receptor regulates Kv7 channels via Gi/o protein signalling and modulates excitability of human induced pluripotent stem cell-derived nociceptive-like neurons.

BACKGROUND AND PURPOSE: Neuropathic pain, a debilitating condition with unmet medical needs, can be characterised as hyperexcitability of nociceptive neurons caused by dysfunction of ion channels. Voltage-gated potassium channels type 7 (Kv7), responsible for maintaining neuronal resting membrane potential and thus excitability, reside under tight control of G protein-coupled receptors (GPCRs). Calcium-sensing receptor (CaSR) is a GPCR that regulates the activity of numerous ion channels, but whether CaSR can control Kv7 channel function has been unexplored until now. EXPERIMENTAL APPROACH: Experiments were conducted in recombinant cell models, mouse dorsal root ganglia (DRG) neurons and human induced pluripotent stem cell (hiPSC)-derived nociceptive-like neurons using patch-clamp electrophysiology and molecular biology techniques. KEY RESULTS: Our results demonstrate that CaSR is expressed in recombinant cell models, hiPSC-derived nociceptive-like neurons and mouse DRG neurons, and its activation induced depolarisation via Kv7.2/7.3 channel inhibition. The CaSR-Kv7.2/7.3 channel crosslink was mediated via the Gi/o protein-adenylate cyclase-cyclicAMP-protein kinase A signalling cascade. Suppression of CaSR function demonstrated a potential to rescue hiPSC-derived nociceptive-like neurons from algogenic cocktail-induced hyperexcitability. CONCLUSION AND IMPLICATIONS: This study demonstrates that the CaSR-Kv7.2/7.3 channel crosslink, via a Gi/o protein signalling pathway, effectively regulates neuronal excitability, providing a feasible pharmacological target for neuronal hyperexcitability management in neuropathic pain.

Ezogabine impacts seizures and development in patients with KCNQ2 developmental and epileptic encephalopathy.

Genetic variants in KCNQ2 are associated with a range of epilepsies, from self- limited (familial) neonatal-infantile epilepsy to developmental and epileptic encephalopathy (DEE). We retrospectively reviewed clinical data from eight patients with KCNQ2-related DEE who were treated with ezogabine. Treatment was initiated at a median age of 8 months (range, 7 weeks to 2.5 years) and continued for a median of 2.6 years (range, 7 months to 4.5 years). Five individuals had daily seizures at baseline and experienced at least 50% seizure reduction with treatment, sustained in four. One individual with two to four yearly seizures improved to rare events. Two individuals were seizure-free; treatment targeted cognition and development. Developmental improvements were reported in all eight patients. Weaning of ezogabine was associated with increased seizure frequency (N = 4), agitation and irritability (N = 2), poor sleep (N = 1), and developmental regression (N = 2). These data suggest that treatment with ezogabine is effective at reducing seizure burden and is associated with improved development. Minimal side effects were observed. Weaning was associated with increased seizures and behavioral disturbances in a subset. An approach targeting potassium channel dysfunction with ezogabine is warranted in patients with KCNQ2-related DEE.

A graphite furnace-atomic absorption spectrometry-based rubidium efflux assay for screening activators of the Kv 7.2/3 channel.

For the characterization of Kv 7.2/3 channel activators, several analytical methods are available that vary in effort and cost. In addition to the technically elaborate patch-clamp method, which serves as a reference method, there exist several medium to high-throughput screening methods including a rubidium efflux flame-atomic absorption spectrometry (F-AAS) assay and a commercial thallium uptake fluorescence-based assay. In this study, the general suitability of a graphite furnace atomic absorption spectrometry (GF-AAS)-based rubidium efflux assay as a screening method for Kv 7.2/3 channel activators was demonstrated. With flupirtine serving as a reference compound, 16 newly synthesizedcompounds and the known Kv 7.2/3 activator retigabine were first classified as either active or inactive by using the GF-AAS-based rubidium (Rb) efflux assay. Then, the results were compared with a thallium (Tl) uptake fluorescence-based fluorometric imaging plate reader (FLIPR) potassium assay. Overall, 16 of 17 compounds were classified by the GF-AAS-based assay in agreement with their channel-activating properties determined by the more expensive Tl uptake, fluorescence-based assay. Thus, the performance of the GF-AAS-based Rb assay for primary drug screening of Kv 7.2/3-activating compounds was clearly demonstrated, as documented by the calculated Z'-factor of the GF-AAS-based method. Moreover, method development included optimization of the coating of the microtiter plates and the washing procedure, which extended the range of this assay to poorly adherent cells such as the HEK293 cells used in this study.

Function of KCNQ2 channels at nodes of Ranvier of lumbar spinal ventral nerves of rats.

Previous immunohistochemical studies have shown the expression of KCNQ2 channels at nodes of Ranvier (NRs) of myelinated nerves. However, functions of these channels at NRs remain elusive. In the present study, we addressed this issue by directly applying whole-cell patch-clamp recordings at NRs of rat lumbar spinal ventral nerves in ex vivo preparations. We show that depolarizing voltages evoke large non-inactivating outward currents at NRs, which are partially inhibited by KCNQ channel blocker linopirdine and potentiated by KCNQ channel activator retigabine. Furthermore, linopirdine significantly alters intrinsic electrophysiological properties of NRs to depolarize resting membrane potential, increase input resistance, prolong AP width, reduce AP threshold, and decrease AP amplitude. On the other hand, retigabine significantly decreases input resistance and increases AP rheobase at NRs. Moreover, linopirdine increases excitability at NRs by converting single AP firing into multiple AP firing at many NRs. Saltatory conduction velocity is significantly reduced by retigabine, and AP success rate at high stimulation frequency is significantly increased by linopirdine. Collectively, KCNQ2 channels play a significant role in regulating intrinsic electrophysiological properties and saltatory conduction at NRs of motor nerve fibers of rats. These findings may provide insights into how the loss-of-function mutation in KCNQ2 channels can lead to neuromuscular disorders in human patients.

Distinctive mechanisms of epilepsy-causing mutants discovered by measuring S4 movement in KCNQ2 channels.

Neuronal KCNQ channels mediate the M-current, a key regulator of membrane excitability in the central and peripheral nervous systems. Mutations in KCNQ2 channels cause severe neurodevelopmental disorders, including epileptic encephalopathies. However, the impact that different mutations have on channel function remains poorly defined, largely because of our limited understanding of the voltage-sensing mechanisms that trigger channel gating. Here, we define the parameters of voltage sensor movements in wt-KCNQ2 and channels bearing epilepsy-associated mutations using cysteine accessibility and voltage clamp fluorometry (VCF). Cysteine modification reveals that a stretch of eight to nine amino acids in the S4 becomes exposed upon voltage sensing domain activation of KCNQ2 channels. VCF shows that the voltage dependence and the time course of S4 movement and channel opening/closing closely correlate. VCF reveals different mechanisms by which different epilepsy-associated mutations affect KCNQ2 channel voltage-dependent gating. This study provides insight into KCNQ2 channel function, which will aid in uncovering the mechanisms underlying channelopathies.

Functional Characterization of Two Variants at the Intron 6-Exon 7 Boundary of the KCNQ2 Potassium Channel Gene Causing Distinct Epileptic Phenotypes.

Pathogenic variants in KCNQ2 encoding for Kv7.2 potassium channel subunits have been found in patients affected by widely diverging epileptic phenotypes, ranging from Self-Limiting Familial Neonatal Epilepsy (SLFNE) to severe Developmental and Epileptic Encephalopathy (DEE). Thus, understanding the pathogenic molecular mechanisms of KCNQ2 variants and their correlation with clinical phenotypes has a relevant impact on the clinical management of these patients. In the present study, the genetic, biochemical, and functional effects prompted by two variants, each found in a non-familial SLNE or a DEE patient but both affecting nucleotides at the KCNQ2 intron 6-exon 7 boundary, have been investigated to test whether and how they affected the splicing process and to clarify whether such mechanism might play a pathogenetic role in these patients. Analysis of KCNQ2 mRNA splicing in patient-derived lymphoblasts revealed that the SLNE-causing intronic variant (c.928-1G > C) impeded the use of the natural splice site, but lead to a 10-aa Kv7.2 in frame deletion (Kv7.2 p.G310Δ10); by contrast, the DEE-causing exonic variant (c.928G > A) only had subtle effects on the splicing process at this site, thus leading to the synthesis of a full-length subunit carrying the G310S missense variant (Kv7.2 p.G310S). Patch-clamp recordings in transiently-transfected CHO cells and primary neurons revealed that both variants fully impeded Kv7.2 channel function, and exerted strong dominant-negative effects when co-expressed with Kv7.2 and/or Kv7.3 subunits. Notably, Kv7.2 p.G310S, but not Kv7.2 p.G310Δ10, currents were recovered upon overexpression of the PIP2-synthesizing enzyme PIP5K, and/or CaM; moreover, currents from heteromeric Kv7.2/Kv7.3 channels incorporating either Kv7.2 mutant subunits were differentially regulated by changes in PIP2 availability, with Kv7.2/Kv7.2 G310S/Kv7.3 currents showing a greater sensitivity to PIP2 depletion when compared to those from Kv7.2/Kv7.2 G310Δ10/Kv7.3 channels. Altogether, these results suggest that the two variants investigated differentially affected the splicing process at the intron 6-exon 7 boundary, and led to the synthesis of Kv7.2 subunits showing a differential sensitivity to PIP2 and CaM regulation; more studies are needed to clarify how such different functional properties contribute to the widely-divergent clinical phenotypes.

Clinical Study of 30 Novel KCNQ2 Variants/Deletions in KCNQ2-Related Disorders.

Background: KCNQ2-related disorder is typically characterized as neonatal onset seizure and epileptic encephalopathy. The relationship between its phenotype and genotype is still elusive. This study aims to provide clinical features, management, and prognosis of patients with novel candidate variants of the KCNQ2 gene. Methods: We enrolled patients with novel variants in the KCNQ2 gene from the China Neonatal Genomes Project between January 2018 and January 2021. All patients underwent next-generation sequencing tests and genetic data were analyzed by an in-house pipeline. The pathogenicity of variants was classified according to the guideline of the American College of Medical Genetics. Each case was evaluated by two geneticists back to back. Patients' information was acquired from clinical records. Results: A total of 30 unrelated patients with novel variants in the KCNQ2 gene were identified, including 19 patients with single-nucleotide variants (SNVs) and 11 patients with copy number variants (CNVs). For the 19 SNVs, 12 missense variants and 7 truncating variants were identified. Of them, 36.8% (7/19) of the KCNQ2 variants were located in C-terminal regions, 15.7% (3/19) in segment S2, and 15.7% (3/19) in segment S4. Among them, 18 of 19 patients experienced seizures in the early neonatal period. However, one patient presented neurodevelopmental delay (NDD) as initial phenotype when he was 2 months old, and he had severe NDD when he was 3 years old. This patient did not present seizure but had abnormal electrographic background activity and brain imaging. Moreover, for the 11 patients with CNVs, 20q13.3 deletions involving EEF1A2, KCNQ2, and CHRNA4 genes were detected. All of them presented neonatal-onset seizures, responded to antiepileptic drugs, and had normal neurological development. Conclusion: In this study, patients with novel KCNQ2 variants have variable phenotypes, whereas patients with 20q13.3 deletion involving EEF1A2, KCNQ2, and CHRNA4 genes tend to have normal neurological development.

Molecular Insights Into Binding and Activation of the Human KCNQ2 Channel by Retigabine.

Voltage-gated potassium channels of the Kv7.x family are involved in a plethora of biological processes across many tissues in animals, and their misfunctioning could lead to several pathologies ranging from diseases caused by neuronal hyperexcitability, such as epilepsy, or traumatic injuries and painful diabetic neuropathy to autoimmune disorders. Among the members of this family, the Kv7.2 channel can form hetero-tetramers together with Kv7.3, forming the so-called M-channels, which are primary regulators of intrinsic electrical properties of neurons and of their responsiveness to synaptic inputs. Here, prompted by the similarity between the M-current and that in Kv7.2 alone, we perform a computational-based characterization of this channel in its different conformational states and in complex with the modulator retigabine. After validation of the structural models of the channel by comparison with experimental data, we investigate the effect of retigabine binding on the two extreme states of Kv7.2 (resting-closed and activated-open). Our results suggest that binding, so far structurally characterized only in the intermediate activated-closed state, is possible also in the other two functional states. Moreover, we show that some effects of this binding, such as increased flexibility of voltage sensing domains and propensity of the pore for open conformations, are virtually independent on the conformational state of the protein. Overall, our results provide new structural and dynamic insights into the functioning and the modulation of Kv7.2 and related channels.

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.

KV7 channels are potential regulators of the exercise pressor reflex.

The exercise pressor reflex (EPR) originates in skeletal muscle and is activated by exercise-induced signals to increase arterial blood pressure and cardiac output. Muscle ischemia can elicit the EPR, which can be inappropriately activated in patients with peripheral vascular disease or heart failure to increase the incidence of myocardial infarction. We seek to better understand the receptor/channels that control excitability of group III and group IV muscle afferent fibers that give rise to the EPR. Bradykinin (BK) is released within contracting muscle and can evoke the EPR. However, the mechanism is incompletely understood. KV7 channels strongly regulate neuronal excitability and are inhibited by BK. We have identified KV7 currents in muscle afferent neurons by their characteristic activation/deactivation kinetics, enhancement by the KV7 activator retigabine, and block by KV7 specific inhibitor XE991. The blocking of KV7 current by different XE991 concentrations suggests that the KV7 current is generated by both KV7.2/7.3 (high affinity) and KV7.5 (low affinity) channels. The KV7 current was inhibited by 300 nM BK in neurons with diameters consistent with both group III and group IV afferents. The inhibition of KV7 by BK could be a mechanism by which this metabolic mediator generates the EPR. Furthermore, our results suggest that KV7 channel activators such as retigabine, could be used to reduce cardiac stress resulting from the exacerbated EPR in patients with cardiovascular disease.NEW & NOTEWORTHY KV7 channels control neuronal excitability. We show that these channels are expressed in muscle afferents and generate currents that are blocked by XE991 and bradykinin (BK). The XE991 block suggests that KV7 current is generated by KV7.2/3 and KV7.5 channels. The BK inhibition of KV7 channels may explain how BK activates the exercise pressor reflex (EPR). Retigabine can enhance KV7 current, which could help control the inappropriately activated EPR in patients with cardiovascular disease.

Peripheral Voltage-Gated Cation Channels in Neuropathic Pain and Their Potential as Therapeutic Targets.

The persistence of increased excitability and spontaneous activity in injured peripheral neurons is imperative for the development and persistence of many forms of neuropathic pain. This aberrant activity involves increased activity and/or expression of voltage-gated Na+ and Ca2+ channels and hyperpolarization activated cyclic nucleotide gated (HCN) channels as well as decreased function of K+ channels. Because they display limited central side effects, peripherally restricted Na+ and Ca2+ channel blockers and K+ channel activators offer potential therapeutic approaches to pain management. This review outlines the current status and future therapeutic promise of peripherally acting channel modulators. Selective blockers of Nav1.3, Nav1.7, Nav1.8, Cav3.2, and HCN2 and activators of Kv7.2 abrogate signs of neuropathic pain in animal models. Unfortunately, their performance in the clinic has been disappointing; some substances fail to meet therapeutic end points whereas others produce dose-limiting side effects. Despite this, peripheral voltage-gated cation channels retain their promise as therapeutic targets. The way forward may include (i) further structural refinement of K+ channel activators such as retigabine and ASP0819 to improve selectivity and limit toxicity; use or modification of Na+ channel blockers such as vixotrigine, PF-05089771, A803467, PF-01247324, VX-150 or arachnid toxins such as Tap1a; the use of Ca2+ channel blockers such as TTA-P2, TTA-A2, Z 944, ACT709478, and CNCB-2; (ii) improving methods for assessing "pain" as opposed to nociception in rodent models; (iii) recognizing sex differences in pain etiology; (iv) tailoring of therapeutic approaches to meet the symptoms and etiology of pain in individual patients via quantitative sensory testing and other personalized medicine approaches; (v) targeting genetic and biochemical mechanisms controlling channel expression using anti-NGF antibodies such as tanezumab or re-purposed drugs such as vorinostat, a histone methyltransferase inhibitor used in the management of T-cell lymphoma, or cercosporamide a MNK 1/2 inhibitor used in treatment of rheumatoid arthritis; (vi) combination therapy using drugs that are selective for different channel types or regulatory processes; (vii) directing preclinical validation work toward the use of human or human-derived tissue samples; and (viii) application of molecular biological approaches such as clustered regularly interspaced short palindromic repeats (CRISPR) technology.

The Role of Kv7.2 in Neurodevelopment: Insights and Gaps in Our Understanding.

Kv7.2 subunits encoded by the KCNQ2 gene constitute a critical molecular component of the M-current, a subthreshold voltage-gated potassium current controlling neuronal excitability by dampening repetitive action potential firing. Pathogenic loss-of-function variants in KCNQ2 have been linked to epilepsy since 1998, and there is ample functional evidence showing that dysfunction of the channel indeed results in neuronal hyperexcitability. The recent description of individuals with severe developmental delay with or without seizures due to pathogenic variants in KCNQ2 (KCNQ2-encephalopathy) reveals that Kv7.2 channels also have an important role in neurodevelopment. Kv7.2 channels are expressed already very early in the developing brain when key developmental processes such as proliferation, differentiation, and synaptogenesis play a crucial role in brain morphogenesis and maturation. In this review, we will discuss the available evidence for a role of Kv7.2 channels in these neurodevelopmental processes, focusing in particular on insights derived from KCNQ2-related human phenotypes, from the spatio-temporal expression of Kv7.2 and other Kv7 family member, and from cellular and rodent models, highlighting critical gaps and research strategies to be implemented in the future. Lastly, we propose a model which divides the M-current activity in three different developmental stages, correlating with the cell characteristics during these particular periods in neuronal development, and how this can be linked with KCNQ2-related disorders. Understanding these mechanisms can create opportunities for new targeted therapies for KCNQ2-encephalopathy.

Two KCNQ2 Encephalopathy Variants in the Calmodulin-Binding Helix A Exhibit Dominant-Negative Effects and Altered PIP2 Interaction.

Heterozygous missense variants in KCNQ2, which encodes the potassium channel subunit Kv7.2, are among the most common genetic causes of severe neonatal-onset epileptic encephalopathy. Because about 20% of known severe Kv7.2 missense changes lie within the intracellular C-terminal region, improving understanding of the underlying pathogenic mechanisms is important. We analyzed the basis for the severe phenotypes of Kv7.2 A337T and A337G, variants in the C-terminal's calmodulin (CaM)-binding Helix A. When expressed heterologously in mammalian cells, alone or in combination with wild type Kv7.2 or with wild type Kv7.2 and Kv7.3, both variants strongly suppressed channel currents. A337T channels expressed alone exhibited significantly reduced protein half-life and surface trafficking and co-immunoprecipitated less CaM. For both variants, increasing cellular phosphatidylinositol 4,5-bisphosphate (PIP2) by overexpression of PI(4)P5-kinase restored current densities. For both variants, the fraction of current suppressed by activation of M1 muscarinic receptors with 10 µM oxotremorine methiodide, which depletes PIP2, was less than for controls. During voltage-sensitive phosphatase-induced transient PIP2 depletion and resynthesize, potassium current inhibition and recovery kinetics were both markedly slowed. These results suggest that these variants may reduce currents by a mechanism not previously described: slowing of PIP2 migration between the bulk membrane and binding sites mediating channel electromechanical coupling. A novel Kv7.2/3-selective opener, SF0034, rescued current amplitudes. Our findings show that these two Helix A variants suppress channel current density strongly, consistent with their severe heterozygous phenotypes, implicate impairment of CaM and PIP2 regulation in KCNQ2 encephalopathy pathogenesis, and highlight the potential usefulness of selective Kv7 openers for this distinctive pathogenic mechanism and patient subgroup.

In Vivo Attenuation of M-Current Suppression Impairs Consolidation of Object Recognition Memory.

The M-current is a low voltage-activated potassium current generated by neuronal Kv7 channels. A prominent role of the M-current is to a create transient increase of neuronal excitability in response to neurotransmitters through the suppression of this current. Accordingly, M-current suppression is assumed to be involved in higher brain functions including learning and memory. However, there is little evidence supporting such a role to date. To address this gap, we examined behavioral tasks to assess learning and memory in homozygous Kv7.2 knock-in mice, Kv7.2(S559A), which show reduced M-current suppression while maintaining a normal basal M-current activity in neurons. We found that Kv7.2(S559A) mice had normal object location memory and contextual fear memory, but impaired long-term object recognition memory. Furthermore, short-term memory for object recognition was intact in Kv7.2(S559A) mice. The deficit in long-term object recognition memory was restored by the administration of a selective Kv7 channel inhibitor, XE991, when delivered during the memory consolidation phase. Lastly, c-Fos induction 2 h after training in Kv7.2(S559A) mice was normal in the hippocampus, which corresponds to intact object location memory, but was reduced in the perirhinal cortex, which corresponds to impaired long-term object recognition memory. Together, these results support the overall conclusion that M-current suppression is important for memory consolidation of specific types of memories.SIGNIFICANCE STATEMENT Dynamic regulation of neuronal excitation is a fundamental mechanism for information processing in the brain, which is mediated by changes in synaptic transmissions or by changes in ion channel activity. Some neurotransmitters can facilitate action potential firing by suppression of a low voltage-activated potassium current, M-current. We demonstrate that M-current suppression is critical for establishment of long-term object recognition memory, but is not required for establishment of hippocampus-dependent location memory or contextual memory. This study suggests that M-current suppression is important for stable encoding of specific types of memories.

RNA interference-mediated silencing of Kv7.2 in rat dorsal root ganglion neurons abolishes the anti-nociceptive effect of a selective channel opener.

INTRODUCTION: Development of agonistic analgesic drugs requires proof of selectivity in vivo attainable by selective antagonists or several knockdown strategies. The Kv7.2 potassium channel encoded by the KCNQ2 gene regulates neuronal excitability and its activation inhibits nociceptive transmission. Although it is a potentially attractive target for analgesics, no clinically approved Kv7.2 agonists are currently available and selectivity of drug candidates is hard to demonstrate in vivo due to the expenditure to generate KCNQ2 knockout animals and the lack of Kv7.2 selective antagonists. The present study describes the set-up of an RNA interference-based model that allows studying the selectivity of Kv7.2 openers. METHODS: Adeno-associated virus (AAV) vectors were used to deliver the expression cassette for a short hairpin RNA targeting KCNQ2. Heat nociception was tested in rats after intrathecal AAV treatment. RESULTS: Surprisingly, screening of AAV serotypes revealed serotype 7, which has rarely been explored, to be best suited for transduction of dorsal root ganglia neurons following intrathecal injection. Knockdown of the target gene was confirmed by qRT-PCR and the anti-nociceptive effect of a Kv7.2 agonist was found to be completely abolished by the treatment. DISCUSSION: We consider this approach not only to be suitable to study the selectivity of novel analgesic drugs targeting Kv7.2, but rather to serve as a general fast and simple method to generate functional and phenotypic knockdown animals during drug discovery for central and peripheral pain targets.

Ethanol Elevates Excitability of Superior Cervical Ganglion Neurons by Inhibiting Kv7 Channels in a Cell Type-Specific and PI(4,5)P2-Dependent Manner.

Alcohol causes diverse acute and chronic symptoms that often lead to critical health problems. Exposure to ethanol alters the activities of sympathetic neurons that control the muscles, eyes, and blood vessels in the brain. Although recent studies have revealed the cellular targets of ethanol, such as ion channels, the molecular mechanism by which alcohol modulates the excitability of sympathetic neurons has not been determined. Here, we demonstrated that ethanol increased the discharge of membrane potentials in sympathetic neurons by inhibiting the M-type or Kv7 channel consisting of the Kv7.2/7.3 subunits, which were involved in determining the membrane potential and excitability of neurons. Three types of sympathetic neurons, classified by their threshold of activation and firing patterns, displayed distinct sensitivities to ethanol, which were negatively correlated with the size of the Kv7 current that differs depending on the type of neuron. Using a heterologous expression system, we further revealed that the inhibitory effects of ethanol on Kv7.2/7.3 currents were facilitated or diminished by adjusting the amount of plasma membrane phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). These results suggested that ethanol and PI(4,5)P2 modulated gating of the Kv7 channel in superior cervical ganglion neurons in an antagonistic manner, leading to regulation of the membrane potential and neuronal excitability, as well as the physiological functions mediated by sympathetic neurons.