Kv7 potassium channels generate slowly activating, non-inactivating outward potassium currents and are critical regulators of cellular excitability. While cholesterol is known to modulate multiple ion channels, its concentration-dependent effects and the influence of cholesterol-derived molecules on Kv7 channels remain insufficiently characterized. In this study, we investigated the effects of cholesterol and cholesterol-derived molecules, including steroid hormones and chemically modified imidazolium-based cholesterol derivatives (CHIMs) on Kv7.2/7.3 channels heterologously expressed in HEK293FT cells using conventional whole-cell patch-clamp recordings. Application of high cholesterol concentrations (1 mM) significantly reduced Kv7.2/7.3 current amplitudes over a wide range of potentials without changing voltage-dependent current characteristics. CHIMs also reduced currents, whereas a fluorescent derivative, CHIM-L-NBD, unexpectedly enhanced Kv7.2/7.3 currents. In contrast, the NBD moiety alone had no effect. Progesterone and 17β-estradiol inhibition of Kv7.2/7.3 currents was most evident at strongly depolarized potentials. For progesterone this was associated with a change in the slope of the activation curve. These findings demonstrate that Kv7.2/7.3 channels respond differentially to structural modifications of cholesterol and to steroid hormones thereby suggesting that different cholesterol-derived molecules may serve as tool compounds with potentially opposing modulatory effects on Kv7.2/7.3 channels.

Potassium (K+) channels are one of the key regulators of neuronal excitability and network stability. Among all the potassium channels, the voltage-gated Kv7 (KCNQ) and the two-pore domain potassium (K2P) channels play an important role in stabilizing and maintaining the resting potential of neurons. They are involved in controlling the input resistance and in the generation of rhythmic activity in the thalamocortical network. The main functions of the thalamocortical system are the regulation of sensory processing and the generation of brain rhythms underlying sleep and wakefulness. By this, it is essential to many aspects of cognition. The thalamocortical system is explicitly sensitive to fine changes in potassium conductance. The dysregulation of thalamocortical oscillation, also known as thalamocortical dysrhythmia, has been known to be involved in several psychiatric and neurological disorders. A prototypical thalamocortical dysrhythmia is childhood absence epilepsy (CAE), in which hypersynchronous activity in the form of spike and wave discharges (SWD) induces sudden losses of conscious awareness in patients. This review will summarize the current knowledge on Kv7 and K2P channels in the regulation of synchrony and excitability in the brain and the thalamocortical system in particular, outlining future research directions, highlighting the therapeutic potential of these channels for the treatment of thalamocortical dysrhythmias in general and CAE specifically.

Ion channels are classically regarded as regulators of electrical excitability, but their role in immune activation and barrier homeostasis extends far beyond global transmembrane ion flux. This functional diversity is, among other things, the result of noncanonical signaling pathways. In this review, we examine the neurovascular endothelium as a paradigm for barrier-associated neuroimmune pathology and discuss three mechanistic principles: (1) permeation-independent scaffolding via cytoplasmic interaction domains, (2) spatio-temporally restricted ion flux within signaling microdomains, and (3) compartmentalized or cargo-mediated signaling. These mechanisms shape endothelial barrier integrity, inflammatory junctional remodeling, and the diapedesis of immune cells across the blood-brain barrier. A deeper understanding of such noncanonical channel functions opens new therapeutic perspectives beyond conventional pore blockade, including the targeted modulation of signaling domains, protein interactions, and subcellular channel localization.

BACKGROUND:Electroencephalography (EEG) allows a versatile recording of neuronal activity in neurological disorders. Signal analytical techniques like time-frequency analysis (TFA) can uncover latent information in the EEG of patients. Classically, EEG and TFA analysis relies on predefined frequency bands. Cluster-based permutation statistics provides an unbiased statistical comparison of high dimensional neurophysiological data, stabilizing the measured effect size and increasing the probability of uncovering the main effect present. METHODS:Resting state surface EEG recordings from 51 Multiple Sclerosis (MS) and 51 control patients were analyzed using TFA and compared by cluster-based permutation statistics. We further correlated results with disease characteristics, retinal nerve fiber layer (RNFL) thickness assessed by optical coherence tomography, evoked potentials (EPs), and neuropsychological data. RESULTS:We detected increased power in the low and high beta frequency bands in MS compared to control patients in a subset of recording sites. Spectral power in the high beta band of resting state EEG recorded at O1 negatively correlated with the RNFL thickness. Furthermore, differences in low and high beta power were dependent on the EP score of MS patients. CONCLUSION:Our results suggest a more desynchronized EEG activity in MS patients compared to controls, correlating with clinical markers of disease severity. The findings indicate differential brain network recruitment that may be the result of compensatory mechanisms and the basis for clinical impairment in MS.

K2P18.1 (TRESK) displays one of the most unusual two pore domain potassium channels, that is associated with different neurological and immune-related diseases emphasizing the therapeutic potential of K2P18.1 activators. Previous studies identified cloxyquin and nitroxoline as lead compounds that do not alter the function of other K2P channels. Although their 8-OH group is crucial for channel activation, 8-hydroxyquinolines are known for various effects including metal chelation and antibacterial properties increasing the risk of side effects. Therefore, we synthesized 14 quinoline derivatives to search for other tolerated substitutions in 8-position. Activity determination using two-electrode voltage clamp (TEVC) revealed that four derivatives achieved moderate inhibitory or stimulatory activity. Among these, benzaldimine 3d represents a new extended scaffold that unexpectedly increases K2P18.1 channel activity by about 60% at 100 µM. Docking studies suggest that despite the enlarged structure, 3d is still able to achieve in silico interactions with important amino acids from the cloxyquin binding site. However, its usability in vitro is limited by hydrolysis leading to a compound half-life of 39 min in aqueous solutions. Nevertheless, the identification of a possible scaffold extension for K2P18.1 channel activators opens a new opportunity for future compound diversification.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system that is frequently accompanied by major depressive symptoms, which substantially worsen disease burden and quality of life. Increasing evidence suggests that this comorbidity is not solely a psychological reaction to neurological disability but may arise from shared neurobiological mechanisms. Ion channels play a central role in regulating neuronal excitability, synaptic transmission and immune cell function, and their dysregulation has emerged as a key feature of MS pathology. Demyelination and neuroinflammation induce profound alterations in the expression, distribution and function of several ion channel families, including hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, voltage-gated potassium channels such as Kv7 and two-pore domain potassium (K2P) channels. These changes contribute to neuronal hyperexcitability, altered thalamocortical network activity and immune cell activation. Importantly, several ion channel systems implicated in MS are also associated with affective regulation, including TRPA1, ASIC1a, N-methyl-d-aspartate receptors and serotonin 5-HT3 receptors. This review summarizes current evidence linking ion channel dysfunction to both MS pathology and depression and highlights emerging pharmacological strategies targeting these channels. Understanding ion channel-mediated mechanisms may provide novel therapeutic opportunities to address both neuroinflammation and neuropsychiatric comorbidities in MS.

The sinoatrial node pacemaker channel HCN4 plays a central role in cardiac automaticity, and disease-associated variants can predispose to atrial arrhythmias. Here, we investigated the functional interplay between the HCN4 variant P883R and the potassium channel β-subunit KCNE1, focusing on the common atrial fibrillation-associated KCNE1 variant G38S and its regulation by the iron-induced serine protease TMPRSS6. Electrophysiological analyses revealed that HCN4-P883R decreases net HCN4 currents If, consistent with impaired automaticity. Co-expression of KCNE1, either wild-type or polymorphic KCNE1-G38S, restored functional properties of the mutant channel, indicating that KCNE1 is a key modulator of HCN4 activity. Importantly, TMPRSS6-mediated proteolytic processing of KCNE1 reduced HCN4 currents, whereas HCN4 expressed alone was insensitive to TMPRSS6, identifying KCNE1 as the direct regulatory target. Notably, KCNE1-G38S altered the HCN4-KCNE1 complex to TMPRSS6-dependent downregulation, resulting in a reduced suppression of HCN4-P883R-mediated currents compared with wild-type KCNE1. Mechanistically, differential TMPRSS6 cleavage depended on the membrane positioning of the KCNE1-32RRSPRSS38 motif. These findings reveal a protease-dependent buffering mechanism that counteracts HCN4 loss-of-function and establish TMPRSS6 as a molecular switch controlling pacemaker activity in a KCNE1 genotype-dependent manner. This dynamic regulatory framework may contribute to the phenotypic variability of sinoatrial node dysfunction and atrial fibrillation.

Upregulation of K(Ca)3.1 channels was observed in highly aggressive tumor cells, such as non small cell lung cancer cells of the A549 line. In order to visualize K(Ca)3.1 channels in these cells, novel fluorescent probes with increased polarity were designed. Key step of the synthesis was a 1,3-dipolar cycloaddition of senicapoc propargyl ether 4 with various azide substituted bodipy dyes. Due to their reduced lipophilicity and promising photophysical properties, the senicapoc-bodipy conjugates 7a (logP = 4.3) and 16 (logP = 4.4) were able to stain K(Ca)3.1 ion channels in fixed, living, and permeabilized A549-3R tumor cells. The apparent size of the observed fluorescent dots indicates labeling of single K(Ca)3.1 channels. The recorded density is in good accordance with literature values. The specificity of K(Ca)3.1 labeling by the senicapoc-bodipy conjugates 7a and 16 was shown with HEK293 cells, blocking experiments and azide precursors. Subsequent staining of K(Ca)3.1 ion channels with hydroxyphenyl derivative 16 and antibodies did not lead to overlapping (yellow) dots, as different states of the ion channel were stained by 16 (open state) and antibody (closed state). In patch clamp experiments, both senicapoc-bodipy conjugates 7a and 16 reduced the current density, although less efficiently than senicapoc. MD simulations showed weaker interactions of the amide moiety of 16 with Thr250, explaining the lower channel inhibition of the open-pore blocker 16 compared to senicapoc (1). Due to their optimal imaging properties, high specificity, balanced lipophilicity/hydrophilicity, and sufficient water solubility, senicapoc-bodipy conjugates 7a and 16 represent innovative diagnostic tools to image K(Ca)3.1 channels.

Background/Objectives: The Ca2+-activated K+ channel K(Ca)3.1 is not only involved in physiological processes such as immune reactions and control of vascular tone, but is highly expressed in various tumor entities. Thus, imaging of K(Ca)3.1 channels comes into focus for the localization of high channel density, i.e., for tumor diagnosis. In particular, the physicochemical properties of the fluorescent probes should be improved compared to existing probes. Methods: The small molecule inhibitor of the K(Ca)3.1 channel, senicapoc, was used as a warhead and was coupled with different fluorescent dyes. After synthesis of the novel probes, their physicochemical properties (lipophilicity, photophysical properties) and their ability to image K(Ca)3.1 channels in A549-3R lung tumor cells were determined. Results: In order to increase the polarity and quantum yield of reported fluorescent probes, three strategies were followed: (1) An F-atom at the B-atom of bodipy-labeled senicapoc derivatives 9a, 9b, and 15a was replaced by a OCH3 moiety, which decreased the logP value by one log-unit. (2) The p-phenylene moiety of the linker was replaced by an aliphatic tetramethylene linker decreasing the lipophilicity by 0.3-0.5 log-units. (3) Instead of bodipy dyes, fluorescein was coupled with the senicapoc warhead resulting in very polar probes 21a and 21b with low logP values of 1.5 and 1.3, respectively. Introduction of an ethyl moiety at the bodipy core increased the quantum yield, which resulted in the best punctate staining pattern of fixed and living A549-3R lung tumor cells with the ethylbodipy-labeled senicapoc derivative 10b. The specificity was shown by various control experiments. Co-staining with 10b and an antibody did not result in overlapping signals. Conclusions: The well-balanced lipophilicity and fluorescent quantum yield render the ethylbodipy-labeled senicapoc derivative 10b a very good probe to image selectively K(Ca)3.1 ion channels in fixed and living tumor cells. It was hypothesized that the antibody binds selectively at the closed channel (58.5%), whereas the senicapoc-bodipy conjugate 10b binds selectively at the open channel (41.5%). The ratio 58.5:41.5 reflects the ratio of the ion channel in closed and open conformations.

Although TREK1 channels are widely expressed in several thalamic nuclei, the role of this K2P family member in modulating thalamic cell excitability and physiological thalamocortical oscillatory activity is not well studied. Here we explored the contribution of TREK1 channels to membrane properties of two important building blocks of the thalamocortical (TC) system, namely, GABAergic neurons of reticular thalamic nucleus (RTN) and TC neurons in different sensory thalamic nuclei including the ventrobasal complex (VB; somatosensory system) and the medial geniculate nucleus (MGN; auditory system), using male TREK1 knock-out (TREK1-/-) mice. Furthermore, we show that the loss of TREK1 channels has distinct effects on neuronal function in different thalamic nuclei. Compared with controls, TREK1-/- mice exhibit decreased excitability in RTN neurons, while VB neurons maintain similar excitability levels. Additionally, the absence of TREK1 channels alters the action potential (AP) characteristics in VB TC neurons and affects GABAergic inhibitory tone in RTN neurons. In TREK1-/- mice, the excitability of cortical pyramidal cells is increased. It is tempting to assume that this combination of changes contributes to a high number of sharp, spindle-like oscillations observed in sleep local field potential (LFP) recordings of these mice. In addition, TREK1-/- mice show a lower amount of delta (1-4 Hz) oscillations during slow-wave sleep and a time-of-day-dependent alteration in the amount of sleep and wakefulness. They also show disturbed auditory signal processing and altered excitability in the auditory thalamus. These findings underline the relevance of TREK1 channels' broad contribution to the thalamus and thalamocortical system.

K2P2.1 (gene: Kcnk2), a two-pore-domain potassium channel, regulates leukocyte transmigration across the blood-brain barrier by a yet unknown mechanism. We demonstrate that Kcnk2-/- mouse brain microvascular endothelial cells (MBMECs) exhibit an altered cytoskeletal structure and surface morphology with increased formation of membrane protrusions. Cell adhesion molecules cluster on those protrusions and facilitate leukocyte adhesion and migration in vitro and in vivo. We observe downregulation of K2P2.1 and activation of actin modulating proteins (cofilin 1, Arp2/3) in inflamed wildtype MBMECs. In the mechanosensitive conformation, K2P2.1 shields the phospholipid PI(4,5)P2 from interaction with other actin regulatory proteins, especially cofilin 1. Consequently, after stimulus-related K2P2.1 downregulation and dislocation from PI(4,5)P2, actin rearrangements are induced. Thus, K2P2.1-mediated regulatory processes are essential for actin dynamics, fast, reversible, and pharmacologically targetable.

Spike-wave-discharges (SWD) are the electrophysiological hallmark of absence epilepsy. SWD are generated in the thalamo-cortical network and a seizure onset zone was identified in the somatosensory cortex (S1). We have shown before that inhibition of the centromedian thalamic nucleus (CM) in GAERS rats resulted in a selective suppression of the spike component while rhythmic cortical 5-9 Hz oscillations remained present. Such oscillations are often seen to precede SWD activity in this well-validated genetic rat model of absence epilepsy, but are also seen in seizure-free periods. The present study characterizes the profile of 5-9 Hz oscillations in thalamocortical circuits during pre- and inter-ictal states. Here we recorded local-field-potentials in S1, CM and the secondary motor cortex of GAERS. Time-frequency analysis was used to assess spectral power and non-linear-association analysis was used to determine coupling strength and directionality between brain areas. Phase-specific electrical stimulation was used to compare cortical excitability and to assess the risk for epileptic afterdischarges. Coupling strength and spectral power were higher for the inter-ictal compared to the pre-ictal 5-9 Hz oscillations. However, coupling strength during pre-ictal oscillations was higher than during passive wakefulness. Double pulse stimulation during 5-9 Hz oscillations was more likely to induce epileptic afterdischarges compared to stimulation during passive wakefulness. While no overall differences in cortical excitability were revealed, phase-specific differences in excitability were noticed during the oscillation. Our findings indicate that intermediate coupling between S1 and CM favors SWD generation, thereby adding to the previous notion that 5-9 Hz oscillations represent high-risk periods for seizure generation. In general, preictal oscillations display a unique electrophysiological profile in GAERS that might pave the way for qualification as biomarker for SWD generation and seizure prediction.

K2P2.1 (TREK1), a two-pore domain potassium channel, has emerged as regulator of leukocyte transmigration into the central nervous system. In the context of skeletal muscle, immune cell infiltration constitutes the pathogenic hallmark of idiopathic inflammatory myopathies (IIMs). However, the underlying mechanisms remain to be elucidated. In this study, we investigated the role of K2P2.1 in the autoimmune response of IIMs. We detected K2P2.1 expression in primary skeletal muscle and endothelial cells of murine and human origin. We observed an increased pro-inflammatory cell response, adhesion and transmigration by pharmacological blockade or genetic deletion of K2P2.1 in vitro and in in vivo myositis mouse models. Of note, our findings were not restricted to endothelial cells as skeletal muscle cells with impaired K2P2.1 function also demonstrated a strong pro-inflammatory response. Conversely, these features were abrogated by activation of K2P2.1 and improved the disease course of a myositis mouse model. In humans, K2P2.1 expression was diminished in IIM patients compared to non-diseased controls arguing for the translatability of our findings. In summary, K2P2.1 may regulate the inflammatory response of skeletal muscle. Further research is required to understand whether K2P2.1 could serve as novel therapeutic target.

Iron storage disease is associated with cardiovascular manifestations, including various forms of cardiac arrhythmias of unknown origin. In this study, cardiac arrhythmias associated with iron overload were investigated in human iPSC-derived cardiomyocytes (hiPSC-CM) and hiPSC-derived sinus node-like pacemaker cells. Among other effects, iron overload leads to an increase in the plasma membrane-anchored protease TMPRSS6. TMPRSS6 cleaves the auxiliary subunit KCNE1 N-terminally and thus modulates the function of both the IKs (KCNQ1/KCNE1 current) and the If (HCN4/KCNE1) ion channels. Furthermore, TMPRSS6 induces a reduction of electric field potential (EFP) count and increased duration in hiPSC-derived ventricular-like cells and in hiPSC-derived pacemaker-like cells. In accordance with these in vitro generated results, TMPRSS6-mediated interactions show pro-arrhythmic effects in silico . Therefore, the TMPRSS6 - KCNE1-KCNQ1 and TMPRSS6 - KCNE1-HCN4 cascades may represent new clinically relevant pro-arrhythmic mechanisms in iron overload diseases. ### Competing Interest Statement The authors have declared no competing interest.

IntroductionGenetic Absence Epilepsy Rats from Strasbourg (GAERS) represent a model of genetic generalized epilepsy. The present longitudinal study in GAERS and age-matched non-epileptic controls (NEC) aimed to characterize the epileptic brain network using two functional measures, resting state-functional magnetic resonance imaging (rs-fMRI) and manganese-enhanced MRI (MEMRI) combined with morphometry, and to investigate potential brain network alterations, following long-term seizure activity.MethodsRepeated rs-fMRI measurements at 9.4 T between 3 and 8 months of age were combined with MEMRI at the final time point of the study. We used graph theory analysis to infer community structure and global and local network parameters from rs-fMRI data and compared them to brain region-wise manganese accumulation patterns and deformation-based morphometry (DBM).ResultsFunctional connectivity (FC) was generally higher in GAERS when compared to NEC. Global network parameters and community structure were similar in NEC and GAERS, suggesting efficiently functioning networks in both strains. No progressive FC changes were observed in epileptic animals. Network-based statistics (NBS) revealed stronger FC within the cortical community, including regions of association and sensorimotor cortex, and with basal ganglia and limbic regions in GAERS, irrespective of age. Higher manganese accumulation in GAERS than in NEC was observed at 8 months of age, consistent with higher overall rs-FC, particularly in sensorimotor cortex and association cortex regions. Functional measures showed less similarity in subcortical regions. Whole brain volumes of 8 months-old GAERS were higher when compared to age-matched NEC, and DBM revealed increased volumes of several association and sensorimotor cortex regions and of the thalamus.Discussionrs-fMRI, MEMRI, and volumetric data collectively suggest the significance of cortical networks in GAERS, which correlates with an increased fronto-central connectivity in childhood absence epilepsy (CAE). Our findings also verify involvement of basal ganglia and limbic regions. Epilepsy-related network alterations are already present in juvenile animals. Consequently, this early condition seems to play a greater role in dynamic brain function than chronic absence seizures.

The development of in vitro pharmacological assays relies on creating genetically modified cell lines that overexpress the target protein of interest. However, the choice of the host cell line can significantly impact the experimental outcomes. This study explores the functional characterization of P2X7 and P2X4 receptor modulators through cellular assays and advanced electrophysiological techniques. The influence of different host cell lines (HEK-293, HEK-293FT, and 1321N1) on the activity of reference agonists and antagonists targeting human and murine P2X4 and P2X7 receptors was systematically investigated, highlighting the significant impact of the host cell on experimental results. The 1321N1 cell line was identified as the preferred host cell line when investigating the human P2X4 receptor due to more consistent agonist activities, antagonist potencies, and a more stable assay signal window. Furthermore, a patch-clamp protocol that allows for the repetitive recording of ATP-mediated inward currents from isolated human CD4+ T-cells was established, revealing that both P2X7 and P2X4 receptors are crucial for immune cell regulation, positioning them as promising therapeutic targets for managing inflammatory disorders.

Background Multiple Sclerosis (MS) is an autoimmune neurodegenerative disease, whose primary hallmark is the occurrence of inflammatory lesions in white and grey matter structures. Increasing evidence in MS patients and respective murine models reported an impaired ionic homeostasis driven by inflammatory-demyelination, thereby profoundly affecting signal propagation. However, the impact of a focal inflammatory lesion on single-cell and network functionality has hitherto not been fully elucidated. Objectives In this study, we sought to determine the consequences of a localized cortical inflammatory lesion on the excitability and firing pattern of thalamic neurons in the auditory system. Moreover, we tested the neuroprotective effect of Retigabine (RTG), a specific Kv7 channel opener, on disease outcome. Methods To resemble the human disease, we focally administered pro-inflammatory cytokines, TNF-α and IFN-γ, in the primary auditory cortex (A1) of MOG35-55 immunized mice. Thereafter, we investigated the impact of the induced inflammatory milieu on afferent thalamocortical (TC) neurons, by performing ex vivo recordings. Moreover, we explored the effect of Kv7 channel modulation with RTG on auditory information processing, using in vivo electrophysiological approaches. Results Our results revealed that a cortical inflammatory lesion profoundly affected the excitability and firing pattern of neighboring TC neurons. Noteworthy, RTG restored control-like values and TC tonotopic mapping. Conclusion Our results suggest that RTG treatment might robustly mitigate inflammation-induced altered excitability and preserve ascending information processing.

Abstract The Phosphatidylinositol 3-phosphate 5-kinase Type III PIKfyve is the main source for selectively generated phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), a known regulator of membrane protein trafficking. PI(3,5)P2 facilitates the cardiac KCNQ1/KCNE1 channel plasma membrane abundance and therewith increases the macroscopic current amplitude. Functional-physical interaction of PI(3,5)P2 with membrane proteins and its structural impact is not sufficiently understood. This study aimed to identify molecular interaction sites and stimulatory mechanisms of the KCNQ1/KCNE1 channel via the PIKfyve-PI(3,5)P2 axis. Mutational scanning at the intracellular membrane leaflet and nuclear magnetic resonance (NMR) spectroscopy identified two PI(3,5)P2 binding sites, the known PIP2 site PS1 and the newly identified N-terminal α–helix S0 as relevant for functional PIKfyve effects. Cd2+ coordination to engineered cysteines and molecular modeling suggest that repositioning of S0 stabilizes the channel s open state, an effect strictly dependent on parallel binding of PI(3,5)P2 to both sites.