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Glucagon-like peptide-1 (GLP-1) is an endogenous gut hormone and a key regulator in maintaining glucose homeostasis by stimulating insulin secretion. Its natural cleavage product GLP-1 (9-36), used to be considered a "bio-inactive" metabolite mainly because of its lack of insulinotropic effects and low affinity for GLP-1 receptors, possesses unique properties such as anti-oxidant and cardiovascular protection. Little is known about the role of GLP-1 (9-36) in central nervous system. Here we report that chronic, systemic application of GLP-1 (9-36) in adult mice facilitated both the induction and maintenance phases of hippocampal long-term potentiation (LTP), a major form of synaptic plasticity. In contrast, spatial learning and memory, as assessed by the Morris water maze test, was not altered by GLP-1 (9-36) administration. At the molecular level, GLP-1 (9-36) reduced protein levels of the potassium channel Kv4.2 in hippocampus, which is linked to elevated dendritic membrane excitability. Moreover, GLP-1(9-36) treatment inhibited phosphorylation of mRNA translational factor eEF2, which is associated with increased capacity for de novo protein synthesis. Finally, we showed that the LTP-enhancing effects by GLP-1 (9-36) treatment in vivo were blunted by application of exendin(9-39)amide [EX(9-39)], the GLP-1 receptor (GLP-1R) antagonist, suggesting its role as a GLP-1R agonist. These findings demonstrate that GLP-1 (9-36), which was considered a "bio-inactive" peptide, clearly exerts physiological effects on neuronal plasticity in the hippocampus, a brain region critical for learning and memory.

α-Synuclein overexpression (ASOX) drives the formation of toxic aggregates in neurons vulnerable in Parkinson's disease (PD), including dopaminergic neurons of the substantia nigra (SN) and cholinergic neurons of the dorsal motor nucleus of the vagus (DMV). Just as these populations differ in when they exhibit α-synucleinopathies during PD pathogenesis, they could also differ in their physiological responses to ASOX. An ASOX-mediated hyperactivity of SN dopamine neurons, which was caused by oxidative dysfunction of Kv4.3 potassium channels, was recently identified in transgenic (A53T-SNCA) mice overexpressing mutated human α-synuclein. Noting that DMV neurons display extensive α-synucleinopathies earlier than SN dopamine neurons while exhibiting milder cell loss in PD, we aimed to define the electrophysiological properties of DMV neurons inA53T-SNCAmice. We found that DMV neurons maintain normal firing rates in response to ASOX. Moreover, Kv4.3 channels in DMV neurons exhibit no oxidative dysfunction in theA53T-SNCAmice, which could only be recapitulated in wild-type mice by glutathione dialysis. Two-photon imaging of redox-sensitive GFP corroborated the finding that mitochondrial oxidative stress was diminished in DMV neurons in theA53T-SNCAmice. This reduction in oxidative stress resulted from a transcriptional downregulation of voltage-activated (Cav) calcium channels in DMV neurons, which led to a reduction in activity-dependent calcium influx via Cav channels. Thus, ASOX induces a homeostatic remodeling with improved redox signaling in DMV neurons, which could explain the differential vulnerability of SN dopamine and DMV neurons in PD and could promote neuroprotective strategies that emulate endogenous homeostatic responses to ASOX (e.g., stressless pacemaking) in DMV neurons.SIGNIFICANCE STATEMENTOverexpression of mutant α-synuclein causes Parkinson's disease, presumably by driving neurodegeneration in vulnerable neuronal target populations. However, the extent of α-synuclein pathology (e.g., Lewy bodies) is not directly related to the degree of neurodegeneration across various vulnerable neuronal populations. Here, we show that, in contrast to dopamine neurons in the substantia nigra, vagal motoneurons do not enhance their excitability and oxidative load in response to chronic mutant α-synuclein overexpression. Rather, by downregulating their voltage-activated calcium channels, vagal motoneurons acquire a stressless form of pacemaking that diminishes mitochondrial and cytosolic oxidative stress. Emulating this endogenous adaptive response to α-synuclein overexpression could lead to novel strategies to protect dopamine neurons and perhaps delay the onset of Parkinson's disease.

Leptin, is a 16 kDa pleiotropic peptide not only primary secreted by adipocytes, but also produced by other tissues, including the heart. Evidence indicates that leptin may have either adverse or beneficial effects on the heart. To obtain further insights, in the present study, we analysed the effect of leptin treatment on cardiac function, [Ca2+]i handling and cellular electrophysiology, which participate in the genesis of pump failure and related arrhythmias, both in control mice and in mice subjected to chronic pressure‐overload by transverse aorta constriction (TAC). Three weeks after surgery, animals received either leptin (0.36 mg kg–1 day–1) or vehicle via osmotic minipumps for 3 weeks. Echocardiographic measurements showed that, although leptin treatment was deleterious on cardiac function in sham, leptin had a cardioprotective effect following TAC. [Ca2+]i transient in cardiomyocytes followed similar pattern. Patch clamp experiments showed prolongation of action potential duration (APD) in TAC and leptin‐treated sham animals, whereas, following TAC, leptin reduced the APD towards control values. APD variations were associated with decreased transient outward potassium current and Kv4.2 and KChIP2 protein expression. TAC myocytes showed a higher incidence of triggered activities and spontaneous Ca2+ waves. These proarrhythmic manifestations, related to Ca2+/calmodulin‐dependent protein kinase II and ryanodine receptor phosphorylation, were reduced by leptin. The results of the present study demonstrate that, although leptin treatment was deleterious on cardiac function in control animals, leptin had a cardioprotective effect following TAC, normalizing cardiac function and reducing arrhythmogeneity at the cellular level., SAF‐2010‐16377 and SAF2014‐57190R from Ministerio de Economía y Competitividad (MINECO); Agence Nationale de la Recherche (ANR‐13‐BSV1‐0023‐01 and ANR‐15‐CE14‐0005); Fondation pour la recherche médicale (Programme cardiovasculaire 2011); CORDDIM‐Région Ile de France (Équipement jeune équipe 2010; Petit et moyen équipement 2011); ISCIII PI14/01078 and Fondos Feder. The laboratory UMR‐S 1180 is a member of the Laboratory of Excellence LERMIT supported by a grant from the Agence Nationale de la Recherche (ANR‐10‐LABX‐33). NGH was a FPU fellow of the Spanish Ministry of Education.

The transient outward current (Ito) in cardiomyocytes is largely mediated by Kv4 channels associated with Kv Channel Interacting Protein 2 (KChIP2). A knockout model has documented the critical role of KChIP2 in Ito expression. The present study was conducted to characterize in both sexes the dependence of Ito properties, including current magnitude, inactivation kinetics, recovery from inactivation and voltage dependence of inactivation, on the number of functional KChIP2 alleles. For this purpose we performed whole-cell patch-clamp experiments on isolated left ventricular cardiomyocytes from male and female mice which had different KChIP2 genotypes; i.e., wild-type (KChIP2+/+), heterozygous knockout (KChIP2+/-) or complete knockout of KChIP2 (KChIP2-/-). We found in both sexes a KChIP2 gene dosage effect (i.e., a proportionality between number of alleles and phenotype) on Ito magnitude, however, concerning other Ito properties, KChIP2+/- resembled KChIP2+/+. Only in the total absence of KChIP2 (KChIP2-/-) we observed a slowing of Ito kinetics, a slowing of recovery from inactivation and a negative shift of a portion of the voltage dependence of inactivation. In a minor fraction of KChIP2-/- myocytes Ito was completely lost. The distinct KChIP2 genotype dependences of Ito magnitude and inactivation kinetics, respectively, seen in cardiomyocytes were reproduced with two-electrode voltage-clamp experiments on Xenopus oocytes expressing Kv4.2 and different amounts of KChIP2. Our results corroborate the critical role of KChIP2 in controlling Ito properties. They demonstrate that the Kv4.2/KChIP2 interaction in cardiomyocytes is highly dynamic, with a clear KChIP2 gene dosage effect on Kv4 channel surface expression but not on inactivation gating.

Background Hydrogen sulfide (H2S) has been shown to have an excitatory effect on gastric motility, but the underlying molecular mechanism is unclear. In this study, we aimed to investigate the possible targets of H2S and determine how H2S affects its target proteins during H2S-induced contraction. Methods Patch-clamp and potentiometric fluorescence dye were utilized to measure the electrophysiological changes. The Biotin-switch assay was utilized to detect the protein S-sulfhydration. The isometric tension measurement was conducted too. Key Results Exogenous H2S enhanced the tonic contraction of gastric antral smooth muscle, and voltage-dependent potassium channel (KV) blocker and Dithiothreitol (DTT, a reducing agent) abolished the excitatory effect of NaHS. Exogenous H2S inhibited the fast inactivation component of the voltage-dependent potassium channel current (IKVfast) in isolated gastric antral smooth muscle cells. H2S inhibited the KV4.3 current in H293 cells with heterologous expression of KV4.3, but did not inhibit the KV4.1 and KV4.2 currents, which together contribute greatly to IKVfast. NaHS significantly decreased the membrane potential in cultured gastric smooth muscle cells, but the NaHS-induced depolarization was suppressed by knockdown of KV4.3 and N-ethylamaleimide (NEM), a free thiol group blocker. In addition, NaHS sulfhydrated KV4.3 in H293 cells and in gastric smooth muscle tissue. However, this S-sulfhydration was inhibited by NEM and DTT. Meanwhile the NaHS-induced inhibition of IKVfast and KV4.3 was also blocked by NEM and DTT. Conclusions & Inferences These results suggest that exogenous H2S sulfhydrates KV4.3 to decrease the membrane potential, thereby enhancing the basal tension of gastric antral smooth muscle.

Parkinson disease (PD) is an α-synucleinopathy resulting in the preferential loss of highly vulnerable dopamine (DA) substantia nigra (SN) neurons. Mutations (e.g., A53T) in the α-synuclein gene (SNCA) are sufficient to cause PD, but the mechanism of their selective action on vulnerable DA SN neurons is unknown. In a mouse model overexpressing mutant α-synuclein (A53T-SNCA), we identified a SN-selective increase ofin vivofiring frequencies in DA midbrain neurons, which was not observed in DA neurons in the ventral tegmental area. The selective and age-dependent gain-of-function phenotype of A53T-SCNA overexpressing DA SN neurons was in part mediated by an increase of their intrinsic pacemaker frequency caused by a redox-dependent impairment of A-type Kv4.3 potassium channels. This selective enhancement of “stressful pacemaking” of DA SN neuronsin vivodefines a functional response to mutant α-synuclein that might be useful as a novel biomarker for the “DA system at risk” before the onset of neurodegeneration in PD.

Haloperidol is commonly used in clinical practice to treat acute and chronic psychosis, but it also has been associated with adverse cardiovascular events. We investigated the effects of haloperidol on Kv4.3 currents stably expressed in CHO cells using a whole-cell patch-clamp technique. Haloperidol did not significantly inhibit the peak amplitude of Kv4.3, but accelerated the decay rate of inactivation of Kv4.3 in a concentration-dependent manner. Thus, the effects of haloperidol on Kv4.3 were estimated from the integral of the Kv4.3 currents during the depolarization pulse. The Kv4.3 was decreased by haloperidol in a concentration-dependent manner with an IC50 value of 3.6 μM. Haloperidol accelerated the decay rate of Kv4.3 inactivation and activation kinetics in a concentration-dependent manner, thereby decreasing the time-to-peak. Haloperidol shifted the voltage dependence of the steady-state activation and inactivation of Kv4.3 in a hyperpolarizing direction. Haloperidol also caused an acceleration of the closed-state inactivation of Kv4.3. Haloperidol produced a use-dependent block of Kv4.3, which was accompanied by a slowing of recovery from the inactivation of Kv4.3. These results suggest that haloperidol blocks Kv4.3 by both interacting with the open state of Kv4.3 channels during depolarization and accelerating the closed-state inactivation at subthreshold membrane potentials.

Background— The PR interval on the ECG reflects atrial depolarization and atrioventricular nodal delay which can be partially differentiated by P wave duration and PR segment, respectively. Genome-wide association studies have identified several genetic loci for PR interval, but it remains to be determined whether this is driven by P wave duration, PR segment, or both. Methods and Results— We replicated 7 of the 9 known PR interval loci in 16 468 individuals of European ancestry. Four loci were unambiguously associated with PR segment, while the others were shared for P wave duration and PR segment. Next, we performed a genome-wide analysis on P wave duration and PR segment separately and identified 5 novel loci. Single-nucleotide polymorphisms in KCND3 ( P =8.3×10 –11 ) and FADS2 ( P =2.7×10 –8 ) were associated with P wave duration, whereas single-nucleotide polymorphisms near IL17D (P =2.3×10 –8 ), in EFHA1 ( P =3.3×10 −10 ), and in LRCH1 ( P =2.1×10 –8 ) were associated with PR segment. Analysis on DNA elements indicated that genome-wide significant single-nucleotide polymorphisms were enriched at genomic regions suggesting active gene transcription in the human right atrium. Quantitative polymerase chain reaction showed that genes were significantly higher expressed in the right atrium and atrioventricular node compared with left ventricle ( P =5.6×10 –6 ). Conclusions— Genetic associations of PR interval seem to be mainly driven by genetic determinants of the PR segment. Some of the PR interval associations are strengthened by a directional consistent effect of genetic determinants of P wave duration. Through genome-wide association we also identified genetic variants specifically associated with P wave duration which might be relevant for cardiac biology.

SNX-482, a peptide toxin isolated from tarantula venom, has become widely used as an inhibitor of Cav2.3 voltage-gated calcium channels. Unexpectedly, we found that SNX-482 dramatically reduced the A-type potassium current in acutely dissociated dopamine neurons from mouse substantia nigra pars compacta. The inhibition persisted when calcium was replaced by cobalt, showing that it was not secondary to a reduction of calcium influx. Currents from cloned Kv4.3 channels expressed in HEK-293 cells were inhibited by SNX-482 with an IC50 of

In the nucleus of the tractus solitarii (NTS), a large proportion of neurones express transient A-type potassium currents (I KA) having deep influence on the fidelity of the synaptic transmission of the visceral primary afferent inputs to second-order neurones. Up to now, the strong impact of I KA within the NTS was considered to result exclusively from its variation in amplitude, and its molecular correlate(s) remained unknown. In order to identify which Kv channels underlie I KA in NTS neurones, the gating properties and the pharmacology of this current were determined using whole cell patch clamp recordings in slices. Complementary information was brought by immunohistochemistry. Strikingly, two neurone subpopulations characterized by fast or slow inactivation time courses (respectively about 50 and 200 ms) were discriminated. Both characteristics matched those of the Kv4 channel subfamily. The other gating properties, also matching the Kv4 channel ones, were homogeneous through the NTS. The activation and inactivation occurred at membrane potentials around the threshold for generating action potentials, and the time course of recovery from inactivation was rapid. Pharmacologically, I KA in NTS neurones was found to be resistant to tetraethylammonium (TEA), sea anemone toxin blood-depressing substance (BDS) and dendrotoxin (DTX), whereas Androctonus mauretanicus mauretanicus toxin 3 (AmmTX3), a scorpion toxin of the α-KTX 15 family that has been shown to block all the members of the Kv4 family, inhibited 80 % of I KA irrespectively of its inactivation time course. Finally, immunohistochemistry data suggested that, among the Kv4 channel subfamily, Kv4.3 is the prevalent subunit expressed in the NTS.

Lipid rafts are cholesterol-enriched microdomains of the cell membrane. Here we investigate the localization of the pore forming K+-channel α-subunit Kv4.2 and the β-subunit KChIP2, underlying the transient outward K+ current (I to), in lipid rafts in left ventricular myocytes. Furthermore, we explored the impact of membrane cholesterol depletion (using 20 mM methyl-beta-cyclodextrin (MBCD)) on K+ outward currents. Cholesterol-saturated MBCD (20 mM) served as control. Myocytes were isolated from the left ventricular free wall of Wistar rats. The Triton X-100 (4 °C) insoluble fraction of whole cell protein was analyzed by sucrose density gradient centrifugation followed by Western blot. Kv4.2 and KChIP2 were partially detected in low-density fractions (lipid rafts). MBCD treatment (5 min) resulted in a shift of Kv4.2 and KChIP2 towards high-density fractions. K+ currents were assessed by whole-cell patch-clamp. MBCD treatment resulted in a 29 ± 3 % decrease in I to (20.0 ± 1.6pApF-1 vs. 28.5 ± 2.0pApF-1, n = 15, p

The distribution and functional presence of three voltage-dependent potassium channels, K v 2.1, K v 3.4, K v 4.3, respectively, were studied in the central nervous system of the snail Helix pomatia by immunohistochemical and electrophysiological methods. Cell clusters displaying immunoreactivity for the different channels were observed in all parts of the CNS, although their localization and number partly varied. Differences were also found in their intracellular, perikaryonal and axonal localization, as well as in their presence in non-neuronal tissues nearby the CNS, such as the perineurium and the aorta wall. At ultrastructural level K v 4.3 channel immunolabeling was observed in axon profiles containing large 80–100 nm granular vesicles. Blotting analyses revealed specific signals for the K v 2.1, K v 3.4 and K v 4.3 channels, confirming the presence of the channels in the Helix CNS. Voltage-clamp recordings proved that outward currents obtained from neurons displaying K v 3.4 or K v 4.3 immunoreactivity contained transient components while K v 2.1 immunoreactive cells were characterized by delayed currents. The distribution of the K + -channels containing neurons suggests specific roles in intercellular signaling processes in the Helix CNS, most probably related to well-defined, partly local events. The cellular localization of the K + -channels studied supports their involvement in both pre- and postsynaptic events at perikaryonal and axonal levels.

Transient outward K+ current (I(to)) plays a crucial role in the early phase of cardiac action potential repolarization. Kv4.3 K(+) channel is an important component of I(to). The function and expression of Kv4.3 K(+) channel decrease in variety of heart diseases, especially in heart hypertrophy/heart failure. Int his review, we summarized the changes of cardiac Kv4.3 K(+) channel in heart diseases and discussed the potential role of Kv4.3 K(+) channel in heart hypertrophy/heart failure. In heart hypertrophy/heart failure of mice and rats, down regulation of Kv4.3 K(+) channel leads to prolongation of action potential duration (APD), which is associated with increased [Ca(2+)](I), activation of calcineurin and heart hypertrophy/heart failure.However, in canine and human, Kv4.3 K(+) channel does not play a major role in setting cardiac APD. So, in addition to Kv4.3 K(+) channel/APD/[Ca(2+)](I) pathway, there exits another mechanism of Kv4.3 K(+) channel in heart hypertrophy and heart failure: downregulation of Kv4.3 K(+) channels leads to CaMKII dissociation from Kv4.3–CaMKII complex and subsequent activation of the dissociated CaMKII , which induces heart hypertrophy/heart failure. Upregulation of Kv4.3K(+) channel inhibits CaMKII activation and its related harmful consequences. We put forward a new point-of-view that Kv4.3 K(+) channel is involved in heart hypertrophy/heart failure independently of its electric function, and drugs inhibiting or upregulating Kv4.3 K(+) channel might be potentially harmful or beneficial to hearts through CaMKII.

To identify genes that promote facial skin youthfulness (SY), a genome-wide association study on an Ashkenazi Jewish discovery group (n=428) was performed using Affymetrix 6.0 Single-Nucleotide Polymorphism (SNP) Array. After SNP quality controls, 901,470 SNPs remained for analysis. The eigenstrat method showed no stratification. Cases and controls were identified by global facial skin aging severity including intrinsic and extrinsic parameters. Linear regression adjusted for age and gender, with no significant differences in smoking history, body mass index, menopausal status, or personal or family history of centenarians. Six SNPs met the Bonferroni threshold with P allele

Diabetes mellitus increases the risk for cardiac dysfunction, heart failure, and sudden death. The wide array of neurohumoral changes associated with diabetes pose a challenge to understanding the roles of specific pathways that alter cardiac function. Here, we use a mouse model with cardiomyocyte-restricted deletion of insulin receptors (CIRKO, cardiac-specific insulin receptor knockout) to study the specific effects of impaired cardiac insulin signaling on ventricular repolarization, independent of the generalized metabolic derangements associated with diabetes. Impaired insulin action caused a reduction in mRNA and protein expression of several key K+ channels that dominate ventricular repolarization. Specifically, components of transient outward K+ current fast component ( Ito,fast; Kv4.2 and KChiP2) were reduced, consistent with a reduction in the amplitude of Ito,fast in isolated left ventricular CIRKO myocytes, compared with littermate controls. The reduction in Ito,fast resulted in ventricular action potential prolongation and prolongation of the QT interval on the surface ECG. These results support the notion that the lack of insulin signaling in the heart is sufficient to cause the repolarization abnormalities described in other animal models of diabetes.

The specific binding of auxiliary Kv channel-interacting proteins (KChIPs) to the N terminus of Kv4 pore-forming α-subunits results in modulation of gating properties, surface expression, and subunit assembly of Kv4 channels. However, the interactions between KChIPs and Kv4 remain elusive. Thus, affinity capillary electrophoresis (ACE) was employed to quantitatively evaluate the interactions between KChIPs and Kv4.3 N terminus (KvN) and between KChIP4a/related mutants and Ca2+ for the first time. The mobility ratio, derivatives calculated from the mobility shift method, was used to deduce the binding constants (Kb). As a result, the binding constants for KChIP4a/KvN and KChIP1/KvN complexes were (8.32 ± 1.66) × 106 L mol–1 and (5.26 ± 0.71) × 106 L mol–1, respectively. In addition, in the presence of calcium (10 μmol L–1), the binding constant of KChIP4a/KvN increased to (6.72 ± 1.66) × 107 L mol–1. In addition, the binding constant of KChIP4a with Ca2+ was (7.1 ± 1.5) × 107 L mol–1. Besides, studies on the effect of truncated mutants revealed that the third EF hand of KChIP4a was related to high-affinity binding with Ca2+, and the integrity of the molecular structure of KChIP4a was important for Ca2+ binding. This method profits from small samples, rapid analysis, and simple operation without being time-consuming.

The present study aimed to determine the effect of chronic treatment with losartan on transient outward potassium current (Ito) and the expression levels of potassium voltage-gated channel subfamily D members 2 and 3 (Kv4.2 and 3) and voltage-gated potassium channel-interacting protein 2 (KChIP2) in rats. Spontaneously hypertensive (SH) rats and Wistar-Kyoto (WKY) rats were used in the study. The rats were divided into lo-SH and SH groups and los-WKY and WKY groups, respectively. Ito was recorded and expression levels of Kv4.2, Kv4.3 and KChIP2 were measured by western blot analysis and quantitative polymerase chain reaction. Ito current density was smaller in SH compared with WKY, los-WKY and los-SH groups (P

Numerous studies and case reports show comorbidity of autism and epilepsy, suggesting some common molecular underpinnings of the two phenotypes. However, the relationship between the two, on the molecular level, remains unclear. Here, whole exome sequencing was performed on a family with identical twins affected with autism and severe, intractable seizures.A de novo variant was identified in theKCND2 gene, which encodes the Kv4.2 potassium channel. Kv4.2 is a major pore-forming subunit in somatodendritic subthreshold A-type potassium current (ISA) channels. The de novo mutation p.Val404Met is novel and occurs at a highly conserved residue within the C-terminal end of the transmembrane helix S6 region of the ion permeation pathway. Functional analysis revealed the likely pathogenicity of the variant in that the p.Val404Met mutant construct showed significantly slowed inactivation, either by itself or after equimolar coexpression with the wild-type Kv4.2 channel construct consistent with a dominant effect. Further, the effect of the mutation on closed-state inactivation was evident in the presence of auxiliary subunits that associate with Kv4 subunits to form ISA channels in vivo. Discovery ofa functionally relevant novel de novo variant, coupled with physiological evidence that the mutant protein disrupts potassium current inactivation, strongly supports KCND2 as the causal gene for epilepsy in this family. Interaction of KCND2 with other genes implicated in autism and the role of KCND2 in synaptic plasticity provide suggestive evidence of an etiological role in autism. © The Author 2014. Published by Oxford University Press.

The neuropathological features associated with Alzheimer’s disease (AD) include the presence of extracellular amyloid-βpeptide-containing plaques and intracellular tau positive neurofibrillary tangles and the loss of synapses and neurons in defined regions of the brain. Dipeptidyl peptidase 10 (DPP10) is a protein that facilitates Kv4 channel surface expression and neuronal excitability. This study aims to explore DPP10789protein distribution in human brains and its contribution to the neurofibrillary pathology of AD and other tauopathies. Immunohistochemical analysis revealed predominant neuronal staining of DPP10789in control brains, and the CA1 region of the hippocampus contained strong reactivity in the distal dendrites of the pyramidal cells. In AD brains, robust DPP10789reactivity was detected in neurofibrillary tangles and plaque-associated dystrophic neurites, most of which colocalized with the doubly phosphorylated Ser-202/Thr-205 tau epitope. DPP10789positive neurofibrillary tangles and plaque-associated dystrophic neurites also appeared in other neurodegenerative diseases such as frontotemporal lobar degeneration, diffuse Lewy body disease, and progressive supranuclear palsy. Occasional DPP10789positive neurofibrillary tangles and neurites were seen in some aged control brains. Western blot analysis showed both full length and truncated DPP10789fragments with the later increasing significantly in AD brains compared to control brains. Our results suggest that DPP10789is involved in the pathology of AD and other neurodegenerative diseases.

Summary Small conductance Ca 2+ -activated K + (SK) channels and voltage-gated A-type K v 4 channels shape dendritic excitatory postsynaptic potentials (EPSPs) in hippocampal CA1 pyramidal neurons. Synaptically evoked Ca 2+ influx through N-methyl-D-aspartate receptors (NMDARs) activates spine SK channels, reducing EPSPs and the associated spine head Ca 2+ transient. However, results using glutamate uncaging implicated Ca 2+ influx through SNX-482-sensitive (SNX-sensitive) Ca v 2.3 (R-type) Ca 2+ channels as the Ca 2+ source for SK channel activation. The present findings show that, using Schaffer collateral stimulation, the effects of SNX and apamin are not mutually exclusive and SNX increases EPSPs independent of SK channel activity. Dialysis with 1,2-bis(o-aminophenoxy)ethane-N'N'N'-tetraacetic acid (BAPTA), application of 4-Aminopyridine (4-AP), expression of a K v 4.2 dominant negative subunit, and dialysis with a KChIPs antibody occluded the SNX-induced increase of EPSPs. The results suggest two distinct Ca 2+ signaling pathways within dendritic spines that link Ca 2+ influx through NMDARs to SK channels and Ca 2+ influx through R-type Ca 2+ channels to K v 4.2-containing channels.

Drug-induced proarrhythmia is a major safety issue in drug development. Developing sensitive in vitro assays that can predict drug-induced cardiotoxicity in humans has been a challenge of toxicology research for decades. Recently, induced pluripotent stem cell-derived human cardiomyocytes (iPSC-hCMs) have become a promising model because they largely replicate the electrophysiological behavior of human ventricular cardiomyocytes. Patient-specific iPSC-hCMs have been proposed for personalized cardiac drug selection and adverse drug response prediction; however, many procedures are involved in cardiomyocytes differentiation and purification process, which may result in large line-to-line and batch-to-batch variations. Here, we examined the purity, cardiac ion channel gene expression profile, and electrophysiological response of 3 batches of iPSC-hCMs from each of 2 major cell suppliers. We found that iPSC-hCMs from both vendors had similar purities. Most of the cardiac ion channel genes were expressed uniformly among different batches of iCells, while larger variations were found in Cor.4U cells, particularly in the expression of CACNA1C, KCND2, and KCNA5 genes, which could underlie the differences in baseline beating rate (BR) and field potential duration (FPD) measurements. Although, in general, the electrophysiological responses of different batches of cells to Na+, Ca2+, Ikr, and Iks channel blockers were similar, with Ikr blocker-induced proarrhythmia, the sensitivities were depended on baseline BR and FPD values: cells that beat slower had longer FPD and greater sensitivity to drug-induced proarrhythmia. Careful evaluation of the performance of iPSC-hCMs and methods of data analysis is warranted for shaping regulatory standards in qualifying iPSC-hCMs for drug safety testing.

Rapidly activating and inactivating A-type K+currents (IA) encoded by Kv4.2 and Kv4.3 pore-forming (α) subunits of the Kv4 subfamily are key regulators of neuronal excitability. Previous studies have suggested a role for Kv4.1 α-subunits in regulating the firing properties of mouse suprachiasmatic nucleus (SCN) neurons. To test this, we utilized an RNA-interference strategy to knockdown Kv4.1, acutely and selectively, in the SCN. Current-clamp recordings revealed that thein vivoknockdown of Kv4.1 significantly (p< 0.0001) increased mean ± SEM repetitive firing rates in SCN neurons during the day (6.4 ± 0.5 Hz) and at night (4.3 ± 0.6 Hz), compared with nontargeted shRNA-expressing SCN neurons (day: 3.1 ± 0.5 Hz; night: 1.6 ± 0.3 Hz). IAwas also significantly (p< 0.05) reduced in Kv4.1-targeted shRNA-expressing SCN neurons (day: 80.3 ± 11.8 pA/pF; night: 55.3 ± 7.7 pA/pF), compared with nontargeted shRNA-expressing (day: 121.7 ± 10.2 pA/pF; night: 120.6 ± 16.5 pA/pF) SCN neurons. The magnitude of the effect of Kv4.1-targeted shRNA expression on firing rates and IAwas larger at night. In addition, Kv4.1-targeted shRNA expression significantly (p< 0.001) increased mean ± SEM nighttime input resistance (Rin; 2256 ± 166 MΩ), compared to nontargeted shRNA-expressing SCN neurons (1143 ± 93 MΩ). Additional experiments revealed that acute knockdown of Kv4.1 significantly (p< 0.01) shortened, by ∼0.5 h, the circadian period of spontaneous electrical activity, clock gene expression and locomotor activity demonstrating a physiological role for Kv4.1-encoded IAchannels in regulating circadian rhythms in neuronal excitability and behavior.

The present study investigates the physiological role of Kvβ1 subunit for sensing pyridine nucleotide (NADH/NAD+) changes in the heart. We used Kvβ1.1 knockout (KO) or wild-type (WT) mice and established that Kvβ1.1 preferentially binds with Kv4.2 and senses the pyridine nucleotide changes in the heart. The cellular action potential duration (APD) obtained from WT cardiomyocytes showed longer APDs with lactate perfusion, which increases intracellular NADH levels, while the APDs remained unaltered in the Kvβ1.1 KO. Ex vivo monophasic action potentials showed a similar response, in which the APDs were prolonged in WT mouse hearts with lactate perfusion; however, the Kvβ1.1 KO mouse hearts did not show APD changes upon lactate perfusion. COS-7 cells coexpressing Kv4.2 and Kvβ1.1 were used for whole cell patch-clamp recordings to evaluate changes caused by NADH (lactate). These data reveal that Kvβ1.1 is required in the mediated inactivation of Kv4.2 currents, when NADH (lactate) levels are increased. In vivo, isoproterenol infusion led to increased NADH in the heart along with QTc prolongation in wild-type mice; regardless of the approach, our data show that Kvβ1.1 recognizes NADH changes and modulates Kv4.2 currents affecting AP and QTc durations. Overall, this study uses multiple levels of investigation, including the heterologous overexpression system, cardiomyocyte, ex vivo, and ECG, and clearly depicts that Kvβ1.1 is an obligatory sensor of NADH/NAD changes in vivo, with a physiological role in the heart. NEW & NOTEWORTHY Cardiac electrical activity is mediated by ion channels, and Kv4.2 plays a significant role, along with its binding partner, the Kvβ1.1 subunit. In the present study, we identify Kvβ1.1 as a sensor of pyridine nucleotide changes and as a modulator of Kv4.2 gating, action potential duration, and ECG in the mouse heart.

In vertebrate neurons, the axon initial segment (AIS) is specialized for action potential initiation. It is organized by a giant 480 Kd variant of ankyrin G (AnkG) that serves as an anchor for ion channels and is required for a plasma membrane diffusion barrier that excludes somatodendritic proteins from the axon. An unusually long exon required to encode this 480Kd variant is thought to have been inserted only recently during vertebrate evolution, so the giant ankyrin-based AIS scaffold has been viewed as a vertebrate adaptation for fast, precise signaling. We re-examined AIS evolution through phylogenomic analysis of ankyrins and by testing the role of ankyrins in proximal axon organization in a model multipolar Drosophila neuron (ddaE). We find giant isoforms of ankyrin in all major bilaterian phyla, and present evidence in favor of a single common origin for giant ankyrins and the corresponding long exon in a bilaterian ancestor. This finding raises the question of whether giant ankyrin isoforms play a conserved role in AIS organization throughout the Bilateria. We examined this possibility by looking for conserved ankyrin-dependent AIS features in Drosophila ddaE neurons via live imaging. We found that ddaE neurons have an axonal diffusion barrier proximal to the cell body that requires a giant isoform of the neuronal ankyrin Ank2. Furthermore, the potassium channel shal concentrates in the proximal axon in an Ank2-dependent manner. Our results indicate that the giant ankyrin-based cytoskeleton of the AIS may have evolved prior to the radiation of extant bilaterian lineages, much earlier than previously thought., Author Summary The axon initial segment (AIS) is currently thought to be a distinguishing feature of vertebrate neurons that adapts them for rapid, precise signaling. It serves as a hub for the regulation of neuronal excitability as the site of action potential initiation and also acts as the boundary between the highly-specialized axon and the rest of the cell. Here we show that the giant ankyrins that structurally organize the AIS, and were thought to be vertebrate-specific, instead have an ancient origin in a bilaterian ancestor. We further show the presence of a giant ankyrin-dependent AIS-like plasma membrane boundary between the axon and soma in a Drosophila sensory neuron. These results suggest that the cytoskeletal backbone for the AIS is not unique to vertebrates, but instead may be an evolutionarily conserved feature of bilaterian neurons.

Voltage-gated potassium (Kv) channels comprise pore-forming α subunits and a multiplicity of regulatory proteins, including the cardiac-expressed and cardiac arrhythmia-linked transmembrane KCNE subunits. After recently uncovering novel, N-terminally extended (L) KCNE3 and KCNE4 isoforms and detecting their transcripts in human atrium, reported here are their functional effects on human cardiac Kv channel α subunits expressed in Xenopus laevis oocytes. As previously reported for short isoforms KCNE3S and KCNE4S, KCNE3L inhibited hERG; KCNE4L inhibited Kv1.1; neither form regulated the HCN1 pacemaker channel. Unlike KCNE4S, KCNE4L was a potent inhibitor of Kv4.2 and Kv4.3; co-expression of cytosolic β subunit KChIP2, which regulates Kv4 channels in cardiac myocytes, partially relieved Kv4.3 but not Kv4.2 inhibition. Inhibition of Kv4.2 and Kv4.3 by KCNE3L was weaker, and its inhibition of Kv4.2 abolished by KChIP2. KCNE3L and KCNE4L also exhibited subunit-specific effects on Kv4 channel complex inactivation kinetics, voltage dependence and recovery. Further supporting the potential physiological significance of the robust functional effects of KCNE4L on Kv4 channels, KCNE4L protein was detected in human atrium, where it co-localized with Kv4.3. The findings establish functional effects of novel human cardiac-expressed KCNE isoforms and further contribute to our understanding of the potential mechanisms influencing cardiomyocyte repolarization.

Widely expressed in the adult central nervous system, the cellular prion protein (PrP(C)) is implicated in a variety of processes, including neuronal excitability. Dipeptidyl aminopeptidase-like protein 6 (DPP6) was first identified as a PrP(C) interactor using in vivo formaldehyde cross-linking of wild type (WT) mouse brain. This finding was confirmed in three cell lines and, because DPP6 directs the functional assembly of K(+) channels, we assessed the impact of WT and mutant PrP(C) upon Kv4.2-based cell surface macromolecular complexes. Whereas a Gerstmann-Sträussler-Scheinker disease version of PrP with eight extra octarepeats was a loss of function both for complex formation and for modulation of Kv4.2 channels, WT PrP(C), in a DPP6-dependent manner, modulated Kv4.2 channel properties, causing an increase in peak amplitude, a rightward shift of the voltage-dependent steady-state inactivation curve, a slower inactivation, and a faster recovery from steady-state inactivation. Thus, the net impact of wt PrP(C) was one of enhancement, which plays a critical role in the down-regulation of neuronal membrane excitability and is associated with a decreased susceptibility to seizures. Insofar as previous work has established a requirement for WT PrP(C) in the Aβ-dependent modulation of excitability in cholinergic basal forebrain neurons, our findings implicate PrP(C) regulation of Kv4.2 channels as a mechanism contributing to the effects of oligomeric Aβ upon neuronal excitability and viability.

Ventricular arrhythmias account for high mortality in cardiopulmonary patients in intensive care units. Cardiovascular alterations and molecular-level changes in response to the commonly used oxygen treatment remains unknown. In the present study we investigated cardiac hypertrophy and cardiac complications in mice subjected to hyperoxia. Results demonstrate that there is a significant increase in average heart weight to tibia length (22%) in mice subjected to hyperoxia treatment vs. normoxia. Functional assessment was performed in mice subjected to hyperoxic treatment, and results demonstrate impaired cardiac function with decreased cardiac output and heart rate. Staining of transverse cardiac sections clearly demonstrates an increase in the cross-sectional area from hyperoxic hearts compared with control hearts. Quantitative real-time RT-PCR and Western blot analysis indicated differential mRNA and protein expression levels between hyperoxia-treated and control left ventricles for ion channels including Kv4.2 (−2 ± 0.08), Kv2.1 (2.54 ± 0.48), and Scn5a (1.4 ± 0.07); chaperone KChIP2 (−1.7 ± 0.06); transcriptional factors such as GATA4 (−1.5 ± 0.05), Irx5 (5.6 ± 1.74), NFκB1 (4.17 ± 0.43); hypertrophy markers including MHC-6 (2.17 ± 0.36) and MHC-7 (4.62 ± 0.76); gap junction protein Gja1 (4.4 ± 0.8); and microRNA processing enzyme Drosha (4.6 ± 0.58). Taken together, the data presented here clearly indicate that hyperoxia induces left ventricular remodeling and hypertrophy and alters the expression of Kv4.2 and MHC6/7 in the heart.

The contribution of transient outward current ( Ito) to changes in ventricular action potential (AP) repolarization induced by acidosis is unresolved, as is the indirect effect of these changes on calcium handling. To address this issue we measured intracellular pH (pHi), Ito, L-type calcium current ( ICa,L), and calcium transients (CaTs) in rabbit ventricular myocytes. Intracellular acidosis [pHi 6.75 with extracellular pH (pHo) 7.4] reduced Ito by ∼50% in myocytes with both high (epicardial) and low (papillary muscle) Ito densities, with little effect on steady-state inactivation and activation. Of the two candidate α-subunits underlying Ito, human (h)Kv4.3 and hKv1.4, only hKv4.3 current was reduced by intracellular acidosis. Extracellular acidosis (pHo 6.5) shifted Ito inactivation toward less negative potentials but had negligible effect on peak current at +60 mV when initiated from −80 mV. The effects of low pHi-induced inhibition of Ito on AP repolarization were much greater in epicardial than papillary muscle myocytes and included slowing of phase 1, attenuation of the notch, and elevation of the plateau. Low pHi increased AP duration in both cell types, with the greatest lengthening occurring in epicardial myocytes. The changes in epicardial AP repolarization induced by intracellular acidosis reduced peak ICa,L, increased net calcium influx via ICa,L, and increased CaT amplitude. In summary, in contrast to low pHo, intracellular acidosis has a marked inhibitory effect on ventricular Ito, perhaps mediated by Kv4.3. By altering the trajectory of the AP repolarization, low pHi has a significant indirect effect on calcium handling, especially evident in epicardial cells.

In the brain and heart, auxiliary Kv channel-interacting proteins (KChIPs) co-assemble with pore-forming Kv4 α-subunits to form a native K+ channel complex and regulate the expression and gating properties of Kv4 currents. Among the KChIP1–4 members, KChIP4a exhibits a unique N terminus that is known to suppress Kv4 function, but the underlying mechanism of Kv4 inhibition remains unknown. Using a combination of confocal imaging, surface biotinylation, and electrophysiological recordings, we identified a novel endoplasmic reticulum (ER) retention motif, consisting of six hydrophobic and aliphatic residues, 12–17 (LIVIVL), within the KChIP4a N-terminal KID, that functions to reduce surface expression of Kv4-KChIP complexes. This ER retention capacity is transferable and depends on its flanking location. In addition, adjacent to the ER retention motif, the residues 19–21 (VKL motif) directly promote closed-state inactivation of Kv4.3, thus leading to an inhibition of channel current. Taken together, our findings demonstrate that KChIP4a suppresses A-type Kv4 current via ER retention and enhancement of Kv4 closed-state inactivation. Background: Compared with other auxiliary KChIPs that enhance Kv4 current, KChIP4a inhibits Kv4 function. Results: We identified an ER retention motif and an adjacent VKL motif within the KChIP4a N terminus that reduces Kv4.3 surface expression and promotes closed-state inactivation (CSI), respectively. Conclusion: ER retention and CSI enhancement are two distinct mechanisms by which the N terminus of KChIP4a suppresses Kv4 function. Significance: This study provides mechanistic insight into auxiliary KChIP4a-induced inhibition of A-type Kv4 current.

Trazodone, a triazolopyridine antidepressant, is commonly used in the treatment of depression and insomnia. Kv4.3 channels are transiently, and rapidly, inactivating Kv channels that are highly expressed in cardiac myocytes and neurons. To determine the electrophysiological basis for the cardiac and neuronal actions of trazodone, we studied the effects of trazodone on Kv4.3 currents stably expressed in Chinese hamster ovary cells using the whole-cell patch-clamp technique. Trazodone decreased the peak amplitude of Kv4.3 in a concentration-dependent manner with an IC50 of 55.4 μM. Under control conditions, the time course of inactivation of Kv4.3 at +40 mV was fitted to a double exponential function. Trazodone produced a concentration-dependent slowing of the fast and slow components of Kv4.3 inactivation during a voltage step to +40 mV. The inhibition of Kv4.3 by trazodone was voltage independent over the entire voltage range tested. Trazodone shifted the voltage dependence of the steady-state inactivation of Kv4.3 to a hyperpolarizing direction. However, the slope factor of the steady-state inactivation was not affected by trazodone. Under control conditions, the closed-state inactivation of Kv4.3 was fitted to a single exponential function. Trazodone significantly accelerated the closed-state inactivation of Kv4.3. Trazodone produced a weak use-dependent inhibition of Kv4.3 at frequencies of 1 and 2 Hz. m-Chlorophenylpiperazine (m-CPP), a major metabolite of trazodone, inhibited Kv4.3 less potently than trazodone, with an IC50 of 118.6 μM. These results suggest that trazodone preferentially inhibited Kv4.3 by both binding to the closed state and accelerating the closed-state inactivation of the channel.

In human atrial myocytes the transient outward current Ito develops a conspicuous faster inactivation with increasing temperatures. Since β-subunits are known to modulate Ito current kinetics, we hypothesized that the temperature sensitivity of Ito is not only determined by the property of the ion-passing α-subunit Kv4.3 but also by its interaction with accessory β-subunits. We therefore studied the influence of the transmembrane β-subunits KCNE1, KCNE2 and DPP6 on Kv4.3/KChIP2 channels in CHO cells at room temperature and at physiological temperature. Exposure to 37 °C caused a significant acceleration of the channel kinetics, whereas current densities and voltage dependences remained unaltered at 37 °C compared to 23 °C. However, Kv4.3/KChIP2 channels without transmembrane β-subunits showed the strongest temperature sensitivity with considerably increased rates of activation and inactivation at 37 °C. KCNE2 significantly slowed the current kinetics at 37 °C compared to Kv4.3/KChIP2 channels, whereas KCNE1 did not influence the channel properties at both temperatures. Interestingly, the accelerating effects of DPP6 on current kinetics described at 23 °C were diminished at physiological temperature, thus at 37 °C current kinetics became remarkably similar for channel complexes Kv4.3/KChIP2 with and without DPP6 isoforms. A Markov state model was developed on the basis of experimental measurements to simulate the influence of β-subunits on Kv4.3 channel complex at both temperatures. In conclusion, the remarkably fast kinetics of the native Ito at 37 °C could be reproduced by co-expressing Kv4.3, KChIP2, KCNE2 and DPP6 in CHO cells, whereas the high temperature sensitivity of human Ito could be not mimicked.

Aims Atrial fibrillation (AF) is the most common cardiac arrhythmia, and early-onset lone AF has been linked to mutations in genes encoding ion channels. Mutations in the pore forming subunit KV4.3 leading to an increase in the transient outward potassium current ( I to) have previously been associated with the Brugada Syndrome. Here we aim to determine if mutations in KV4.3 or in the auxiliary subunit K+ Channel-Interacting Protein (KChIP) 2 are associated with early-onset lone AF. Methods and results Two hundred and nine unrelated early-onset lone AF patients (

Background/Aims: In diabetic ventricular myocytes, transient outward potassium current (I-to) amplitude is severely reduced because of the impaired catecholamine release that characterizes diabetic autonomic neuropathy. Sympathetic nervous system exhibits a trophic effect on I-to since incubation of myocytes with noradrenaline restores current amplitude via beta-adrenoceptor (beta AR) stimulation. Here, we investigate the intracellular signalling pathway though which incubation of diabetic cardiomyocytes with the beta AR agonist isoproterenol recovers I-to amplitude to normal values. Methods: Experiments were performed in ventricular myocytes isolated from streptozotocin-diabetic rats. I-to current was recorded by using the patch-clamp technique. Kv4 channel expression was determined by immunofluorescence. Protein-protein interaction was determined by coimmunoprecipitation. Results: Stimulation of beta AR activates first a G alpha s protein, adenylyl cyclase and Protein Kinase A. PKA-phosphorylated receptor then switches to the G alpha i protein. This leads to the activation of the beta AR-Kinase-1 and further receptor phosphorylation and arrestin dependent internalization. The internalized receptor-arrestin complex recruits and activates cSrc and the MAPK cascade, where Ras, c-Raf1 and finally ERK1/2 mediate the increase in Kv4.2 and Kv4.3 protein abundance in the plasma membrane. Conclusion: beta(2)AR stimulation activates a G alpha s and G alpha i protein dependent pathway where the ERK1/2 modulates the Ito current amplitude and the density of the Kv4.2 and Kv4.2 channels in the plasma membrane upon sympathetic stimulation in diabetic heart. This work has been supported by grants from the MICIN (SAF2010-16120), and the Industry and Education Departments of Basque Government (SA-2010/00012; IT331-10).

Rationale: A chromosomal haplotype producing cardiac overexpression of dipeptidyl peptidase-like protein-6 (DPP6) causes familial idiopathic ventricular fibrillation. The molecular basis of transient outward current ( I to ) in Purkinje fibers (PFs) is poorly understood. We hypothesized that DPP6 contributes to PF I to and that its overexpression might specifically alter PF I to properties and repolarization. Objective: To assess the potential role of DPP6 in PF I to . Methods and Results: Clinical data in 5 idiopathic ventricular fibrillation patients suggested arrhythmia origin in the PF-conducting system. PF and ventricular muscle I to had similar density, but PF I to differed from ventricular muscle in having tetraethylammonium sensitivity and slower recovery. DPP6 overexpression significantly increased, whereas DPP6 knockdown reduced, I to density and tetraethylammonium sensitivity in canine PF but not in ventricular muscle cells. The K + -channel interacting β-subunit K + -channel interacting protein type-2, essential for normal expression of I to in ventricular muscle, was weakly expressed in human PFs, whereas DPP6 and frequenin (neuronal calcium sensor-1) were enriched. Heterologous expression of Kv4.3 in Chinese hamster ovary cells produced small I to ; I to amplitude was greatly enhanced by coexpression with K + -channel interacting protein type-2 or DPP6. Coexpression of DPP6 with Kv4.3 and K + -channel interacting protein type-2 failed to alter I to compared with Kv4.3/K + -channel interacting protein type-2 alone, but DPP6 expression with Kv4.3 and neuronal calcium sensor-1 (to mimic PF I to composition) greatly enhanced I to compared with Kv4.3/neuronal calcium sensor-1 and recapitulated characteristic PF kinetic/pharmacological properties. A mathematical model of cardiac PF action potentials showed that I to enhancement can greatly accelerate PF repolarization. Conclusions: These results point to a previously unknown central role of DPP6 in PF I to , with DPP6 gain of function selectively enhancing PF current, and suggest that a DPP6-mediated PF early-repolarization syndrome might be a novel molecular paradigm for some forms of idiopathic ventricular fibrillation.

Background/Aims: Potassium channels are tetrameric proteins providing potassium selective passage through lipid embedded proteinaceous pores with highest fidelity. The selectivity results from binding to discrete potassium binding sites and stabilization of a hydrated potassium ion in a central internal cavity. The four potassium binding sites, generated by the conserved TTxGYGD signature sequence are formed by the backbone carbonyls of the amino acids TXGYG. Residues KV1.5-Val481, KV4.3-Leu368 and KV7.1- Ile 313 represent the amino acids in the X position of the respective channels. Methods: Here, we study the impact of these residues on ion selectivity, permeation and inactivation kinetics as well as the modulation by β-subunits using site-specific mutagenesis, electrophysiological analyses and molecular dynamics simulations. Results: We identify this position as key in modulation of slow inactivation by structurally dissimilar β-subunits in different KV channels. Conclusion: We propose a model in which structural changes accompanying activation and β-subunit modulation allosterically constrain the backbone carbonyl oxygen atoms via the side chain of the respective X-residue in the signature sequence to reduce conductance during slow inactivation.

To report a novel cell surface autoantigen of encephalitis that is a critical regulatory subunit of the Kv4.2 potassium channels.Four patients with encephalitis of unclear etiology and antibodies with a similar pattern of neuropil brain immunostaining were selected for autoantigen characterization. Techniques included immunoprecipitation, mass spectrometry, cell-base experiments with Kv4.2 and several dipeptidyl-peptidase-like protein-6 (DPPX) plasmid constructs, and comparative brain immunostaining of wild-type and DPPX-null mice.Immunoprecipitation studies identified DPPX as the target autoantigen. A cell-based assay confirmed that all 4 patients, but not 210 controls, had DPPX antibodies. Symptoms included agitation, confusion, myoclonus, tremor, and seizures (1 case with prominent startle response). All patients had pleocytosis, and 3 had severe prodromal diarrhea of unknown etiology. Given that DPPX tunes up the Kv4.2 potassium channels (involved in somatodendritic signal integration and attenuation of dendritic back-propagation of action potentials), we determined the epitope distribution in DPPX, DPP10 (a protein homologous to DPPX), and Kv4.2. Patients' antibodies were found to be specific for DPPX, without reacting with DPP10 or Kv4.2. The unexplained diarrhea led to a demonstration of a robust expression of DPPX in the myenteric plexus, which strongly reacted with patients' antibodies. The course of neuropsychiatric symptoms was prolonged and often associated with relapses during decreasing immunotherapy. Long-term follow-up showed substantial improvement in 3 patients (1 was lost to follow-up).Antibodies to DPPX are associated with a protracted encephalitis characterized by central nervous system hyperexcitability (agitation, myoclonus, tremor, seizures), pleocytosis, and frequent diarrhea at symptom onset. The disorder is potentially treatable with immunotherapy.

To identify the causative gene in spinocerebellar ataxia (SCA) 22, an autosomal dominant cerebellar ataxia mapped to chromosome 1p21-q23.We previously characterized a large Chinese family with progressive ataxia designated SCA22, which overlaps with the locus of SCA19. The disease locus in a French family and an Ashkenazi Jewish American family was also mapped to this region. Members from all 3 families were enrolled. Whole exome sequencing was performed to identify candidate mutations, which were narrowed by linkage analysis and confirmed by Sanger sequencing and cosegregation analyses. Mutational analyses were also performed in 105 Chinese and 55 Japanese families with cerebellar ataxia. Mutant gene products were examined in a heterologous expression system to address the changes in protein localization and electrophysiological functions.We identified heterozygous mutations in the voltage-gated potassium channel Kv4.3-encoding gene KCND3: an in-frame 3-nucleotide deletion c.679_681delTTC p.F227del in both the Chinese and French pedigrees, and a missense mutation c.1034GT p.G345V in the Ashkenazi Jewish family. Direct sequencing of KCND3 further identified 3 mutations, c.1034GT p.G345V, c.1013TC p.V338E, and c.1130CT p.T377M, in 3 Japanese kindreds. Immunofluorescence analyses revealed that the mutant p.F227del Kv4.3 subunits were retained in the cytoplasm, consistent with the lack of A-type K(+) channel conductance in whole cell patch-clamp recordings.Our data identify the cause of SCA19/22 in patients of diverse ethnic origins as mutations in KCND3. These findings further emphasize the important role of ion channels as key regulators of neuronal excitability in the pathogenesis of cerebellar degeneration.

We examined the effects of the sulfonylurea compound NS5806 on neuronal A-type channel function. Using whole-cell patch-clamp we studied the effects of NS5806 on the somatodendritic A-type current (ISA) in cultured hippocampal neurons and the currents mediated by Kv4.2 channels coexpressed with different auxiliary β-subunits, including both Kv channel interacting proteins (KChIPs) and dipeptidyl aminopeptidase-related proteins (DPPs), in HEK 293 cells. The amplitude of the ISA component in hippocampal neurons was reduced in the presence of 20 μM NS5806. ISA decay kinetics were slowed and the recovery kinetics accelerated, but the voltage dependence of steady-state inactivation was shifted to more negative potentials by NS5806. The peak amplitudes of currents mediated by ternary Kv4.2 channel complexes, associated with DPP6-S (short splice-variant) and either KChIP2, KChIP3 or KChIP4, were potentiated and their macroscopic inactivation slowed by NS5806, whereas the currents mediated by binary Kv4.2 channels, associated only with DPP6-S, were suppressed, and the NS5806-mediated slowing of macroscopic inactivation was less pronounced. Neither potentiation nor suppression and no effect on current decay kinetics in the presence of NS5806 were observed for Kv4.2 channels associated with KChIP3 and the N-type inactivation-conferring DPP6a splice-variant. For all recombinant channel complexes, NS5806 slowed the recovery from inactivation and shifted the voltage dependence of steady-state inactivation to more negative potentials. Our results demonstrate the activity of NS5806 on native ISA and possible molecular correlates in the form of recombinant Kv4.2 channels complexed with different KChIPs and DPPs, and they shed some light on the mechanism of NS5806 action.

OBJECTIVE: To identify the causative gene for the neurodegenerative disorder spinocerebellar ataxia type 19 (SCA19) located on chromosomal region 1p21-q21.METHODS: Exome sequencing was used to identify the causal mutation in a large SCA19 family. We then screened 230 ataxia families for mutations located in the same gene (KCND3, also known as Kv4.3) using high-resolution melting. SCA19 brain autopsy material was evaluated, and in vitro experiments using ectopic expression of wild-type and mutant Kv4.3 were used to study protein localization, stability, and channel activity by patch-clamping.RESULTS: We detected a T352P mutation in the third extracellular loop of the voltage-gated potassium channel KCND3 that cosegregated with the disease phenotype in our original family. We identified 2 more novel missense mutations in the channel pore (M373I) and the S6 transmembrane domain (S390N) in 2 other ataxia families. T352P cerebellar autopsy material showed severe Purkinje cell degeneration, with abnormal intracellular accumulation and reduced protein levels of Kv4.3 in their soma. Ectopic expression of all mutant proteins in HeLa cells revealed retention in the endoplasmic reticulum and enhanced protein instability, in contrast to wild-type Kv4.3 that was localized on the plasma membrane. The regulatory β subunit Kv channel interacting protein 2 was able to rescue the membrane localization and the stability of 2 of the 3 mutant Kv4.3 complexes. However, this either did not restore the channel function of the membrane-located mutant Kv4.3 complexes or restored it only partially.INTERPRETATION: KCND3 mutations cause SCA19 by impaired protein maturation and/or reduced channel function.

Accumulation of amyloid-β (Aβ) is widely believed to be an early event in the pathogenesis of Alzheimer's disease (AD). K

Voltage-gated K+ (Kv) channel activation depends on interactions between voltage sensors and an intracellular activation gate that controls access to a central pore cavity. Here, we hypothesize that this gate is additionally responsible for closed-state inactivation (CSI) in Kv4.x channels. These Kv channels undergo CSI by a mechanism that is still poorly understood. To test the hypothesis, we deduced the state of the Kv4.1 channel intracellular gate by exploiting the trap-door paradigm of pore blockade by internally applied quaternary ammonium (QA) ions exhibiting slow blocking kinetics and high-affinity for a blocking site. We found that inactivation gating seemingly traps benzyl-tributylammonium (bTBuA) when it enters the central pore cavity in the open state. However, bTBuA fails to block inactivated Kv4.1 channels, suggesting gated access involving an internal gate. In contrast, bTBuA blockade of a Shaker Kv channel that undergoes open-state P/C-type inactivation exhibits fast onset and recovery inconsistent with bTBuA trapping. Furthermore, the inactivated Shaker Kv channel is readily blocked by bTBuA. We conclude that Kv4.1 closed-state inactivation modulates pore blockade by QA ions in a manner that depends on the state of the internal activation gate.

Mossy fiber afferents to cerebellar granule cells form the primary synaptic relay into cerebellum, providing an ideal site to process signal inputs differentially. Mossy fiber input is known to exhibit a long-term potentiation (LTP) of synaptic efficacy through a combination of presynaptic and postsynaptic mechanisms. However, the specific postsynaptic mechanisms contributing to LTP of mossy fiber input is unknown. The current study tested the hypothesis that LTP induces a change in intrinsic membrane excitability of rat cerebellar granule cells through modification of Kv4 A-type potassium channels. We found that theta-burst stimulation of mossy fiber input in lobule 9 granule cells lowered the current threshold to spike and increases the gain of spike firing by 2- to 3-fold. The change in postsynaptic excitability was traced to hyperpolarizing shifts in both the half-inactivation and half-activation potentials of Kv4 that occurred upon coactivating NMDAR and group I metabotropic glutamatergic receptors. The effects of theta-burst stimulation on Kv4 channel control of the gain of spike firing depended on a signaling cascade leading to extracellular signal-related kinase activation. Under physiological conditions, LTP of synaptically evoked spike output was expressed preferentially for short bursts characteristic of sensory input, helping to shape signal processing at the mossy fiber–granule cell relay.SIGNIFICANCE STATEMENTCerebellar granule cells receive mossy fiber inputs that convey information on different sensory modalities and feedback from descending cortical projections. Recent work suggests that signal processing across multiple cerebellar lobules is controlled differentially by postsynaptic ionic mechanisms at the level of granule cells. We found that long-term potentiation (LTP) of mossy fiber input invoked a large increase in granule cell excitability by modifying the biophysical properties of Kv4 channels through a specific signaling cascade. LTP of granule cell output became evident in response to bursts of mossy fiber input, revealing that Kv4 control of intrinsic excitability is modified to respond most effectively to patterns of afferent input that are characteristic of physiological sensory patterns.

Background The Brugada syndrome is an inherited cardiac arrhythmia associated with high risk of sudden death. Although 20% of patients with Brugada syndrome carry mutations in SCN 5A , the molecular mechanisms underlying this condition are still largely unknown. Methods and Results We combined whole‐exome sequencing and linkage analysis to identify the genetic variant likely causing Brugada syndrome in a pedigree for which SCN 5A mutations had been excluded. This approach identified 6 genetic variants cosegregating with the Brugada electrocardiographic pattern within the pedigree. In silico gene prioritization pointed to 1 variant residing in KCNAB 2 , which encodes the voltage‐gated K + channel β2‐subunit (Kvβ2‐R12Q). Kvβ2 is widely expressed in the human heart and has been shown to interact with the fast transient outward K + channel subunit Kv4.3, increasing its current density. By targeted sequencing of the KCNAB 2 gene in 167 unrelated patients with Brugada syndrome, we found 2 additional rare missense variants (L13F and V114I). We then investigated the physiological effects of the 3 KCNAB 2 variants by using cellular electrophysiology and biochemistry. Patch‐clamp experiments performed in COS ‐7 cells expressing both Kv4.3 and Kvβ2 revealed a significant increase in the current density in presence of the R12Q and L13F Kvβ2 mutants. Although biotinylation assays showed no differences in the expression of Kv4.3, the total and submembrane expression of Kvβ2‐R12Q were significantly increased in comparison with wild‐type Kvβ2. Conclusions Altogether, our results indicate that Kvβ2 dysfunction can contribute to the Brugada electrocardiographic pattern.

Tetrameric assembly of channel subunits in the endoplasmic reticulum (ER) is essential for surface expression and function of K

Pituitary adenylate cyclase (PAC)-activating polypeptide (PACAP) peptides ( Adcyap1) signaling at the selective PAC1 receptor ( Adcyap1r1) participate in multiple homeostatic and stress-related responses, yet the cellular mechanisms underlying PACAP actions remain to be completely elucidated. PACAP/PAC1 receptor signaling increases excitability of neurons within the guinea pig cardiac ganglia, and as these neurons are readily accessible, this neuronal system is particularly amenable to study of PACAP modulation of ionic conductances. The present study investigated how PACAP activation of MEK/ERK signaling contributed to the peptide-induced increase in cardiac neuron excitability. Treatment with the MEK inhibitor PD 98059 blocked PACAP-stimulated phosphorylated ERK and, in parallel, suppressed the increase in cardiac neuron excitability. However, PD 98059 did not blunt the ability of PACAP to enhance two inward ionic currents, one flowing through hyperpolarization-activated nonselective cationic channels ( Ih) and another flowing through low-voltage-activated calcium channels ( IT), which support the peptide-induced increase in excitability. Thus a PACAP - and MEK/ERK-sensitive, voltage-dependent conductance(s), in addition to Ih and IT, modulates neuronal excitability. Despite prior work implicating PACAP downregulation of the KV4.2 potassium channel in modulation of excitability in other cells, treatment with the KV4.2 current blocker 4-aminopyridine did not replicate the PACAP-induced increase in excitability in cardiac neurons. However, cardiac neurons express the ERK target, the NaV1.7 sodium channel, and treatment with the selective NaV1.7 channel inhibitor PF-04856264 decreased the PACAP modulation of excitability. From these results, PACAP/PAC1 activation of MEK/ERK signaling may phosphorylate the NaV1.7 channel, enhancing sodium currents near the threshold, an action contributing to repetitive firing of the cardiac neurons exposed to PACAP.

Background Exposure to chronic ethanol (EtOH) results in changes in the expression of proteins that regulate neuronal excitability. This study examined whether chronic EtOH alters the hippocampal expression and function of fragile X mental retardation protein (FMRP) and the role of FMRP in the modulation of chronic EtOH-induced changes in the expression of NMDA receptors and Kv4.2 channels. Methods For in vivo studies, C57BL/6J mice underwent a chronic intermittent EtOH (CIE) vapor exposure procedure. After CIE, hippocampal tissue was collected and subjected to immunoblot blot analysis of NMDA receptor subunits (GluN1, GluN2B), Kv4.2, and its accessory protein KChIP3. For in vitro studies, hippocampal slice cultures were exposed to 75 mM EtOH for 8 days. Following EtOH exposure, mRNAs bound to FMRP was measured. In a separate set of studies, cultures were exposed to an inhibitor of S6K1 (PF-4708671 [PF], 6 μM) in order to assess whether EtOH-induced homeostatic changes in protein expression depend upon changes in FMRP activity. Results Immunoblot blot analysis revealed increases in GluN1 and GluN2B but reductions in Kv4.2 and KChIP3. Analysis of mRNAs bound to FMRP revealed a similar bidirectional change observed as reduction of GluN2B and increase in Kv4.2 and KChIP3 mRNA transcripts. Analysis of FMRP further revealed that while chronic EtOH did not alter the expression of FMRP, it significantly increased phosphorylation of FMRP at the S499 residue that is known to critically regulate its activity. Inhibition of S6K1 prevented the chronic EtOH-induced increase in phospho-FMRP and changes in NMDA subunits, Kv4.2, and KChIP3. In contrast, PF had no effect in the absence of alcohol, indicating it was specific for the chronic EtOH-induced changes. Conclusions These findings demonstrate that chronic EtOH exposure enhances translational control of plasticity-related proteins by FMRP, and that S6K1 and FMRP activities are required for expression of chronic EtOH-induced homeostatic plasticity at glutamatergic synapses in the hippocampus.

The venom from the scorpion Tityus serrulatus (Ts) has been extensively studied mainly because of its rich cocktail of neurotoxins. Neurotoxins are the major and the most known components based on their modulation of voltage-gated ion channels. Until now, electrophysiological studies demonstrated that the Ts venom comprises toxins that affect Nav and Kv channels. However, although many studies have been conducted in this field, many peptides from Ts venom await further studies, including Ts8 toxin. Here we report the isolation and electrophysiological study of Ts8. The toxin Ts19 Frag-II was used as negative control. Ts8 demonstrates, among 20 tested channels, to be a selective modulator of Kv4.2 channels. Based on studies investigating the involvement of Kv4.2 on controlling nociception, we further investigated the modulation of pain by Ts8. Using intraplantar injections, Ts8 induced overt nociception (licking and lifting behaviors) and decreased the mechanical nociceptive threshold (hyperalgesia). Furthermore, the hyperalgesia was prolonged when intrathecal injections were performed. Independent of the severity, most of the victims stung by Ts scorpions report an intense and persistent pain as the major manifestation. The new role of Ts8 on nociception could explain, at least partially, this phenomenon. Additionally, our study also stresses the involvement of toxins specific to Nav channels and inflammatory mediators on the Ts painful sting. This work provides useful insights for a better understanding of the prolonged and intense pain associated with Ts envenoming for the development of specific therapies.

The roles of several small GTPases in the expression of an endogenous potassium current, Ito,f, in adult rat ventricular myocytes have been investigated. The results indicate that forward trafficking of newly synthesized Kv4.2, which underlies Ito,fin these cells, requires both Rab1 and Sar1 function. Expression of a Rab1 dominant negative (DN) reduced Ito,fcurrent density by roughly one-half relative to control, mCherry-transfected myocytes. Similarly, expression of a Sar1DN nearly halved Ito,fcurrent density. Rab11 is not essential to trafficking of Kv4.2, as expression of a Rab11DN had no effect on Ito,fover the time frames investigated here. In a process dependent on intact endoplasmic reticulum (ER)-to-Golgi transport, however, overexpression of wild-type Rab11 resulted in a doubling of Ito,fdensity; block of ER-to-Golgi traffic by Brefeldin A completely abrogated the effect. Also implicated in the trafficking of Kv4.2 are Rab5 and Rab4. Rab5DN expression increased endogenous Ito,fby two- to threefold, nonadditively with inhibition of dynamin-dependent endocytosis. And, in a phenomenon similar to that previously reported for myoblast-expressed Kv1.5, Rab4DN expression roughly doubled endogenous peak transient currents. Colocalization experiments confirmed the involvement of Rab4 in postinternalization trafficking of Kv4.2. There was little role evident for the lysosome in the degradation of internalized Kv4.2, as overexpression of neither wild-type nor DN isoforms of Rab7 had any effect on Ito,f. Instead, degradation may depend largely on the proteasome; the proteasome inhibitor MG132 significantly increased Ito,fdensity.