Oxidative stress-related cellular aging causes dysfunction of the Kv3.1/KCNC1 channel reverted by melatonin.

The voltage-gated Kv3.1/KCNC1 channel is abundantly expressed in fast-spiking principal neurons and GABAergic inhibitory interneurons throughout the ascending auditory pathway and in various brain regions. Inactivating mutations in the KCNC1 gene lead to forms of epilepsy and a decline in the expression of the Kv3.1 channel is involved in age-related hearing loss. As oxidative stress plays a fundamental role in the pathogenesis of epilepsy and age-related hearing loss, we hypothesized that an oxidative insult might affect the function of this channel. To verify this hypothesis, the activity and expression of endogenous and ectopic Kv3.1 were measured in models of oxidative stress-related aging represented by cell lines exposed to 100 mM d-galactose. In these models, intracellular reactive oxygen species, thiobarbituric acid reactive substances, sulfhydryl groups of cellular proteins, and the activity of catalase and superoxide dismutase were dysregulated, while the current density of Kv3.1 was significantly reduced. Importantly, the antioxidant melatonin reverted all these effects. The reduction of function of Kv3.1 was not determined by direct oxidation of amino acid side chains of the protein channel or reduction of transcript or total protein levels but was linked to reduced trafficking to the cell surface associated with Src phosphorylation as well as metabolic and endoplasmic reticulum stress. The data presented here specify Kv3.1 as a novel target of oxidative stress and suggest that Kv3.1 dysfunction might contribute to age-related hearing loss and increased prevalence of epilepsy during aging. The pharmacological use of the antioxidant melatonin can be protective in this setting.

Myoclonus improvement after seizures in progressive myoclonic epilepsy type 7: a case report.

BACKGROUND: Progressive Myoclonic Epilepsy (PME) is a group of rare diseases that are difficult to differentiate from one another based on phenotypical characteristics. CASE REPORT: We report a case of PME type 7 due to a pathogenic variant in KCNC1 with myoclonus improvement after epileptic seizures. DISCUSSION: Myoclonus improvement after seizures may be a clue to the diagnosis of Progressive Myoclonic Epilepsy type 7.

A structurally precise mechanism links an epilepsy-associated KCNC2 potassium channel mutation to interneuron dysfunction.

De novo heterozygous variants in KCNC2 encoding the voltage-gated potassium (K+) channel subunit Kv3.2 are a recently described cause of developmental and epileptic encephalopathy (DEE). A de novo variant in KCNC2 c.374G > A (p.Cys125Tyr) was identified via exome sequencing in a patient with DEE. Relative to wild-type Kv3.2, Kv3.2-p.Cys125Tyr induces K+ currents exhibiting a large hyperpolarizing shift in the voltage dependence of activation, accelerated activation, and delayed deactivation consistent with a relative stabilization of the open conformation, along with increased current density. Leveraging the cryogenic electron microscopy (cryo-EM) structure of Kv3.1, molecular dynamic simulations suggest that a strong π-π stacking interaction between the variant Tyr125 and Tyr156 in the α-6 helix of the T1 domain promotes a relative stabilization of the open conformation of the channel, which underlies the observed gain of function. A multicompartment computational model of a Kv3-expressing parvalbumin-positive cerebral cortex fast-spiking γ-aminobutyric acidergic (GABAergic) interneuron (PV-IN) demonstrates how the Kv3.2-Cys125Tyr variant impairs neuronal excitability and dysregulates inhibition in cerebral cortex circuits to explain the resulting epilepsy.

Novel KCNC2 variant associated with developmental and epileptic encephalopathy.

The KCNC2 gene encodes Kv3.2, which is a member of the voltage-gated potassium channel subfamily. It is crucial for the generation of fast-spiking properties in cortical GABAergic interneurons. Recently, KCNC2 variations were found to be associated with epileptic encephalopathy in unrelated individuals. Here, we report a Chinese patient with developmental and epileptic encephalopathy (DEE) and motor development delay. Whole-exome sequencing (WES) revealed a novel heterozygous variant in the KCNC2 gene NM_139137.4:c.1163T>C (p.Phe388Ser), and subsequent Sanger sequencing showed that it was a de novo mutation. We identified the KCNC2 likely pathogenic variant in a DEE patient by reanalysis of WES data in a Chinese family. Our study enriched the variation spectrum of the KCNC2 gene and promoted the application of WES technology and data reanalysis in the diagnosis of epilepsy.

A KCNC1-related neurological disorder due to gain of Kv3.1 function.

OBJECTIVE: To further clarify genotype:phenotype correlations associated with variants in KCNC1 encoding the voltage-gated potassium (K+) channel subunit Kv3.1 and which are an emerging cause of a spectrum of neurological disease including intellectual disability, isolated myoclonus, progressive myoclonus epilepsy, and developmental and epileptic encephalopathy. METHODS: We describe the clinical and genetic characteristics of a series of three patients with de novo heterozygous missense variants in KCNC1 associated with nonspecific developmental delay/intellectual disability and central hypotonia without epilepsy or ataxia. All three variants lead to amino acids alterations with mild predicted differences in physicochemical properties yet are localized to the S6 pore region of the Kv3.1 protein between the selectivity filter and PXP motif important for K+ channel gating. We performed whole-cell voltage clamp electrophysiological recording of wild-type versus variants in a heterologous mammalian expression system. RESULTS: We demonstrate a prominent leftward (hyperpolarized) shift in the voltage dependence of activation and slowed deactivation of all variants in the clinically defined series. INTERPRETATION: Electrophysiological recordings are consistent with a gain of K+ channel function that is predicted to exert a loss of function on the excitability of Kv3-expressing high frequency- firing neurons based on the unique electrophysiological properties of Kv3 channels. These results define a clinical-genetic syndrome within the spectrum of KCNC1-related neurological disorders.

Potassium channels and epilepsy.

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

Investigation of novel de novo KCNC2 variants causing severe developmental and early-onset epileptic encephalopathy.

Purpose The voltage-gated potassium channel Kv3.2, encoded by KCNC2, facilitates fast-spiking GABAergic interneurons to fire action potentials at high frequencies. It is pivotal to maintaining excitation/inhibition balance in mammalian brains. This study identified two novel de novo KCNC2 variants, p.Pro470Ser (P470S) and p.Phe382Leu (F382L), in patients with early onset developmental and epileptic encephalopathy (DEE). Methods To examine the molecular basis of DEE, we studied the functional characteristics of variant channels using patch-clamp techniques and computational modeling. Results Whole-cell patch clamp recordings from infected HEK293 cells revealed that channel activation and deactivation kinetics strongly decreased in both Kv3.2 P470S and F382L variant channels. This decrease also occurred in Kv3.2 p.Val471Leu (V471L) channels, known to be associated with DEE. In addition, Kv3.2 F382L and V471L variants exhibited a significant increase in channel conductance and a ∼20 mV negative shift in the threshold for voltage-dependent activation. Simulations of model GABAergic interneurons revealed that all variants decreased neuronal firing frequency. Thus, the variants' net loss-of-function effects disinhibited neural networks. Conclusion Our findings provide compelling evidence supporting the role of KCNC2 as a disease-causing gene in human neurodevelopmental delay and epilepsy.

Cryo-EM structure of the human Kv3.1 channel reveals gating control by the cytoplasmic T1 domain.

Kv3 channels have distinctive gating kinetics tailored for rapid repolarization in fast-spiking neurons. Malfunction of this process due to genetic variants in the KCNC1 gene causes severe epileptic disorders, yet the structural determinants for the unusual gating properties remain elusive. Here, we present cryo-electron microscopy structures of the human Kv3.1a channel, revealing a unique arrangement of the cytoplasmic tetramerization domain T1 which facilitates interactions with C-terminal axonal targeting motif and key components of the gating machinery. Additional interactions between S1/S2 linker and turret domain strengthen the interface between voltage sensor and pore domain. Supported by molecular dynamics simulations, electrophysiological and mutational analyses, we identify several residues in the S4/S5 linker which influence the gating kinetics and an electrostatic interaction between acidic residues in α6 of T1 and R449 in the pore-flanking S6T helices. These findings provide insights into gating control and disease mechanisms and may guide strategies for the design of pharmaceutical drugs targeting Kv3 channels.

A missense mutation in Kcnc3 causes hippocampal learning deficits in mice.

Although a wide variety of genetic tools has been developed to study learning and memory, the molecular basis of memory encoding remains incompletely understood. Here, we undertook an unbiased approach to identify novel genes critical for memory encoding. From a large-scale, in vivo mutagenesis screen using contextual fear conditioning, we isolated in mice a mutant, named Clueless, with spatial learning deficits. A causative missense mutation (G434V) was found in the voltage-gated potassium channel, subfamily C member 3 (Kcnc3) gene in a region that encodes a transmembrane voltage sensor. Generation of a Kcnc3G434V CRISPR mutant mouse confirmed this mutation as the cause of the learning defects. While G434V had no effect on transcription, translation, or trafficking of the channel, electrophysiological analysis of the G434V mutant channel revealed a complete loss of voltage-gated conductance, a broadening of the action potential, and decreased neuronal firing. Together, our findings have revealed a role for Kcnc3 in learning and memory.

Predicting the functional effects of voltage-gated potassium channel missense variants with multi-task learning.

BACKGROUND: Variants in genes encoding voltage-gated potassium channels are associated with a broad spectrum of neurological diseases including epilepsy, ataxia, and intellectual disability. Knowledge of the resulting functional changes, characterized as overall ion channel gain- or loss-of-function, is essential to guide clinical management including precision medicine therapies. However, for an increasing number of variants, little to no experimental data is available. New tools are needed to evaluate variant functional effects. METHODS: We catalogued a comprehensive dataset of 959 functional experiments across 19 voltage-gated potassium channels, leveraging data from 782 unique disease-associated and synthetic variants. We used these data to train a taxonomy-based multi-task learning support vector machine (MTL-SVM), and compared performance to several baseline methods. FINDINGS: MTL-SVM maintains channel family structure during model training, improving overall predictive performance (mean balanced accuracy 0·718 ± 0·041, AU-ROC 0·761 ± 0·063) over baseline (mean balanced accuracy 0·620 ± 0·045, AU-ROC 0·711 ± 0·022). We can obtain meaningful predictions even for channels with few known variants (KCNC1, KCNQ5). INTERPRETATION: Our model enables functional variant prediction for voltage-gated potassium channels. It may assist in tailoring current and future precision therapies for the increasing number of patients with ion channel disorders. FUNDING: This work was supported by intramural funding of the Medical Faculty, University of Tuebingen (PATE F.1315137.1), the Federal Ministry for Education and Research (Treat-ION, 01GM1907A/B/G/H) and the German Research Foundation (FOR-2715, Le1030/16-2, He8155/1-2).

Germline mosaicism of a missense variant in KCNC2 in a multiplex family with autism and epilepsy characterized by long-read sequencing.

Currently, protein-coding de novo variants and large copy number variants have been identified as important for ~30% of individuals with autism. One approach to identify relevant variation in individuals who lack these types of events is by utilizing newer genomic technologies. In this study, highly accurate PacBio HiFi long-read sequencing was applied to a family with autism, epileptic encephalopathy, cognitive impairment, and mild dysmorphic features (two affected female siblings, unaffected parents, and one unaffected male sibling) with no known clinical variant. From our long-read sequencing data, a de novo missense variant in the KCNC2 gene (encodes Kv3.2) was identified in both affected children. This variant was phased to the paternal chromosome of origin and is likely a germline mosaic. In silico assessment revealed the variant was not in controls, highly conserved, and predicted damaging. This specific missense variant (Val473Ala) has been shown in both an ortholog and paralog of Kv3.2 to accelerate current decay, shift the voltage dependence of activation, and prevent the channel from entering a long-lasting open state. Seven additional missense variants have been identified in other individuals with neurodevelopmental disorders (p = 1.03 × 10-5 ). KCNC2 is most highly expressed in the brain; in particular, in the thalamus and is enriched in GABAergic neurons. Long-read sequencing was useful in discovering the relevant variant in this family with autism that had remained a mystery for several years and will potentially have great benefits in the clinic once it is widely available.

Spectrum of Phenotypic, Genetic, and Functional Characteristics in Patients With Epilepsy With KCNC2 Pathogenic Variants.

BACKGROUND AND OBJECTIVES: KCNC2 encodes Kv3.2, a member of the Shaw-related (Kv3) voltage-gated potassium channel subfamily, which is important for sustained high-frequency firing and optimized energy efficiency of action potentials in the brain. The objective of this study was to analyze the clinical phenotype, genetic background, and biophysical function of disease-associated Kv3.2 variants. METHODS: Individuals with KCNC2 variants detected by exome sequencing were selected for clinical, further genetic, and functional analysis. Cases were referred through clinical and research collaborations. Selected de novo variants were examined electrophysiologically in Xenopus laevis oocytes. RESULTS: We identified novel KCNC2 variants in 18 patients with various forms of epilepsy, including genetic generalized epilepsy (GGE), developmental and epileptic encephalopathy (DEE) including early-onset absence epilepsy, focal epilepsy, and myoclonic-atonic epilepsy. Of the 18 variants, 10 were de novo and 8 were classified as modifying variants. Eight drug-responsive patients became seizure-free using valproic acid as monotherapy or in combination, including severe DEE cases. Functional analysis of 4 variants demonstrated gain of function in 3 severely affected DEE cases and loss of function in 1 case with a milder phenotype (GGE) as the underlying pathomechanisms. DISCUSSION: These findings implicate KCNC2 as a novel causative gene for epilepsy and emphasize the critical role of KV3.2 in the regulation of brain excitability.

Suppression of Kv3.3 channels by antisense oligonucleotides reverses biochemical effects and motor impairment in spinocerebellar ataxia type 13 mice.

Mutations in KCNC3, the gene that encodes the Kv3.3 voltage dependent potassium channel, cause Spinocerebellar Ataxia type 13 (SCA13), a disease associated with disrupted motor behaviors, progressive cerebellar degeneration, and abnormal auditory processing. The Kv3.3 channel directly binds Hax-1, a cell survival protein. A disease-causing mutation, Kv3.3-G592R, causes overstimulation of Tank Binding Kinase 1 (Tbk1) in the cerebellum, resulting in the degradation of Hax-1 by promoting its trafficking into multivesicular bodies and then to lysosomes. We have now tested the effects of antisense oligonucleotides (ASOs) directed against the Kv3.3 channel on both wild type mice and those bearing the Kv3.3-G592R-encoding mutation. Intracerebroventricular infusion of the Kcnc3-specific ASO suppressed both mRNA and protein levels of the Kv3.3 channel. In wild-type animals, this produced no change in levels of activated Tbk1, Hax-1 or Cd63, a tetraspanin marker for late endosomes/multivesicular bodies. In contrast, in mice homozygous for the Kv3.3-G592R-encoding mutation, the same ASO reduced Tbk1 activation and levels of Cd63, while restoring the expression of Hax-1 in the cerebellum. The motor behavior of the mice was tested using a rotarod assay. Surprisingly, the active ASO had no effects on the motor behavior of wild type mice but restored the behavior of the mutant mice to those of age-matched wild type animals. Our findings indicate that, in mature intact animals, suppression of Kv3.3 expression can reverse the deleterious effects of a SCA13 mutation while having little effect on wild type animals. Thus, targeting Kv3.3 expression may prove a viable therapeutic approach for SCA13.

A recurrent de novo variant supports KCNC2 involvement in the pathogenesis of developmental and epileptic encephalopathy.

Developmental and epileptic encephalopathies (DEE) are a heterogenous group of conditions characterized by the co-occurrence of epilepsy and intellectual/developmental disability. Despite several known DEE-related genes, including these encoding ion channels, still many cases remain without molecular diagnosis. Here, we present a 2-year-old girl with severe DEE in whom whole exome sequencing revealed de novo p.(Val471Leu) variant in the KCNC2 encoding Kv3.2, a voltage-gated potassium channel. To the best of our knowledge, this is the third DEE case due to KCNC2 mutation. Our clinical and molecular findings, particularly the recurrence of p.(Val471Leu) in patient with similar clinical phenotype, further support KCNC2 as a novel DEE-associated gene.

A KCNC1 mutation in epilepsy of infancy with focal migrating seizures produces functional channels that fail to be regulated by PKC phosphorylation.

Channelopathies caused by mutations in genes encoding ion channels generally produce a clear change in channel function. Accordingly, mutations in KCNC1, which encodes the voltage-dependent Kv3.1 potassium channel, result in progressive myoclonus epilepsy as well as other developmental and epileptic encephalopathies, and these have been shown to reduce or fully abolish current amplitude. One exception to this is the mutation A513V Kv3.1b, located in the cytoplasmic C-terminal domain of the channel protein. This de novo variant was detected in a patient with epilepsy of infancy with focal migrating seizures (EIFMS), but no difference could be detected between A513V Kv3.1 current and that of wild-type Kv3.1. Using both biochemical and electrophysiological approaches, we have now confirmed that this variant produces functional channels but find that the A513V mutation renders the channel completely insensitive to regulation by phosphorylation at S503, a nearby regulatory site in the C-terminus. In this respect, the mutation resembles those in another channel, KCNT1, which are the major cause of EIFMS. Because the amplitude of Kv3.1 current is constantly adjusted by phosphorylation in vivo, our findings suggest that loss of such regulation contributes to EIFMS phenotype and emphasize the role of channel modulation for normal neuronal function.NEW & NOTEWORTHY Ion channel mutations that cause serious human diseases generally alter the biophysical properties or expression of the channel. We describe a de novo mutation in the Kv3.1 potassium channel that causes severe intellectual disability with early-onset epilepsy. The properties of this channel appear identical to those of wild-type channels, but the mutation prevents phosphorylation of the channel by protein kinase C. Our findings emphasize the role of channel modulation in normal brain function.

Methylation gene KCNC1 is associated with overall survival in patients with seminoma.

The aim of the present study was to explore and verify the potential mechanism of seminoma progression. Data on 132 RNA­seq and 156 methylation sites from stage II/III and I seminoma specimens were downloaded from The Cancer Genome Atlas database. An initial filter of |fold­change| >2 and false discovery rate <0.05 were used to identify differentially expressed genes (DEGs) which were associated with differential methylation site genes; these genes were considered potential candidates for further investigation by survival analysis. Potassium voltage­gated channel subfamily C member 1 (KCNC1) expression was verified in seminoma human tissues and three seminoma cell lines. The invasive, proliferative and apoptotic abilities of the human testicular tumor Ntera­2 and normal human testis Hs1.Tes cell lines were assessed following aberrant KCNC1 expression. KCNC1 was identified as a DEG, in which hypermethylation inhibited its expression and it was associated with poor overall survival in patients with seminoma. The present results demonstrated that KCNC1 is negatively correlated with methylation. Due to the abnormal expression of KCNC1 in seminoma cells, it was suggested that KCNC1 could be used as a diagnostic indicator and therapeutic target for the progression of seminoma.

Kv3.1 channels regulate the rate of critical period plasticity.

Experience-dependent plasticity within visual cortex is controlled by postnatal maturation of inhibitory circuits, which are both morphologically diverse and precisely connected. Gene-targeted disruption of the voltage-dependent potassium channel Kv3.1 broadens action potentials and reduces net inhibitory function of parvalbumin (PV)-positive GABA subtypes within the neocortex. In mice lacking Kv3.1, the rate of input loss from an eye deprived of vision was slowed two-fold, despite otherwise normal critical period timecourse and receptive field properties. Rapid ocular dominance plasticity was restored by local or systemic enhancement of GABAergic transmission with acute benzodiazepine infusion. Diazepam instead exacerbated a global suppression of slow-wave oscillations during sleep described previously in these mutant mice, which therefore did not account for the rescued plasticity. Rapid ocular dominance shifts closely reflected Kv3.1 gene dosage that prevented prolonged spike discharge of their target pyramidal cells in vivo or the spike amplitude decrement of fast-spiking cells during bouts of high-frequency firing in vitro. Late postnatal expression of this unique channel in fast-spiking interneurons thus subtly regulates the speed of critical period plasticity with implications for mental illnesses.

Progressive myoclonus epilepsy KCNC1 variant causes a developmental dendritopathy.

OBJECTIVE: Mutations in KCNC1 can cause severe neurological dysfunction, including intellectual disability, epilepsy, and ataxia. The Arg320His variant, which occurs in the voltage-sensing domain of the channel, causes a highly penetrant and specific form of progressive myoclonus epilepsy with severe ataxia, designated myoclonus epilepsy and ataxia due to potassium channel mutation (MEAK). KCNC1 encodes the voltage-gated potassium channel KV 3.1, a channel that is important for enabling high-frequency firing in interneurons, raising the possibility that MEAK is associated with reduced interneuronal function. METHODS: To determine how this variant triggers MEAK, we expressed KV 3.1bR320H in cortical interneurons in vitro and investigated the effects on neuronal function and morphology. We also performed electrophysiological recordings of oocytes expressing KV 3.1b to determine whether the mutation introduces gating pore currents. RESULTS: Expression of the KV 3.1bR320H variant profoundly reduced excitability of mature cortical interneurons, and cells expressing these channels were unable to support high-frequency firing. The mutant channel also had an unexpected effect on morphology, severely impairing neurite development and interneuron viability, an effect that could not be rescued by blocking KV 3 channels. Oocyte recordings confirmed that in the adult KV 3.1b isoform, R320H confers a dominant negative loss-of-function effect by slowing channel activation, but does not introduce potentially toxic gating pore currents. SIGNIFICANCE: Overall, our data suggest that, in addition to the regulation of high-frequency firing, KV 3.1 channels play a hitherto unrecognized role in neuronal development. MEAK may be described as a developmental dendritopathy.

Cerebellar Kv3.3 potassium channels activate TANK-binding kinase 1 to regulate trafficking of the cell survival protein Hax-1.

Mutations in KCNC3, which encodes the Kv3.3 potassium channel, cause degeneration of the cerebellum, but exactly how the activity of an ion channel is linked to the survival of cerebellar neurons is not understood. Here, we report that Kv3.3 channels bind and stimulate Tank Binding Kinase 1 (TBK1), an enzyme that controls trafficking of membrane proteins into multivesicular bodies, and that this stimulation is greatly increased by a disease-causing Kv3.3 mutation. TBK1 activity is required for the binding of Kv3.3 to its auxiliary subunit Hax-1, which prevents channel inactivation with depolarization. Hax-1 is also an anti-apoptotic protein required for survival of cerebellar neurons. Overactivation of TBK1 by the mutant channel leads to the loss of Hax-1 by its accumulation in multivesicular bodies and lysosomes, and also stimulates exosome release from neurons. This process is coupled to activation of caspases and increased cell death. Our studies indicate that Kv3.3 channels are directly coupled to TBK1-dependent biochemical pathways that determine the trafficking of cellular constituents and neuronal survival.

A high-affinity, partial antagonist effect of 3,4-diaminopyridine mediates action potential broadening and enhancement of transmitter release at NMJs.

3,4-Diaminopyridine (3,4-DAP) increases transmitter release from neuromuscular junctions (NMJs), and low doses of 3,4-DAP (estimated to reach ∼1 µM in serum) are the Food and Drug Administration (FDA)-approved treatment for neuromuscular weakness caused by Lambert-Eaton myasthenic syndrome. Canonically, 3,4-DAP is thought to block voltage-gated potassium (Kv) channels, resulting in prolongation of the presynaptic action potential (AP). However, recent reports have shown that low millimolar concentrations of 3,4-DAP have an off-target agonist effect on the Cav1 subtype ("L-type") of voltage-gated calcium (Cav) channels and have speculated that this agonist effect might contribute to 3,4-DAP effects on transmitter release at the NMJ. To address 3,4-DAP's mechanism(s) of action, we first used the patch-clamp electrophysiology to characterize the concentration-dependent block of 3,4-DAP on the predominant presynaptic Kv channel subtypes found at the mammalian NMJ (Kv3.3 and Kv3.4). We identified a previously unreported high-affinity (1-10 µM) partial antagonist effect of 3,4-DAP in addition to the well-known low-affinity (0.1-1 mM) antagonist activity. We also showed that 1.5-µM DAP had no effects on Cav1.2 or Cav2.1 current. Next, we used voltage imaging to show that 1.5- or 100-µM 3,4-DAP broadened the AP waveform in a dose-dependent manner, independent of Cav1 calcium channels. Finally, we demonstrated that 1.5- or 100-µM 3,4-DAP augmented transmitter release in a dose-dependent manner and this effect was also independent of Cav1 channels. From these results, we conclude that low micromolar concentrations of 3,4-DAP act solely on Kv channels to mediate AP broadening and enhance transmitter release at the NMJ.