POPDC2 a novel susceptibility gene for conduction disorders.

Despite recent progress in the understanding of cardiac ion channel function and its role in inherited forms of ventricular arrhythmias, the molecular basis of cardiac conduction disorders often remains unresolved. We aimed to elucidate the genetic background of familial atrioventricular block (AVB) using a whole exome sequencing (WES) approach. In monozygotic twins with a third-degree AVB and in another, unrelated family with first-degree AVB, we identified a heterozygous nonsense mutation in the POPDC2 gene causing a premature stop at position 188 (POPDC2W188⁎), deleting parts of its cAMP binding-domain. Popeye-domain containing (POPDC) proteins are predominantly expressed in the skeletal muscle and the heart, with particularly high expression of POPDC2 in the sinoatrial node of the mouse. We now show by quantitative PCR experiments that in the human heart the POPDC-modulated two-pore domain potassium (K2P) channel TREK-1 is preferentially expressed in the atrioventricular node. Co-expression studies in Xenopus oocytes revealed that POPDC2W188⁎ causes a loss-of-function with impaired TREK-1 modulation. Consistent with the high expression level of POPDC2 in the murine sinoatrial node, POPDC2W188⁎ knock-in mice displayed stress-induced sinus bradycardia and pauses, a phenotype that was previously also reported for POPDC2 and TREK-1 knock-out mice. We propose that the POPDC2W188⁎ loss-of-function mutation contributes to AVB pathogenesis by an aberrant modulation of TREK-1, highlighting that POPDC2 represents a novel arrhythmia gene for cardiac conduction disorders.

Stress-Kinase Regulation of TASK-1 and TASK-3.

BACKGROUND/AIMS: TASK channels belong to the two-pore-domain potassium (K2P) channel family. TASK-1 is discussed to contribute to chronic atrial fibrillation (AFib) and has been together with uncoupling protein 1 found as a marker protein of brown adipose tissue (BAT) fat. In addition, TASK-1 was linked in a genome-wide association study to an increased body mass index. A recent study showed that TASK-1 inhibition is causing obesity in mice by a BAT whitening and that these effects are linked to the mineralocorticoid receptor pathway, albeit the mechanism remained elusive. Therefore, we aimed to probe whether K2P channels are regulated by serum- and glucocorticoid-inducible kinases (SGKs) which are known to modify many cellular functions by modulating ion channels. METHODS: To this end we used functional co-expression studies and chemiluminescence-assays in Xenopus oocytes, together with fluorescence imaging and quantitative PCR experiments. RESULTS: SGKs and proteinkinase B (PKB) induced a strong, dose- and time-dependent current reduction of TASK-1 and TASK-3. SGK co-expression reduced the surface expression of TASK-1/3, leading to a predominant localization of the channels into late endosomes. The down regulation of TASK-3 channels was abrogated by the dynamin inhibitor dynasore, confirming a role of SGKs in TASK-1/3 channel endocytosis. CONCLUSION: Stress-mediated changes in SGK expression pattern or activation is likely to alter TASK-1/3 expression at the surface membrane. The observed TASK-1 regulation might contribute to the pathogenesis of chronic AFib and provide a mechanistic link between increased mineralocorticoid levels and TASK-1 reduction, both linked to BAT whitening.

Electrophysiological characterization of a large set of novel variants in the SCN5A-gene: identification of novel LQTS3 and BrS mutations.

SCN5A encodes for the α-subunit of the cardiac voltage-gated sodium channel Nav1.5. Gain-of-function mutations in SCN5A are related to congenital long QT syndrome (LQTS3) characterized by delayed cardiac repolarization, leading to a prolonged QT interval in the ECG. Loss-of-function mutations in SCN5A are related to Brugada syndrome (BrS), characterized by an ST-segment elevation in the right precordial leads (V1-V3). The aim of this study was the characterization of a large set of novel SCN5A variants found in patients with different cardiac phenotypes, mainly LQTS and BrS. SCN5A variants of 13 families were functionally characterized in Xenopus laevis oocytes using the two-electrode voltage-clamp technique. We found in most of the cases, but not all, that the electrophysiology of the variants correlated with the clinically diagnosed phenotype. A susceptibility to develop LQTS can be suggested in patients carrying the variants S216L, K480N, A572D, F816Y, and G983D. However, taking the phenotype into account, the presence of the variants in genomic data bases, the mutational segregation, combined with our in vitro and in silico experiments, the variants S216L, S262G, K480N, A572D, F816Y, G983D, and T1526P remain as variants of unknown significance. However, the SCN5A variants R568H and A993T can be classified as pathogenic LQTS3 causing mutations, while R222stop and R2012H are novel BrS causing mutations.

Gain-of-function mutations in the calcium channel CACNA1C (Cav1.2) cause non-syndromic long-QT but not Timothy syndrome.

Gain-of-function mutations in CACNA1C, encoding the L-type Ca(2+) channel Cav1.2, cause Timothy syndrome (TS), a multi-systemic disorder with dysmorphic features, long-QT syndrome (LQTS) and autism spectrum disorders. TS patients have heterozygous mutations (G402S and G406R) located in the alternatively spliced exon 8, causing a gain-of-function by reduced voltage-dependence of inactivation. Screening 540 unrelated patients with non-syndromic forms of LQTS, we identified six functional relevant CACNA1C mutations in different regions of the channel. All these mutations caused a gain-of-function combining different mechanisms, including changes in current amplitude, rate of inactivation and voltage-dependence of activation or inactivation, similar as in TS. Computer simulations support the theory that the novel CACNA1C mutations prolong action potential duration. We conclude that genotype-negative LQTS patients should be investigated for mutations in CACNA1C, as a gain-of-function in Cav1.2 is likely to cause LQTS and only specific and rare mutations, i.e. in exon 8, cause the multi-systemic TS.

Popeye domain containing proteins modulate the voltage-gated cardiac sodium channel Nav1.5.

Popeye domain containing (POPDC) proteins are predominantly expressed in the heart and skeletal muscle, modulating the K2P potassium channel TREK-1 in a cAMP-dependent manner. POPDC1 and POPDC2 variants cause cardiac conduction disorders with or without muscular dystrophy. Searching for POPDC2-modulated ion channels using a functional co-expression screen in Xenopus oocytes, we found POPDC proteins to modulate the cardiac sodium channel Nav1.5. POPDC proteins downregulate Nav1.5 currents in a cAMP-dependent manner by reducing the surface expression of the channel. POPDC2 and Nav1.5 are both expressed in different regions of the murine heart and consistently POPDC2 co-immunoprecipitates with Nav1.5 from native cardiac tissue. Strikingly, the knock-down of popdc2 in embryonic zebrafish caused an increased upstroke velocity and overshoot of cardiac action potentials. The POPDC modulation of Nav1.5 provides a new mechanism to regulate cardiac sodium channel densities under sympathetic stimulation, which is likely to have a functional impact on cardiac physiology and inherited arrhythmias.

Sodium permeable and "hypersensitive" TREK-1 channels cause ventricular tachycardia.

In a patient with right ventricular outflow tract (RVOT) tachycardia, we identified a heterozygous point mutation in the selectivity filter of the stretch-activated K2P potassium channel TREK-1 (KCNK2 or K2P2.1). This mutation introduces abnormal sodium permeability to TREK-1. In addition, mutant channels exhibit a hypersensitivity to stretch-activation, suggesting that the selectivity filter is directly involved in stretch-induced activation and desensitization. Increased sodium permeability and stretch-sensitivity of mutant TREK-1 channels may trigger arrhythmias in areas of the heart with high physical strain such as the RVOT We present a pharmacological strategy to rescue the selectivity defect of the TREK-1 pore. Our findings provide important insights for future studies of K2P channel stretch-activation and the role of TREK-1 in mechano-electrical feedback in the heart.

Functional mutagenesis screens reveal the 'cap structure' formation in disulfide-bridge free TASK channels.

Two-pore-domain potassium (K2P) channels have a large extracellular cap structure formed by two M1-P1 linkers, containing a cysteine for dimerization. However, this cysteine is not present in the TASK-1/3/5 subfamily. The functional role of the cap is poorly understood and it remained unclear whether K2P channels assemble in the domain-swapped orientation or not. Functional alanine-mutagenesis screens of TASK-1 and TRAAK were used to build an in silico model of the TASK-1 cap. According to our data the cap structure of disulfide-bridge free TASK channels is similar to that of other K2P channels and is most likely assembled in the domain-swapped orientation. As the conserved cysteine is not essential for functional expression of all K2P channels tested, we propose that hydrophobic residues at the inner leaflets of the cap domains can interact with each other and that this way of stabilizing the cap is most likely conserved among K2P channels.

POPDC1(S201F) causes muscular dystrophy and arrhythmia by affecting protein trafficking.

The Popeye domain-containing 1 (POPDC1) gene encodes a plasma membrane-localized cAMP-binding protein that is abundantly expressed in striated muscle. In animal models, POPDC1 is an essential regulator of structure and function of cardiac and skeletal muscle; however, POPDC1 mutations have not been associated with human cardiac and muscular diseases. Here, we have described a homozygous missense variant (c.602C>T, p.S201F) in POPDC1, identified by whole-exome sequencing, in a family of 4 with cardiac arrhythmia and limb-girdle muscular dystrophy (LGMD). This allele was absent in known databases and segregated with the pathological phenotype in this family. We did not find the allele in a further screen of 104 patients with a similar phenotype, suggesting this mutation to be family specific. Compared with WT protein, POPDC1(S201F) displayed a 50% reduction in cAMP affinity, and in skeletal muscle from patients, both POPDC1(S201F) and WT POPDC2 displayed impaired membrane trafficking. Forced expression of POPDC1(S201F) in a murine cardiac muscle cell line (HL-1) increased hyperpolarization and upstroke velocity of the action potential. In zebrafish, expression of the homologous mutation (popdc1(S191F)) caused heart and skeletal muscle phenotypes that resembled those observed in patients. Our study therefore identifies POPDC1 as a disease gene causing a very rare autosomal recessive cardiac arrhythmia and LGMD, expanding the genetic causes of this heterogeneous group of inherited rare diseases.