Familial ST-depression syndrome (FSTD) is a recently identified inherited cardiac disease associated with arrhythmias and systolic dysfunction. The underlying genetic aetiology has remained elusive. This study aimed at finding the causative variant.

A total of 67 FSTD patients (20 families) were studied. Linkage analysis and whole-genome sequencing (WGS) were initially performed. An identified non-coding variant was functionally characterized in AC16 human cardiomyocytes, muscle tissue, and human myocardium. In silico analyses, luciferase and dCas9-activator/repressor assays, protein-DNA experiments, chromosome conformation capture (4C), and RNA sequencing were also performed.

The electrocardiographic (ECG) phenotype was inherited in an autosomal dominant manner in all families. Linkage analysis revealed a single peak on chromosome 20, and WGS identified a single, rare, non-coding variant located 18 kb downstream of KCNB1 on chromosome 20 in all affected individuals. Perfect co-segregation with the ECG phenotype was observed together with full penetrance in all families. The variant creates a MEF2-binding site and presence of the variant allele or MEF2 co-expression enhanced transcriptional activity. dCas9-activator/repressor assays showed that KCNB1 was the only gene consistently regulated by the locus and 4C experiments in AC16 cells and human muscle tissue confirmed the locus-KCNB1 promoter interaction. Expression analysis in human endocardial tissue did not document any change in gene expression likely explained by expressional heterogeneity.

A gain-of-function enhancer variant creates a hyperactive regulatory locus that interacts with the KCNB1 promoter and causes FSTD. This is the first time that KCNB1 has been implicated in human cardiac electrophysiology and arrhythmogenesis.

Migraines are a common type of headache affecting around 15% of the population. The signalling pathways leading to migraines have not been fully understood, but neuronal voltage-gated ion channels, such as KCNG4, have been linked to this pathology. KCNG4 (Kv6.4) is a silent member of the superfamily of voltage-gated potassium (Kv) channels, which expresses in heterotetramers with members of the KCNB (Kv2) family. The genetic variant Kv6.4-L360P has previously been linked to migraines, but their mode of action remains unknown. Here, we characterized the molecular characteristics of Kv6.4-L360P when co-expressed with Kv2.1. We found that Kv6.4-L360P almost completely abolishes Kv2 currents, and we propose that this mechanism in the trigeminal system, linked to the initiation of migraine, leads to the pathology.

Voltage-gated K+ (KV) channels govern K+ ion flux across cell membranes in response to changes in membrane potential. They are formed by the assembly of four subunits, typically from the same family. Electrically silent KV channels (KVS), however, are unable to conduct currents on their own. It has been assumed that these KVS must obligatorily assemble with subunits from the KV2 family into heterotetrameric channels, thereby giving rise to currents distinct from those of homomeric KV2 channels. Herein, we show that KVS subunits indeed also modulate the activity, biophysical properties and surface expression of recombinant KV7 isoforms in a subunit-specific manner. Employing co-immunoprecipitation, and proximity labelling, we unveil the spatial coexistence of KVS and KV7 within a single protein complex. Electrophysiological experiments further indicate functional interaction and probably heterotetramer formation. Finally, single-cell transcriptomic analyses identify native cell types in which this KVS and KV7 interaction may occur. Our findings demonstrate that KV cross-family interaction is much more versatile than previously thought-possibly serving nature to shape potassium conductance to the needs of individual cell types.

The brain ventricular system (BVS) consists of brain ventricles and channels filled with cerebrospinal fluid (CSF). Disturbance of CSF flow has been linked to scoliosis and neurodegenerative diseases, including hydrocephalus. This could be due to defects of CSF production by the choroid plexus or impaired CSF movement over the ependyma dependent on motile cilia. Most vertebrates have horizontal body posture. They retain additional evolutionary innovations assisting CSF flow, such as the Reissner fiber. The causes of hydrocephalus have been studied using animal models including rodents (mice, rats, hamsters) and zebrafish. However, the horizontal body posture reduces the effect of gravity on CSF flow, which limits the use of mammalian models for scoliosis. In contrast, fish swim against the current and experience a forward-to-backward mechanical force akin to that caused by gravity in humans. This explains the increased popularity of the zebrafish model for studies of scoliosis. "Slit-ventricle" syndrome is another side of the spectrum of BVS anomalies. It develops because of insufficient inflation of the BVS. Recent advances in zebrafish functional genetics have revealed genes that could regulate the development of the BVS and CSF circulation. This review will describe the BVS of zebrafish, a typical teleost, and vertebrates in general, in comparative perspective. It will illustrate the usefulness of the zebrafish model for developmental studies of the choroid plexus (CP), CSF flow and the BVS.

The Kv family of voltage-gated potassium channels regulate neuronal excitability. The biophysical characteristics of Kv channels can be matched to the needs of different neurons by forming homotetrameric or heterotetrameric channels within one of four subfamilies. The cytoplasmic tetramerization (T1) domain plays a major role in dictating the compatibility of different Kv subunits. The only Kv subfamily lacking a representative structure of the T1 domain is the Kv2 family. Here, X-ray crystallography was used to solve the structure of the human Kv2.1 T1 domain. The structure is similar to those of other T1 domains, but surprisingly formed a pentamer instead of a tetramer. In solution the Kv2.1 T1 domain also formed a pentamer, as determined by inline SEC-MALS-SAXS and negative-stain electron microscopy. The Kv2.1 T1-T1 interface involves electrostatic interactions, including a salt bridge formed by the negative charges in a previously described CDD motif, and inter-subunit coordination of zinc. It is shown that zinc binding is important for stability. In conclusion, the Kv2.1 T1 domain behaves differently from the other Kv T1 domains, which may reflect the versatility of Kv2.1, which can assemble with the regulatory KvS subunits and scaffold ER-plasma membrane contacts.

Neuronal autoantibodies and favorable response to immunosuppressive treatment have been described in patients with chronic epilepsy of unknown cause, suggesting autoimmune etiology. Our aim was to identify novel epilepsy-specific autoantibodies reactive with neuronal surface antigens.

Sera of 172 epilepsy patients with unknown cause and 30 healthy controls were screened with indirect immunofluorescence to identify IgG reacting with primary rat neuronal cultures. Putative target autoantigens were investigated with immunoprecipitation (IP) and liquid chromatography-mass/mass spectrometry (LC-MS/MS) studies using SH-SY5Y cells. Validation of LC-MS/MS results was carried out by IP and immunocytochemistry assays.

Antibodies to neuronal cell surface antigens were detected in 18 epilepsy patients. LC-MS/MS analysis identified voltage-gated potassium channel modifier subfamily F member 1 (KCNF1, Kv5.1) as the single common cell surface antigen in 4 patients with Lennox-Gastaut syndrome (n = 2), focal epilepsy of unknown cause (n = 1) and mesial temporal lobe epilepsy with hippocampal sclerosis (n = 1). These patients had the common features of early seizure onset and treatment-resistance. IP assays and co-localization (serum IgG and commercial Kv5.1-antibody) studies done with non-fixed Kv5.1-transfected HEK293 cells and primary neuronal cultures confirmed the presence of Kv5.1-antibody in 4 epilepsy patients identified by LC-MS/MS. Similar findings were not obtained by sera of other patients with epilepsy, patients with autoimmune encephalitis and healthy controls.

The herein described novel neuronal surface antibody to Kv5.1 appears to be associated with treatment-resistant epilepsy of unknown cause. Exact clinical and pathogenic significance of this antibody remains to be elucidated.

The electrically silent (KvS) members of the voltage-gated potassium (Kv) subfamilies Kv5, Kv6, Kv8, and Kv9 selectively modulate Kv2 subunits by forming heterotetrameric Kv2/KvS channels. Based on the reported 3:1 stoichiometry of Kv2.1/Kv9.3 channels, we tested the hypothesis that Kv2.1/Kv6.4 channels express, in contrast to the assumed 3:1, in a 2:2 stoichiometry. We investigate the Kv2.1/Kv6.4 stoichiometry using single subunit counting and functional characterization of tetrameric concatemers. For selecting the most probable stoichiometry, we introduce a model-selection method that is applicable for any multimeric complex by investigating the stoichiometry of Kv2.1/Kv6.4 channels. Weighted likelihood calculations bring rigor to a powerful technique. Using the weighted-likelihood model-selection method and analysis of electrophysiological data, we show that Kv2.1/Kv6.4 channels express, in contrast to the assumed 3:1, in a 2:2 stoichiometry. Within this stoichiometry, the Kv6.4 subunits have to be positioned alternating with Kv2.1 to express functional channels. The variability in Kv2/KvS assembly increases the diversity of heterotetrameric configurations and extends the regulatory possibilities of KvS by allowing the presence of more than one silent subunit.

The brain ventricular system (BVS) consists of brain ventricles and channels connecting ventricles filled with cerebrospinal fluid (CSF). The disturbance of CSF flow has been linked to neurodegenerative disease including hydrocephalus, which manifests itself as an abnormal expansion of BVS. This relatively common developmental disorder has been observed in human and domesticated animals and linked to functional deficiency of various cells lineages facing BVS, including the choroid plexus or ependymal cells that generate CSF or the ciliated cells that cilia beating generates CSF flow. To understand the underlying causes of hydrocephalus, several animal models were developed, including rodents (mice, rat, and hamster) and zebrafish. At another side of a spectrum of BVS anomalies there is the "slit-ventricle" syndrome, which develops due to insufficient inflation of BVS. Recent advances in functional genetics of zebrafish brought to light novel genetic elements involved in development of BVS and circulation of CSF. This review aims to reveal common elements of morphologically different BVS of zebrafish as a typical representative of teleosts and other vertebrates and illustrate useful features of the zebrafish model for studies of BVS. Along this line, recent analyses of the two novel zebrafish mutants affecting different subunits of the potassium voltage-gated channels allowed to emphasize an important functional convergence of the evolutionarily conserved elements of protein transport essential for BVS development, which were revealed by the zebrafish and mouse studies.

The brain ventricular system is essential for neurogenesis and brain homeostasis. Its neuroepithelial lining effects these functions, but the underlying molecular pathways remain to be understood. We found that the potassium channels expressed in neuroepithelial cells determine the formation of the ventricular system. The phenotype of a novel zebrafish mutant characterized by denudation of neuroepithelial lining of the ventricular system and hydrocephalus is mechanistically linked to Kcng4b, a homologue of the 'silent' voltage-gated potassium channel α-subunit K_v6.4. We demonstrated that Kcng4b modulates proliferation of cells lining the ventricular system and maintains their integrity. The gain of Kcng4b function reduces the size of brain ventricles. Electrophysiological studies suggest that Kcng4b mediates its effects via an antagonistic interaction with Kcnb1, the homologue of the electrically active delayed rectifier potassium channel subunit K_v2.1. Mutation of kcnb1 reduces the size of the ventricular system and its gain of function causes hydrocephalus, which is opposite to the function of Kcng4b. This demonstrates the dynamic interplay between potassium channel subunits in the neuroepithelium as a novel and crucial regulator of ventricular development in the vertebrate brain.

The diversity of the voltage-gated K(+) (Kv) channel subfamily Kv2 is increased by interactions with auxiliary β-subunits and by assembly with members of the modulatory so-called silent Kv subfamilies (Kv5-Kv6 and Kv8-Kv9). However, it has not yet been investigated whether these two types of modulating subunits can associate within and modify a single channel complex simultaneously. Here, we demonstrate that the transmembrane β-subunit KCNE5 modifies the Kv2.1/Kv6.4 current extensively, whereas KCNE2 and KCNE4 only exert minor effects. Co-expression of KCNE5 with Kv2.1 and Kv6.4 did not alter the Kv2.1/Kv6.4 current density but modulated the biophysical properties significantly; KCNE5 accelerated the activation, slowed the deactivation and steepened the slope of the voltage-dependence of the Kv2.1/Kv6.4 inactivation by accelerating recovery of the closed-state inactivation. In contrast, KCNE5 reduced the current density ~2-fold without affecting the biophysical properties of Kv2.1 homotetramers. Co-localization of Kv2.1, Kv6.4 and KCNE5 was demonstrated with immunocytochemistry and formation of Kv2.1/Kv6.4/KCNE5 and Kv2.1/KCNE5 complexes was confirmed by Fluorescence Resonance Energy Transfer experiments performed in HEK293 cells. These results suggest that a triple complex consisting of Kv2.1, Kv6.4 and KCNE5 subunits can be formed. In vivo, formation of such tripartite Kv2.1/Kv6.4/KCNE5 channel complexes might contribute to tissue-specific fine-tuning of excitability.

The KCNE single-span transmembrane subunits are encoded by five-member gene families in the human and mouse genomes. Primarily recognized for co-assembling with and functionally regulating the voltage-gated potassium channels, the broad influence of KCNE subunits in mammalian physiology belies their small size. KCNE2 has been widely studied since we first discovered one of its roles in the heart and its association with inherited and acquired human Long QT syndrome. Since then, physiological analyses together with human and mouse genetics studies have uncovered a startling array of functions for KCNE2, in the heart, stomach, thyroid and choroid plexus. The other side of this coin is the variety of interconnected disease manifestations caused by KCNE2 disruption, involving both excitable cells such as cardiomyocytes, and non-excitable, polarized epithelia. Kcne2 deletion in mice has been particularly instrumental in illustrating the potential ramifications within a monogenic arrhythmia syndrome, with removal of one piece revealing the unexpected complexity of the puzzle. Here, we review current knowledge of the function and pathobiology of KCNE2.

The "silent" voltage-gated potassium (KvS) channel subunit Kv6.4 does not form electrically functional homotetramers at the plasma membrane but assembles with Kv2.1 subunits, generating functional Kv2.1/Kv6.4 heterotetramers. The N-terminal T1 domain determines the subfamily-specific assembly of Kv1-4 subunits by preventing interactions between subunits that belong to different subfamilies. For Kv6.4, yeast-two-hybrid experiments showed an interaction of the Kv6.4 N-terminus with the Kv2.1 N-terminus, but unexpectedly also with the Kv3.1 N-terminus. We confirmed this interaction by Fluorescence Resonance Energy Transfer (FRET) and co-immunoprecipitation (co-IP) using N-terminal Kv3.1 and Kv6.4 fragments. However, full-length Kv3.1 and Kv6.4 subunits do not form heterotetramers at the plasma membrane. Therefore, additional interactions between the Kv6.4 and Kv2.1 subunits should be important in the Kv2.1/Kv6.4 subfamily-specificity. Using FRET and co-IP approaches with N- and C-terminal fragments we observed that the Kv6.4 C-terminus physically interacts with the Kv2.1 N-terminus but not with the Kv3.1 N-terminus. The N-terminal amino acid sequence CDD which is conserved between Kv2 and KvS subunits appeared to be a key determinant since charge reversals with arginine substitutions abolished the interaction between the N-terminus of Kv2.1 and the C-terminus of both Kv2.1 and Kv6.4. In addition, the Kv6.4(CKv3.1) chimera in which the C-terminus of Kv6.4 was replaced by the corresponding domain of Kv3.1, disrupted the assembly with Kv2.1. These results indicate that the subfamily-specific Kv2.1/Kv6.4 heterotetramerization is determined by interactions between Kv2.1 and Kv6.4 that involve both the N- and C-termini in which the conserved N-terminal CDD sequence plays a key role.

KCNB1, a voltage-gated potassium (K(+)) channel that conducts a major delayed rectifier current in the brain, pancreas and cardiovascular system is a key player in apoptotic programs associated with oxidative stress. As a result, this protein represents a bona fide drug target for limiting the toxic effects of oxygen radicals. Until recently the consensus view was that reactive oxygen species trigger a pro-apoptotic surge in KCNB1 current via phosphorylation and SNARE-dependent incorporation of KCNB1 channels into the plasma membrane. However, new evidence shows that KCNB1 can be modified by oxidants and that oxidized KCNB1 channels can directly activate pro-apoptotic signaling pathways. Hence, a more articulated picture of the pro-apoptotic role of KCNB1 is emerging in which the protein induces cell's death through distinct molecular mechanisms and activation of multiple pathways. In this review article we discuss the diverse functional, toxic and protective roles that KCNB1 channels play in the major organs where they are expressed.

Membrane proteins that respond to changes in transmembrane voltage are critical in regulating the function of living cells. The voltage-sensing domains (VSDs) of voltage-gated ion channels are extensively studied to elucidate voltage-sensing mechanisms, and yet many aspects of their structure-function relationship remain elusive. Here, we transplanted homologous amino acid motifs from the tetrameric voltage-activated potassium channel Kv3.1 to the monomeric VSD of Ciona intestinalis voltage-sensitive phosphatase (Ci-VSP) to explore which portions of Kv3.1 subunits depend on the tetrameric structure of Kv channels and which properties of Kv3.1 can be transferred to the monomeric Ci-VSP scaffold. By attaching fluorescent proteins to these chimeric VSDs, we obtained an optical readout to establish membrane trafficking and kinetics of voltage-dependent structural rearrangements. We found that motifs extending from 10 to roughly 100 amino acids can be readily transplanted from Kv3.1 into Ci-VSP to form engineered VSDs that efficiently incorporate into the plasma membrane and sense voltage. Some of the functional features of these engineered VSDs are reminiscent of Kv3.1 channels, indicating that these properties do not require interactions between Kv subunits or between the voltage sensing and the pore domains of Kv channels.

Mutations in KCNV2 have been proposed as the molecular basis for cone dystrophy with supernormal rod electroretinogram. KCNV2 codes for the modulatory voltage-gated potassium channel α-subunit, Kv8.2, which is incapable of forming functional channels on its own. Functional heteromeric channels are however formed with Kv2.1 in heterologous expression systems, with both α-subunit genes expressed in rod and cone photoreceptors. Of the 30 mutations identified in the KCNV2 gene, we have selected three missense mutations localized in the potassium channel pore and two missense mutations localized in the tetramerization domain for analysis. We characterized the differences between homomeric Kv2.1 and heteromeric Kv2.1/Kv8.2 channels and investigated the influence of the selected mutations on the function of heteromeric channels. We found that two pore mutations (W467G and G478R) led to the formation of nonconducting heteromeric Kv2.1/Kv8.2 channels, whereas the mutations localized in the tetramerization domain prevented heteromer generation and resulted in the formation of homomeric Kv2.1 channels only. Consequently, our study suggests the existence of two distinct molecular mechanisms involved in the disease pathology.

The voltage-gated K(+) (Kv) channel subunit Kv6.4 does not form functional homotetrameric channels but co-assembles with Kv2.1 to form functional Kv2.1/Kv6.4 heterotetrameric channels. Compared to Kv2.1 homotetramers, Kv6.4 exerts a ~40 mV hyperpolarizing shift in the voltage-dependence of Kv2.1/Kv6.4 channel inactivation, without a significant effect on activation gating. However, the underlying mechanism of this Kv6.4-induced modulation of Kv2.1 channel inactivation, and whether the Kv6.4 subunit participates in the voltage-dependent gating of heterotetrameric channels is not well understood. Here we report distinct gating charge movement of Kv2.1/Kv6.4 heterotetrameric channels, compared to Kv2.1 homotetramers, as revealed by gating current recordings from mammalian cells expressing these channels. The gating charge movement of Kv2.1/Kv6.4 heterotetrameric channels displayed an extra component around the physiological K(+) equilibrium potential, characterized by a second sigmoidal relationship of the voltage-dependence of gating charge movement. This distinct gating charge displacement reflects movement of the Kv6.4 voltage-sensing domain and has a voltage-dependency that matches the hyperpolarizing shift in Kv2.1/Kv6.4 channel inactivation. These results provide a mechanistic basis for the modulation of Kv2.1 channel inactivation gating kinetics by silent Kv6.4 subunits.

Cerebral vascular smooth muscle contractility plays a crucial role in controlling arterial diameter and, thereby, blood flow regulation in the brain. A number of K(+) channels have been suggested to contribute to the regulation of diameter by controlling smooth muscle membrane potential (E(m)) and Ca(2+) influx. Previous studies indicate that stromatoxin (ScTx1)-sensitive, Kv2-containing channels contribute to the control of cerebral arterial diameter at 80 mmHg, but their precise role and molecular composition were not determined. Here, we tested if Kv2 subunits associate with 'silent' subunits from the Kv5, Kv6, Kv8 or Kv9 subfamilies to form heterotetrameric channels that contribute to control of diameter of rat middle cerebral arteries (RMCAs) over a range of intraluminal pressure from 10 to 100 mmHg. The predominant mRNAs expressed by RMCAs encode Kv2.1 and Kv9.3 subunits. Co-localization of Kv2.1 and Kv9.3 proteins at the plasma membrane of dissociated single RMCA myocytes was detected by proximity ligation assay. ScTx1-sensitive native current of RMCA myocytes and Kv2.1/Kv9.3 currents exhibited functional identity based on the similarity of their deactivation kinetics and voltage dependence of activation that were distinct from those of homomultimeric Kv2.1 channels. ScTx1 treatment enhanced the myogenic response of pressurized RMCAs between 40 and 100 mmHg, but this toxin also caused constriction between 10 and 40 mmHg that was not previously observed following inhibition of large conductance Ca(2+)-activated K(+) (BK(Ca)) and Kv1 channels. Taken together, this study defines the molecular basis of Kv2-containing channels and contributes to our understanding of the functional significance of their expression in cerebral vasculature. Specifically, our findings provide the first evidence of heteromultimeric Kv2.1/Kv9.3 channel expression in RMCA myocytes and their distinct contribution to control of cerebral arterial diameter over a wider range of E(m) and transmural pressure than Kv1 or BK(Ca) channels owing to their negative range of voltage-dependent activation.