Calcium-Dependent Regulation of Neuronal Excitability Is Rescued in Fragile X Syndrome by a Tat-Conjugated N-Terminal Fragment of FMRP.

Fragile X syndrome (FXS) arises from the loss of fragile X messenger ribonucleoprotein (FMRP) needed for normal neuronal excitability and circuit functions. Recent work revealed that FMRP contributes to mossy fiber long-term potentiation by adjusting the Kv4 A-type current availability through interactions with a Cav3-Kv4 ion channel complex, yet the mechanism has not yet been defined. In this study using wild-type and Fmr1 knock-out (KO) tsA-201 cells and cerebellar sections from male Fmr1 KO mice, we show that FMRP associates with all subunits of the Cav3.1-Kv4.3-KChIP3 complex and is critical to enabling calcium-dependent shifts in Kv4.3 inactivation to modulate the A-type current. Specifically, upon depolarization Cav3 calcium influx activates dual-specific phosphatase 1/6 (DUSP1/6) to deactivate ERK1/2 (ERK) and lower phosphorylation of Kv4.3, a signaling pathway that does not function in Fmr1 KO cells. In Fmr1 KO mouse tissue slices, cerebellar granule cells exhibit a hyperexcitable response to membrane depolarizations. Either incubating Fmr1 KO cells or in vivo administration of a tat-conjugated FMRP N-terminus fragment (FMRP-N-tat) rescued Cav3-Kv4 function and granule cell excitability, with a decrease in the level of DUSP6. Together these data reveal a Cav3-activated DUSP signaling pathway critical to the function of a FMRP-Cav3-Kv4 complex that is misregulated in Fmr1 KO conditions. Moreover, FMRP-N-tat restores function of this complex to rescue calcium-dependent control of neuronal excitability as a potential therapeutic approach to alleviating the symptoms of FXS.

E3 ubiquitin ligase rififylin has yin and yang effects on rabbit cardiac transient outward potassium currents (Ito) and corresponding channel proteins.

Genome-wide association studies have reported a correlation between a SNP of the RING finger E3 ubiquitin protein ligase rififylin (RFFL) and QT interval variability in humans (Newton-Cheh et al., 2009). Previously, we have shown that RFFL downregulates expression and function of the human-like ether-a-go-go-related gene potassium channel and corresponding rapidly activating delayed rectifier potassium current (IKr) in adult rabbit ventricular cardiomyocytes. Here, we report that RFFL also affects the transient outward current (Ito), but in a peculiar way. RFFL overexpression in adult rabbit ventricular cardiomyocytes significantly decreases the contribution of its fast component (Ito,f) from 35% to 21% and increases the contribution of its slow component (Ito,s) from 65% to 79%. Since Ito,f in rabbits is mainly conducted by Kv4.3, we investigated the effect of RFFL on Kv4.3 expressed in HEK293A cells. We found that RFFL overexpression reduced Kv4.3 expression and corresponding Ito,f in a RING domain-dependent manner in the presence or absence of its accessory subunit Kv channel-interacting protein 2. On the other hand, RFFL overexpression in Kv1.4-expressing HEK cells leads to an increase in both Kv1.4 expression level and Ito,s, similarly in a RING domain-dependent manner. Our physiologically detailed rabbit ventricular myocyte computational model shows that these yin and yang effects of RFFL overexpression on Ito,f, and Ito,s affect phase 1 of the action potential waveform and slightly decrease its duration in addition to suppressing IKr. Thus, RFFL modifies cardiac repolarization reserve via ubiquitination of multiple proteins that differently affect various potassium channels and cardiac action potential duration.

Design of Ultrapotent Genetically Encoded Inhibitors of Kv4.2 for Gating Neural Plasticity.

The Kv4.2 potassium channel plays established roles in neuronal excitability, while also being implicated in plasticity. Current means to study the roles of Kv4.2 are limited, motivating us to design a genetically encoded membrane tethered Heteropodatoxin-2 (MetaPoda). We find that MetaPoda is an ultrapotent and selective gating-modifier of Kv4.2. We narrow its site of contact with the channel to two adjacent residues within the voltage sensitive domain (VSD) and, with docking simulations, suggest that the toxin binds the VSD from within the membrane. We also show that MetaPoda does not require an external linker of the channel for its activity. In neurons (obtained from female and male rat neonates), MetaPoda specifically, and potently, inhibits all Kv4 currents, leaving all other A-type currents unaffected. Inhibition of Kv4 in hippocampal neurons does not promote excessive excitability, as is expected from a simple potassium channel blocker. We do find that MetaPoda's prolonged expression (1 week) increases expression levels of the immediate early gene cFos and prevents potentiation. These findings argue for a major role of Kv4.2 in facilitating plasticity of hippocampal neurons. Lastly, we show that our engineering strategy is suitable for the swift engineering of another potent Kv4.2-selective membrane-tethered toxin, Phrixotoxin-1, denoted MetaPhix. Together, we provide two uniquely potent genetic tools to study Kv4.2 in neuronal excitability and plasticity.

An E280K Missense Variant in KCND3/Kv4.3-Case Report and Functional Characterization.

A five-year-old girl presented with headache attacks, clumsiness, and a history of transient gait disturbances. She and her father, mother, twin sister, and brother underwent neurological evaluation, neuroimaging, and exome sequencing covering 357 genes associated with movement disorders. Sequencing revealed the new variant KCND3 c.838G>A, p.E280K in the father and sisters, but not in the mother and brother. KCND3 encodes voltage-gated potassium channel D3 (Kv4.3) and mutations have been associated with spinocerebellar ataxia type 19/22 (SCA19/22) and cardiac arrhythmias. SCA19/22 is characterized by ataxia, Parkinsonism, peripheral neuropathy, and sometimes, intellectual disability. Neuroimaging, EEG, and ECG were unremarkable. Mild developmental delay with impaired fluid reasoning was observed in both sisters, but not in the brother. None of the family members demonstrated ataxia or parkinsonism. In Xenopus oocyte electrophysiology experiments, E280K was associated with a rightward shift in the Kv4.3 voltage-activation relationship of 11 mV for WT/E280K and +17 mV for E280K/E280K relative to WT/WT. Steady-state inactivation was similarly right-shifted. Maximal peak current amplitudes were similar for WT/WT, WT/E280K, and E280K/E280K. Our data indicate that Kv4.3 E280K affects channel activation and inactivation and is associated with developmental delay. However, E280K appears to be relatively benign considering it does not result in overt ataxia.

Promoting proliferation and tumorigenesis of breast cancer: KCND2's significance as a prognostic factor.

In recent years, the potassium voltage-gated channel subfamily D (KCND) channels, particularly KCND2 (also known as Kv4.2), have been suggested to play a role in a variety of cancers, but their role in breast cancer has not yet been revealed. We analyzed RNA sequencing data from The Cancer Genome Atlas database and the Genotype-Tissue Expression database to investigate the differential expression of KCND2 in breast cancer and normal breast tissue. In addition, we leveraged GO and KEGG analysis techniques to gain a better understanding of the potential functional enrichment of 500 genes related to KCND2. Our findings were validated using collected tissue samples and clinical data from hospitals showed that KCND2 is a crucial independent factor in the prognosis of breast cancer patients. The higher the expression of KCND2, the shorter the survival time of breast cancer patients. Colony formation assay confirmed that KCND2 promotes the proliferation of breast cancer cells, whereas transwell assay and wound healing assay verified that KCND2 promoted breast cancer invasion and migration. In addition, 5-Ethynyl-2'-deoxyuridine (EdU) and flow cytometry revealed that KCND2 affected the cycle changes of breast cancer cells and contributed to the G1/S phase transition of breast cancer cells. Overall, our study demonstrates that KCND2 holds a promising potential as a significant target for breast cancer diagnosis and therapy.

Modulation of KV4.3-KChIP2 Channels by IQM-266: Role of DPP6 and KCNE2.

The transient outward potassium current (Itof) is generated by the activation of KV4 channels assembled with KChIP2 and other accessory subunits (DPP6 and KCNE2). To test the hypothesis that these subunits modify the channel pharmacology, we analyzed the electrophysiological effects of (3-(2-(3-phenoxyphenyl)acetamido)-2-naphthoic acid) (IQM-266), a new KChIP2 ligand, on the currents generated by KV4.3/KChIP2, KV4.3/KChIP2/DPP6 and KV4.3/KChIP2/KCNE2 channels. CHO cells were transiently transfected with cDNAs codifying for different proteins (KV4.3/KChIP2, KV4.3/KChIP2/DPP6 or KV4.3/KChIP2/KCNE2), and the potassium currents were recorded using the whole-cell patch-clamp technique. IQM-266 decreased the maximum peak of KV4.3/KChIP2, KV4.3/KChIP2/DPP6 and KV4.3/KChIP2/KCNE2 currents, slowing their time course of inactivation in a concentration-, voltage-, time- and use-dependent manner. IQM-266 produced an increase in the charge in KV4.3/KChIP2 channels that was intensified when DPP6 was present and abolished in the presence of KCNE2. IQM-266 induced an activation unblocking effect during the application of trains of pulses to cells expressing KV4.3/KChIP2 and KV4.3/KChIP2/KCNE2, but not in KV4.3/KChIP2/DPP6 channels. Overall, all these results are consistent with a preferential IQM-266 binding to an active closed state of Kv4.3/KChIP2 and Kv4.3/KChIP2/KCNE2 channels, whereas in the presence of DPP6, IQM-266 binds preferentially to an inactivated state. In conclusion, DPP6 and KCNE2 modify the pharmacological response of KV4.3/KChIP2 channels to IQM-266.

Acacetin, a Potent Transient Outward Current Blocker, May Be a Novel Therapeutic for KCND3-Encoded Kv4.3 Gain-of-Function-Associated J-Wave Syndromes.

BACKGROUND: The transient outward current (Ito) that mediates early (phase 1) repolarization is conducted by the KCND3-encoded Kv4.3 pore-forming α-subunit. KCND3 gain-of-function mutations have been reported previously as a pathogenic substrate for J wave syndromes (JWS), including the Brugada syndrome and early repolarization syndrome, as well as autopsy-negative sudden unexplained death (SUD). Acacetin, a natural flavone, is a potent Ito current blocker. Acacetin may be a novel therapeutic for KCND3-mediated J wave syndrome. METHODS: KCND3-V392I was identified in an 18-year-old male with J wave syndrome/early repolarization syndrome, and a history of cardiac arrest including ventricular tachycardia/ventricular fibrillation and atrial fibrillation/atrial flutter. Pathogenic KCND3 mutation was engineered by site-directed mutagenesis and co-expressed with wild-type KChIP2 in TSA201 cells. Gene-edited/variant-corrected isogenic control and patient-specific pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from the p. Val392Ile-KCND3-positive patient were generated. Ito currents and action potentials were recorded before and after treatment with Acacetin using the whole cell patch-clamp and multielectrode array technique. Western blot and immunocytochemistry were performed to investigate KCND3 expression. RESULTS: KCND3-V392I demonstrated a marked gain-of-function phenotype, increasing peak Ito current density by 92.2% (P<0.05 versus KCND3-WT). KCND3 expression was significantly increased in KCND3-V392I-derived iPSC-CMs (P<0.05 versus isogenic control). While KCND3-WT revealed an IC50 of 7.2±1.0 µmol/L for acacetin effect, 30 µmol/L acacetin dramatically inhibited KCND3-V392I peak Ito current density by 96.2% (P<0.05 versus before Acacetin). Ito was also increased by 60.9% in Kv4.3-V392I iPSC-CM (P<0.05 versus isogenic control iPSC-CM). Ten micromoles per liter acacetin, a concentration approaching its IC50 value, inhibited Ito by ≈50% in patient-derived iPSC-CMs and reduced the accentuated action potential notch displayed in KCND3-V392I-derived iPSC-CMs. CONCLUSIONS: This preclinical study provides pharmacological and functional evidence to suggest that Acacetin may be a novel therapeutic for patients with KCND3 gain-of-function-associated J wave syndrome by inhibiting Ito and abolishing the accentuated action potential notch in patient-derived iPSC-CMs.

Altered closed state inactivation gating in Kv4.2 channels results in developmental and epileptic encephalopathies in human patients.

Kv4.2 subunits, encoded by KCND2, serve as the pore-forming components of voltage-gated, inactivating ISA K+ channels expressed in the brain. ISA channels inactivate without opening in response to subthreshold excitatory input, temporarily increasing neuronal excitability, the back propagation of action potentials, and Ca2+ influx into dendrites, thereby regulating mechanisms of spike timing-dependent synaptic plasticity. As previously described, a de novo variant in Kv4.2, p.Val404Met, is associated with an infant-onset developmental and epileptic encephalopathy in monozygotic twin boys. The p.Val404Met variant enhances inactivation directly from closed states, but dramatically impairs inactivation after channel opening. We now report the identification of a closely related, novel, de novo variant in Kv4.2, p.Val402Leu, in a boy with an early-onset pharmacoresistant epilepsy that evolved to an epileptic aphasia syndrome (Continuous Spike Wave during Sleep Syndrome). Like p.Val404Met, the p.Val402Leu variant increases the rate of inactivation from closed states, but significantly slows inactivation after the pore opens. Although quantitatively the p.Val402Leu mutation alters channel kinetics less dramatically than p.Val404Met, our results strongly support the conclusion that p.Val402Leu and p.Val404Met cause the clinical features seen in the affected individuals and underscore the importance of closed state inactivation in ISA channels in normal brain development and function.

Kv4.2 phosphorylation by PKA drives Kv4.2-KChIP2 dissociation, leading to Kv4.2 out of lipid rafts and internalization.

Sympathetic regulation of the Kv4.2 transient outward potassium current (Ito) is critical for the acute electrical and contractile response of the myocardium under physiological and pathological conditions. Previous studies have suggested that KChIP2, the key auxiliary subunit of Kv4 channels, is required for the sympathetic regulation of Kv4.2 current densities. Of interest, Kv4.2 and KChIP2, and key components mediating acute sympathetic signaling transduction are present in lipid rafts, which are profoundly involved in regulation of Ito densities in rat ventricular myocytes. However, little is known about the mechanisms of Kv4.2-raft association and its connection with acute sympathetic regulation. With the aid of high-resolution fluorescent microscope, we demonstrated that KChIP2 assisted Kv4.2 localization in lipid rafts in HEK293 cells. Moreover, PKA-mediated Kv4.2 phosphorylation, the downstream signaling event of acute sympathetic stimulation, induced dissociation between Kv4.2 and KChIP2, resulting in Kv4.2 shifting out of lipid rafts in KChIP2-expressed HEK293. The mutation that mimics Kv4.2 phosphorylation by PKA (K4.2-S552D) similarly disrupted Kv4.2 interaction with KChIP2 and also decreased the surface stability of Kv4.2. The attenuated Kv4.2-KChIP2 interaction was also observed in native neonatal rat ventricular myocytes (NRVMs) upon acute adrenergic stimulation with phenylephrine (PE). Furthermore, PE stimulation decreased Kv4.2 location at lipid rafts and induced internalization of Kv4.2 as well as the effect of lipid rafts disruption. In conclusion, KChIP2 contributes to targeting Kv4.2 to lipid rafts. Acute adrenergic stimulation induces Kv4.2-KChIP2 dissociation, leading to Kv4.2 out of lipid rafts and internalization, reinforcing the critical role of Kv4.2-lipid raft association in the essential physiological response of Ito to acute sympathetic regulation.

Activation and closed-state inactivation mechanisms of the human voltage-gated KV4 channel complexes.

The voltage-gated ion channel activity depends on both activation (transition from the resting state to the open state) and inactivation. Inactivation is a self-restraint mechanism to limit ion conduction and is as crucial to membrane excitability as activation. Inactivation can occur when the channel is open or closed. Although open-state inactivation is well understood, the molecular basis of closed-state inactivation has remained elusive. We report cryo-EM structures of human KV4.2 channel complexes in inactivated, open, and closed states. Closed-state inactivation of KV4 involves an unprecedented symmetry breakdown for pore closure by only two of the four S4-S5 linkers, distinct from known mechanisms of open-state inactivation. We further capture KV4 in a putative resting state, revealing how voltage sensor movements control the pore. Moreover, our structures provide insights regarding channel modulation by KChIP2 and DPP6 auxiliary subunits. Our findings elucidate mechanisms of closed-state inactivation and voltage-dependent activation of the KV4 channel.

Early repolarization syndrome, epilepsy, and atrial fibrillation in a young girl with novel KCND3 mutation managed with quinidine.

A 6-year-old girl presented with a difficult to control epilepsy syndrome. On evaluation, additional presyncope episodes associated with polymorphic ventricular tachycardia were also noted. A diagnosis of early repolarization syndrome (ERS) was made with an early repolarization pattern on electrocardiogram, documented VT episodes, and clinical presyncope (proposed Shanghai score 7). Paroxysmal atrial fibrillation (AF) was also noted on 24-h Holter recordings. The child was stabilized with isoprenaline infusion and was later discharged with arrhythmia control on quinidine and cilostazol. The genetic evaluation revealed a potassium channel KCND3 gene missense mutation. The case highlights the association of epilepsy syndrome and AF with ERS; the possible association of KCND3 gene mutation with a malignant phenotype; and management issues in a small child.

Irx5 and transient outward K+ currents contribute to transmural contractile heterogeneities in the mouse ventricle.

Previous studies have established that transmural gradients of the fast transient outward K+ current (Ito,f) correlate with regional differences in action potential (AP) profile and excitation-contraction coupling (ECC) with high Ito,f expression in the epimyocardium (EPI) being associated with short APs and low contractility and vice versa. Herein, we investigated the effects of altering the Ito,f gradients on transmural contractile properties using mice lacking Irx5 (Irx5-KO) or lacking Kcnd2 (KV4.2-KO) or both. Irx5-KO mice exhibited decreased global LV contractility in association with elevated Ito,f, as well as reduced cell shortening and Ca2+ transient amplitudes in cardiomyocytes isolated from the endomyocardium (ENDO) but not in cardiomyocytes from the EPI. Transcriptional profiling revealed that the primary effect of Irx5 ablation on ECC-related genes was to increase Ito,f gene expression (i.e., Kcnd2 and Kcnip2) in the ENDO, but not the EPI. By contrast, KV4.2-KO mice showed selective increases in cell shortening and Ca2+ transients in isolated EPI cardiomyocytes, leading to enhanced ventricular contractility and mice lacking both Irx5 and Kcnd2 displayed elevated ventricular contractility, comparable to KV4.2-KO mice, demonstrating a dominant role of Irx5-dependent modulation of Ito,f in the regulation of contractility. Our findings show that the transmural electromechanical heterogeneities in the healthy ventricles depend on the Irx5-dependent Ito,f gradients. These observations provide a useful framework for assessing the molecular mechanisms underlying the alterations in contractile heterogeneity seen in the diseased heart.NEW & NOTEWORTHY Irx5 is a vital transcription factor that establishes the transmural heterogeneity of ventricular myocyte contractility, thereby ensuring proper contractile function in the healthy heart. Regional differences in excitation-contraction coupling in the ventricular myocardium are primarily mediated through the inverse relationship between Irx5 and the fast transient outward K+ current (Ito,f) across the ventricular wall.

Inducing Ito,f and phase 1 repolarization of the cardiac action potential with a Kv4.3/KChIP2.1 bicistronic transgene.

The fast transient outward potassium current (Ito,f) plays a key role in phase 1 repolarization of the human cardiac action potential (AP) and its reduction in heart failure (HF) contributes to the loss of contractility. Therefore, restoring Ito,f might be beneficial for treating HF. The coding sequence of a P2A peptide was cloned, in frame, between Kv4.3 and KChIP2.1 genes and ribosomal skipping was confirmed by Western blotting. Typical Ito,f properties with slowed inactivation and accelerated recovery from inactivation due to the association of KChIP2.1 with Kv4.3 was seen in transfected HEK293 cells. Both bicistronic components trafficked to the plasmamembrane and in adenovirus transduced rabbit cardiomyocytes both t-tubular and sarcolemmal construct labelling appeared. The resulting current was similar to Ito,f seen in human ventricular cardiomyocytes and was 50% blocked at ~0.8 mmol/l 4-aminopyridine and increased ~30% by 5 µmol/l NS5806 (an Ito,f agonist). Variation in the density of the expressed Ito,f, in rabbit cardiomyocytes recapitulated typical species-dependent variations in AP morphology. Simultaneous voltage recording and intracellular Ca2+ imaging showed that modification of phase 1 to a non-failing human phenotype improved the rate of rise and magnitude of the Ca2+ transient. Ito,f expression also reduced AP triangulation but did not affect ICa,L and INa magnitudes. This raises the possibility for a new gene-based therapeutic approach to HF based on selective phase 1 modification.

Age-related changes in Kv4/Shal and Kv1/Shaker expression in Drosophila and a role for reactive oxygen species.

Age-related changes in ion channel expression are likely to affect neuronal signaling. Here, we examine how age affects Kv4/Shal and Kv1/Shaker K+ channel protein levels in Drosophila. We show that Kv4/Shal protein levels decline sharply from 3 days to 10 days, then more gradually from 10 to 40 days after eclosion. In contrast, Kv1/Shaker protein exhibits a transient increase at 10 days that then stabilizes and eventually declines at 40 days. We present data that begin to show a relationship between reactive oxygen species (ROS), Kv4/Shal, and locomotor performance. We show that Kv4/Shal levels are negatively affected by ROS, and that over-expression of Catalase or RNAi knock-down of the ROS-generating enzyme, Nicotinamide Adenine Dinucleotide Phosphate (NADPH) Oxidase (NOX), can attenuate the loss of Kv4/Shal protein. Finally, we compare levels of Kv4.2 and Kv4.3 in the hippocampus, olfactory bulb, cerebellum, and motor cortex of mice aged 6 weeks and 1 year. While there was no global decline in Kv4.2/4.3 that parallels what we report in Drosophila, we did find that Kv4.2/4.3 are differentially affected in various brain regions; this survey of changes may help inform mammalian studies that examine neuronal function with age.

Structural basis of gating modulation of Kv4 channel complexes.

Modulation of voltage-gated potassium (Kv) channels by auxiliary subunits is central to the physiological function of channels in the brain and heart1,2. Native Kv4 tetrameric channels form macromolecular ternary complexes with two auxiliary ß-subunits-intracellular Kv channel-interacting proteins (KChIPs) and transmembrane dipeptidyl peptidase-related proteins (DPPs)-to evoke rapidly activating and inactivating A-type currents, which prevent the backpropagation of action potentials1-5. However, the modulatory mechanisms of Kv4 channel complexes remain largely unknown. Here we report cryo-electron microscopy structures of the Kv4.2-DPP6S-KChIP1 dodecamer complex, the Kv4.2-KChIP1 and Kv4.2-DPP6S octamer complexes, and Kv4.2 alone. The structure of the Kv4.2-KChIP1 complex reveals that the intracellular N terminus of Kv4.2 interacts with its C terminus that extends from the S6 gating helix of the neighbouring Kv4.2 subunit. KChIP1 captures both the N and the C terminus of Kv4.2. In consequence, KChIP1 would prevent N-type inactivation and stabilize the S6 conformation to modulate gating of the S6 helices within the tetramer. By contrast, unlike the reported auxiliary subunits of voltage-gated channel complexes, DPP6S interacts with the S1 and S2 helices of the Kv4.2 voltage-sensing domain, which suggests that DPP6S stabilizes the conformation of the S1-S2 helices. DPP6S may therefore accelerate the voltage-dependent movement of the S4 helices. KChIP1 and DPP6S do not directly interact with each other in the Kv4.2-KChIP1-DPP6S ternary complex. Thus, our data suggest that two distinct modes of modulation contribute in an additive manner to evoke A-type currents from the native Kv4 macromolecular complex.

Voltage-dependent potassium channel Kv4.2 alleviates the ischemic stroke impairments through activating neurogenesis.

As well as their ion transportation function, the voltage-dependent potassium channels could act as the cell signal inducer in a variety of pathogenic processes. However, their roles in neurogenesis after stroke insults have not been clearly illustrated. In our preliminary study, the expressions of voltage-dependent potassium channels Kv4.2 was significantly decreased after stroke in cortex, striatum and hippocampus by real-time quantitative PCR assay. To underlie the neuroprotection of Kv4.2 in stroke rehabilitation, recombinant plasmids encoding the cDNAs of mouse Kv4.2 was constructed. Behavioral tests showed that the increased Kv4.2 could be beneficial to the recovery of the sensory, the motor functions and the cognitive deficits after stroke. Temozolomide (TMZ), an inhibitor of neurogenesis, could partially abolish the mentioned protections of Kv4.2. The immunocytochemical staining showed that Kv4.2 could promote the proliferations of neural stem cells and induce the neural stem cells to differentiate into neurons in vitro and in vivo. And Kv4.2 could up-regulate the expressions of ERK1/2, p-ERK1/2, p-STAT3, NGF, p-TrkA, and BDNF, CAMKII and the concentration of intracellular Ca2+. Namely, we concluded that Kv4.2 promoted neurogenesis through ERK1/2/STAT3, NGF/TrkA, Ca2+/CAMKII signal pathways and rescued the ischemic impairments. Kv4.2 might be a potential drug target for ischemic stroke intervention.

Rare Gain-of-Function KCND3 Variant Associated with Cerebellar Ataxia, Parkinsonism, Cognitive Dysfunction, and Brain Iron Accumulation.

Loss-of-function mutations in the KV4.3 channel-encoding KCND3 gene are linked to neurodegenerative cerebellar ataxia. Patients suffering from neurodegeneration associated with iron deposition may also present with cerebellar ataxia. The mechanism underlying brain iron accumulation remains unclear. Here, we aim to ascertain the potential pathogenic role of KCND3 variant in iron accumulation-related cerebellar ataxia. We presented a patient with slowly progressive cerebellar ataxia, parkinsonism, cognitive impairment, and iron accumulation in the basal ganglia and the cerebellum. Whole exome sequencing analyses identified in the patient a heterozygous KCND3 c.1256G>A (p.R419H) variant predicted to be disease-causing by multiple bioinformatic analyses. In vitro biochemical and immunofluorescence examinations revealed that, compared to the human KV4.3 wild-type channel, the p.R419H variant exhibited normal protein abundance and subcellular localization pattern. Electrophysiological investigation, however, demonstrated that the KV4.3 p.R419H variant was associated with a dominant increase in potassium current amplitudes, as well as notable changes in voltage-dependent gating properties leading to enhanced potassium window current. These observations indicate that, in direct contrast with the loss-of-function KCND3 mutations previously reported in cerebellar ataxia patients, we identified a rare gain-of-function KCND3 variant that may expand the clinical and molecular spectra of neurodegenerative cerebellar disorders associated with brain iron accumulation.

Characterization of the A-type potassium current in murine gastric fundus smooth muscles.

Transient outward, or "A-type," currents are rapidly inactivating voltage-gated potassium currents that operate at negative membrane potentials. A-type currents have not been reported in the gastric fundus, a tonic smooth muscle. We used whole cell voltage clamp to identify and characterize A-type currents in smooth muscle cells (SMCs) isolated from murine fundus. A-type currents were robust in these cells with peak amplitudes averaging 1.5 nA at 0 mV. Inactivation was rapid with a time constant of 71 ms at 0 mV; recovery from inactivation at -80 mV was similarly rapid with a time constant of 75 ms. A-type currents in fundus were blocked by 4-aminopyridine (4-AP), flecainide, and phrixotoxin-1 (PaTX1). Remaining currents after 4-AP and PaTX1 displayed half-activation potentials that were shifted to more positive potentials and showed incomplete inactivation. Currents after tetraethylammonium (TEA) displayed half inactivation at -48.1 ± 1.0 mV. Conventional microelectrode and contractile experiments on intact fundus muscles showed that 4-AP depolarized membrane potential and increased tone under conditions in which enteric neurotransmission was blocked. These data suggest that A-type K+ channels in fundus SMCs are likely active at physiological membrane potentials, and sustained activation of A-type channels contributes to the negative membrane potentials of this tonic smooth muscle. Quantitative analysis of Kv4 expression showed that Kcnd3 was dominantly expressed in fundus SMCs. These data were confirmed by immunohistochemistry, which revealed Kv4.3-like immunoreactivity within the tunica muscularis. These observations indicate that Kv4 channels likely form the A-type current in murine fundus SMCs.

KCND2 variants associated with global developmental delay differentially impair Kv4.2 channel gating.

Here, we report on six unrelated individuals, all presenting with early-onset global developmental delay, associated with impaired motor, speech and cognitive development, partly with developmental epileptic encephalopathy and physical dysmorphisms. All individuals carry heterozygous missense variants of KCND2, which encodes the voltage-gated potassium (Kv) channel α-subunit Kv4.2. The amino acid substitutions associated with the variants, p.(Glu323Lys) (E323K), p.(Pro403Ala) (P403A), p.(Val404Leu) (V404L) and p.(Val404Met) (V404M), affect sites known to be critical for channel gating. To unravel their likely pathogenicity, recombinant mutant channels were studied in the absence and presence of auxiliary ß-subunits under two-electrode voltage clamp in Xenopus oocytes. All channel mutants exhibited slowed and incomplete macroscopic inactivation, and the P403A variant in addition slowed activation. Co-expression of KChIP2 or DPP6 augmented the functional expression of both wild-type and mutant channels; however, the auxiliary ß-subunit-mediated gating modifications differed from wild type and among mutants. To simulate the putative setting in the affected individuals, heteromeric Kv4.2 channels (wild type + mutant) were studied as ternary complexes (containing both KChIP2 and DPP6). In the heteromeric ternary configuration, the E323K variant exhibited only marginal functional alterations compared to homomeric wild-type ternary, compatible with mild loss-of-function. By contrast, the P403A, V404L and V404M variants displayed strong gating impairment in the heteromeric ternary configuration, compatible with loss-of-function or gain-of-function. Our results support the etiological involvement of Kv4.2 channel gating impairment in early-onset monogenic global developmental delay. In addition, they suggest that gain-of-function mechanisms associated with a substitution of V404 increase epileptic seizure susceptibility.

Voltage-gated potassium channel dysfunction in dorsal root ganglia contributes to the exaggerated exercise pressor reflex in rats with chronic heart failure.

An exaggerated exercise pressor reflex (EPR) causes excessive sympathoexcitation and exercise intolerance during physical activity in the chronic heart failure (CHF) state. Muscle afferent sensitization contributes to the genesis of the exaggerated EPR in CHF. However, the cellular mechanisms underlying muscle afferent sensitization in CHF remain unclear. Considering that voltage-gated potassium (Kv) channels critically regulate afferent neuronal excitability, we examined the potential role of Kv channels in mediating the sensitized EPR in male rats with CHF. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting experiments demonstrate that both mRNA and protein expressions of multiple Kv channel isoforms (Kv1.4, Kv3.4, Kv4.2, and Kv4.3) were downregulated in lumbar dorsal root ganglions (DRGs) of CHF rats compared with sham rats. Immunofluorescence data demonstrate significant decreased Kv channel staining in both NF200-positive and IB4-positive lumbar DRG neurons in CHF rats compared with sham rats. Data from patch-clamp experiments demonstrate that the total Kv current, especially IA, was dramatically decreased in medium-sized IB4-negative muscle afferent neurons (a subpopulation containing mostly Aδ neurons) from CHF rats compared with sham rats, indicating a potential functional loss of Kv channels in muscle afferent Aδ neurons. In in vivo experiments, adenoviral overexpression of Kv4.3 in lumbar DRGs for 1 wk attenuated the exaggerated EPR induced by muscle static contraction and the mechanoreflex by passive stretch without affecting the blunted cardiovascular response to hindlimb arterial injection of capsaicin in CHF rats. These data suggest that Kv channel dysfunction in DRGs plays a critical role in mediating the exaggerated EPR and muscle afferent sensitization in CHF.NEW & NOTEWORTHY The primary finding of this manuscript is that voltage-gated potassium (Kv) channel dysfunction in DRGs plays a critical role in mediating the exaggerated EPR and muscle afferent sensitization in chronic heart failure (CHF). We propose that manipulation of Kv channels in DRG neurons could be considered as a potential new approach to reduce the exaggerated sympathoexcitation and to improve exercise intolerance in CHF, which can ultimately facilitate an improved quality of life and reduce mortality.