Genes Encoding Ion Channel Subunits Research Articles

Investigation of the Genetic Etiology in Idiopathic Generalized Epileptic Disorders by Targeted Next-generation Sequencing Technique

Idiopathic generalized epilepsy is the most common group of epilepsy disorders in children and adolescents. Various types of genetic abnormality were identified among the hereditary factors that explain epilepsy. To determine the variations in the etiopathogenesis, treatment protocol planning, and prognosis of idiopathic generalized epilepsy using the next-generation sequencing method. A cross-sectional study. This study included 32 patients with idiopathic generalized epilepsy. Genomic DNA was obtained from peripheral venous blood samples taken from the patients. A total of 18 genes encoding ion channel subunits that are involved in monogenic disorders and are associated with idiopathic generalized epilepsy were included. The targeted custom next-generation sequencing panel was designed to cover all coding exons and all exon/intron splice site regions of 18 genes. We detected 9 (28%) variations, including 1 likely pathogenic (a variant in the SCN1A gene) and 8 of unknown clinical significance (2 in the CLCN2 genes, GABBR2, SCN1B, SLC2A1, SLC4A10 genes, and 2 in the TBC1D24 gene). Study results should be supported by functional advanced studies, with increased existing knowledge in the relevant variations.

Ion Channel Gene Mutations Causing Skeletal Muscle Disorders: Pathomechanisms and Opportunities for Therapy

Skeletal muscle ion channelopathies (SMICs) are a large heterogeneous group of rare genetic disorders caused by mutations in genes encoding ion channel subunits in the skeletal muscle mainly characterized by myotonia or periodic paralysis, potentially resulting in long-term disabilities. However, with the development of new molecular technologies, new genes and new phenotypes, including progressive myopathies, have been recently discovered, markedly increasing the complexity in the field. In this regard, new advances in SMICs show a less conventional role of ion channels in muscle cell division, proliferation, differentiation, and survival. Hence, SMICs represent an expanding and exciting field. Here, we review current knowledge of SMICs, with a description of their clinical phenotypes, cellular and molecular pathomechanisms, and available treatments.

Epilepsy and brain channelopathies from infancy to adulthood.

Genetic brain channelopathies result from inherited or de novo mutations of genes encoding ion channel subunits within the central nervous system. Most neurological channelopathies arise in childhood with paroxysmal or episodic symptoms, likely because of a transient impairment of homeostatic mechanisms regulating membrane excitability, and the prototypical expression of this impairment is epilepsy. Migraine, episodic ataxia and alternating hemiplegia can also occur, as well as chronic phenotypes, such as spinocerebellar ataxias, intellectual disability and autism spectrum disorder. Voltage-gated and ligand-gated channels may be involved. In most cases, a single gene may be associated with a phenotypical spectrum that shows variable expressivity. Different clinical features may arise at different ages and the adult phenotype may be remarkably modified from the syndrome onset in childhood or adolescence. Recognizing the prominent phenotypical traits of brain channelopathies is essential to perform appropriate diagnostic investigations and to provide the better care not only in the paediatric setting but also for adult patients and their caregivers. Herein, we provide an overview of genetic brain channelopathies associated with epilepsy, highlight the different molecular mechanisms and describe the different clinical characteristics which may prompt the clinician to suspect specific syndromes and to possibly establish tailored treatments.

Trafficking and localisation to the plasma membrane of Nav 1.5 promoted by the β2 subunit is defective due to a β2 mutation associated with Brugada syndrome.

Cardiac channelopathies arise by mutations in genes encoding ion channel subunits. One example is Brugada Syndrome (BrS), which causes arrhythmias and sudden death. BrS is often associated with mutations in SCN5A, encoding Nav 1.5, the α subunit of the major cardiac voltage-gated sodium channel. This channel forms a protein complex including one or two associated β subunits as well as other proteins. We analysed regulation of Nav 1.5 localisation and trafficking by β2, specifically, Nav 1.5 arrival to the cell surface. We used polarised Madin-Darby canine kidney (MDCK) cells and mouse atria-derived HL-1 cells, which retain phenotypic features of adult cardiomyocytes. In both, Nav 1.5 was found essentially intracellular, mainly in the endoplasmic reticulum, whereas β2 localised to the plasma membrane, and was restricted to the apical surface in MDCK cells. A fraction of β2 interacted with Nav 1.5, despite their limited overlap. Importantly, β2 promoted Nav 1.5 localisation to the cell surface. Both β2 WT and the BrS-associated mutation D211G (substitution of Asp for Gly) effectively reached the plasma membrane. Strikingly, however, β2 D211G was defective in promoting Nav 1.5 surface localisation. Our data sustain that β2 promotes surface localisation of Nav 1.5, which can be affected due to β2 mutations associated with channelopathies. Our findings add to the understanding of β2 role in Nav 1.5 trafficking and localisation, which must influence cell excitability and electrical coupling in the heart. This study will contribute to knowledge on development of arrhythmias.

Therapeutic Approaches to Genetic Ion Channelopathies and Perspectives in Drug Discovery.

In the human genome more than 400 genes encode ion channels, which are transmembrane proteins mediating ion fluxes across membranes. Being expressed in all cell types, they are involved in almost all physiological processes, including sense perception, neurotransmission, muscle contraction, secretion, immune response, cell proliferation, and differentiation. Due to the widespread tissue distribution of ion channels and their physiological functions, mutations in genes encoding ion channel subunits, or their interacting proteins, are responsible for inherited ion channelopathies. These diseases can range from common to very rare disorders and their severity can be mild, disabling, or life-threatening. In spite of this, ion channels are the primary target of only about 5% of the marketed drugs suggesting their potential in drug discovery. The current review summarizes the therapeutic management of the principal ion channelopathies of central and peripheral nervous system, heart, kidney, bone, skeletal muscle and pancreas, resulting from mutations in calcium, sodium, potassium, and chloride ion channels. For most channelopathies the therapy is mainly empirical and symptomatic, often limited by lack of efficacy and tolerability for a significant number of patients. Other channelopathies can exploit ion channel targeted drugs, such as marketed sodium channel blockers. Developing new and more specific therapeutic approaches is therefore required. To this aim, a major advancement in the pharmacotherapy of channelopathies has been the discovery that ion channel mutations lead to change in biophysics that can in turn specifically modify the sensitivity to drugs: this opens the way to a pharmacogenetics strategy, allowing the development of a personalized therapy with increased efficacy and reduced side effects. In addition, the identification of disease modifiers in ion channelopathies appears an alternative strategy to discover novel druggable targets.

Louis Ptáček receives the 2015 ASCI/Stanley J. Korsmeyer Award. Interview by Sarah Jackson.

Channelopathies encompass a diverse set of diseases that are caused by mutations in genes encoding ion channel subunits or their regulators. The first recognition of a channelopathy came from Louis Ptacek (Figure 1), whose lab cloned a mutated muscle sodium channel gene found in a family with hyperkalemic periodic paralysis. His subsequent work extends far beyond channelopathies, as he has continued studying a number of inherited, familial disorders. Ptacek’s research has identified a wide range of genes that are mutated in patients with migraine, epilepsy, cardiac arrhythmias, neurodegeneration, and circadian rhythm disorders. This year, the ASCI honors Ptacek’s outstanding work using human genetics to provide fundamental insights into the molecular mechanisms that drive disease pathophysiology. The JCI recently spoke with Ptacek about his path to success as a physician-scientist.

DELETION OF THE POTASSIUM CHANNEL GENE KCNC2 IN A FAMILY WITH NEURODEVELOPMENTAL DELAY AND ATAXIA

Mutations in genes encoding ion channel subunits have been described in association with a growing number of central and peripheral nervous system phenotypes. The Kv3 subfamily of potassium channels are...

Ion Channel Drug Discovery: Challenges And Future Directions

Ion channels are targets of many therapeutically useful agents, and worldwide sales of ion channel-targeted drugs are estimated to be approximately US$12 billion. Nevertheless, considering that over 400 genes encoding ion channel subunits have been identified, ion channels remain significantly under-exploited as therapeutic targets. This is at least partly due to limitations in high-throughput assay technologies that support screening and lead optimization. Will the recent developments in automated electrophysiology rectify this situation? What are the other major limitations and can they be overcome? In this article, we review the status of ion channel drug discovery, discuss current challenges and propose alternative approaches that may facilitate the discovery of new drugs in the future.

Triggering of cardiac arrhythmic events in long QT syndrome: lessons from funny bunnies

Triggering of cardiac arrhythmic events in long QT syndrome: lessons from funny bunnies

Early ion-channel remodeling and arrhythmias precede hypertrophy in a mouse model of complete atrioventricular block

Complete atrioventricular block (CAVB) and related ventricular bradycardia are known to induce ventricular hypertrophy and arrhythmias. Different animal models of CAVB have been established with the most common being the dog model. Related studies were mainly focused on the consequences on the main repolarizing currents in these species, i.e. IKr and IKs, with a limited time point kinetics post-AVB. In order to explore at a genomic scale the electrical remodeling induced by AVB and its chronology, we have developed a novel model of CAVB in the mouse using a radiofrequency-mediated ablation procedure. We investigated transcriptional changes in ion channels and contractile proteins in the left ventricles as a function of time (12 h, 1, 2 and 5 days after CAVB), using high-throughput real-time RT-PCR. ECG in conscious and anesthetized mice, left ventricular pressure recordings and patch-clamp were used for characterization of this new mouse model. As expected, CAVB was associated with a lengthening of the QT interval. Moreover, polymorphic ventricular tachycardia was recorded in 6/9 freely-moving mice during the first 24 h post-ablation. Remarkably, myocardial hypertrophy was only evident 48 h post-ablation and was associated with increased heart weight and altered expression of contractile proteins. During the first 24 hours post-CAVB, genes encoding ion channel subunits were either up-regulated (such as Nav1.5, + 74%) or down-regulated (Kv4.2, −43%; KChIP2, −47%; Navβ1, −31%; Cx43, −29%). Consistent with the transient alteration of Kv4.2 expression, I to was reduced at day 1, but restored at day 5. In conclusion, CAVB induces two waves of molecular remodeling: an early one (≤ 24 h) leading to arrhythmias, a later one related to hypertrophy. These results provide new molecular basis for ventricular tachycardia induced by AV block.

Genetics of the epilepsies: Channelopathies and beyond

Genetics of the epilepsies: Channelopathies and beyond

Grand challenge for ion channels: an underexploited resource for therapeutics

OPINION article Front. Pharmacol., 20 August 2010Sec. Pharmacology of Ion Channels and Channelopathies Volume 1 - 2010 | https://doi.org/10.3389/fphar.2010.00113

Mutation in Nav1.5 Associated with Brugada Syndrome - a Mutational Hotspot?

Several studies have demonstrated an association between Brugada syndrome (BrS) and mutations in genes encoding ion channel subunits including SCN5A, CACNA1C, CACNB2b, SCN1B, and KCNE3. Mutations in SCN5A, encoding the voltage-gated sodium channel Nav1.5, represent the majority with greater than 293 mutations in SCN5A linked to the syndrome (Kapplinger et al, Heart Rhythm, In press 2009). We identified a missense mutation (G1408R) in SCN5A in a large BrS family. Intriguingly, this mutation had been reported earlier in two independent studies and has also shown to be associated with Sick Sinus Syndrome (SSS) and Cardiac Conduction Defect (CCD) (Kyndt et al, 2001; Benson et al, 2003). Nav1.5-G1408R channels heterologously expressed in CHO cells and studied using patch-clamp techniques failed to generate any sodium channel current (INa). Co-expression of the mutated channels with wild-type (WT) channels resulted in a 50% reduction of current amplitudes with no changes in kinetic properties when compared with WT channels. The residue resides in the DIII pore region and is conserved among species. We addressed the importance of this amino acid at position 1408 by replacing it with another small neutral amino acid (G1408A) and by substituting a negatively charged aspartic acid (G1408D). Our results show that substituting glycine with alanine retains WT behavior while exchange to the positively charged arginine (G1408R) or negatively charged aspartic acid leads to a complete loss-of-function. In conclusion, we describe a SCN5A mutation associated with BrS which results in a loss of function of INa important for action potential generation. Further, we show that the presence of a neutral hydrophobic amino acid at this position is crucial for normal channel function.

Confirmation of associations between ion channel gene SNPs and QTc interval duration in healthy subjects

Population-based association studies have identified several polymorphic variants in genes encoding ion channel subunits associated with the electrocardiographic heart-rate-corrected QT (QTc) length in healthy populations of Caucasian origin (KCNH2 rs1,805,123 (K897 T) and rs3,815,459, SCN5A rs1,805,126 (D1,819D), 1,141-3 C>A, rs1,805,124 (H558R), and IVS24+116 G>A, KCNQ1 rs757,092, KCNE1 IVS2-128 G>A and rs1,805,127 (G38S), and KCNE2 rs2,234,916 (T8A)). However, few of these results have been replicated in independent populations. We tested the association of SNPs KCNQ1 rs757,092, KCNH2 rs3,815,459, SCN5A IVS24+116 G>A, KCNE1 IVS2-128 G>A and KCNE2 rs2,234,916 with QTc length in two groups of 200 subjects presenting the shortest and the longest QTc from a cohort of 2,008 healthy subjects. All polymorphisms were in Hardy-Weinberg equilibrium in both groups. The minor allele SCN5A IVS24+116 A was more frequent in the group of subjects with the shortest QTc, whereas the minor alleles KCNQ1 rs757,092 G and KCNH2 rs3,815,459 A were more frequent in the group with the longest QTc. There was no significant difference for KCNE1 IVS2-128 G>A and KCNE2 rs2,234,916 between the two groups. Haplotype analysis showed a twofold increased risk of QTc lengthening for carriers of the haplotype, combining alleles C and A of the two common KCNE1 SNPs, IVS2-129 C>T (rs2,236,609) and rs1,805,127 (G38S), respectively. In conclusion, our study confirms the reported associations between QTc length and KCNQ1 rs757,092 and KCNH2 rs3,815,459.

P3-3: Ionic remodeling and ventricular arrhythmias precede hypertrophy after complete atrioventricular block in the mouse

Complete atrioventricular block (cAVB) and related ventricular bradycardia are known to induce cardiac hypertrophy and arrhythmias. We have developed a mouse model of catheter-mediated radiofrequency AV node ablation, which we used to investigate ionic remodeling and its functional consequences in relation to time after cAVB. Using high-throughput real time RT-PCR, the expression of 96 genes encoding ion channel subunits and contractile proteins was evaluated in the left ventricle of 10-week-old control (C) mice and at 12 hours (H12), 1, 2 and 5 days (D1-5) after cAVB.

Long-Term Amiodarone Administration Remodels Expression of Ion Channel Transcripts in the Mouse Heart

The basis for the unique effectiveness of long-term amiodarone treatment on cardiac arrhythmias is incompletely understood. The present study investigated the pharmacogenomic profile of amiodarone on genes encoding ion-channel subunits. Adult male mice were treated for 6 weeks with vehicle or oral amiodarone at 30, 90, or 180 mg x kg(-1) x d(-1). Plasma and myocardial levels of amiodarone and N-desethylamiodarone increased dose-dependently, reaching therapeutic ranges observed in human. Plasma triiodothyronine levels decreased, whereas reverse triiodothyronine levels increased in amiodarone-treated animals. In ECG recordings, amiodarone dose-dependently prolonged the RR, PR, QRS, and corrected QT intervals. Specific microarrays containing probes for the complete ion-channel repertoire (IonChips) and real-time reverse transcription-polymerase chain reaction experiments demonstrated that amiodarone induced a dose-dependent remodeling in multiple ion-channel subunits. Genes encoding Na+ (SCN4A, SCN5A, SCN1B), connexin (GJA1), Ca2+ (CaCNA1C), and K+ channels (KCNA5, KCNB1, KCND2) were downregulated. In patch-clamp experiments, lower expression of K+ and Na+ channel genes was associated with decreased I(to,f), I(K,slow), and I(Na) currents. Inversely, other K+ channel alpha- and beta-subunits, such as KCNA4, KCNK1, KCNAB1, and KCNE3, were upregulated. Long-term amiodarone treatment induces a dose-dependent remodeling of ion-channel expression that is correlated with the cardiac electrophysiologic effects of the drug. This profile cannot be attributed solely to the amiodarone-induced cardiac hypothyroidism syndrome. Thus, in addition to the direct effect of the drug on membrane proteins, part of the therapeutic action of long-term amiodarone treatment is likely related to its effect on ion-channel transcripts.

Microarray analysis reveals complex remodeling of cardiac ion channel expression with altered thyroid status: relation to cellular and integrated electrophysiology.

Although electrophysiological remodeling occurs in various myocardial diseases, the underlying molecular mechanisms are poorly understood. cDNA microarrays containing probes for a large population of mouse genes encoding ion channel subunits ("IonChips") were developed and exploited to investigate remodeling of ion channel transcripts associated with altered thyroid status in adult mouse ventricle. Functional consequences of hypo- and hyperthyroidism were evaluated with patch-clamp and ECG recordings. Hypothyroidism decreased heart rate and prolonged QTc duration. Opposite changes were observed in hyperthyroidism. Microarray analysis revealed that hypothyroidism induces significant reductions in KCNA5, KCNB1, KCND2, and KCNK2 transcripts, whereas KCNQ1 and KCNE1 expression is increased. In hyperthyroidism, in contrast, KCNA5 and KCNB1 expression is increased and KCNQ1 and KCNE1 expression is decreased. Real-time RT-PCR validated these results. Consistent with microarray analysis, Western blot experiments confirmed those modifications at the protein level. Patch-clamp recordings revealed significant reductions in I(to,f) and I(K,slow) densities, and increased I(Ks) density in hypothyroid myocytes. In addition to effects on K+ channel transcripts, transcripts for the pacemaker channel HCN2 were decreased and those encoding the alpha1C Ca2+ channel (CaCNA1C) were increased in hypothyroid animals. The expression of Na+, Cl-, and inwardly rectifying K+ channel subunits, in contrast, were unaffected by thyroid hormone status. Taken together, these data demonstrate that thyroid hormone levels selectively and differentially regulate transcript expression for at least nine ion channel alpha- and beta-subunits. Our results also document the potential of cDNA microarray analysis for the simultaneous examination of ion channel transcript expression levels in the diseased/remodeled myocardium.