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Atrial Fibrillation (AF) is the most common cardiac arrhythmia. Its pathogenesis is complex and poorly understood. Whole exome sequencing of Danish families with AF revealed a novel four nucleotide deletion c.1041_1044del in CLCN2 shared by affected individuals. We aimed to investigate the role of genetic variation of CLCN2 encoding the inwardly rectifying chloride channel ClC-2 as a risk factor for the development of familiar AF. The effect of the CLCN2 variant was evaluated by electrophysiological recordings on transiently transfected cells. We used quantitative PCR to assess CLCN2 mRNA expression levels in human atrial and ventricular tissue samples. The nucleotide deletion CLCN2 c.1041_1044del results in a frame-shift and premature stop codon. The truncated ClC-2 p.V347fs channel does not conduct current. Co-expression with wild-type ClC-2, imitating the heterozygote state of the patients, resulted in a 50% reduction in macroscopic current, suggesting an inability of truncated ClC-2 protein to form channel complexes with wild type channel subunits. Quantitative PCR experiments using human heart tissue from healthy donors demonstrated that CLCN2 is expressed across all four heart chambers. Our genetic and functional data points to a possible link between loss of ClC-2 function and an increased risk of developing AF.

Background Dysfunction of NaV 1.5 encoded by SCN5A accounts for approximately half of the channelopathic SIDS cases. We investigated the functional effect of two gene variants identified in the same patient, one in SCN5A and one in SCN1Bb. The aim of the study was to risk stratify the proband's family. Methods The family was referred for cardiovascular genetic evaluation to assess familial risk of cardiac disease. Functional analysis of the identified variants was performed with patch-clamp electrophysiology in HEK293 cells. Results A 16-month-old healthy boy died suddenly in the context of nonspecific illness and possible fever. Postmortem genetic testing revealed variants in the SCN5A and SCN1Bb genes. The proband's father carries the same variants but is asymptomatic. Electrophysiological analysis of the NaV 1.5_1281X truncation revealed complete loss-of-function of the channel. Coexpression of NaV 1.5 with NaV β1b significantly increased INa density when compared to NaV 1.5 alone. The NaV β1b _V268I variant abolished this INa density increase. Moreover, it shifted the activation curve toward more depolarized potentials. Conclusions Genetic variation of both sodium channel and its modifiers may contribute to sudden unexplained death in childhood. However, the asymptomatic father suggests that genetic variation of these genes is not sufficient to cause sudden death or clinically detectable SCN5A phenotypes.

BACKGROUND/AIMS: The voltage-gated potassium channel KV11.1 has been originally cloned from the brain and is expressed in a variety of tissues. The role of phosphorylation for channel function is a matter of debate. In this study, we aimed to elucidate the extent and role of protein kinase D mediated phosphorylation.METHODS: We employed mass spectrometry, whole-cell patch clamp electrophysiology, confocal microscopy, site-directed mutagenesis, and western blotting.RESULTS: Using brain tissue from rat and mouse, we mapped several phosphorylated KV11.1 residues by LC-MS mass spectrometry and identified protein kinase D (PKD1) as possible regulatory kinase. Co-expression of KV11.1 with PKD1 reduced current amplitudes without altering protein levels or surface expression of the channel. Based on LC-MS results from in vivo and HEK293 cell experiments we chose four KV11.1 mutant candidates for further functional analysis. Ablation of the putative PKD phosphorylation site in the mutant S284A increased the maximal current indicating S284 as a main PKD target in KV11.1.CONCLUSIONS: Our data might help mitigating a long-standing controversy in the field regarding PKC regulation of KV11.1. We propose that PKD1 mediates the PKC effects on KV11.1 and we found that PKD targets S284 in the N-terminus of the channel.

Aim The voltage-gated potassium channel KV11.1 is the molecular basis for the IKr current which plays an important role in cardiac physiology. Its malfunction is associated with both inherited and acquired cardiac arrhythmias. Native currents differ from those in experimental models, suggesting additional regulatory mechanisms. We hypothesised that the post-translational modification sumoylation finetunes channel activity. Methods The functional effects of sumoylation on KV11.1 were addressed by employing two-electrode voltage-clamp (TEVC) experiments in Xenopus laevis oocytes. Site-directed mutagenesis enabled a further analyse of the SUMO-target amino acids. We assessed protein expression levels and used confocal imaging for localization studies. Results Co-expression with Ubc9 and SUMO alters the electrophysiological properties of KV11.1 leading to a decrease in steady-state current amplitude largely due to faster inactivation and alteration of deactivation kinetics. We identified three lysines (K21, K93, and K116) in the PAS-domain as the putative SUMO-targets. Conclusion This study indicates KV11.1 as a sumoylation target, and offers the three main targets; K21, K93, and K116. Furthermore, it proposes an underlying mechanism for the observed kinetic impact of the PAS domain. This article is protected by copyright. All rights reserved.