KCNQ Channels Research Articles

Dopamine drives neuronal excitability via KCNQ channel phosphorylation for reward behavior.

Dysfunctional dopamine signaling is implicated in various neuropsychological disorders. Previously, we reported that dopamine increases D1 receptor (D1R)-expressing medium spiny neuron (MSN) excitability and firing rates in the nucleus accumbens (NAc) via the PKA/Rap1/ERK pathway to promote reward behavior. Here, the results show that the D1R agonist, SKF81297, inhibits KCNQ-mediated currents and increases D1R-MSN firing rates in murine NAc slices, which is abolished by ERK inhibition. Invitro ERK phosphorylates KCNQ2 at Ser414 and Ser476; invivo, KCNQ2 is phosphorylated downstream of dopamine signaling in NAc slices. Conditional deletion of Kcnq2 in D1R-MSNs reduces the inhibitory effect of SKF81297 on KCNQ channel activity, while enhancing neuronal excitability and cocaine-induced reward behavior. These effects are restored by wild-type, but not phospho-deficient KCNQ2. Hence, D1R-ERK signaling controls MSN excitability via KCNQ2 phosphorylation to regulate reward behavior, making KCNQ2 a potential therapeutical target for psychiatric diseases with a dysfunctional reward circuit.

CENTIPEDE VENOM TOXINS AND ITS BIOMEDICAL AND PHARMACOLOGICAL PROPERTIES

The present review article explains venoms from various centipede species with their biomedical and pharmacological properties. Centipede venom is a natural source of bioactive proteins, peptides and other small molecules. These use venoms as defense arsenals to paralyze prey. This review paper sketch out important physiological effects like platelet aggregation, anticoagulant, phospholipase A2 and trypsin inhibiting activity. Centipede venom toxins selectively bind Kv2.1 channel and block them. Centipede venom disrupts cardiovascular, respiratory, muscular and nervous systems by targeting the broadly distributed KCNQ channels. It also signifies toxin-voltage-gated integrations and its inhibition. These peptides can be used for developing drugs for treatments as well as bio-insecticides for insect control.

Clinically Relevant KCNQ1 Variants Causing KCNQ1-KCNE2 Gain-of-Function Affect the Ca2+ Sensitivity of the Channel.

Dominant KCNQ1 variants are well-known for underlying cardiac arrhythmia syndromes. The two heterozygous KCNQ1 missense variants, R116L and P369L, cause an allelic disorder characterized by pituitary hormone deficiency and maternally inherited gingival fibromatosis. Increased K+ conductance upon co-expression of KCNQ1 mutant channels with the beta subunit KCNE2 is suggested to underlie the phenotype; however, the reason for KCNQ1-KCNE2 (Q1E2) channel gain-of-function is unknown. We aimed to discover the genetic defect in a single individual and three family members with gingival overgrowth and identified the KCNQ1 variants P369L and V185M, respectively. Patch-clamp experiments demonstrated increased constitutive K+ conductance of V185M-Q1E2 channels, confirming the pathogenicity of the novel variant. To gain insight into the pathomechanism, we examined all three disease-causing KCNQ1 mutants. Manipulation of the intracellular Ca2+ concentration prior to and during whole-cell recordings identified an impaired Ca2+ sensitivity of the mutant KCNQ1 channels. With low Ca2+, wild-type KCNQ1 currents were efficiently reduced and exhibited a pre-pulse-dependent cross-over of current traces and a high-voltage-activated component. These features were absent in mutant KCNQ1 channels and in wild-type channels co-expressed with calmodulin and exposed to high intracellular Ca2+. Moreover, co-expression of calmodulin with wild-type Q1E2 channels and loading the cells with high Ca2+ drastically increased Q1E2 current amplitudes, suggesting that KCNE2 normally limits the resting Q1E2 conductance by an increased demand for calcified calmodulin to achieve effective channel opening. Our data link impaired Ca2+ sensitivity of the KCNQ1 mutants R116L, V185M and P369L to Q1E2 gain-of-function that is associated with a particular KCNQ1 channelopathy.

Chronic Cerebral Hypoperfusion-induced Dysregulations of Hyperpolarization- activated Cyclic Nucleotide-gated, KCNQ and G Protein-coupled Inwardly Rectifying Potassium Channels Correlated with Susceptibility and Unsusceptibility to Anxiety Behaviors.

Cerebrovascular lesions could induce affective disorders; however, the depression- and anxiety-related symptoms caused by Chronic Cerebral Hypoperfusion (CCH) and the roles of different Hyperpolarization-activated Cyclic Nucleotide-gated (HCN), KCNQ and G protein-coupled inwardly rectifying potassium (GirK) channel subunits in these pathological processes have been poorly elucidated so far. To investigate the behavioral change and the alteration of HCN, KCNQ, and GirK subunits in amygdale rats suffering from CCH. Permanent bilateral occlusion of the common carotid arteries was used to induce CCH. Anxiety and depression levels were assessed by the elevated plus maze test, sucrose preference test and forced swimming test to classify rats as highly anxious or depressive 'susceptibility' vs. 'unsusceptibility'. The expression of brain-derived neurotrophic factor (BDNF), tyrosine kinase receptor B (TrKB), HCN1/2, KCNQ2/3, and GirK1/2/3 were quantified by Western blotting. The main emotional change caused by 4 weeks of CCH is likely to be anxiety-like behavior (50%), accompanied by a down-regulation of BDNF and TrKB expression in amygdale. The increase of HCN1 and decrease of KCNQ3 expression in amygdale may be factors to blame for anxiety- like symptom caused by CCH, and the increase of KCNQ2 and Girk1 expression in amygdale may play a role in resilience to the anxiety induced by CCH. The different subunits of HCN, KCNQ and GirK channels in amygdale may contribute to distinct response to aversive stimuli or stress induced by CCH that evokes divergent influences on anxiety-like behavior in rats.

Two small-molecule activators share similar effector sites in the KCNQ1 channel pore but have distinct effects on voltage sensor movements.

ML277 and R-L3 are two small-molecule activators of KCNQ1, the pore-forming subunit of the slowly activating potassium channel IKs. KCNQ1 loss-of-function mutations prolong cardiac action potential duration and are associated with long QT syndrome, which predispose patients to lethal ventricular arrhythmia. ML277 and R-L3 enhance KCNQ1 current amplitude and slow deactivation. However, the presence of KCNE1, an auxiliary subunit of IKs channels, renders the channel insensitive to both activators. We found that ML277 effects are dependent on several residues in the KCNQ1 pore domain. Some of these residues are also necessary for R-L3 effects. These residues form a putative hydrophobic pocket located between two adjacent KCNQ1 subunits, where KCNE1 subunits are thought to dwell, thus providing an explanation for how KCNE1 renders the IKs channel insensitive to these activators. Our experiments showed that the effect of R-L3 on voltage sensor movement during channel deactivation was much more prominent than that of ML277. Simulations using a KCNQ1 kinetic model showed that the effects of ML277 and R-L3 could be reproduced through two different effects on channel gating: ML277 enhances KCNQ1 channel function through a pore-dependent and voltage sensor–independent mechanism, while R-L3 affects both channel pore and voltage sensor.

Electro-mechanical coupling of KCNQ channels is a target of epilepsy-associated mutations and retigabine.

KCNQ2 and KCNQ3 form the M-channels that are important in regulating neuronal excitability. Inherited mutations that alter voltage-dependent gating of M-channels are associated with neonatal epilepsy. In the homolog KCNQ1 channel, two steps of voltage sensor activation lead to two functionally distinct open states, the intermediate-open (IO) and activated-open (AO), which define the gating, physiological, and pharmacological properties of KCNQ1. However, whether the M-channel shares the same mechanism is unclear. Here, we show that KCNQ2 and KCNQ3 feature only a single conductive AO state but with a conserved mechanism for the electro-mechanical (E-M) coupling between voltage sensor activation and pore opening. We identified some epilepsy-linked mutations in KCNQ2 and KCNQ3 that disrupt E-M coupling. The antiepileptic drug retigabine rescued KCNQ3 currents that were abolished by a mutation disrupting E-M coupling, suggesting that modulating the E-M coupling in KCNQ channels presents a potential strategy for antiepileptic therapy.

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.

Venom resistance mechanisms in centipede show tissue specificity

Venomous animals utilize venom glands to secrete and store powerful toxins for intraspecific and/or interspecific antagonistic interactions, implying that tissue-specific resistance is essential for venom glands to anatomically separate toxins from other tissues. Here, we show the mechanism of tissue-specific resistance in centipedes (Scolopendra subspinipes mutilans), where the splice variant of the receptor repels its own toxin. Unlike the well-known resistance mechanism by mutation in a given exon, we found that the KCNQ1 channel is highly expressed in the venom gland as a unique splice variant in which the pore domain and transmembrane domain six, partially encoded by exon 6 (rather than 7 as found in other tissues), contain eleven mutated residues. Such a splice variant is sufficient to gain resistance to SsTx (a lethal toxin for giant preycapture) in the venom gland due to a partially buried binding site. Therefore, the tissue-specific KCNQ1 modification confers resistance to the toxins, establishing a safe zone in the venom-storing/secreting environment.

Structural and electrophysiological basis for the modulation of KCNQ1 channel currents by ML277

The KCNQ1 ion channel plays critical physiological roles in electrical excitability and K+ recycling in organs including the heart, brain, and gut. Loss of function is relatively common and can cause sudden arrhythmic death, sudden infant death, epilepsy and deafness. Here, we report cryogenic electron microscopic (cryo-EM) structures of Xenopus KCNQ1 bound to Ca2+/Calmodulin, with and without the KCNQ1 channel activator, ML277. A single binding site for ML277 was identified, localized to a pocket lined by the S4-S5 linker, S5 and S6 helices of two separate subunits. Several pocket residues are not conserved in other KCNQ isoforms, explaining specificity. MD simulations and point mutations support this binding location for ML277 in open and closed channels and reveal that prevention of inactivation is an important component of the activator effect. Our work provides direction for therapeutic intervention targeting KCNQ1 loss of function pathologies including long QT interval syndrome and seizures.

A disease-associated mutation in thyroid hormone receptor α1 causes hearing loss and sensory hair cell patterning defects in mice.

Resistance to thyroid hormone due to mutations in THRA, which encodes the thyroid hormone receptor α (TRα1), shows variable clinical presentation. Mutations affecting TRβ1 and TRβ2 cause deafness in mice and have been associated with deafness in humans. To test whether TRα1 also affects hearing function, we used mice heterozygous for a frameshift mutation in Thra that is similar to human THRA mutations (ThraS1/+ mice) and reduces tissue sensitivity to thyroid hormone. Compared to wild-type littermates, ThraS1/+ mice showed moderate high-frequency sensorineural hearing loss as juveniles and increased age-related hearing loss. Ultrastructural examination revealed aberrant orientation of ~20% of sensory outer hair cells (OHCs), as well as increased numbers of mitochondria with fragmented morphology and autophagic vacuoles in both OHCs and auditory nerve fibers. Molecular dissection of the OHC lateral wall components revealed that the potassium ion channel Kcnq4 was aberrantly targeted to the cytoplasm of mutant OHCs. In addition, mutant cochleae showed increased oxidative stress, autophagy, and mitophagy associated with greater age-related cochlear cell damage, demonstrating that TRα1 is required for proper development of OHCs and for maintenance of OHC function. These findings suggest that patients with THRA mutations may present underdiagnosed, mild hearing loss and may be more susceptible to age-related hearing loss.

The KCNQ channel inhibitor XE991 suppresses nicotinic acetylcholine receptor-mediated responses in rat intracardiac ganglion neurons.

XE991 (10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone) is reportedly a potent and selective Kv7 (KCNQ) channel inhibitor. This study aimed to evaluate how XE991 affects nicotinic responses in intracardiac ganglion neurons. We studied how the KCNQ channel inhibitor XE991 could affect nicotinic responses in acutely isolated rat intracardiac ganglion neurons using a perforated patch-clamp recording configuration and Ca2+ imaging. XE991 reversibly and concentration-dependently inhibited the nicotine (10μM)-induced current with an IC50 of 14.4μM. The EC50 values for nicotine-induced currents in the absence and presence of 10μM XE991 were 8.7 and 12.0μM, respectively. Because XE991 suppressed the maximum response of the nicotine concentration-response curve, the inhibitory effect of this drug appears to be noncompetitive. In addition, linopirdine reduced the amplitude of 10µM nicotine-induced currents with an IC50 value of 16.9μM. The inorganic KCNQ channel inhibitor Ba2+ affected neither the nicotine-induced current nor the inhibitory effect of XE991 on the nicotinic response. The KCNQ activator flupirtine at a concentration of 10μM slightly but markedly inhibited the nicotine-induced current. Finally, XE991 inhibited the nicotine-induced elevation of intracellular calcium concentration and the nicotine-induced firing of action potentials. We propose that XE991 inhibits nicotinic acetylcholine receptors in intracardiac ganglion neurons, which in turn attenuate nicotine-induced neuronal excitation.

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.

Diversity-oriented synthesis of marine polybrominated diphenyl ethers as potential KCNQ potassium channel activators

Natural polybrominated diphenyl ethers, often isolated from marine sponges, have been reported to possess various biological activities, such as antibacterial, antioxidant and antidiabetic effects. Via a high throughput screening of our marine natural product library, the polybrominated diphenyl ether 3 was found to display a KCNQ potassium channel activation effect. To obtain more compound 3 related natural products and their derivatives for further bioactivity study, a diversity-oriented synthesis was conducted, leading to the successful synthesis of five polybrominated diphenyl ether natural products (1–4, 6) and 30 new derivatives. Compound 3 was found to preferentially potentiate KCNQ1 potassium channel, whereas 17h relatively activated KCNQ2 potassium channel. The structure–activity relationship was analyzed assisted by molecular docking and 17h was further conducted for its agonistic mechanism study on KCNQ2 channel. This research work may give an insight for the discovery of marine polybrominated diphenyl ether derived new drug leads.

Human KCNQ5 de novo mutations underlie epilepsy and intellectual disability.

We identified six novel de novo human KCNQ5 variants in children with motor/language delay, intellectual disability (ID), and/or epilepsy by whole exome sequencing. These variants, comprising two nonsense and four missense alterations, were functionally characterized by electrophysiology in HEK293/CHO cells, together with four previously reported KCNQ5 missense variants (Lehman A, Thouta S, Mancini GM, Naidu S, van Slegtenhorst M, McWalter K, Person R, Mwenifumbo J, Salvarinova R; CAUSES Study; EPGEN Study; Guella I, McKenzie MB, Datta A, Connolly MB, Kalkhoran SM, Poburko D, Friedman JM, Farrer MJ, Demos M, Desai S, Claydon T. Am J Hum Genet 101: 65-74, 2017). Surprisingly, all eight missense variants resulted in gain of function (GOF) due to hyperpolarized voltage dependence of activation or slowed deactivation kinetics, whereas the two nonsense variants were confirmed to be loss of function (LOF). One severe GOF allele (P369T) was tested and found to extend a dominant GOF effect to heteromeric KCNQ5/3 channels. Clinical presentations were associated with altered KCNQ5 channel gating: milder presentations with LOF or smaller GOF shifts in voltage dependence [change in voltage at half-maximal conduction (ΔV50) = ∼-15 mV] and severe presentations with larger GOF shifts in voltage dependence (ΔV50 = ∼-30 mV). To examine LOF pathogenicity, two Kcnq5 LOF mouse lines were created with CRISPR/Cas9. Both lines exhibited handling- and thermal-induced seizures and abnormal cortical EEGs consistent with epileptiform activity. Our study thus provides evidence for in vivo KCNQ5 LOF pathogenicity and strengthens the contribution of both LOF and GOF mutations to global pediatric neurological impairment, including ID/epilepsy.NEW & NOTEWORTHY Six novel de novo human KCNQ5 variants were identified from children with neurodevelopmental delay, intellectual disability, and/or epilepsy. Expression of these variants along with four previously reported KCNQ5 variants from a similar cohort revealed GOF potassium channels, negatively shifted in V50 of activation and/or delayed deactivation kinetics. GOF is extended to KCNQ5/3 heteromeric channels, making these the predominant channels affected in heterozygous de novo patients. Kcnq5 LOF mice exhibited seizures, consistent with in vivo pathogenicity.

P166 “LONG QT SYNDROME:AN ENTIRE FAMILY WITH KCNQ1 MUTATION”

Abstract Introduction Congenital long QT syndrome (LQTS) is characterized by prolongation of the QT interval that can trigger life–threatening arrhythmias leading to syncope and sudden death. Mutations in genes encoding for ion channels have been identified in 75% of LQTS cases. Long QT syndrome type 1 (LQTS1) is one of three subtypes of LQTS identified based on genetic substrate. LQTS1 is linked to a “loss–of–function” mutation in the gene encoding the potassium channel KCNQ1, and arrhythmias tend to occur during childhood with prevalence in the male sex. Adrenergic triggers are the most important trigger for arrhythmic events. Clinical Case Extensive family with long QT syndrome with positive genetics for mutation in the KCNQ1 gene. The index case in the family appears to be a woman who at the age of approximately 30 years died suddenly after surgery. The first patient subjected to genetic analysis, with positive result, was the daughter of this woman who, at the age of about 25 years has experienced a ventricular arrhythmia during induction of anesthesia for surgery with baseline ECG finding of prolonged QTc. Set beta–blocker therapy, arrhythmias no longer occurred. Subsequently, a cardiological screening including electrocardiogram, transthoracic echocardiography and genetic analysis was undertaken in first–degree relatives and, in cases of positivity, this screening was progressively extended to second–, third– and fourth–degree relatives. All the women in the family analyzed, eight in total, including two girls aged 5 and 12 years, tested positive for genetic analysis, and one of the women had a syncopal episode at the age of 50 years. Of the men analyzed, four in total, only two (father and son) tested positive on genetic analysis. In all patients with positive genetics a QTc >480 msec was detected on ECG in clinostatism and/or orthostatism, for which a diagnosis of LQTS1 (mean Schwartz–score 4) was made and beta–blocker therapy was started. Conclusions This clinical case describes an entire family with LQTS with positive genetics for KCNQ1 with phenotypic manifestations different from those described in the literature, in particular with prevalence in the female sex and with onset of arrhythmias in adulthood.

Neuropathology findings in KCNQ2 neonatal epileptic encephalopathy.

PurposeKCNQ2-epileptic encephalopathy (EE) is a neonatal epilepsy syndrome characterized by a typical clinical presentation and EEG recording, but without any brain or cortical abnormal development on MRI. Most of the patients have a severe developmental impairment. The epileptogenic mechanisms are thought to be the result of the changes of the M-current density causing a change of brain excitability. Although recent studies allow us to better understand the physiopathology of KCNQ2-EE, the neuropathology of this ion channel dysfunction has only been previously described in a single case report. MethodsWe report the neuropathology study of a case of KCNQ2-EE with a typical electro-phenotype due to a de novo heterozygous single nucleotide pathogenic variant in the exon 5 of the KCNQ2 gene (NM_172107.2:c.802C>T; p.Leu268Phe). ResultsAt the macroscopic level, the brain had a normal structure with a normal neocortical gyral pattern. At the histological level, the cortex had a usual six-layer lamination in all lobes but blurred gray-white matter boundaries due to excessive heterotopic neurons in deep white matter were observed. This diffuse mild malformation of cortical development is suggestive of a neuronal migration disorder. ConclusionIn recent years, our understanding of the role of ion channel dysfunctions in early brain development has expanded from the occurrence of EE to brain malformation. Through this rare neuropathological report, we emphasize the role of KCNQ2 channels in the process of cortical development. As for other genetic neonatal onset epilepsies, more reports are needed to further delineate the range of neuropathological abnormalities for KCNQ2-EE.

Behavior of KCNQ Channels in Neural Plasticity and Motor Disorders.

The broad distribution of voltage-gated potassium channels (VGKCs) in the human body makes them a critical component for the study of physiological and pathological function. Within the KCNQ family of VGKCs, these aqueous conduits serve an array of critical roles in homeostasis, especially in neural tissue. Moreover, the greater emphasis on genomic identification in the past century has led to a growth in literature on the role of the ion channels in pathological disease as well. Despite this, there is a need to consolidate the updated findings regarding both the pharmacotherapeutic and pathological roles of KCNQ channels, especially regarding neural plasticity and motor disorders which have the largest body of literature on this channel. Specifically, KCNQ channels serve a remarkable role in modulating the synaptic efficiency required to create appropriate plasticity in the brain. This role can serve as a foundation for clinical approaches to chronic pain. Additionally, KCNQ channels in motor disorders have been utilized as a direction for contemporary pharmacotherapeutic developments due to the muscarinic properties of this channel. The aim of this study is to provide a contemporary review of the behavior of these channels in neural plasticity and motor disorders. Upon review, the behavior of these channels is largely dependent on the physiological role that KCNQ modulatory factors (i.e., pharmacotherapeutic options) serve in pathological diseases.

Cellular and Molecular Mechanisms Regulating the Normal Physiological Function of the Epithelial Sodium Channel

High blood pressure is a significant risk factor for cardiovascular disease. Many details about the molecular mechanisms that regulate blood pressure are unknown. The Epithelial Na+ Channel (ENaC) is the final arbiter of Na+ excretion in the kidneys (see attached Figure). Appropriate renal sodium excretion is a key factor in the regulation of blood pressure. Abnormalities in ENaC function have been directly linked to several human diseases of blood pressure. The kinase, casein kinase II (CKII), phosphorylates ENaC but the physiological importance of this is obscure. The CKII phosphorylation site within ENaC resides within a canonical “anchor” ankyrin binding motif. Phosphorylation of Nav and KCNQ channels by CKII acts as a molecular “switch” favoring the binding of ankyrin‐3 (Ank‐3). The binding of Ank‐3 facilitates the proper membrane localization of these channels increasing their activity. Here, we tested the premise that phosphorylation of ENaC by CKII within “anchor” motifs is necessary and sufficient for Ank‐3 binding to the channel, which is required for normal channel locale and function, and the proper regulation of renal Na+ excretion and blood pressure (see attached Figure). We tested this hypothesis using endpoint measurements including analyses of ENaC activity and trafficking to the plasma membrane, and quantification of CKII‐dependent ENaC‐Ank3 binding. This was addressed by combining total internal reflection fluorescence microscopy with fluorescence recovery after photo bleaching (TIRF/FRAP) to follow movement of ENaC toward the plasma membrane in living cells and electrophysiology of ENaC activity in split‐open collecting ducts from principal cell‐specific CKII and Ank3 knockout mice. We also used whole animal physiological studies of sodium excretion and blood pressure in knockout mice to understand the physiological consequences of CKII and Ank3 regulation of ENaC. In addition, scanning mutagenesis was used to identify key residues in the overlapping CKII and Ank3 sites in ENaC. Furthermore, we tested whether CKII influences Ank‐3 binding to β‐ENaC subunit using fluorescence resonance energy transfer (FRET). Findings showed that disrupting CKII signaling and abrogation of the CKII phosphorylation and Ank3 binding site within ENaC decreases channel trafficking toward the plasma membrane and activity, promotes improper sodium excretion, and decreases blood pressure in KO mice. These results showed how and when CKII phosphorylation of ENaC functions as a “switch” to favor Ank‐3 binding to increase channel activity to include having a detailed understanding of the mechanisms mediating this regulation, and a rich appreciation of the physiological consequences of such regulation.

PO-629-02 NEW EVIDENCE TO CHALLENGE CLINGEN'S “DISPUTED EVIDENCE” DESIGNATION FOR AKAP9 AS A BONA FIDE SUSCEPTIBILITY GENE FOR CONGENITAL LONG QT SYNDROME

A-kinase-anchoring protein-9 (AKAP9) mediates the phosphorylation state of the KCNQ1-encoded Kv7.1 potassium channel. Loss-of-function (LOF) variants in KCNQ1 cause type 1 long QT syndrome (LQT1). In 2007, we published data suggesting AKAP9-S1570L as a novel LQTS-causative variant and AKAP9 as a novel LQTS-susceptibility gene. However, in a recent re-appraisal of LQTS genes, the Clinical Genome Resource (ClinGen) demoted AKAP9 to a “disputed evidence” gene due to apparent lack of sufficient evidence to support causation of LQTS. To characterize the electrophysiology of a LQTS patient-derived induced pluripotent stem cell-cardiomyocyte (iPSC-CM) AKAP9-S1570L model and its corresponding CRISPR/Cas9 gene-edited/variant-corrected isogenic control (IC). iPSC-CMs were generated from an AKAP9-S1570L positive female diagnosed with LQTS at 14 years of age with a QTc > 480 ms, history of syncope, a LQT1-suggestive treadmill stress test, and a positive family history of QT-prolongation. Standard whole-cell patch clamp technique was performed to measure the action potential duration (APD) of both the AKAP9-S1570L and IC iPSC-CMs. To determine if KCNQ1 contributes to the APD prolongation of the variant iPSC-CMs, the KCNQ1 channel specific activator, ML277 was used in additional APD measurements. APD50 (457 ± 21) and APD90 (549 ± 22 ms) were significantly prolonged in the AKAP9-S1570L iPSC-CMs compared to the IC iPSC-CMs (APD50, 335 ± 15 ms; APD90, 413 ± 16 ms; p<0.05). Addition of ML277 resulted in a greater degree of APD shortening in AKAP9-S1570L iPSC-CMs (APD50 shortening, 349 ± 40 ms (p<0.05); APD90 shortening, 397 ± 42 ms (p<0.05) than in IC iPSC-CMs (APD50 shortening, 219 ± 31 ms; APD90 shortening, 242 ± 33 ms). This indicates KCNQ1 LOF may contribute to the mechanism underlying APD prolongation in the patient-specific AKAP9-S1570L iPSC-CMs, consistent with our previously published data. Using patient specific iPSC-CMs and isogenic controls, we provide new evidence bolstering AKAP9-S1570L as the pathogenic LQTS-causative variant responsible for this patient’s LQTS. Whether ClinGen deems this sufficient evidence to rescue AKAP9 from genetic purgatory and re-classify it as a sufficient evidence LQTS-susceptibility gene, remains to be seen.

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.