Nicole Schmitt

Loss-of-function mutations in K_v7.1 often lead to long QT syndrome (LQTS), a cardiac repolarization disorder associated with arrhythmia and subsequent sudden cardiac death. The discovery of agonisticI_Ksmodulators may offer a new potential strategy in pharmacological treatment of this disorder. The benzodiazepine derivative (R)-L3 potently activates K_v7.1 channels and shortens action potential duration, thus may represent a starting point for drug development. However, the molecular mechanisms underlying modulation by (R)-L3 are still unknown. By combining alanine scanning mutagenesis, non-canonical amino acid incorporation, voltage-clamp electrophysiology and fluorometry, andin silicoprotein modelling, we show that (R)-L3 not only stimulates currents by allosteric modulation of the pore domain but also alters the kinetics independently from the pore domain effects. We identify novel (R)-L3-interacting key residues in the lower S4-segment of K_v7.1 and observed an uncoupling of the outer S4 segment with the inner S5, S6 and selectivity filter segments.

Atrial fibrillation (AF) is predicted to affect around 17.9 million individuals in Europe by 2060. The disease is associated with severe electrical and structural remodelling of the heart, and increased the risk of stroke and heart failure. In order to improve treatment and find new drug targets, the field needs to better comprehend the exact molecular mechanisms in these remodelling processes. This study aims to identify gene and miRNA networks involved in the remodelling of AF hearts in AF patients with mitral valve regurgitation (MVR). Total RNA was extracted from right atrial biopsies from patients undergoing surgery for mitral valve replacement or repair with AF and without history of AF to test for differentially expressed genes and miRNAs using RNA-sequencing and miRNA microarray. In silico predictions were used to construct a mRNA-miRNA network including differentially expressed mRNAs and miRNAs. Gene and chromosome enrichment analysis were used to identify molecular pathways and high-density AF loci. We found 644 genes and 43 miRNAs differentially expressed in AF patients compared to controls. From these lists, we identified 905 pairs of putative miRNA-mRNA interactions, including 37 miRNAs and 295 genes. Of particular note, AF-associated miR-130b-3p, miR-338-5p and miR-208a-3p were differentially expressed in our AF tissue samples. These miRNAs are predicted regulators of several differentially expressed genes associated with cardiac conduction and fibrosis. We identified two high-density AF loci in chromosomes 14q11.2 and 6p21.3. AF in MVR patients is associated with down-regulation of ion channel genes and up-regulation of extracellular matrix genes. Other AF related genes are dysregulated and several are predicted to be targeted by miRNAs. Our novel miRNA-mRNA regulatory network provides new insights into the mechanisms of AF.

Background The slow delayed rectifier potassium current I-Ks is crucial for the repolarization of the cardiac action potential. It is conducted by the voltage-gated channel K(V)7.1 encoded by KCNQ1, together with its beta-subunit KCNE1. Loss-of-function (LOF) mutations in KCNQ1 have been associated with heritable cardiac arrhythmias such as Long QT syndrome (LQTS). This disease is characterized by prolonged ventricular repolarization and propensity to ventricular tachyarrhythmia that may lead to syncope, cardiac arrest, and sudden death. We aimed to functionally characterize two K(V)7.1 mutations (p.A150T and p.L374H) identified in two independent LQTS patients with different severity of disease phenotype, family history, and co-segregation of LQTS. Methods We performed whole-cell patch clamp recordings in CHO-K1 cells, and confocal imaging in Madin-Darby Canine Kidney (MDCK) cells. Results I-Ks-A150T showed significantly decreased current amplitudes from above +20 mV (approximately 52% decrease at +40 mV), but demonstrated cell membrane localization similar to wild-type (WT). I-Ks-L374H, however, exhibited a complete LOF compared to WT channels. Confocal imaging showed endoplasmic reticulum retention of the channel in MDCK cells. Mimicking the heterozygous state of the patients by co-expressing WT and mutant subunits resulted in an approximately 22% decrease in current at +40 mV for A150T. The L374H mutation showed a more pronounced effect (62% reduction at +40 mV compared to WT channel). Conclusion Both mutations, K(V)7.1 A150T and L374H, led to loss of channel function. The degree of LOF may mirror the disease phenotype observed in the patients.

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/Aims: The voltage-gated potassium channel K_V11.1 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 K_V11.1 residues by LC-MS mass spectrometry and identified protein kinase D (PKD1) as possible regulatory kinase. Co-expression of K_V11.1 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 K_V11.1 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 K_V11.1. Conclusions: Our data might help mitigating a long-standing controversy in the field regarding PKC regulation of K_V11.1. We propose that PKD1 mediates the PKC effects on K_V11.1 and we found that PKD targets S284 in the N-terminus of the channel.

Background: Dysfunction of Na_V1.5 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 Na_V1.5_1281X truncation revealed complete loss‐of‐function of the channel. Coexpression of Na_V1.5 with Na_Vβ1b significantly increased I_Na density when compared to Na_V1.5 alone. The Na_Vβ1b _V268I variant abolished this I_Na 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.

Aim: We investigated the effect of variants in genes encoding sodium channel modifiers SNTA1 and GPD1L found in early onset atrial fibrillation (AF) patients. Patients & methods: Genetic screening in patients with early onset lone AF revealed three variants in GPD1L and SNTA1 in three AF patients. Functional analysis was performed by patch-clamp electrophysiology. Results: Co-expression of GPD1L or its p.A326E variant with NaV1.5 did not alter INa density or current kinetics. SNTA1 shifted the peak-current by -5 mV. The SNTA1-p.A257G variant significantly increased INa. SNTA1-p.P74L did not produce functional changes. Conclusion: Although genetic variation of sodium channel modifiers may contribute to development of AF at a molecular level, it is unlikely a monogenic cause of the disease.

Kv2.1 is a major delayed-rectifier voltage-gated potassium channel widely expressed in neurons of the CNS. Kv2.1 localizes in high-density cell-surface clusters in the soma and proximal dendrites as well as in the axon initial segment (AIS). Given the crucial roles of both of these compartments in integrating signal input and then generating output, this localization of Kv2.1 is ideal for regulating the overall excitability of neurons. Here we used fluorescence recovery after photobleaching imaging, mutagenesis, and pharmacological interventions to investigate the molecular mechanisms that control the localization of Kv2.1 in these two different membrane compartments in cultured rat hippocampal neurons of mixed sex. Our data uncover a unique ability of Kv2.1 channels to use two molecularly distinct trafficking pathways to accomplish this. Somatodendritic Kv2.1 channels are targeted by the conventional secretory pathway, whereas axonal Kv2.1 channels are targeted by a nonconventional trafficking pathway independent of the Golgi apparatus. We further identified a new AIS trafficking motif in the C-terminus of Kv2.1, and show that putative phosphorylation sites in this region are critical for the restricted and clustered localization in the AIS. These results indicate that neurons can regulate the expression and clustering of Kv2.1 in different membrane domains independently by using two distinct localization mechanisms, which would allow neurons to precisely control local membrane excitability.SIGNIFICANCE STATEMENTOur study uncovered a novel mechanism that targets the Kv2.1 voltage-gated potassium channel to two distinct trafficking pathways and two distinct subcellular destinations: the somatodendritic plasma membrane and that of the axon initial segment. We also identified a distinct motif, including putative phosphorylation sites, that is important for the AIS localization. This raises the possibility that the destination of a channel protein can be dynamically regulated via changes in post-translational modification, which would impact the excitability of specific membrane compartments.

Aim: The voltage‐gated potassium channel K_V11.1 is the molecular basis for the I_Kr 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 hypothesized that the post‐translational modification sumoylation fine‐tunes channel activity.Methods: The functional effects of sumoylation on K_V11.1 were addressed by employing two‐electrode voltage‐clamp (TEVC) experiments in Xenopus laevis oocytes. Site‐directed mutagenesis enabled a further analysis 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 K_V11.1 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 K_V11.1 as a sumoylation target and offers three main targets: K21, K93, and K116. Furthermore, it proposes an underlying mechanism for the observed kinetic impact of the PAS domain.

A recent experimental study investigating patients with lone atrial fibrillation identified six novel mutations in the KCNA5 gene. The mutants exhibited both gain- and loss-of-function of the atrial specific ultra-rapid delayed rectifier K⁺ current, I_Kur. The aim of this study is to elucidate and quantify the functional impact of these KCNA5 mutations on atrial electrical activity. A multi-scale model of the human atria was updated to incorporate detailed experimental data on I_Kur from both wild-type and mutants. The effects of the mutations on human atrial action potential and rate dependence were investigated at the cellular level. In tissue, we assessed the effects of the mutations on the vulnerability to unidirectional conduction patterns and dynamics of re-entrant excitation waves. Gain-of-function mutations shortened the action potential duration in single cells, and stabilised and accelerated re-entrant excitation in tissue. Loss-of-function mutations had heterogeneous effects on action potential duration and promoted early-after-depolarisations following beta-adrenergic stimulation. In the tissue model, loss-of-function mutations facilitated breakdown of excitation waves at more physiological excitation rates than the wild-type, and the generation of early-after-depolarisations promoted unidirectional patterns of excitation. Gain- and loss-of-function I_Kur mutations produced multiple mechanisms of atrial arrhythmogenesis, with significant differences between the two groups of mutations. This study provides new insights into understanding the mechanisms by which mutant I_Kur contributes to atrial arrhythmias. In addition, as I_Kur is an atrial-specific channel and a number of I_Kur-selective blockers have been developed as anti-AF agents, this study also helps to understand some contradictory results on both pro- and anti-arrhythmic effects of blocking I_Kur. In a recent study, six mutations resulting in either gain-of-function or loss-of-function in the ultra-rapid delayed rectifier potassium current I_Kur, were identified to be associated with atrial fibrillation (AF). However, the causative link between the mutant I_Kur (either gain- or loss-of-function) and AF genesis, especially the difference and similarity between the two mutant groups, has not been elucidated. In our study, we used multiscale computational models to investigate the mechanism of arrhythmogenesis mediated by the two groups of mutations. The results suggest that the gain-of-function mutations shortened atrial action potential duration, stabilised and accelerated re-entrant excitation waves in tissue; the loss-of-function mutation promoted early-after-depolarisations following beta-adrenergic stimulation and thus wave breaks in tissue. We show these two groups of mutants carrying I_Kur produced multiple mechanisms of atrial arrhythmogenesis, with significant differences between the two groups. Our study provides new insights into understanding the mechanisms by which mutant I_Kur contributes to atrial arrhythmias. In addition, as I_Kur is an atrial-specific channel and a number of I_Kur-selective blockers have been developed as anti-AF agents, this study also helps to understand some contradictory results on both pro- and anti-arrhythmic effects of blocking I_Kur.