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Cardiac arrhythmias significantly contribute to cardiovascular morbidity and mortality. The rabbit heart serves as an accepted model system for studying cardiac cell excitation and arrhythmogenicity. Accordingly, primary cultures of adult rabbit ventricular cardiomyocytes serve as a preferable model to study molecular mechanisms of human cardiac excitation. However, the use of adult rabbit cardiomyocytes is often regarded as excessively costly. Therefore, we developed and characterized a novel low-cost rabbit cardiomyocyte model, namely, 3-week-old ventricular cardiomyocytes (3wRbCMs). Ventricular myocytes were isolated from whole ventricles of 3-week-old New Zealand White rabbits of both sexes by standard enzymatic techniques. Using wheat germ agglutinin, we found a clear T-tubule structure in acutely isolated 3wRbCMs. Cells were adenovirally infected (multiplicity of infection of 10) to express Green Fluorescent Protein (GFP) and cultured for 48 h. The cells showed action potential duration (APD90 = 253 ± 24 ms) and calcium transients similar to adult rabbit cardiomyocytes. Freshly isolated and 48-h-old-cultured cells expressed critical ion channel proteins: calcium voltage-gated channel subunit alpha1 C (Cavα1c), sodium voltage-gated channel alpha subunit 5 (Nav1.5), potassium voltage-gated channel subfamily D member 3 (Kv4.3), and subfamily A member 4 (Kv1.4), and also subfamily H member 2 (RERG. Kv11.1), KvLQT1 (K7.1) protein and inward-rectifier potassium channel (Kir2.1). The cells displayed an appropriate electrophysiological phenotype, including fast sodium current (I Na), transient outward potassium current (I to), L-type calcium channel peak current (I Ca,L), rapid and slow components of the delayed rectifier potassium current (I Kr and I Ks), and inward rectifier (I K1). Although expression of the channel proteins and some currents decreased during the 48 h of culturing, we conclude that 3wRbCMs are a new, low-cost alternative to the adult-rabbit-cardiomyocytes system, which allows the investigation of molecular mechanisms of cardiac excitation on morphological, biochemical, genetic, physiological, and biophysical levels. [ABSTRACT FROM AUTHOR]

Introduction: DEND syndrome is a rare channelopathy characterized by a combination of developmental delay, epilepsy and severe neonatal diabetes. Gain of function mutations in the KCNJ11 gene, encoding the KIR6.2 subunit of the IKATP potassium channel, stand at the basis of most forms of DEND syndrome. In a previous search for existing drugs with the potential of targeting Cantú Syndrome, also resulting from increased IKATP, we found a set of candidate drugs that may also possess the potential to target DEND syndrome. In the current work, we combined Molecular Modelling including Molecular Dynamics simulations, with single cell patch clamp electrophysiology, in order to test the effect of selected drug candidates on the KIR6.2 WT and DEND mutant channels.Methods: Molecular dynamics simulations were performed to investigate potential drug binding sites. To conduct in vitro studies, KIR6.2 Q52R and L164P mutants were constructed. Inside/out patch clamp electrophysiology on transiently transfected HEK293T cells was performed for establishing drug-channel inhibition relationships.Results: Molecular Dynamics simulations provided insight in potential channel interaction and shed light on possible mechanisms of action of the tested drug candidates. Effective IKIR6.2/SUR2a inhibition was obtained with the pore-blocker betaxolol (IC50 values 27–37 μM). Levobetaxolol effectively inhibited WT and L164P (IC50 values 22 μM) and Q52R (IC50 55 μM) channels. Of the SUR binding prostaglandin series, travoprost was found to be the best blocker of WT and L164P channels (IC50 2–3 μM), while Q52R inhibition was 15–20% at 10 μM.Conclusion: Our combination of MD and inside-out electrophysiology provides the rationale for drug mediated IKATP inhibition, and will be the basis for 1) screening of additional existing drugs for repurposing to address DEND syndrome, and 2) rationalized medicinal chemistry to improve IKATP inhibitor efficacy and specificity.

Abstract This paper introduces a new technique combining microdialysis with patch clamp to detect the changes in ATP (adenosine triphosphate) concentration in acupoints during acupuncture. The microdialysis probe was implanted into the Zusanli acupoint (ST 36) of adult SD (Sprague Dawley) rats to sample acupoint fluid containing ATP released during acupuncture. Then, the fluid with ATP was delivered in real time to 293 T cells overexpressing P2X3 receptors, with which we could carry out patch clamp experiments. The results showed that changes in membrane currents could reflect changes in the concentration of ATP. Thus, we can successfully detect ATP released in acupoints during acupuncture in real time. This technique provides us with a new way to study the mechanism of acupuncture signal initiation.

Achieving the distinctive complex behaviors of adult mammals requires the development of a great variety of specialized neural circuits. Although the development of these circuits begins during the embryonic stage, they remain immature at birth, requiring a postnatal maturation process to achieve these complex tasks. Understanding how the neuronal membrane properties and circuits change during development is the first step to understand their transition into efficient ones. Thus, using whole cell patch clamp recordings, we have studied the changes in the electrophysiological properties of layer V pyramidal neurons of the rat primary motor cortex during postnatal development. Among all the parameters studied, only the voltage threshold was established at birth and, although some of the changes occurred mainly during the second postnatal week, other properties such as membrane potential, capacitance, duration of the post-hyperpolarization phase or the maximum firing rate were not defined until the beginning of adulthood. Those modifications lead to a decrease in neuronal excitability and to an increase in the working range in young adult neurons, allowing more sensitive and accurate responses. This maturation process, that involves an increase in neuronal size and changes in ionic conductances, seems to be influenced by the neuronal type and by the task that neurons perform as inferred from the comparison with other pyramidal and motor neuron populations.

Cardiac arrhythmias significantly contribute to cardiovascular morbidity and mortality. The rabbit heart serves as an accepted model system for studying cardiac cell excitation and arrhythmogenicity. Accordingly, primary cultures of adult rabbit ventricular cardiomyocytes serve as a preferable model to study molecular mechanisms of human cardiac excitation. However, the use of adult rabbit cardiomyocytes is often regarded as excessively costly. Therefore, we developed and characterized a novel low-cost rabbit cardiomyocyte model, namely, 3-week-old ventricular cardiomyocytes (3wRbCMs). Ventricular myocytes were isolated from whole ventricles of 3-week-old New Zealand White rabbits of both sexes by standard enzymatic techniques. Using wheat germ agglutinin, we found a clear T-tubule structure in acutely isolated 3wRbCMs. Cells were adenovirally infected (multiplicity of infection of 10) to express Green Fluorescent Protein (GFP) and cultured for 48 h. The cells showed action potential duration (APD90 = 253 ± 24 ms) and calcium transients similar to adult rabbit cardiomyocytes. Freshly isolated and 48-h-old-cultured cells expressed critical ion channel proteins: calcium voltage-gated channel subunit alpha1 C (Cavα1c), sodium voltage-gated channel alpha subunit 5 (Nav1.5), potassium voltage-gated channel subfamily D member 3 (Kv4.3), and subfamily A member 4 (Kv1.4), and also subfamily H member 2 (RERG. Kv11.1), KvLQT1 (K7.1) protein and inward-rectifier potassium channel (Kir2.1). The cells displayed an appropriate electrophysiological phenotype, including fast sodium current (INa), transient outward potassium current (Ito), L-type calcium channel peak current (ICa,L), rapid and slow components of the delayed rectifier potassium current (IKr and IKs), and inward rectifier (IK1). Although expression of the channel proteins and some currents decreased during the 48 h of culturing, we conclude that 3wRbCMs are a new, low-cost alternative to the adult-rabbit-cardiomyocytes system, which allows the investigation of molecular mechanisms of cardiac excitation on morphological, biochemical, genetic, physiological, and biophysical levels.

Cellular electrophysiology is the foundation of many fields, from basic science in neurology, cardiology, oncology to safety critical applications for drug safety testing, clinical phenotyping, etc. Patch-clamp voltage clamp is the gold standard technique for studying cellular electrophysiology. Yet, the quality of these experiments is not always transparent, which may lead to erroneous conclusions for studies and applications. Here, we have developed a new computational approach that allows us to explain and predict the experimental artefacts in voltage-clamp experiments. The computational model captures the experimental procedure and its inadequacies, including: voltage offset, series resistance, membrane capacitance and (imperfect) amplifier compensations, such as series resistance compensation and supercharging. The computational model was validated through a series of electrical model cell experiments. Using this computational approach, the artefacts in voltage-clamp experiments of cardiac fast sodium current, one of the most challenging currents to voltage clamp, were able to be resolved and explained through coupling the observed current and the simulated membrane voltage, including some typically observed shifts and delays in the recorded currents. We further demonstrated that the typical way of averaging data for current-voltage relationships would lead to biases in the peak current and shifts in the peak voltage, and such biases can be in the same order of magnitude as those differences reported for disease-causing mutations. Therefore, the presented new computational pipeline will provide a new standard of assessing the voltage-clamp experiments and interpreting the experimental data, which may be able to rectify and provide a better understanding of ion channel mutations and other related applications., Competing Interests: Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The voltage-gated Kv1.5 potassium channel, conducting the ultra-rapid delayed rectifier K + current (I Kur ) in human cells, plays important roles in the repolarization of atrial action potentials and regulation of the vascular tone. We previously reported that activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) induces endocytic degradation of cell-surface Kv1.5 channels, and a point mutation removing the phosphorylation site, T15A, in the N terminus of Kv1.5 abolished the PMA-effect. In the present study, using mutagenesis, patch clamp recording, Western blot analysis, and immunocytochemical staining, we demonstrate that ubiquitination is involved in the PMA-mediated degradation of mature Kv1.5 channels. Since the expression of the Kv1.4 channel is unaffected by PMA treatment, we swapped the N- and/or C-termini between Kv1.5 and Kv1.4. We found that the N-terminus alone did not but both N- and C-termini of Kv1.5 did confer PMA sensitivity to mature Kv1.4 channels, suggesting the involvement of Kv1.5 C-terminus in the channel ubiquitination. Removal of each of the potential ubiquitination residue Lysine at position 536, 565, and 591 by Arginine substitution (K536R, K565R, and K591R) had little effect, but removal of all three Lysine residues with Arginine substitution (3K-R) partially reduced PMA-mediated Kv1.5 degradation. Furthermore, removing the cysteine residue at position 604 by Serine substitution (C604S) drastically reduced PMA-induced channel degradation. Removal of the three Lysines and Cys604 with a quadruple mutation (3K-R/C604S) or a truncation mutation (Δ536) completely abolished the PKC activation-mediated degradation of Kv1.5 channels. These results provide mechanistic insight into PKC activation-mediated Kv1.5 degradation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Together with its β-subunit OSTM1, ClC-7 performs 2Cl - /H + exchange across lysosomal membranes. Pathogenic variants in either gene cause lysosome-related pathologies, including osteopetrosis and lysosomal storage. CLCN7 variants can cause recessive or dominant disease. Different variants entail different sets of symptoms. Loss of ClC-7 causes osteopetrosis and mostly neuronal lysosomal storage. A recently reported de novo CLCN7 mutation (p.Tyr715Cys) causes widespread severe lysosome pathology (hypopigmentation, organomegaly, and delayed myelination and development, "HOD syndrome"), but no osteopetrosis. We now describe two additional HOD individuals with the previously described p.Tyr715Cys and a novel p.Lys285Thr mutation, respectively. Both mutations decreased ClC-7 inhibition by PI(3,5)P 2 and affected residues lining its binding pocket, and shifted voltage-dependent gating to less positive potentials, an effect partially conferred to WT subunits in WT/mutant heteromers. This shift predicts augmented pH gradient-driven Cl - uptake into vesicles. Overexpressing either mutant induced large lysosome-related vacuoles. This effect depended on Cl - /H + -exchange, as shown using mutants carrying uncoupling mutations. Fibroblasts from the p.Y715C patient also displayed giant vacuoles. This was not observed with p.K285T fibroblasts probably due to residual PI(3,5)P 2 sensitivity. The gain of function caused by the shifted voltage-dependence of either mutant likely is the main pathogenic factor. Loss of PI(3,5)P 2 inhibition will further increase current amplitudes, but may not be a general feature of HOD. Overactivity of ClC-7 induces pathologically enlarged vacuoles in many tissues, which is distinct from lysosomal storage observed with the loss of ClC-7 function. Osteopetrosis results from a loss of ClC-7, but osteoclasts remain resilient to increased ClC-7 activity., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The voltage-gated potassium Kv1.3 channel is an essential component of vital cellular processes which is also involved in the pathogenesis of some autoimmune, neuroinflammatory and oncological diseases. Pore blockers of the Kv1.3 channel are considered as potential drugs and are used to study Kv1 channels’ structure and functions. Screening and study of the blockers require the assessment of their ability to bind the channel. Expanding the variety of methods used for this, we report on the development of the fluorescent competitive binding assay for measuring affinities of pore blockers to Kv1.3 at the membrane of mammalian cells. The assay constituents are hongotoxin 1 conjugated with Atto488, fluorescent mKate2-tagged Kv1.3 channel, which was designed to improve membrane expression of the channel in mammalian cells, confocal microscopy, and a special protocol of image processing. The assay is implemented in the “mix and measure”, format and allows the screening of Kv1.3 blockers, such as peptide toxins, that bind to the extracellular vestibule of the K+-conducting pore, and analyzing their affinity.

Parkinson's disease (PD) is characterized by motor impairments caused by degeneration of dopamine neurons in the substantia nigra pars compacta. In addition to these symptoms, PD patients often suffer from non-motor comorbidities including sleep and psychiatric disturbances, which are thought to depend on concomitant alterations of serotonergic and noradrenergic transmission. A primary locus of serotonergic neurons is the dorsal raphe nucleus (DRN), providing brain-wide serotonergic input. Here, we identified electrophysiological and morphological parameters to classify serotonergic and dopaminergic neurons in the murine DRN under control conditions and in a PD model, following striatal injection of the catecholamine toxin, 6-hydroxydopamine (6-OHDA). Electrical and morphological properties of both neuronal populations were altered by 6-OHDA. In serotonergic neurons, most changes were reversed when 6-OHDA was injected in combination with desipramine, a noradrenaline (NA) reuptake inhibitor, protecting the noradrenergic terminals. Our results show that the depletion of both NA and dopamine in the 6-OHDA mouse model causes changes in the DRN neural circuitry., Competing Interests: LB, YJ, RT, RM, GF, GS No competing interests declared, (© 2023, Boi, Johansson et al.)

Mutations in more than half of human connexin genes encoding gap junction (GJ) subunits have been linked to inherited human diseases. Functional studies of human GJ channels are essential for revealing mechanistic insights into the etiology of disease-linked connexin mutants. However, the commonly used Xenopus oocytes, N2A, HeLa, and other model cells for recombinant expression of human connexins have different and significant limitations. Here we developed a human cell line (HEK293) with each of the endogenous connexins (Cx43 and Cx45) knocked out using the CRISPR-Cas9 system. Double knockout HEK293 cells showed no background GJ coupling, were easily transfected with several human connexin genes (such as those encoding Cx46, Cx50, Cx37, Cx45, Cx26, and Cx36) which successfully formed functional GJs and were readily accessible for dual patch clamp analysis. Single knockout Cx43 or Cx45 HEK cell lines could also be used to characterize human GJ channels formed by Cx45 or Cx43, respectively, with an expression level suitable for studying macroscopic and single channel GJ channel properties. A cardiac arrhythmia linked Cx45 mutant R184G failed to form functional GJs in DKO HEK293 cells with impaired localizations. These genetically engineered HEK293 cells are well suited for patch clamp study of human GJ channels., (© 2024 The Author(s).)

We investigate role of ATP sensitive potassium (K ATP ) channel in cytotoxic effect of cypermethrin on rat aortic smooth muscle cells. Cytotoxicity analysis was performed at 0, 0.1, 0.5, 10, 50, and 100 µM concentrations of cypermethrin and the cell index (CI) was calculated. K ATP currents were recorded using patch clamp technique for 50 and 100 µM concentrations and channel conductivity was determined by obtaining current-voltage characteristics. No cytotoxic effect was observed in the first 72 hours. At the 96th hour, only at 100 µM concentration, the CI value decreased significantly compared to control group and at 120 and 144th hours, it was observed that the CI value decreased significantly at all concentrations. Currents and conductivities were significantly decreased at 50 and 100 µM concentrations. Results gave clues that cypermethrin causes a cytotoxic effect on vascular smooth muscles and that K ATP channels may have a role in the emergence of this effect.

Mammalian SLC26 proteins are membrane-based anion transporters that belong to the large SLC26/SulP family, and many of their variants are associated with hereditary diseases. Recent structural studies revealed a strikingly similar homodimeric molecular architecture for several SLC26 members, implying a shared molecular principle. Now a new question emerges as to how these structurally similar proteins execute diverse physiological functions. In this study, we sought to identify the common versus distinct molecular mechanism among the SLC26 proteins using both naturally occurring and artificial missense changes introduced to SLC26A4, SLC26A5, and SLC26A9. We found: (i) the basic residue at the anion binding site is essential for both anion antiport of SLC26A4 and motor functions of SLC26A5, and its conversion to a nonpolar residue is crucial but not sufficient for the fast uncoupled anion transport in SLC26A9; (ii) the conserved polar residues in the N- and C-terminal cytosolic domains are likely involved in dynamic hydrogen-bonding networks and are essential for anion antiport of SLC26A4 but not for motor (SLC26A5) and uncoupled anion transport (SLC26A9) functions; (iii) the hydrophobic interaction between each protomer's last transmembrane helices, TM14, is not of functional significance in SLC26A9 but crucial for the functions of SLC26A4 and SLC26A5, likely contributing to optimally orient the axis of the relative movements of the core domain with respect to the gate domains within the cell membrane. These findings advance our understanding of the molecular mechanisms underlying the diverse physiological roles of the SLC26 family of proteins., Competing Interests: Conflicts of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

SCN5A gene variants are associated with both Brugada syndrome and conduction disturbances, sometimes expressing an overlapping phenotype. Functional consequences of SCN5A variants assessed by patch‐clamp electrophysiology are particularly beneficial for correct pathogenic classification and are related to disease penetrance and severity. Here, we identify a novel SCN5A loss of function variant, p.1449Y>H, which presented with high penetrance and complete left bundle branch block, totally masking the typical findings on the electrocardiogram. We highlight the possibility of this overlap combination that makes impossible an electrocardiographic diagnosis and, through a functional analysis, associate the p.1449Y>H variant to SCN5A pathogenicity. [ABSTRACT FROM AUTHOR]

Background and Purpose: Quercetin is a well‐known plant flavonoid with neuroprotective properties. Earlier work suggested it may relieve psychiatric disorders, cognition deficits and memory dysfunction through anti‐oxidant and/or radical scavenging mechanisms. In addition, quercetin modulated the physiological function of some ion channels. However, the detailed ionic mechanisms of the bioeffects of quercetin remain unknown. Experimental Approach Effects of quercetin on neuronal activities in the prefrontal cortex (PFC) and its ionic mechanisms were analysed by calcium imaging using mice bearing a green fluorescent protein, calmodulin, and M13 fusion protein and patch clamp in acute brain slices from C57BL/6 J mice and in HEK 293 cells. The possible ionic mechanism of action of quercetin on D‐amphetamine‐induced manic‐like effects in mice was explored with c‐fos staining and the open field behaviour test. Key Results: Quercetin reduced calcium influx triggered by PFC pyramidal neuronal activity. This effect involved increasing the rheobase of neuronal firing through decreasing membrane resistance following quercetin treatment. Spadin, a blocker of TREK‐1 potassium channels, also blocked the effect of quercetin on the membrane resistance and neuronal firing. Further, spadin blocked the neuroprotective effects of quercetin. The effects of quercetin on TREK‐1 channels could be mimicked by GF109203X, a protein kinase C inhibitor. In vivo, injection of quercetin relieved the manic hyperlocomotion in mice, induced by D‐amphetamine. This action was partly alleviated by spadin. Conclusion and Implications: TREK‐1 channels are a novel target for quercetin, by inhibiting PKC. This action could contribute to both the neuroprotective and anti‐manic‐like effects. [ABSTRACT FROM AUTHOR]

The bacterial mechanosensitive channel of small conductance (MscS) has been extensively studied to understand how mechanical forces are converted into the conformational changes that underlie mechanosensitive (MS) channel gating. We showed that lipid removal by β-cyclodextrin can mimic membrane tension. Here, we show that all cyclodextrins (CDs) can activate reconstituted Escherichia coli MscS, that MscS activation by CDs depends on CD-mediated lipid removal, and that the CD amount required to gate MscS scales with the channel's sensitivity to membrane tension. Importantly, cholesterol-loaded CDs do not activate MscS. CD-mediated lipid removal ultimately causes MscS desensitization, which we show is affected by the lipid environment. While many MS channels respond to membrane forces, generalized by the "force-from-lipids" principle, their different molecular architectures suggest that they use unique ways to convert mechanical forces into conformational changes. To test whether CDs can also be used to activate other MS channels, we chose to investigate the mechanosensitive channel of large conductance (MscL) and demonstrate that CDs can also activate this structurally unrelated channel. Since CDs can open the least tension-sensitive MS channel, MscL, they should be able to open any MS channel that responds to membrane tension. Thus, CDs emerge as a universal tool for the structural and functional characterization of unrelated MS channels. [ABSTRACT FROM AUTHOR]

Background: Mutations in ATP1A2, the gene encoding the α2 subunit of Na+/K+-ATPase, are the main cause of familial hemiplegic migraine type 2 (FHM2). The clinical presentation of FHM2 with mutations in the same gene varies from pure FHM to severe forms with epilepsy and intellectual disability, but the correlation of these symptoms with different ATP1A2 mutations is still unclear. Methods: Ten ATP1A2 missense mutations were selected according to different phenotypes of FHM patients. They caused pure FHM (FHM: R65W, R202Q, R593W, G762S), FHM with epilepsy (FHME: R548C, E825K, R938P), or FHM with epilepsy and intellectual disability (FHMEI: T378N, G615R, D718N). After ouabain resistance and fluorescence modification, plasmids carrying those mutations were transiently transfected into HEK293T and HeLa cells. The biochemical functions were studied including cell survival assays, membrane protein extraction, western blotting, and Na+/K+-ATPase activity tests. The electrophysiological functions of G762S, R938P, and G615R mutations were investigated in HEK293T cells using whole-cell patch-clamp. Homology modeling was performed to determine the locational distribution of ATP1A2 mutations. Results: Compared with wild-type pumps, all mutations showed a similar level of protein expression and decreased cell viability in the presence of 1 µM ouabain, and there was no significant difference among the mutant groups. The changes in Na+/K+-ATPase activity were correlated with the severity of FHM phenotypes. In the presence of 100 µM ouabain, the Na+/K+-ATPase activity was FHM > FHME > FHMEI. The ouabain-sensitive Na+/K+-ATPase activity of each mutant was significantly lower than that of the wild-type protein, and there was no significant difference among all mutant groups. Whole-cell voltage-clamp recordings in HEK293T cells showed that the ouabain-sensitive pump currents of G615R were significantly reduced, while those of G762S and R938P were comparable to those of the wild-type strain. Conclusions: ATP1A2 mutations cause phenotypes ranging from pure FHM to FHM with epilepsy and intellectual disability due to varying degrees of deficits in biochemical and electrophysiological properties of Na+/K+-ATPase. Mutations associated with intellectual disability presented with severe impairment of Na+/K+-ATPase. Whether epilepsy is accompanied, or the type of epilepsy did not seem to affect the degree of impairment of pump function. [ABSTRACT FROM AUTHOR]

Preterm infants frequently suffer from pulmonary complications due to a physiological and structural lung immaturity resulting in significant morbidity and mortality. Novel in vitro and in vivo models are required to study the underlying mechanisms of late lung maturation and to facilitate the development of new therapeutic strategies. Organoids recapitulate essential aspects of structural organization and possibly organ function, and can be used to model developmental and disease processes. We aimed at generating fetal lung organoids (LOs) and to functionally characterize this in vitro model in comparison to primary lung epithelial cells and lung explants ex vivo. LOs were generated with alveolar and endothelial cells from fetal rat lung tissue, using a Matrigel-gradient and air-liquid-interface culture conditions. Immunocytochemical analysis showed that the LOs consisted of polarized epithelial cell adhesion molecule (EpCAM)-positive cells with the apical membrane compartment facing the organoid lumen. Expression of the alveolar type 2 cell marker, RT2-70, and the Club cell marker, CC-10, were observed. Na+ transporter and surfactant protein mRNA expression were detected in the LOs. First time patch clamp analyses demonstrated the presence of several ion channels with specific electrophysiological properties, comparable to vital lung slices. Furthermore, the responsiveness of LOs to glucocorticoids was demonstrated. Finally, maturation of LOs induced by mesenchymal stem cells confirmed the convenience of the model to test and establish novel therapeutic strategies. The results showed that fetal LOs replicate key biological lung functions essential for lung maturation and therefore constitute a suitable in vitro model system to study lung development and related diseases.

Voltage-gated Kv1.3 potassium channels are key regulators of T lymphocyte activation, proliferation and cytokine production, by providing the necessary membrane hyper-polarization for calcium influx following immune stimulation. It is noteworthy that an accumulating body of in vivo and in vitro evidence links these channels to multiple sclerosis pathophysiology. Here we studied the electrophysiological properties and the transcriptional and translational expression of T lymphocyte Kv1.3 channels in multiple sclerosis, by combining patch clamp recordings, reverse transcription polymerase chain reaction and flow cytometry on freshly isolated peripheral blood T lymphocytes from two patient cohorts with multiple sclerosis, as well as from healthy and disease controls. Our data demonstrate that T lymphocytes in MS, manifest a significant up-regulation of Kv1.3 mRNA, Kv1.3 membrane protein and Kv1.3 current density and therefore of functional membrane channel protein, compared to control groups (p < 0.001). Interestingly, patient sub-grouping shows that Kv1.3 channel density is significantly higher in secondary progressive, compared to relapsing-remitting multiple sclerosis (p < 0.001). Taking into account the tight connection between Kv1.3 channel activity and calcium-dependent processes, our data predict and could partly explain the reported alterations of T lymphocyte function in multiple sclerosis, while they highlight Kv1.3 channels as potential therapeutic targets and peripheral biomarkers for the disease.

Ca2+ entry into mitochondria is through the mitochondrial calcium uniporter complex (MCUcx), a Ca2+-selective channel composed of five subunit types. Two MCUcx subunits (MCU and EMRE) span the inner mitochondrial membrane, while three Ca2+-regulatory subunits (MICU1, MICU2, and MICU3) reside in the intermembrane space. Here, we provide rigorous analysis of Ca2+ and Na+ fluxes via MCUcx in intact isolated mitochondria to understand the function of MICU subunits. We also perform direct patch clamp recordings of macroscopic and single MCUcx currents to gain further mechanistic insights. This comprehensive analysis shows that the MCUcx pore, composed of the EMRE and MCU subunits, is not occluded nor plugged by MICUs during the absence or presence of extramitochondrial Ca2+ as has been widely reported. Instead, MICUs potentiate activity of MCUcx as extramitochondrial Ca2+ is elevated. MICUs achieve this by modifying the gating properties of MCUcx allowing it to spend more time in the open state.

BackgroundThe ion transport stoichiometry (q) of electrogenic transporters is an important determinant of their function. q can be determined by the reversal potential (Erev) if the transporter under study is the only electrogenic transport mechanism or a specific inhibitor is available. An alternative approach is to calculate delta reversal potential (ΔErev) by altering the concentrations of the transported substrates. This approach is based on the hypothesis that the contributions of other channels and transporters on the membrane to Erev are additive. However, Erev is a complicated function of the sum of different conductances rather than being additive.ResultsWe propose a new delta current (ΔI) method based on a simplified model for electrogenic secondary active transport by Heinz (Electrical Potentials in Biological Membrane Transport, 1981). ΔI is the difference between two currents obtained from altering the external concentration of a transported substrate thereby eliminating other currents without the need for a specific inhibitor. q is determined by the ratio of ΔI at two different membrane voltages (V1 and V2) where q = 2RT/(F(V2 -V1))ln(ΔI2/ΔI1) + 1. We tested this ΔI methodology in HEK-293 cells expressing the elctrogenic SLC4 sodium bicarbonate cotransporters NBCe2-C and NBCe1-A, the results were consistent with those obtained with the Erev inhibitor method. Furthermore, using computational simulations, we compared the estimates of q with the ΔErev and ΔI methods. The results showed that the ΔErev method introduces significant error when other channels or electrogenic transporters are present on the membrane and that the ΔI equation accurately calculates the stoichiometric ratio.ConclusionsWe developed a ΔI method for estimating transport stoichiometry of electrogenic transporters based on the Heinz model. This model reduces to the conventional reversal potential method when the transporter under study is the only electrogenic transport process in the membrane. When there are other electrogenic transport pathways, ΔI method eliminates their contribution in estimating q. Computational simulations demonstrated that the ΔErev method introduces significant error when other channels or electrogenic transporters are present and that the ΔI equation accurately calculates the stoichiometric ratio. This new ΔI method can be readily extended to the analysis of other electrogenic transporters in other tissues.

Aripiprazole is an atypical antipsychotic drug, which is prescribed for many psychiatric diseases such as schizophrenia and mania in bipolar disorder. It primarily acts as an agonist of dopaminergic and other G-protein coupled receptors. So far, an interaction with ligand- or voltage-gated ion channels has been classified as weak. Meanwhile, we identified aripiprazole in a preliminary test as a potent blocker of voltage-gated sodium channels. Here, we present a detailed analysis about the interaction of aripiprazole with the dominant voltage-gated sodium channel of heart muscle (hNav1.5). Electrophysiological experiments were performed by means of the patch clamp technique at human heart muscle sodium channels (hNav1.5), heterologously expressed in human TsA cells. Aripiprazole inhibits the hNav1.5 channel in a state- but not use-dependent manner. The affinity for the resting state is weak with an extrapolated Kr of about 55 µM. By contrast, the interaction with the inactivated state is strong. The affinities for the fast and slow inactivated state are in the low micromolar range (0.5–1 µM). Kinetic studies indicate that block development for the inactivated state must be described with a fast (ms) and a slow (s) time constant. Even though the time constants differ by a factor of about 50, the resulting affinity constants were nearly identical (in the range of 0.5 µM). Besides this, aripirazole also interacts with the open state of the channel. Using an inactivation deficit mutant, an affinity of about 1 µM was estimated. In summary, aripiprazole inhibits voltage-gated sodium channels at low micromolar concentrations. This property might add to its possible anticancer and neuroprotective properties.

Ouabain is a cardiac glycoside, initially isolated from plants, and currently thought to be a hormone since some mammals synthesize it endogenously. It has been shown that in epithelial cells, it induces changes in properties and components related to apical–basolateral polarity and cell–cell contacts. In this work, we used a whole-cell patch clamp to test whether ouabain affects the properties of the voltage-gated potassium currents (Ik) of epithelial cells (MDCK). We found that: (1) in cells arranged as mature monolayers, ouabain induced changes in the properties of Ik; (2) it also accelerated the recovery of Ik in cells previously trypsinized and re-seeded at confluence; (3) in cell–cell contact-lacking cells, ouabain did not produce a significant change; (4) Na+/K+ ATPase might be the receptor that mediates the effect of ouabain on Ik; (5) the ouabain-induced changes in Ik required the synthesis of new nucleotides and proteins, as well as Golgi processing and exocytosis, as evidenced by treatment with drugs inhibiting those processes; and (5) the signaling cascade included the participation of cSrC, PI3K, Erk1/2, NF-κB and β-catenin. These results reveal a new role for ouabain as a modulator of the expression of voltage-gated potassium channels, which require cells to be in contact with themselves.

Ladybird beetles (Coleoptera: Coccinellidae) possess strong chemical defences that are secreted in response to stress and are also found on the coating of eggs, which are rich in alkaloids that are responsible for their toxicity to other species. Recent studies have shown that alkaloids from several species of ladybird beetle can target nicotinic acetylcholine receptors (nAChRs) acting as receptor antagonists. Here, we have explored the actions of (−)-adaline, found in the 2-spot (Adalia bipunctata) and 10-spot (Adalia decempunctata) ladybirds, on both mammalian (α1β1γδ, α7, α4β2, α3β4) and insect nAChRs using patch-clamp of TE671 cells and locust brain neurons natively expressing nAChRs, as well as two-electrode voltage clamp of Xenopus laevis oocytes recombinantly expressing nAChRs. All nAChR subtypes were antagonised by (−)-adaline in a time-dependent, voltage-dependent and non-competitive manner with the lowest IC50s at rat α3β4 (0.10 μM) and locust neuron (1.28 μM) nAChRs, at a holding potential of −75 mV. The data imply that (−)-adaline acts as an open channel blocker of nAChRs.

Lamotrigine is widely prescribed to treat bipolar neurological disorder and epilepsy. It exerts its antiepileptic action by blocking voltage-gated sodium channels in neurons. Recently, the US Food and Drug Administration issued a warning on the use of Lamotrigine after observations of conduction anomalies and Brugada syndrome patterns on the electrocardiograms of epileptic patients treated with the drug. Brugada syndrome and conduction disturbance are both associated with alterations of the cardiac sodium current (INa) kinetics and amplitude. In this study, we used the patch clamp technique on cardiomyocytes from epileptic rats to test the hypothesis that Lamotrigine also blocks INa in the heart. We found that Lamotrigine inhibited 60% of INa peak amplitude and reduced cardiac excitability in epileptic rats but had little effect in sham animals. Moreover, Lamotrigine inhibited 67% of INaL and, more importantly, prolonged the action potential refractory period in epileptic animals. Our results suggest that enhanced affinity of Lamotrigine for INa may in part explain the clinical phenotypes observed in epileptic patients.

Abstract: Projection neurons in the spinal dorsal horn relay sensory information to higher brain centres. The activation of these populations is shaped by afferent input from the periphery, descending input from the brain, and input from local interneuron circuits. Much of our recent understanding of dorsal horn circuitry comes from studies in transgenic mice; however, information on projection neurons is still based largely on studies in monkey, cat, and rat. We used viral labelling to identify and record from mouse parabrachial nucleus (PBN) projecting neurons located in the dorsal horn of spinal cord slices. Overall, mouse lamina I spinoparabrachial projection neurons (SPBNs) exhibit many electrophysiological and morphological features that overlap with rat. Unbiased cluster analysis distinguished 4 distinct subpopulations of lamina I SPBNs, based on their electrophysiological properties that may underlie different sensory signalling features in each group. We also provide novel information on SPBNs in the deeper lamina (III-V), which have not been previously studied by patch clamp analysis. These neurons exhibited higher action potential discharge frequencies and received weaker excitatory synaptic input than lamina I SPBNs, suggesting this deeper population produces different sensory codes destined for the PBN. Mouse SPBNs from both regions (laminae I and III-V) were often seen to give off local axon collaterals, and we provide neuroanatomical evidence they contribute to excitatory input to dorsal horn circuits. These data provide novel information to implicate excitatory input from parabrachial projection neuron in dorsal horn circuit activity during processing of nociceptive information, as well as defining deep dorsal horn projection neurons that provide an alternative route by which sensory information can reach the PBN. [ABSTRACT FROM AUTHOR]

Key points: Gap junctions formed by different connexins are expressed throughout the body and harbour unique channel properties that have not been fully defined mechanistically.Recent structural studies by cryo‐electron microscopy have produced high‐resolution models of the related but functionally distinct lens connexins (Cx50 and Cx46) captured in a stable open state, opening the door for structure–function comparison.Here, we conducted comparative molecular dynamics simulation and electrophysiology studies to dissect the isoform‐specific differences in Cx46 and Cx50 intercellular channel function.We show that key determinants Cx46 and Cx50 gap junction channel open stability and unitary conductance are shaped by structural and dynamic features of their N‐terminal domains, in particular the residue at the 9th position and differences in hydrophobic anchoring sites.The results of this study establish the open state Cx46/50 structural models as archetypes for structure–function studies targeted at elucidating the mechanism of gap junction channels and the molecular basis of disease‐causing variants. Connexins form intercellular communication channels, known as gap junctions (GJs), that facilitate diverse physiological roles, from long‐range electrical and chemical coupling to coordinating development and nutrient exchange. GJs formed by different connexin isoforms harbour unique channel properties that have not been fully defined mechanistically. Recent structural studies on Cx46 and Cx50 defined a novel and stable open state and implicated the amino‐terminal (NT) domain as a major contributor for isoform‐specific functional differences between these closely related lens connexins. To better understand these differences, we constructed models corresponding to wildtype Cx50 and Cx46 GJs, NT domain swapped chimeras, and point variants at the 9th residue for comparative molecular dynamics (MD) simulation and electrophysiology studies. All constructs formed functional GJ channels, except the chimeric Cx46‐50NT variant, which correlated with an introduced steric clash and increased dynamical behaviour (instability) of the NT domain observed by MD simulation. Single channel conductance correlated well with free‐energy landscapes predicted by MD, but resulted in a surprisingly greater degree of effect. Additionally, we observed significant effects on transjunctional voltage‐dependent gating (Vj gating) and/or open state dwell times induced by the designed NT domain variants. Together, these studies indicate intra‐ and inter‐subunit interactions involving both hydrophobic and charged residues within the NT domains of Cx46 and Cx50 play important roles in defining GJ open state stability and single channel conductance, and establish the open state Cx46/50 structural models as archetypes for structure–function studies targeted at elucidating GJ channel mechanisms and the molecular basis of cataract‐linked connexin variants. Key points: Gap junctions formed by different connexins are expressed throughout the body and harbour unique channel properties that have not been fully defined mechanistically.Recent structural studies by cryo‐electron microscopy have produced high‐resolution models of the related but functionally distinct lens connexins (Cx50 and Cx46) captured in a stable open state, opening the door for structure–function comparison.Here, we conducted comparative molecular dynamics simulation and electrophysiology studies to dissect the isoform‐specific differences in Cx46 and Cx50 intercellular channel function.We show that key determinants Cx46 and Cx50 gap junction channel open stability and unitary conductance are shaped by structural and dynamic features of their N‐terminal domains, in particular the residue at the 9th position and differences in hydrophobic anchoring sites.The results of this study establish the open state Cx46/50 structural models as archetypes for structure–function studies targeted at elucidating the mechanism of gap junction channels and the molecular basis of disease‐causing variants. [ABSTRACT FROM AUTHOR]

Purpose: The present study aimed to evaluate the effects of zinc deprivation on the properties of membrane and spike-discharge features of mesencephalic trigeminal neurons (MTNs), which are important sensory neurons for oral-motor reflexes and rhythmical jaw movements.Methods: Neonatal Sprague Dawley rats (P10-12) were distributed equally into a normal diet group and a zinc-deficient diet (ZD) group. Whole cell patch-clamp recordings were obtained from MTNs from coronal brain slices.Results: Passive membrane properties showed a modest depolarized membrane potential and decreased cell capacitance in the ZD group. Zinc deprivation decreased the minimal current amplitude, which induced an action potential and increased the amplitude of afterhyperpolarization following the action potential. Negligible changes were observed for other action potential properties. A decreased burst duration was observed, accompanied by hastened spike frequency adaptation in the burst discharge. There was no difference in the resonant properties at both the subthreshold depolarized potential and hyperpolarized membrane potential between the control and ZD groups.Conclusion: These results suggests that neither the persistent sodium conductance nor slow inwardly rectifying conductance were altered; however, there appeared to be an increase in Ca2+-dependent K+ conductance in zincdeficient MTNs. [ABSTRACT FROM AUTHOR]

Retinal organoids (ROs) derived from human inducible pluripotent stem cells (hiPSCs) exhibit considerable therapeutic potential. However, current quality control of ROs during in vitro differentiation is largely limited to the detection of molecular markers, often by immunostaining, polymerase chain reaction (PCR) assays and sequencing, often without proper functional assessments. As such, in the current study, we systemically characterized the physiological maturation of photoreceptor‐like cells in hiPSC‐derived ROs. By performing patch‐clamp recordings from photoreceptor‐like cells in ROs at distinct differentiation stages (ie, Differentiation Day [D]90, D150, and D200), we determined the electrophysiological properties of the plasma membrane and several characteristic ion channels closely associated with the physiological functions of the photoreceptors. Ionic hallmarks, such as hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels and cyclic nucleotide‐gated (CNG) channels, matured progressively during differentiation. After D200 in culture, these characteristic currents closely resembled those in macaque or human native photoreceptors. Furthermore, we demonstrated that the hyperpolarization‐activated inward current/depolarization‐activated outward current ratio (I−120/I+40), termed as the inward‐outward current (IOC) ratio hereon, accurately represented the maturity of photoreceptors and could serve as a sensitive indicator of pathological state. Thus, this study provides a comprehensive dataset describing the electrophysiological maturation of photoreceptor‐like cells in hiPSC‐derived ROs for precise and sensitive quality control during RO differentiation. [ABSTRACT FROM AUTHOR]

• BRI led to an increase in GFAP-positive cells 24 h and 72 h after reperfusion. • EPO reversed the effect of BRI on GFAP-positive cells 24 h after reperfusion. • BRI led to an increase in the peak amplitude of action potential and CV 24 h after reperfusion. • EPO could reverse the electrophysiological alterations resulted by BRI 24 h after reperfusion. The high mortality rate associated with acute kidney injury (AKI) is commonly due to progressive, inflammatory multiple organ dysfunction, which often involves neurological complications. The AKI-stimulated mechanisms leading to brain dysfunction are not well understood, which hinders development of new therapeutic avenues to minimize AKI-mediated neural effects. The hippocampal CA1 area is a particularly vulnerable region during AKI but the electrophysiological and inflammatory mechanisms involved in this vulnerability remain largely unknown. Here, we used immunohistochemistry to quantitatively investigate the number of astrocytes expressing glial fibrillary acidic protein (GFAP) as an indicator of inflammation, and whole cell patch clamp to evaluate electrophysiological changes in CA1 at different time points following induction of bilateral renal ischemia (BRI) in male Wistar rats. Further we evaluated the effectiveness of erythropoietin (EPO, 1000 U/kg i.p.) in mitigating BRI-associated changes. Plasma concentrations of blood urea nitrogen (BUN) were significantly enhanced at 24 h, 72 h and 1 week, and creatinine (Cr) was increased at 24 h after reperfusion, which were changes reduced by EPO. BRI led to an increase in CA1 GFAP-positive cells 24 h and 72 h, but not 1 week, after reperfusion, and EPO reversed this effect of BRI at 24 h. Additionally, BRI caused an increase in the peak amplitude and coefficient of variation of CA1 pyramidal neuronal action potentials, which were changes not seen in presence of EPO. When taken together, altered neuronal electrophysiological properties and astrogliosis could contribute to the neurological complications induced by AKI, and EPO offers hope as a potential neuroprotective agent. [ABSTRACT FROM AUTHOR]

[Display omitted] • Octopus cells were labelled with SUR1, SUR2 and Kir6.2 antibodies. • Octopus cells were also strongly labelled with TRPM2 antibody. • Activation of K ATP channels causes membrane hyperpolarizations. • K ATP channel antagonists led to membrane depolarization. • Octopus cells appear to possess both KATP and TRPM2-like channels. ATP-sensitive potassium (K ATP) channels and transient receptor potential melastatin 2 (TRPM2) channels are commonly expressed both pre- and postsynaptically in the central nervous system (CNS). We hypothesized that K ATP and TRPM2 may couple metabolic status to the resting membrane potential of octopus neurons of the mouse ventral cochlear nucleus (VCN). Therefore, we studied the expression of K ATP channels and TRPM2 channels in octopus cells by immunohistochemical techniques and their contribution to neuronal electrical properties by the electrophysiological patch clamp technique. In immunohistochemical staining of octopus cells, labelling with Kir6.2 and SUR1 antibodies was strong, and labelling with the SUR2 antibody was moderate, but labelling with Kir6.1 was very weak. Octopus cells had intense staining with TRPM2 antibodies. In patch clamp recordings, bath application of K ATP channel agonists H 2 O 2 (880 μM), ATZ (1 mM), cromakalim (50 μM), diazoxide (200 μM), NNC 55-0118 and NN 414 separately resulted in hyperpolarizations of resting potential to different extents. Application of 8-Bro-cADPR (50 μM), a specific antagonist of TRPM2 channels, in the presence of H 2 O 2 (880 μM) resulted in further hyperpolarization by approximately 1 mV. The amplitudes of H 2 O 2 -induced outward K ATP currents and ADPR-induced inward currents were 206.1 ± 31.5 pA (n = 4) and 136.8 ± 22.4 pA, respectively, at rest. Their respective reversal potentials were -77 ± 2.6 mV (n = 3) and -6.3 ± 2.9 (n = 3) and -6.3 ± 2.9 (n = 3). In conclusion, octopus cells appear to possess both K ATP channels and TRPM2-like channels. K ATP might largely be constituted by SUR1–Kir6.2 subunits and SUR2–Kir6.2 subunits. Both K ATP and TRPM2-like channels might have a modulatory action in setting the membrane potential. [ABSTRACT FROM AUTHOR]

The slowpoke channel responds to the intracellular calcium concentration and the depolarization of the cell membrane. It plays an important role in maintaining the resting potential and regulating the homeostasis of neurons, but it can also regulate circadian rhythm, sperm capacitation, ethanol tolerance, and other physiological processes in insects. This renders it a potentially useful target for the development of pest control strategies. There are relatively few studies on the slowpoke channels in lepidopteran pests, and their pharmacological properties are still unclear. So, in this study, the slowpoke gene of Plutella xylostella (Pxslo) was heterologous expressed in HEK293T cells, and the I-V curve of the slowpoke channel was measured by whole cell patch clamp recordings. Results showed that the slowpoke channel could be activated at −20 mV with 150 μM Ca2+. The subsequent comparison of the electrophysiological characteristics of the alternative splicing site E and G deletions showed that the deletion of the E site enhances the response of the slowpoke channel to depolarization, while the deletion of the G site weakens the response of the slowpoke channel to depolarization. Meanwhile, the nonspecific inhibitors TEA and 4-AP of the Kv channels, and four pesticides were tested and all showed an inhibition effect on the PxSlo channel at 10 or 100 μM, suggesting that these pesticides also target the slowpoke channel. This study enriches our understanding of the slowpoke channel in Lepidopteran insects and can aid in the development of relevant pest management strategies. [Display omitted] • PxSlo was expressed in HEK293, the calcium ion and voltage dependent channel currents were detected. • PxSlo channel was suppressed by four tested insecticides (cypermethrin, transfluthrin, tebufenozide and flonicamid). • Three types of splices of PxSlo exhibit different electrophysiological characteristics. [ABSTRACT FROM AUTHOR]

Early repolarization syndrome (ERS) is defined as occurring in patients with early repolarization pattern who have survived idiopathic ventricular fibrillation with clinical evaluation unrevealing for other explanations. The pathophysiologic basis of the ERS is currently uncertain. The objective of the present study was to examine the electrophysiological mechanism of ERS utilizing induced pluripotent stem cells (iPSCs) and CRISPR/Cas9 genome editing. Whole genome sequencing was used to identify the DPP6 (c.2561T > C/p.L854P) variant in four families with sudden cardiac arrest induced by ERS. Cardiomyocytes were generated from iPSCs from a 14-year-old boy in the four families with ERS and an unrelated healthy control subject. Patch clamp recordings revealed more significant prolongation of the action potential duration (APD) and increased transient outward potassium current (I to) (103.97 ± 18.73 pA/pF vs 44.36 ± 16.54 pA/pF at +70 mV, P < 0.05) in ERS cardiomyocytes compared with control cardiomyocytes. Of note, the selective correction of the causal variant in iPSC-derived cardiomyocytes using CRISPR/Cas9 gene editing normalized the I to , whereas prolongation of the APD remained unchanged. ERS cardiomyocytes carrying DPP6 mutation increased I to and lengthen APD, which maybe lay the electrophysiological foundation of ERS. [ABSTRACT FROM AUTHOR]

Abstract Background Voltage-gated sodium (Nav) channels have traditionally been considered a trademark of excitable cells. However, recent studies have shown the presence of Nav channels in several non-excitable cells, such as astrocytes and macrophages, demonstrating that the roles of these channels are more diverse than was previously thought. Despite the earlier discoveries, the presence of Nav channel-mediated currents in the cells of retinal pigment epithelium (RPE) has been dismissed as a cell culture artifact. We challenge this notion by investigating the presence and possible role of Nav channels in RPE both ex vivo and in vitro. Results Our work demonstrates that several subtypes of Nav channels are found in human embryonic stem cell (hESC)-derived and mouse RPE, most prominently subtypes Nav1.4, Nav1.6, and Nav1.8. Whole cell patch clamp recordings from the hESC-derived RPE monolayers showed that the current was inhibited by TTX and QX-314 and was sensitive to the selective blockers of the main Nav subtypes. Importantly, we show that the Nav channels are involved in photoreceptor outer segment phagocytosis since blocking their activity significantly reduces the efficiency of particle internalization. Consistent with this role, our electron microscopy results and immunocytochemical analysis show that Nav1.4 and Nav1.8 accumulate on phagosomes and that pharmacological inhibition of Nav channels as well as silencing the expression of Nav1.4 with shRNA impairs the phagocytosis process. Conclusions Taken together, our study shows that Nav channels are present in RPE, giving this tissue the capacity of fast electrical signaling. The channels are critical for the physiology of RPE with an important role in photoreceptor outer segment phagocytosis.

The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) is admitted as a pivotal site of integrating and regulating orofacial nociceptive inputs. Although citral (3,7-dimethyl-2,6-octadienal) is involved in antinociception, the action mechanism of citral on the SG neurons of the Vc has not been fully clarified yet. In this study, we examined the direct membrane effects of citral and how citral mediates responses on the SG neurons of the Vc in juvenile mice using a whole-cell patch-clamp technique. Under high chloride pipette solution, citral showed repeatable inward currents that persisted in the presence of tetrodotoxin, a voltage-gated Na+ channel blocker, and 6-cyano-7-nitro-quinoxaline-2,3-dione, a non-N-methyl-D-aspartate (NMDA) glutamate receptor antagonist, D-2-amino-5-phosphonopentanoic acid, an NMDA receptor antagonist. However, the citral-induced inward currents were partially blocked by picrotoxin, a gamma-aminobutyric acid (GABAA)-receptor antagonist, or by strychnine, a glycine receptor antagonist. Further, the citral-induced responses were almost blocked by picrotoxin with strychnine. We also found that citral exhibited additive effect with GABA-induced inward currents and glycine-induced inward currents were potentiated by citral. In addition, citral suppressed the firing activities by positive current injection on the SG neurons of the Vc. Taken together, these results demonstrate that citral has glycine- and/or GABA-mimetic actions and suggest that citral might be a potential target for orofacial pain modulation by the activation of inhibitory neurotransmission in the SG area of the Vc.

Abstract Background Transcranial ultrasonic stimulation is a novel noninvasive tool for neuromodulation, and has high spatial resolution and deep penetration. Although it can increase excitation of neurons, its effects on neuron are poorly understood. This study was to evaluate effect of ultrasonic stimulation (US) on neurons in vitro. In this paper, the effect of US on the excitability and voltage-dependent $$ K^{ + } $$ K+ currents of CA1 pyramidal neurons in the rat hippocampus was studied using patch clamp. Results Our results suggest that US increased the spontaneous firing rate and inhibited transient outward potassium current ($$ \varvec{I}_{\varvec{A}} $$ IA ) and delayed rectifier potassium current ($$ \varvec{I}_{\varvec{K}} ) $$ IK) . Furthermore, US altered the activation of $$ \varvec{I}_{\varvec{K}} $$ IK channels, inactivation and recovery properties of $$ \varvec{I}_{\varvec{A}} $$ IA channels. After US, the $$ \varvec{I}_{\varvec{K}} $$ IK activation curves significantly moved to the negative voltage direction and increased its slope factor. Moreover, the data showed that US moved the inactivation curve of $$ \varvec{I}_{\varvec{A}} $$ IA to the negative voltage and increased the slope factor. Besides, US delayed the recovery of $$ \varvec{I}_{\varvec{A}} $$ IA channel. Conclusions Our data indicate that US can increase excitation of neurons by inhibiting potassium currents. Different US decreased the voltage sensitivity of $$ \varvec{I}_{\varvec{K}} $$ IK activation differentially. Moreover, the more time is needed for US to make the $$ \varvec{I}_{\varvec{A}} $$ IA channels open again after inactivating. US may play a physiological role by inhibiting voltage-dependent potassium currents in neuromodulation. Our research can provide a theoretical basis for the future clinical application of ultrasound in neuromodulation.

Mutations in the KCNA1 gene, encoding the voltage-gated potassium channel Kv1.1, have been associated with a spectrum of neurological phenotypes, including episodic ataxia type 1 and developmental and epileptic encephalopathy. We have recently identified a de novo variant in KCNA1 in the highly conserved Pro-Val-Pro motif within the pore of the Kv1.1 channel in a girl affected by early onset epilepsy, ataxia and developmental delay. Other mutations causing severe epilepsy are located in Kv1.1 pore domain. The patient was initially treated with a combination of antiepileptic drugs with limited benefit. Finally, seizures and ataxia control were achieved with lacosamide and acetazolamide. The aim of this study was to functionally characterize Kv1.1 mutant channel to provide a genotype–phenotype correlation and discuss therapeutic options for KCNA1-related epilepsy. To this aim, we transfected HEK 293 cells with Kv1.1 or P403A cDNAs and recorded potassium currents through whole-cell patch-clamp. P403A channels showed smaller potassium currents, voltage-dependent activation shifted by +30 mV towards positive potentials and slower kinetics of activation compared with Kv1.1 wild-type. Heteromeric Kv1.1+P403A channels, resembling the condition of the heterozygous patient, confirmed a loss-of-function biophysical phenotype. Overall, the functional characterization of P403A channels correlates with the clinical symptoms of the patient and supports the observation that mutations associated with severe epileptic phenotype cluster in a highly conserved stretch of residues in Kv1.1 pore domain. This study also strengthens the beneficial effect of acetazolamide and sodium channel blockers in KCNA1 channelopathies.

Introduction: Atrial fibrillation (AF) is the most common cardiac arrhythmia. Consequently, novel therapies are being developed. Ultimately, the impact of compounds on the action potential (AP) needs to be tested in freshly isolated human atrial myocytes. However, the frequent depolarized state of these cells upon isolation seriously hampers reliable AP recordings.Purpose: We assessed whether AP recordings from single human atrial myocytes could be improved by providing these cells with a proper inward rectifier K+ current (IK1), and consequently with a regular, non-depolarized resting membrane potential (RMP), through “dynamic clamp”.Methods: Single myocytes were enzymatically isolated from left atrial appendage tissue obtained from patients with paroxysmal AF undergoing minimally invasive surgical ablation. APs were elicited at 1 Hz and measured using perforated patch-clamp methodology, injecting a synthetic IK1 to generate a regular RMP. The injected IK1 had strong or moderate rectification. For comparison, a regular RMP was forced through injection of a constant outward current. A wide variety of ion channel blockers was tested to assess their modulatory effects on AP characteristics.Results: Without any current injection, RMPs ranged from −9.6 to −86.2 mV in 58 cells. In depolarized cells (RMP positive to −60 mV), RMP could be set at −80 mV using IK1 or constant current injection and APs could be evoked upon stimulation. AP duration differed significantly between current injection methods (p < 0.05) and was shortest with constant current injection and longest with injection of IK1 with strong rectification. With moderate rectification, AP duration at 90% repolarization (APD90) was similar to myocytes with regular non-depolarized RMP, suggesting that a synthetic IK1 with moderate rectification is the most appropriate for human atrial myocytes. Importantly, APs evoked using each injection method were still sensitive to all drugs tested (lidocaine, nifedipine, E-4031, low dose 4-aminopyridine, barium, and apamin), suggesting that the major ionic currents of the atrial cells remained functional. However, certain drug effects were quantitatively dependent on the current injection approach used.Conclusion: Injection of a synthetic IK1 with moderate rectification facilitates detailed AP measurements in human atrial myocytes. Therefore, dynamic clamp represents a promising tool for testing novel antiarrhythmic drugs.

The molecular mechanisms underlying the diversity of cortical glutamatergic synapses are still incompletely understood. Here, we tested the hypothesis that presynaptic active zones (AZs) are constructed from molecularly uniform, independent release sites (RSs), the number of which scales linearly with the AZ size. Paired recordings between hippocampal CA1 pyramidal cells and fast-spiking interneurons in acute slices from adult mice followed by quantal analysis demonstrate large variability in the number of RSs (N) at these connections. High-resolution molecular analysis of functionally characterized synapses reveals variability in the content of one of the key vesicle priming factors – Munc13-1 – in AZs that possess the same N. Replica immunolabeling also shows a threefold variability in the total Munc13-1 content of AZs of identical size and a fourfold variability in the size and density of Munc13-1 clusters within the AZs. Our results provide evidence for quantitative molecular heterogeneity of RSs and support a model in which the AZ is built up from variable numbers of molecularly heterogeneous, but independent RSs.

Astrocytes and oligodendrocytes are main players in the brain to ensure ion and neurotransmitter homeostasis, metabolic supply, and fast action potential propagation in axons. These functions are fostered by the formation of large syncytia in which mainly astrocytes and oligodendrocytes are directly coupled. Panglial networks constitute on connexin-based gap junctions in the membranes of neighboring cells that allow the passage of ions, metabolites, and currents. However, these networks are not uniform but exhibit a brain region-dependent heterogeneous connectivity influencing electrical communication and intercellular ion spread. Here, we describe different approaches to analyze gap junctional communication in acute tissue slices that can be implemented easily in most electrophysiology and imaging laboratories. These approaches include paired recordings, determination of syncytial isopotentiality, tracer coupling followed by analysis of network topography, and wide field imaging of ion sensitive dyes. These approaches are capable to reveal cellular heterogeneity causing electrical isolation of functional circuits, reduced ion-transfer between different cell types, and anisotropy of tracer coupling. With a selective or combinatory use of these methods, the results will shed light on cellular properties of glial cells and their contribution to neuronal function.

Atomoxetine, a neuroactive drug, is approved for the treatment of attention-deficit/hyperactivity disorder (ADHD). It is primarily known as a high affinity blocker of the noradrenaline transporter, whereby its application leads to an increased level of the corresponding neurotransmitter in different brain regions. However, the concentrations used to obtain clinical effects are much higher than those which are required to block the transporter system. Thus, off-target effects are likely to occur. In this way, we previously identified atomoxetine as blocker of NMDA receptors. As many psychotropic drugs give rise to sudden death of cardiac origin, we now tested the hypothesis whether atomoxetine also interacts with voltage-gated sodium channels of heart muscle type in clinically relevant concentrations. Electrophysiological experiments were performed by means of the patch-clamp technique at human heart muscle sodium channels (hNav1.5) heterogeneously expressed in human embryonic kidney cells. Atomoxetine inhibited sodium channels in a state- and use-dependent manner. Atomoxetine had only a weak affinity for the resting state of the hNav1.5 (Kr: ∼ 120 µM). The efficacy of atomoxetine strongly increased with membrane depolarization, indicating that the inactivated state is an important target. A hallmark of this drug was its slow interaction. By use of different experimental settings, we concluded that the interaction occurs with the slow inactivated state as well as by slow kinetics with the fast-inactivated state. Half-maximal effective concentrations (2–3 µM) were well within the concentration range found in plasma of treated patients. Atomoxetine also interacted with the open channel. However, the interaction was not fast enough to accelerate the time constant of fast inactivation. Nevertheless, when using the inactivation-deficient hNav1.5_I408W_L409C_A410W mutant, we found that the persistent late current was blocked half maximal at about 3 µM atomoxetine. The interaction most probably occurred via the local anesthetic binding site. Atomoxetine inhibited sodium channels at a similar concentration as it is used for the treatment of ADHD. Due to its slow interaction and by inhibiting the late current, it potentially exerts antiarrhythmic properties.

ABSTRACT Background Myotonia congenita (MC) is a common channelopathy affecting skeletal muscle and which is due to pathogenic variants within the CLCN1 gene. Various alterations in the function of the channel have been reported and we here illustrate a novel one. Methods A patient presenting the symptoms of myotonia congenita was shown to bear a new heterozygous missense variant in exon 9 of the CLCN1 gene (c.1010 T > G, p.(Phe337Cys)). Confocal imaging and patch clamp recordings of transiently transfected HEK293 cells were used to functionally analyze the effect of this variant on channel properties. Results Confocal imaging showed that the F337C mutant incorporated as well as the WT channel into the plasma membrane. However, in patch clamp, we observed a smaller conductance for F337C at −80 mV. We also found a marked reduction of the fast gating component in the mutant channels, as well as an overall reduced voltage dependence. Conclusion To our knowledge, this is the first report of a mixed alteration in the biophysical properties of hClC‐1 consisting of a reduced conductance at resting potential and an almost abolished voltage dependence.

Transient receptor potential melastatin member 8 (TRPM8), a Ca2+‐permeable nonselective cation channel activated by cold and cooling agents, mediates allodynia. Dysfunction or abnormal expression of TRPM8 has been found in several human cancers. The role of ubiquitination in the regulation of TRPM8 function remains poorly understood. Here, we identified the ubiquitin (Ub)‐ligase E3, tripartite motif‐containing 4 (TRIM4), as a novel interaction partner of TRPM8 and confirmed that the TRIM4–TRPM8 interaction was mediated through the SPRY domain of TRIM4. Patch‐clamp assays showed that TRIM4 negatively regulates TRPM8‐mediated currents in HEK293 cells. Moreover, TRIM4 reduced the expression of TRPM8 on the cell surface by promoting the K63‐linked ubiquitination of TRPM8. Further analyses revealed that the TRPM8 N‐terminal lysine residue at 423 was the major ubiquitination site that mediates its functional regulation by TRIM4. A Ub‐activating enzyme E1, Ub‐like modifier‐activating enzyme 1 (UBA1), was also found to interact with TRPM8, thereby regulating its channel function and ubiquitination. In addition, knockdown of UBA1 impaired the regulation of TRPM8 ubiquitination and function by TRIM4. Thus, this study demonstrates that TRIM4 downregulates TRPM8 via K423‐mediated TRPM8 ubiquitination and requires UBA1 to regulate TRPM8. [ABSTRACT FROM AUTHOR]

Birds acquired endothermy and a four-chambered heart independently from mammals in the course of evolution. Though avian embryos are widely used in experiments, little is known about the adult avian heart. Recent studies have shown that, despite a large evolutionary distance, the set of repolarizing potassium currents in avian myocardium resembles that in mammalian heart as well as that in humans. This allows for proposing birds as a potential model in experimental cardiology. The present study for the first time describes inward rectifier currents in working myocardium of quail. Using patch clamp method, we recorded main background inward rectifier current IK1 was recorded in isolated atrial and ventricular cardiomyocytes of quail. Both inward and outward components of IK1 in ventricular cells were larger than those in atrial cells, while there were no differences in voltage dependence of inward rectification. Acetylcholine and carbachol induced activation of acetylcholine-dependent inward rectifier current IKACh in atrial but not in ventricular myocytes. IKACh in atrial myocytes was sensitive to tertiapin. Constitutively active IKACh has not been detected. In multicellular preparations of the quail right atrium, carbachol induced hyperpolarization and shortening of action potentials, while no such effects were observed in preparations of the right ventricle. Activation of IKACh upon application of carbachol was dose-dependent with EC50 = 4.922 × 10–7 М. The described distribution of inward rectifier currents in avian myocardium is similar to that in mammalian species, which are widely used as model objects in experimental cardiology. [ABSTRACT FROM AUTHOR]