Neuronal Channelopathies Research Articles

Exploring K v 1.2 Channel Inactivation Through MD Simulations and Network Analysis.

The KCNA2 gene encodes the K v 1.2 channel, a mammalian Shaker-like voltage-gated K+ channel, whose defections are linked to neuronal deficiency and childhood epilepsy. Despite the important role in the kinetic behavior of the channel, the inactivation remained hereby elusive. Here, we studied the K v 1.2 inactivation via a combined simulation/network theoretical approach that revealed two distinct pathways coupling the Voltage Sensor Domain and the Pore Domain to the Selectivity Filter. Additionally, we mutated some residues implicated in these paths and we explained microscopically their function in the inactivation mechanism by computing a contact map. Interestingly, some pathological residues shown to impair the inactivation lay on the paths. In summary, the presented results suggest two pathways as the possible molecular basis of the inactivation mechanism in the K v 1.2 channel. These pathways are consistent with earlier mutational studies and known mutations involved in neuronal channelopathies.

Inter-Regulation of Kv4.3 and Voltage-Gated Sodium Channels Underlies Predisposition to Cardiac and Neuronal Channelopathies

Background: Genetic variants in voltage-gated sodium channels (Nav) encoded by SCNXA genes, responsible for INa, and Kv4.3 channels encoded by KCND3, responsible for the transient outward current (Ito), contribute to the manifestation of both Brugada syndrome (BrS) and spinocerebellar ataxia (SCA19/22). We examined the hypothesis that Kv4.3 and Nav variants regulate each other’s function, thus modulating INa/Ito balance in cardiomyocytes and INa/I(A) balance in neurons. Methods: Bicistronic and other constructs were used to express WT or variant Nav1.5 and Kv4.3 channels in HEK293 cells. INa and Ito were recorded. Results: SCN5A variants associated with BrS reduced INa, but increased Ito. Moreover, BrS and SCA19/22 KCND3 variants associated with a gain of function of Ito, significantly reduced INa, whereas the SCA19/22 KCND3 variants associated with a loss of function (LOF) of Ito significantly increased INa. Auxiliary subunits Navβ1, MiRP3 and KChIP2 also modulated INa/Ito balance. Co-immunoprecipitation and Duolink studies suggested that the two channels interact within the intracellular compartments and biotinylation showed that LOF SCN5A variants can increase Kv4.3 cell-surface expression. Conclusion: Nav and Kv4.3 channels modulate each other’s function via trafficking and gating mechanisms, which have important implications for improved understanding of these allelic cardiac and neuronal syndromes.

Estimating neuronal conductance model parameters using dynamic action potential clamp

BackgroundParameterization of neuronal membrane conductance models relies on data acquired from current clamp (CC) or voltage clamp (VC) recordings. Although the CC approach provides key information on a neuron’s firing properties, it is often difficult to disentangle the influence of multiple conductances that contribute to the excitation properties of a real neuron. Isolation of a single conductance using pharmacological agents or heterologous expression simplifies analysis but requires extensive VC evaluation to explore the complete state behavior of the channel of interest. New MethodWe present an improved parameterization approach that uses data derived from dynamic action potential clamp (DAPC) recordings to extract conductance equation parameters. We demonstrate the utility of the approach by applying it to the standard Hodgkin-Huxley conductance model although other conductance models could be easily incorporated as well. ResultsUsing a fully simulated setup we show that, with as few as five action potentials previously recorded in DAPC mode, sodium conductance equation parameters can be determined with average parameter errors of less than 4% while action potential firing accuracy approaches 100%. In real DAPC experiments, we show that by “training” our model with five or fewer action potentials, subsequent firing lasting for several seconds could be predicted with ˜96% mean firing rate accuracy and 94% temporal overlap accuracy. Comparison with existing methodsOur DAPC-based approach surpasses the accuracy of VC-based approaches for extracting conductance equation parameters with a significantly reduced temporal overhead. ConclusionDAPC-based approach will facilitate the rapid and systematic characterization of neuronal channelopathies.

Involvement of the delayed rectifier outward potassium channel Kv2.1 in methamphetamine-induced neuronal apoptosis via the p38 mitogen-activated protein kinase signaling pathway.

Methamphetamine (Meth) is an illicit psychostimulant with high abuse potential and severe neurotoxicity. Recent studies have shown that dysfunctions in learning and memory induced by Meth may partially reveal the mechanisms of neuronal channelopathies. Kv2.1, the primary delayed rectifying potassium channel in neurons, is responsible for mediating apoptotic current surge. However, whether Kv2.1 is involved in Meth-mediated neural injury remains unknown. In the present study, the treatment of primary cultured hippocampal neurons with Meth indicated that Meth induced a time- and dose-dependent augmentation of Kv2.1 protein expression, accompanied by elevated cleaved-caspase 3 and declined bcl-2/bax ratio. The blockage of Kv2.1 with the inhibitor GxTx-1E or the knockdown of the channel noticeably abrogated the pro-apoptotic effects mediated by Meth, demonstrating the specific roles of Kv2.1 in Meth-mediated neural damage. Additionally, the p38 mitogen-activated protein kinase (MAPK) signaling was demonstrated to be involved in Meth-mediated Kv2.1 upregulation and in the subsequent pro-apoptotic effects, as treatment with a p38 MAPK inhibitor significantly attenuated Meth-mediated Kv2.1 upregulation and cell apoptosis. Of note, PRE-084, a sigma-1 receptor agonist, obviously attenuated Meth-induced upregulation of Kv2.1 expression, neural apoptosis and p38 MAPK activation. Taken together, these results reveal a novel mechanism involved in Meth-induced neural death with implications for therapeutic interventions for Meth users.

Potassium Channels: A Potential Therapeutic Target for Parkinson's Disease.

The pathogenesis of the second major neurodegenerative disorder, Parkinson's disease (PD), is closely associated with the dysfunction of potassium (K+) channels. Therefore, PD is also considered to be an ion channel disease or neuronal channelopathy. Mounting evidence has shown that K+ channels play crucial roles in the regulations of neurotransmitter release, neuronal excitability, and cell volume. Inhibition of K+ channels enhances the spontaneous firing frequency of nigral dopamine (DA) neurons, induces a transition from tonic firing to burst discharge, and promotes the release of DA in the striatum. Recently, three K+ channels have been identified to protect DA neurons and to improve the motor and non-motor symptoms in PD animal models: small conductance (SK) channels, A-type K+ channels, and KV7/KCNQ channels. In this review, we summarize the physiological and pharmacological effects of the three K+ channels. We also describe in detail the laboratory investigations regarding K+ channels as a potential therapeutic target for PD.

Editorial: Recent Advances in Voltage-Gated Sodium Channels, their Pharmacology and Related Diseases.

Editorial: Recent Advances in Voltage-Gated Sodium Channels, their Pharmacology and Related Diseases.

In vivo impact of presynaptic calcium channel dysfunction on motor axons in episodic ataxia type 2.

Ion channel dysfunction causes a range of neurological disorders by altering transmembrane ion fluxes, neuronal or muscle excitability, and neurotransmitter release. Genetic neuronal channelopathies affecting peripheral axons provide a unique opportunity to examine the impact of dysfunction of a single channel subtype in detail in vivo. Episodic ataxia type 2 is caused by mutations in CACNA1A, which encodes the pore-forming subunit of the neuronal voltage-gated calcium channel Cav2.1. In peripheral motor axons, this channel is highly expressed at the presynaptic neuromuscular junction where it contributes to action potential-evoked neurotransmitter release, but it is not expressed mid-axon or thought to contribute to action potential generation. Eight patients from five families with genetically confirmed episodic ataxia type 2 underwent neurophysiological assessment to determine whether axonal excitability was normal and, if not, whether changes could be explained by Cav2.1 dysfunction. New mutations in the CACNA1A gene were identified in two families. Nerve conduction studies were normal, but increased jitter in single-fibre EMG studies indicated unstable neuromuscular transmission in two patients. Excitability properties of median motor axons were compared with those in 30 age-matched healthy control subjects. All patients had similar excitability abnormalities, including a high electrical threshold and increased responses to hyperpolarizing (P < 0.00007) and depolarizing currents (P < 0.001) in threshold electrotonus. In the recovery cycle, refractoriness (P < 0.0002) and superexcitability (P < 0.006) were increased. Cav2.1 dysfunction in episodic ataxia type 2 thus has unexpected effects on axon excitability, which may reflect an indirect effect of abnormal calcium current fluxes during development.

A novel neuronal channelopathy could provide the trigger for Ca2+ dysregulation and sympathetic impairment seen in the pro-hypertensive rat

A novel neuronal channelopathy could provide the trigger for Ca2+ dysregulation and sympathetic impairment seen in the pro-hypertensive rat

CARDIAC FEATURES IN ADULTS WITH ALTERNATING HEMIPLEGIA

<h3>Introduction</h3> Alternating hemiplegia of childhood (AHC) is a rare neurodevelopmental disorder caused by de novo mutations in the ATP1A3 gene encoding the α3-subunit of the Na+/K+-ATPase transporter, expressed in cardiac and neuronal tissue. AHC is characterised by recurrent plegic attacks, dystonic posturing and seizures. Non-paroxysmal neurological manifestations and developmental delay may occur. Potentially fatal arrhythmias are associated with cardiac channelopathies but also with neurological channelopathies such as Andersen-Tawil syndrome, and postulated in sudden unexplained death in epilepsy. Autonomic dysfunction and one case of asystole have been reported in AHC, but specific cardiac abnormalities have not been defined. <h3>Methods and results</h3> Nine patients from an original UK cohort of twenty-six (adults n=6; children n=3), underwent detailed cardiac and neurological phenotyping, baseline ECG testing and ATP1A3 molecular genetic analysis. Repolarisation abnormalities and varying degrees of intraventricular conduction delay were seen in all six, ATP1A3-mutation bearing, adults. No abnormalities were detected in children. <h3>Conclusions</h3> These cardiac features raise the possibility of an arrhythmogenic potential of a neuronal channelopathy co-expressed in the heart and brain, warranting cardiac surveillance from adolescence into adulthood and further risk prediction strategies in AHC.

Dravet syndrome patient-derived neurons suggest a novel epilepsy mechanism.

Neuronal channelopathies cause brain disorders, including epilepsy, migraine, and ataxia. Despite the development of mouse models, pathophysiological mechanisms for these disorders remain uncertain. One particularly devastating channelopathy is Dravet syndrome (DS), a severe childhood epilepsy typically caused by de novo dominant mutations in the SCN1A gene encoding the voltage-gated sodium channel Na(v) 1.1. Heterologous expression of mutant channels suggests loss of function, raising the quandary of how loss of sodium channels underlying action potentials produces hyperexcitability. Mouse model studies suggest that decreased Na(v) 1.1 function in interneurons causes disinhibition. We aim to determine how mutant SCN1A affects human neurons using the induced pluripotent stem cell (iPSC) method to generate patient-specific neurons. Here we derive forebrain-like pyramidal- and bipolar-shaped neurons from 2 DS subjects and 3 human controls by iPSC reprogramming of fibroblasts. DS and control iPSC-derived neurons are compared using whole-cell patch clamp recordings. Sodium current density and intrinsic neuronal excitability are examined. Neural progenitors from DS and human control iPSCs display a forebrain identity and differentiate into bipolar- and pyramidal-shaped neurons. DS patient-derived neurons show increased sodium currents in both bipolar- and pyramidal-shaped neurons. Consistent with increased sodium currents, both types of patient-derived neurons show spontaneous bursting and other evidence of hyperexcitability. Sodium channel transcripts are not elevated, consistent with a post-translational mechanism. These data demonstrate that epilepsy patient-specific iPSC-derived neurons are useful for modeling epileptic-like hyperactivity. Our findings reveal a previously unrecognized cell-autonomous epilepsy mechanism potentially underlying DS, and offer a platform for screening new antiepileptic therapies.

In vivo loss of slow potassium channel activity in individuals with benign familial neonatal epilepsy in remission

Benign familial neonatal epilepsy is a neuronal channelopathy most commonly caused by mutations in KCNQ2, which encodes the K(v)7.2 subunit of the slow K(+) channel. K(v)7.2 is expressed in both central and peripheral nervous systems. Seizures occur in the neonatal period, often in clusters within the first few days of life, and usually remit by 12 months of age. The mechanism of involvement of K(v)7.2 mutations in the process of seizure generation has not been established in vivo. In peripheral axons, K(v)7.2 contributes to the nodal slow K(+) current. The present study aimed to determine whether axonal excitability studies could detect changes in peripheral nerve function related to dysfunction or loss of slow potassium channel activity. Nerve excitability studies were performed on eight adults with KCNQ2 mutations and a history of benign familial neonatal epilepsy, now in remission. Studies detected distinctive changes in peripheral nerve, indicating a reduction in slow K(+) current. Specifically, accommodation to long-lasting depolarizing currents was reduced in mutation carriers by 24% compared with normal controls, and the threshold undershoot after 100 ms depolarizing currents was reduced by 22%. Additional changes in excitability included a reduction in the relative refractory period, an increase in superexcitability and a tendency towards reduced sub-excitability. Modelling of the nerve excitability changes suggested that peripheral nerve hyperexcitability may have been ameliorated by upregulation of other potassium channels. We conclude that subclinical dysfunction of K(v)7.2 in peripheral axons can be reliably detected non-invasively in adulthood. Related alterations in neuronal excitability may contribute to epilepsy associated with KCNQ2 mutations.

HIV-1gp120 Induces Neuronal Apoptosis through Enhancement of 4-Aminopyridine-Senstive Outward K+ Currents

Human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) usually occurs late in the course of HIV-1 infection and the mechanisms underlying HAD pathogenesis are not well understood. Accumulating evidence indicates that neuronal voltage-gated potassium (Kv) channels play an important role in memory processes and acquired neuronal channelopathies in HAD. To examine whether Kv channels are involved in HIV-1-associated neuronal injury, we studied the effects of HIV-1 glycoprotein 120 (gp120) on outward K+ currents in rat cortical neuronal cultures using whole-cell patch techniques. Exposure of cortical neurons to gp120 produced a dose-dependent enhancement of A-type transient outward K+ currents (IA). The gp120-induced increase of IA was attenuated by T140, a specific antagonist for chemokine receptor CXCR4, suggesting gp120 enhancement of neuronal IA via CXCR4. Pretreatment of neuronal cultures with a protein kinase C (PKC) inhibitor, GF109203X, inhibited the gp120-induced increase of IA. Biological significance of gp120 enhancement of IA was demonstrated by experimental results showing that gp120-induced neuronal apoptosis, as detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and caspase-3 staining, was attenuated by either an IA blocker 4-aminopyridine or a specific CXCR4 antagonist T140. Taken together, these results suggest that gp120 may induce caspase-3 dependent neuronal apoptosis by enhancing IA via CXCR4-PKC signaling.

Nerve excitability studies characterize KV1.1 fast potassium channel dysfunction in patients with episodic ataxia type 1

Episodic ataxia type 1 is a neuronal channelopathy caused by mutations in the KCNA1 gene encoding the fast K(+) channel subunit K(v)1.1. Episodic ataxia type 1 presents with brief episodes of cerebellar dysfunction and persistent neuromyotonia and is associated with an increased incidence of epilepsy. In myelinated peripheral nerve, K(v)1.1 is highly expressed in the juxtaparanodal axon, where potassium channels limit the depolarizing afterpotential and the effects of depolarizing currents. Axonal excitability studies were performed on patients with genetically confirmed episodic ataxia type 1 to characterize the effects of K(v)1.1 dysfunction on motor axons in vivo. The median nerve was stimulated at the wrist and compound muscle action potentials were recorded from abductor pollicis brevis. Threshold tracking techniques were used to record strength-duration time constant, threshold electrotonus, current/threshold relationship and the recovery cycle. Recordings from 20 patients from eight kindreds with different KCNA1 point mutations were compared with those from 30 normal controls. All 20 patients had a history of episodic ataxia and 19 had neuromyotonia. All patients had similar, distinctive abnormalities: superexcitability was on average 100% higher in the patients than in controls (P < 0.00001) and, in threshold electrotonus, the increase in excitability due to a depolarizing current (20% of threshold) was 31% higher (P < 0.00001). Using these two parameters, the patients with episodic ataxia type 1 and controls could be clearly separated into two non-overlapping groups. Differences between the different KCNA1 mutations were not statistically significant. Studies of nerve excitability can identify K(v)1.1 dysfunction in patients with episodic ataxia type 1. The simple 15 min test may be useful in diagnosis, since it can differentiate patients with episodic ataxia type 1 from normal controls with high sensitivity and specificity.

POG07 Natural history trials of neurological channelopathies

Neurological ion channel disorders are Mendelian disorders caused by mutations in genes that encode ion channel subunits. Two broad groups exist: Skeletal muscle and neuronal channelopathies. As a member of...

Gene therapy rescues cone function in congenital achromatopsia

The successful restoration of visual function with recombinant adeno-associated virus (rAAV)-mediated gene replacement therapy in animals and humans with an inherited disease of the retinal pigment epithelium has ushered in a new era of retinal therapeutics. For many retinal disorders, however, targeting of therapeutic vectors to mutant rods and/or cones will be required. In this study, the primary cone photoreceptor disorder achromatopsia served as the ideal translational model to develop gene therapy directed to cone photoreceptors. We demonstrate that rAAV-mediated gene replacement therapy with different forms of the human red cone opsin promoter led to the restoration of cone function and day vision in two canine models of CNGB3 achromatopsia, a neuronal channelopathy that is the most common form of achromatopsia in man. The robustness and stability of the observed treatment effect was mutation independent, but promoter and age dependent. Subretinal administration of rAAV5-hCNGB3 with a long version of the red cone opsin promoter in younger animals led to a stable therapeutic effect for at least 33 months. Our results hold promise for future clinical trials of cone-directed gene therapy in achromatopsia and other cone-specific disorders.

Clinical neurophysiology of the episodic ataxias: Insights into ion channel dysfunction in vivo

Clinical neurophysiology has become an invaluable tool in the diagnosis of muscle channelopathies, but the situation is less clear cut with neuronal channelopathies. The genetic episodic ataxias are a group of disorders with heterogeneous phenotype and genotype, but share in common the feature of intermittent cerebellar dysfunction. Episodic ataxia (EA) types 1 and 2 are the most widely recognised of the autosomal dominant episodic ataxias and are caused by dysfunction of neuronal voltage-gated ion channels. There are central and peripheral nervous system manifestations in both conditions, and they are therefore good models of neuronal channelopathies to study neurophysiologically. To date most work has focussed upon characterising the electrophysiological properties of mutant channels in vitro. This review summarises the role of voltage-gated potassium and calcium channels, mutations of which underlie the main types of episodic ataxia types 1 and 2. The clinical, genetic and electrophysiological features of EA1 and EA2 are outlined, and a protocol for the assessment of these patients is proposed.

Monogenic migraine syndromes highlight novel drug targets

AbstractIn the post‐genomic era, the paradigm for drug discovery has changed, as every gene may become a potential target. Genetic diseases provide a special window into gene target selection. This approach is being applied to migraine making use of the genes and mutations causing familial hemiplegic migraine (FHM). FHM is caused by missense mutations in CACNA1A, altering a neuronal P/Q Ca2+ channel, in ATP1A2, altering α2 Na,K‐ATPase, and in SCN1A, altering a neuronal sodium channel. These genes provide insights into migraine pathogenesis that likely extend to other forms of migraine as well. Since the three FHM genes are only co‐expressed in neurons, FHM is a neuronal, not a vascular, disease and because they all encode ion transport proteins, FHM is a neuronal channelopathy—meaning meta‐stable neuronal hyperexcitability is the substrate of migraine, much as it is for genetic epilepsy syndromes. This similarity is reinforced, since different mutations of all three FHM genes can produce seizure syndromes. This has implications for drug discovery in that seizure medications already known to modulate the FHM channel mechanisms warrant more targeted development, and that drugs targeted to vascular headaches, such as the historically effective triptans, or experimental botulinum toxin, may well work by similar nonvascular mechanisms. Finally, in model neurogenetic systems such as Caenorhabditis elegans, the FHM genes also provide both a comprehensive means to discover all genes involved in their signaling pathway—genes potentially involved in common forms of the disease, and an in vivo whole animal means to screen rapidly for novel therapeutics. Drug Dev Res 68:432–440, 2007. © 2008 Wiley‐Liss, Inc.

Acquired neuronal channelopathies in HIV-associated dementia

A gene expression profile of the human brain cortex was performed in people with HIV-1-associated dementia (HAD) using Affymetrix HG-U133 chips. Messenger RNA transcripts in middle frontal gyrus from subjects with HAD or milder neurocognitive dysfunction were compared to HIV-negative people. The analysis focused on ionic conductance carriers that control membrane excitation. Overexpressed ionic channel genes in brain cortex of subjects with dementia included (1) a calcium-driven K + channel that prolongs afterhyperpolarization (AHP) current, (2) a leak type of K + channel that prolongs the AHP, (3) an adenosine receptor that modulates cationic current via G proteins, (4) a G protein-coupled serotonin receptor that modulates cyclic AMP-linked current transduction, (5) a G protein-coupled dopamine receptor, (6) a GABA receptor subunit that conducts chloride current. Underexpressed current generators in the demented subjects included (1) two voltage-gated K + channels that influence refractory periods and the onset of AHP, (2) a Na + channel subunit that modifies current inactivation and the onset of the AHP, (3) a neuronal type of voltage-sensitive Ca 2+ channel that controls postsynaptic membrane excitability, (4) a metabotropic glutamate receptor that regulates cationic gating via G protein coupling, (5) A specific Gα protein that transduces metabotropic cationic current, (6) an NMDA receptor subunit, (7) a glycine receptor subunit that modulates chloride current. These gene expression shifts probably occurred in neurons because they were not present in gyral white matter. Acquired neuronal channelopathies were not associated with a generalized shift of neuronal or glial cell markers, which suggest that they were not an artifact produced by neurodegeneration and/or glial cell proliferation. Channelopathies were not correlated with a generalized increase of inflammatory cell transcripts and were present in demented people without, and with HIV encephalitis (HIVE). We surveyed experimentally induced perturbations of these channels to determine the implications for brain function. Eleven experimental channelopathies produced decreased neuronal firing frequencies and pacemaker rates in model neurons; seven channelopathies increase neuronal firing rates experimentally. The implied disruption of neuronal excitability is consistent with some features of HAD, including its potential reversibility after HIV-1 replication is suppressed, the abnormal electroencephalographic recordings, the lack of clear-cut correlation with neurodegeneration and the lack of strict correlation with brain inflammation. The channelopathy concept may have wide relevance to the subcortical dementias.

The neuronal channelopathies.

This review addresses the molecular and cellular mechanisms of diseases caused by inherited mutations of ion channels in neurones. Among important recent advances is the elucidation of several dominantly inherited epilepsies caused by mutations of both voltage-gated and ligand-gated ion channels. The neuronal channelopathies show evidence of phenotypic convergence; notably, episodic ataxia can be caused by mutations of either calcium or potassium channels. The channelopathies also show evidence of phenotypic divergence; for instance, different mutations of the same calcium channel gene are associated with familial hemiplegic migraine, episodic or progressive ataxia, coma and epilepsy. Future developments are likely to include the discovery of other ion channel genes associated with inherited and sporadic CNS disorders. The full range of manifestations of inherited ion channel mutations remains to be established.

Teaching course: ion channelopathies in neurology.

In 1990 the molecular basis for a hereditary disorder inhumans, hyperkalemic periodic paralysis, was first genet-ically demonstrated to be impaired ion channel function.Since then over a dozen diseases, now termed as chan-nelopathies, have been described. Most of the disordersaffect excitable tissue such as muscle and nerve; however,kidney diseases have also been described. Basic researchon structure-function relationships and physiology of ex-citation has benefited tremendously from the discovery ofdisease-causing mutations pointing to regions of specialsignificance within the channel proteins.This course focuses mainly on the clinical and geneticfeatures of neurological disturbances in humans caused bygenetic defects in voltage-gated sodium, calcium, potas-sium, and chloride channels. Disorders of skeletal muscleare by far the most studied and therefore more detailed inthis text than the neuronal channelopathies which havebeen discovered only very recently. Review literature maybe found in the attached reference list [1–12].