An Ultrasensitive Genetically Encoded Voltage Indicator Uncovers the Electrical Activity of Non-Excitable Cells.

Most animal cell types are classified as non-excitable because they do not generate action potentials observed in excitable cells, such as neurons and muscle cells. Thus, resolving voltage signals in non-excitable cells demands sensors with exceptionally high voltage sensitivity. In this study, the ultrabright, ultrasensitive, and calibratable genetically encoded voltage sensor rEstus is developed using structure-guided engineering.

KCNA2 IgG autoimmunity in neuropsychiatric diseases.

Background: Autoantibodies against the potassium voltage-gated channel subfamily A member 2 (KCNA2) have been described in a few cases of neuropsychiatric disorders, but their diagnostic and pathophysiological role is currently unknown, imposing challenges to medical practice.

Design / Methods: We retrospectively collected comprehensive clinical and paraclinical data of 35 patients with KCNA2 IgG autoantibodies detected in cell-based and tissue-based assays. Patients' sera and cerebrospinal fluid (CSF) were used for characterization of the antigen, clinical-serological correlations, and determination of IgG subclasses.

[Deep healing-Comments on the ethical risks and chances of artificial intelligence].

Hardly any other field of application of artificial intelligence (AI) needs more ethics by design than medicine; however, if a deep integration of ethical principles succeeds there is a chance of "deep healing", for each individual and also for medicine and the healthcare system itself. Thus, AI in medicine could also become a blueprint for how AI should enter society and culture.

Peripheral temperature dysregulation associated with functionally altered Na1.8 channels.

The voltage-gated sodium channel Na1.8 is prominently expressed in the soma and axons of small-caliber sensory neurons, and pathogenic variants of the corresponding gene SCN10A are associated with peripheral pain and autonomic dysfunction. While most disease-associated SCN10A variants confer gain-of-function properties to Na1.

Selenomethionine mis-incorporation and redox-dependent voltage-gated sodium channel gain of function.

Selenomethionine (SeMet) readily replaces methionine (Met) residues in proteins during translation. Long-term dietary SeMet intake results in the accumulation of the amino acid in tissue proteins. Despite the high rates of SeMet incorporation in proteins and its stronger susceptibility to oxidation compared to Met, little is known about the effect of SeMet mis-incorporation on electrical excitability and ion channels.

Selenomethionine incorporation in proteins of individual mammalian cells determined with a genetically encoded fluorescent sensor.

Selenomethionine (SeMet) randomly replaces methionine (Met) in protein translation. Because of strongly differing redox properties of SeMet and Met, SeMet mis-incorporation may have detrimental effects on protein function, possibly compromising the use of nutritional SeMet supplementation as an anti-oxidant. Studying the functional impact of SeMet in proteins on a cellular level is hampered by the lack of accurate and efficient methods for estimating the SeMet incorporation level in individual viable cells.

Extracellular hemin is a reverse use-dependent gating modifier of cardiac voltage-gated Na channels.

Heme (Fe-protoporphyrin IX) is a well-known protein prosthetic group; however, heme and hemin (Fe-protoporphyrin IX) are also increasingly viewed as signaling molecules. Among the signaling targets are numerous ion channels, with intracellular-facing heme-binding sites modulated by heme and hemin in the sub-µM range. Much less is known about extracellular hemin, which is expected to be more abundant, in particular after hemolytic insults.

Intracellular hemin is a potent inhibitor of the voltage-gated potassium channel Kv10.1.

Heme, an iron-protoporphyrin IX complex, is a cofactor bound to various hemoproteins and supports a broad range of functions, such as electron transfer, oxygen transport, signal transduction, and drug metabolism. In recent years, there has been a growing recognition of heme as a non-genomic modulator of ion channel functions. Here, we show that intracellular free heme and hemin modulate human ether à go-go (hEAG1, Kv10.

A GFP-based ratiometric sensor for cellular methionine oxidation.

Methionine oxidation is a reversible post-translational protein modification, affecting protein function, and implicated in aging and degenerative diseases. The detection of accumulating methionine oxidation in living cells or organisms, however, has not been achieved. Here we introduce a genetically encoded probe for methionine oxidation (GEPMO), based on the super-folder green fluorescent protein (sfGFP), as a specific, versatile, and integrating sensor for methionine oxidation.

Divergent roles of haptoglobin and hemopexin deficiency for disease progression of Shiga-toxin-induced hemolytic-uremic syndrome in mice.

Thrombotic microangiopathy, hemolysis and acute kidney injury are typical clinical characteristics of hemolytic-uremic syndrome (HUS), which is predominantly caused by Shiga-toxin-producing Escherichia coli. Free heme aggravates organ damage in life-threatening infections, even with a low degree of systemic hemolysis. Therefore, we hypothesized that the presence of the hemoglobin- and the heme-scavenging proteins, haptoglobin and hemopexin, respectively impacts outcome and kidney pathology in HUS.

Monitoring of compound resting membrane potentials of cell cultures with ratiometric genetically encoded voltage indicators.

The cellular resting membrane potential (V) not only determines electrical responsiveness of excitable cells but also plays pivotal roles in non-excitable cells, mediating membrane transport, cell-cycle progression, and tumorigenesis. Studying these processes requires estimation of V, ideally over long periods of time. Here, we introduce two ratiometric genetically encoded V indicators, rArc and rASAP, and imaging and analysis procedures for measuring differences in average resting V between cell groups.

Diffraction-Unlimited Photomanipulation at the Plasma Membrane via Specifically Targeted Upconversion Nanoparticles.

Engineered UCNP are used to trigger rapid photoconversion of the fluorescent protein Dendra2 with nanoscopic precision and over longer distances in mammalian cells. By exploiting the synergy of high-level thulium doping with core-shell design and elevated excitation intensities, intense UCNP emission is achieved, allowing fast photoconversion of Dendra2 with <10 nm resolution. A tailored biocompatible surface coating and functionalization with a derivate of green fluorescent protein (GFP) for recognition of antiGFP nanobodies are developed.

Bilirubin Oxidation End Products (BOXes) Induce Neuronal Oxidative Stress Involving the Nrf2 Pathway.

Delayed ischemic neurological deficit (DIND) is a severe complication after subarachnoid hemorrhage (SAH). Previous studies have suggested that bilirubin oxidation end products (BOXes) are probably associated with the DIND after SAH, but there is a lack of direct evidence yet even on cellular levels. In the present study, we aim to explore the potential role of BOXes and the involved mechanisms in neuronal function.

Fe-Mediated Activation of BK Channels by Rapid Photolysis of CORM-S1 Releasing CO and Fe.

Heme catabolism by heme oxygenase (HO) with a decrease in intracellular heme concentration and a concomitant local release of CO and Fe has the potential to regulate BK channels. Here, we show that the iron-based photolabile CO-releasing molecule CORM-S1 [dicarbonyl-bis(cysteamine)iron(II)] coreleases CO and Fe, making it a suitable light-triggered source of these downstream products of HO activity. To investigate the impact of CO, iron, and cysteamine on BK channel activation, human Slo1 (hSlo1) was expressed in HEK293T cells and studied with electrophysiological methods.

Impact of intracellular hemin on N-type inactivation of voltage-gated K channels.

N-type inactivation of voltage-gated K channels is conferred by the N-terminal "ball" domains of select pore-forming α subunits or of auxiliary β subunits, and influences electrical cellular excitability. Here, we show that hemin impairs inactivation of K channels formed by Kv3.4 α subunits as well as that induced by the subunits Kvβ1.

[Only artificial intelligence can heal us? : Artificial intelligence in medicine as deus ex machina, grands recits nouveaux or supertool?].

Background: Modern medicine is becoming digital medicine. This is accompanied by a fundamental change in medicine and its professions and an altered relationship between the actors in the healthcare system. In particular, patients become the vital center again.

Labile heme impairs hepatic microcirculation and promotes hepatic injury.

Sepsis is a life-threatening clinical syndrome defined as a deregulated host response to infection associated with organ dysfunction. Mechanisms underlying the pathophysiology of septic liver dysfunction are incompletely understood. Among others, the iron containing tetrapyrrole heme inflicts hepatic damage when released into the circulation during systemic inflammation and sepsis.

Membrane potential manipulation with visible flash lamp illumination of targeted microbeads.

The electrical membrane potential (V) is a key dynamical variable of excitable membranes. Despite the tremendous success of optogenetic methods to modulate V with light, there are some shortcomings, such as the need of genetic manipulation and limited time resolution. Direct optical stimulation of gold nanoparticles targeted to cells is an attractive alternative because the absorbed energy heats the membrane and, thus, generates capacitive current sufficient to trigger action potentials [1, Carvalho-de-Souza et al.

Linkage Evidence for a Two-Locus Inheritance of LQT-Associated Seizures in a Multigenerational LQT Family With a Novel Loss-of-Function Mutation.

Mutations in several genes encoding ion channels can cause the long-QT (LQT) syndrome with cardiac arrhythmias, syncope and sudden death. Recently, mutations in some of these genes were also identified to cause epileptic seizures in these patients, and the sudden unexplained death in epilepsy (SUDEP) was considered to be the pathologic overlap between the two clinical conditions. For LQT-associated mutations, only few investigations reported the coincidence of cardiac dysfunction and epileptic seizures.

Effect of Conformational Diversity on the Bioactivity of µ-Conotoxin PIIIA Disulfide Isomers.

Cyclic µ-conotoxin PIIIA, a potent blocker of skeletal muscle voltage-gated sodium channel Na1.4, is a 22mer peptide stabilized by three disulfide bonds. Combining electrophysiological measurements with molecular docking and dynamic simulations based on NMR solution structures, we investigated the 15 possible 3-disulfide-bonded isomers of µ-PIIIA to relate their blocking activity at Na1.

Disruption of Membrane Integrity by the Bacterium-Derived Antifungal Jagaricin.

Jagaricin is a lipopeptide produced by the bacterial mushroom pathogen , the causative agent of mushroom soft rot disease. Apart from causing lesions in mushrooms, jagaricin is a potent antifungal active against human-pathogenic fungi. We show that jagaricin acts by impairing membrane integrity, resulting in a rapid flux of ions, including Ca, into susceptible target cells.

Large-conductance Ca- and voltage-gated K channels form and break interactions with membrane lipids during each gating cycle.

Membrane depolarization and intracellular Ca promote activation of the large-conductance Ca- and voltage-gated (Slo1) big potassium (BK) channel. We examined the physical interactions that stabilize the closed and open conformations of the ion conduction gate of the human Slo1 channel using electrophysiological and computational approaches. The results show that the closed conformation is stabilized by intersubunit ion-ion interactions involving negative residues (E321 and E324) and positive residues (RKK) at the cytoplasmic ends of the transmembrane S6 segments ("RKK ring").

ANK2 functionally interacts with KCNH2 aggravating long QT syndrome in a double mutation carrier.

Pathogenic long QT mutations often comprise high phenotypic variability and particularly variants in ANK2 (long QT syndrome 4) frequently lack QT prolongation. We sought to elucidate the genetic and functional background underlying the clinical diversity in a 3-generation family with different cardiac arrhythmias. Next-generation sequencing-based screening of patients with QT prolongation identified the index patient of the family carrying an ANK2-E1813K variant and a previously uncharacterized KCNH2-H562R mutation in a double heterozygous conformation.