Single-channel Recordings Research Articles

A deep learning approach to real-time Markov modeling of ion channel gating

The patch-clamp technique allows us to eavesdrop the gating behavior of individual ion channels with unprecedented temporal resolution. The signals arise from conformational changes of the channel protein as it makes rapid transitions between conducting and non-conducting states. However, unambiguous analysis of single-channel datasets is challenging given the inadvertently low signal-to-noise ratio as well as signal distortions caused by low-pass filtering. Ion channel kinetics are typically described using hidden Markov models (HMM), which allow conclusions on the inner workings of the protein. In this study, we present a Deep Learning approach for extracting models from single-channel recordings. Two-dimensional dwell-time histograms are computed from the idealized time series and are subsequently analyzed by two neural networks, that have been trained on simulated datasets, to determine the topology and the transition rates of the HMM. We show that this method is robust regarding noise and gating events beyond the corner frequency of the low-pass filter. In addition, we propose a method to evaluate the goodness of a predicted model by re-simulating the prediction. Finally, we tested the algorithm with data recorded on a patch-clamp setup. In principle, it meets the requirements for model extraction during an ongoing recording session in real-time.

The gating properties of Drosophila NMJ glutamate receptors and their dependence on Neto.

The Drosophila neuromuscular junction (NMJ) is a powerful genetic system that has revealed numerous conserved mechanisms for synapse development and homeostasis. The fly NMJ uses glutamate as the excitatory neurotransmitter and relies on kainate-type glutamate receptors and their auxiliary protein Neto for synapse assembly and function. However, despite decades of study, the reconstitution of NMJ glutamate receptors using heterologous systems has been achieved only recently, and there are no reports on the gating properties for the recombinant receptors. Here, using outside-out, patch clamp recordings and fast ligand application, we examine for the first time the biophysical properties of native type-A and type-B NMJ receptors in complexes with either Neto-α or Neto-β and compare them with recombinant receptors expressed in HEK293T cells. We found that type-A and type-B receptors have strikingly different gating properties that are further modulated by Neto-α and Neto-β. We captured single-channel events and revealed major differences between type-A and type-B receptors and also between Neto splice variants. Surprisingly, we found that deactivation is extremely fast and that the decay of synaptic currents resembles the rate of ionotropic glutamate receptor (iGluR) desensitization. The functional analyses of recombinant iGluRs that we report here should greatly facilitate the interpretation of compound in vivo phenotypes of mutant animals. KEY POINTS: We report the reconstitution of Drosophila neuromuscular junction ionotropic glutamate receptors (iGluRs) with Neto splice forms. Using outside-out patches and fast ligand application, we examine the deactivation and desensitization of the four iGluR/Neto complexes found in vivo. Expression of functional channels is absolutely dependent on Neto. Single-channel recordings revealed different lifetimes for different receptor complexes. The decay of synaptic currents is controlled by desensitization.

Copper Increases the Cooperative Gating of Rat P2X2a Receptor Channels

Background/Objectives: P2X receptor channels are widely expressed in the CNS, where they have multiple functions in health and disease. The rat P2X2a (rP2X2a) receptor channel is modulated by copper, an essential trace element that plays important roles in synaptic modulation and neurodegenerative disorders. Although essential extracellular amino acids that coordinate copper have been identified, the exact mechanism of copper-induced modulation has not been yet elucidated. Methods: We used HEK293T cells expressing rP2X2a channel(s) and performed outside-out single-channel and whole-cell recordings to explore copper’s effects on rP2X2 currents and determine whether this metal can increase the cooperative gating of rP2X2a channel. Results: In whole-cell recordings and in patches containing 2 or 3 rP2X2a channels, copper enhanced the ATP-induced currents, significantly reducing the ATP EC50 and increasing the Hill coefficient. Moreover, copper increased the apparent Po in patches containing two or three channels. By contrast, in patches containing only one rP2X2a channel, we did not observe any significant changes in ATP EC50, the Hill coefficient, or Po. Conclusions: Copper modulates the gating of rP2X2a channels, enhancing interchannel cooperativity without altering single-channel conductance or Po. This novel regulatory mechanism could be relevant for understanding the role of P2X2 channels in physiological and pathological processes.

Deep learning-based spike sorting: a survey.

Objective.Deep learning is increasingly permeating neuroscience, leading to a rise in signal-processing applications for extracellular recordings. These signals capture the activity of small neuronal populations, necessitating 'spike sorting' to assign action potentials (spikes) to their underlying neurons. With the rise in publications delving into new methodologies and techniques for deep learning-based spike sorting, it is crucial to synthesise these findings critically. This survey provides an in-depth evaluation of the approaches, methodologies and outcomes presented in recent articles, shedding light on the current state-of-the-art.Approach.Twenty-four articles published until December 2023 on deep learning-based spike sorting have been examined. The proposed methods are divided into three sub-problems of spike sorting: spike detection, feature extraction and classification. Moreover, integrated systems, i.e. models that detect spikes and extract features or do classification within a single network, are included.Main results.Although most algorithms have been developed for single-channel recordings, models utilising multi-channel data have already shown promising results, with efficient hardware implementations running quantised models on application-specific integrated circuits and field programmable gate arrays. Convolutional neural networks have been used extensively for spike detection and classification as the data can be processed spatiotemporally while maintaining low-parameter models and increasing generalisation and efficiency. Autoencoders have been mainly utilised for dimensionality reduction, enabling subsequent clustering with standard methods. Also, integrated systems have shown great potential in solving the spike sorting problem from end to end.Significance.This survey explores recent articles on deep learning-based spike sorting and highlights the capabilities of deep neural networks in overcoming associated challenges, but also highlights potential biases of certain models. Serving as a resource for both newcomers and seasoned researchers in the field, this work provides insights into the latest advancements and may inspire future model development.

Stable oxidative posttranslational modifications alter the gating properties of RyR1.

The ryanodine receptor type 1 (RyR1) is a Ca2+ release channel that regulates skeletal muscle contraction by controlling Ca2+ release from the sarcoplasmic reticulum (SR). Posttranslational modifications (PTMs) of RyR1, such as phosphorylation, S-nitrosylation, and carbonylation are known to increase RyR1 open probability (Po), contributing to SR Ca2+ leak and skeletal muscle dysfunction. PTMs on RyR1 have been linked to muscle dysfunction in diseases like breast cancer, rheumatoid arthritis, Duchenne muscle dystrophy, and aging. While reactive oxygen species (ROS) and oxidative stress induce PTMs, the impact of stable oxidative modifications like 3-nitrotyrosine (3-NT) and malondialdehyde adducts (MDA) on RyR1 gating remains unclear. Mass spectrometry and single-channel recordings were used to study how 3-NT and MDA modify RyR1 and affect Po. Both modifications increased Po in a dose-dependent manner, with mass spectrometry identifying 30 modified residues out of 5035 amino acids per RyR1 monomer. Key modifications were found in domains critical for protein interaction and channel activation, including Y808/3NT in SPRY1, Y1081/3NT and H1254/MDA in SPRY2&3, and Q2107/MDA and Y2128/3NT in JSol, near the binding site of FKBP12. Though these modifications did not directly overlap with FKBP12 binding residues, they promoted FKBP12 dissociation from RyR1. These findings provide detailed insights into how stable oxidative PTMs on RyR1 residues alter channel gating, advancing our understanding of RyR1-mediated Ca2+ release in conditions associated with oxidative stress and muscle weakness.

Inactivation induced by pathogenic Cav1.3 L-type Ca2+-channel variants enhances sensitivity for dihydropyridine Ca2+ channel blockers.

Pathogenic gain-of-function mutations in Cav1.3 L-type voltage-gated Ca2+-channels (CACNA1D) cause neurodevelopmental disorders with or without endocrine symptoms. We aimed to confirm a pathogenic gain-of function phenotype of CACNA1D de novo missense mutations A749T and L271H, and investigated the molecular mechanism causing their enhanced sensitivity for the Ca2+-channel blocker isradipine, a potential therapeutic for affected patients. Wildtype and mutant channels were expressed in tsA-201 cells and their gating analysed using whole-cell and single-channel patch-clamp recordings. The voltage-dependence of isradipine action was quantified using protocols inducing variable fractions of inactivated channels. The molecular basis for altered channel gating in the mutants was investigated using in silico modelling and molecular dynamics simulations. Both mutations were confirmed pathogenic due to characteristic shifts of voltage-dependent activation and inactivation towards negative potentials (~20 mV). At negative holding potentials both mutations showed significantly higher isradipine sensitivity compared to wildtype. The affinity for wildtype and mutant channels increased with channel inactivation as predicted by the modulated receptor hypothesis (30- to 40-fold). The IC50 was indistinguishable for wildtype and mutants when >50% of channels were inactivated. Mutations A749T and L271H induce pathogenic gating changes. Like wildtype, isradipine inhibition is strongly voltage-dependent. Our data explains their apparent higher drug sensitivity at a given negative voltage by the availability of more inactivated channels due to their more negative inactivation voltage range. Low nanomolar isradipine concentrations will only inhibit Cav1.3 channels in neurons during prolonged depolarized states without selectivity for mutant channels.

Optimizing wearable single-channel EEG sleep staging in a heterogeneous sleep-disordered population using transfer learning.

While various wearable EEG devices have been developed, performance evaluation of the devices and their associated automated sleep stage classification models is mostly limited to healthy subjects. A major barrier for applying automated wearable EEG sleep staging in clinical populations is the need for large-scale data for model training. We therefore investigated transfer learning as strategy to overcome limited data availability and optimize automated single-channel EEG sleep staging in people with sleep disorders. We acquired 52 single-channel frontopolar headband EEG recordings from a heterogeneous sleep-disordered population with concurrent PSG. We compared three model training strategies: 'pre-training' (i.e., training on a larger dataset of 901 conventional PSGs), 'training-from-scratch' (i.e., training on wearable headband recordings), and 'fine-tuning' (i.e., training on conventional PSGs, followed by training on headband recordings). Performance was evaluated on all headband recordings using 10-fold cross-validation. Highest performance for 5-stage classification was achieved with fine-tuning (κ = .778), significantly higher than with pre-training (κ = .769) and with training-from-scratch (κ = .733). No significant differences or systematic biases were observed with clinically relevant sleep parameters derived from PSG. All sleep disorder categories showed comparable performance. This study emphasizes the importance of leveraging larger available datasets through deep transfer learning to optimize performance with limited data availability. Findings indicate strong similarity in data characteristics between conventional PSG and headband recordings. Altogether, results suggest the combination of the headband, classification model, and training methodology can be viable for sleep monitoring in the heterogeneous clinical population.

A lumen-tunable triangular DNA nanopore for molecular sensing and cross-membrane transport

Synthetic membrane nanopores made of DNA are promising systems to sense and control molecular transport in biosensing, sequencing, and synthetic cells. Lumen-tunable nanopore like the natural ion channels and systematically increasing the lumen size have become long-standing desires in developing nanopores. Here, we design a triangular DNA nanopore with a large tunable lumen. It allows in-situ transition from expanded state to contracted state without changing its stable triangular shape, and vice versa, in which specific DNA bindings as stimuli mechanically pinch and release the three corners of the triangular frame. Transmission electron microscopy images and molecular dynamics simulations illustrate the stable architectures and the high shape retention. Single-channel current recordings and fluorescence influx studies demonstrate the low-noise repeatable readouts and the controllable cross-membrane macromolecular transport. We envision that the proposed DNA nanopores could offer powerful tools in molecular sensing, drug delivery, and the creation of synthetic cells.

Abstract Mo065: An Anti-arrhythmic Action and Novel Molecular Mechanisms of Alda-1 in Holiday Heart Syndrome

Introduction: Holiday Heart Syndrome (HHS) is caused by excessive binge alcohol intake. Atrial fibrillation (AF) is the most common arrhythmia in HHS patients. With a worldwide rising trend in heavy alcohol consumption despite significant prevention efforts, effective drugs against alcohol-evoked AF are in urgent need. Methods: We assessed the effect of Alda-1, a cardiac protective agent, on mitigating binge alcohol-evoked AF and alcohol-activated stress kinase JNK2 in 50mM alcohol-exposed (24hr) H9c2 differentiated myocytes and in a well-characterized HHS mouse model (2g/kg ethanol i.p., 4 injections, every other day). Alda-1’s effect on alcohol-evoked RyR2 channel-mediated Ca 2+ waves/sparks & tachycardia/fibrillation (AT/AF) incidence were measured in intact mouse atria. RyR2 channel open probability was assessed in human RyR2 single-channel recordings. Results: We found that Alda-1 significantly reduced atrial arrhythmia inducibility in HHS mouse atria, as evidenced by a decreased incidence of alcohol-evoked AT/AF (0 vs. 0.21 incidences/pacing attempts/animal; n=6, 8). Alda-1 is an agonist of aldehyde dehydrogenase 2 (ALDH2; a key enzyme in alcohol detoxification), which inhibits cellular apoptosis. We found that ALDH2 was increased by 42%±6 in HHS mice compared to controls, and which remained elevated upon Alda-1 treatment (n=6,7,7; p<0.05). HHS hearts showed unchanged apoptotic signaling pathways, suggesting alternative mechanisms in AF genesis. Our lab recently revealed a critical role of activated cardiac stress kinase JNK2 in AF risk. Intriguingly, Alda-1 suppressed alcohol-activated JNK2 by 70%±13 (immunoprecipitated JNK2-specific activity; n=9,9, p<0.001) (but not JNK1, the other cardiac JNK isoform) in cells, while JNK2 inhibition alleviated alcohol-evoked RyR2 channel dysfunction in human RyR2 channels as well as Ca 2+ -triggered atrial arrhythmic activities and AT/AF (0 out of 8; p<0.05) in intact mouse hearts. Conclusion: Alda-1 effectively mitigates binge alcohol-evoked atrial arrhythmogenesis by suppressing JNK2 activity independently of an ALDH2-mediated anti-apoptosis effect. Our findings highlight the potential of Alda-1 as a therapeutic agent for the increasing incidence of alcohol-evoked AF.

Characterization of a novel Ca2+-Activated potassium channel in rat brain rough endoplasmic reticulum

ObjectivesPotassium channels in the endoplasmic reticulum (ER) are crucial for maintaining calcium balance during calcium fluxes. Disruption in ER calcium balance leads to ER stress, implicated in diseases like diabetes and Alzheimer's disease (AD). However, limited data exists on ER potassium channels in excitable tissues such as the brain. To fill this gap, we aimed to evaluate potassium currents in rat brain rough endoplasmic reticulum (RER). MethodsRats were euthanized under deep anesthesia and their brains were immediately removed. The brains were then homogenized in ice-cold sucrose buffer, followed by the extraction of RER microsomes through a series of centrifugation processes. Purity of sample was evaluated using western blotting technique. Single channel recordings were done in voltage steps from +50 to −60 mV following incorporation of rat brain RER vesicles into planar bilayers. ResultsWe observed a voltage-dependent potassium channel with an approximate conductance of 188 pS. Channel open probability was low at negative voltages, increasing at positive voltages. The channel was blocked by Charybdotoxin but not by Iberiotoxin. Additionally, TRAM-34, a specific KCa3.1 channel blocker, suppressed channel current amplitude and open probability. Western blot analysis revealed specific bands for anti-KCa3.1 antibody, approximately 50 kDa in brain homogenate and RER fraction. ConclusionOur study provides strong evidence for the presence of an KCa3.1 channel on the RER membrane in rat brain, exhibiting distinct electro-pharmacological profile compared to plasma membrane and other organelles.

CaM-dependent modulation of human CaV1.3 whole-cell and single-channel currents by C-terminal CaMKII phosphorylation site S1475.

Phosphorylation enables rapid modulation of voltage-gated calcium channels (VGCC) in physiological and pathophysiological conditions. How phosphorylation modulates human CaV1.3 VGCC, however, is largely unexplored. We characterized modulation of CaV1.3 gating via S1475, the human equivalent of a phosphorylation site identified in the rat. S1475 is highly conserved in CaV1.3 but absent from all other high-voltage activating calcium channel types co-expressed with CaV1.3 in similar tissues. Further, it is located in the C-terminal EF-hand motif, which binds calmodulin (CaM). This is involved in calcium-dependent channel inactivation (CDI). We used amino acid exchanges that mimic either sustained phosphorylation (S1475D) or phosphorylation resistance (S1475A). Whole-cell and single-channel recordings of phosphorylation state imitating CaV1.3 variants in transiently transfected HEK-293 cells revealed functional relevance of S1475 in human CaV1.3. We obtained three main findings: (1) CaV1.3_S1475D, imitating sustained phosphorylation, displayed decreased current density, reduced CDI and (in-) activation kinetics shifted to more depolarized voltages compared with both wildtype CaV1.3 and the phosphorylation-resistant CaV1.3_S1475A variant. Corresponding to the decreased current density, we find a reduced open probability of CaV1.3_S1475D at the single-channel level. (2) Using CaM overexpression or depletion, we find that CaM is necessary for modulating CaV1.3 through S1475. (3) CaMKII activation led to CaV1.3_WT-current properties similar to those of CaV1.3_S1475D, but did not affect CaV1.3_S1475A, confirming that CaMKII modulates human CaV1.3 via S1475. Given the physiological and pathophysiological importance of CaV1.3, our findings on the S1475-mediated interplay of phosphorylation, CaM interaction and CDI provide hints for approaches on specific CaV1.3 modulation under physiological and pathophysiological conditions. KEY POINTS: Phosphorylation modulates activity of voltage-gated L-type calcium channels for specific cellular needs but is largely unexplored for human CaV1.3 channels. Here we report that S1475, a CaMKII phosphorylation site identified in rats, is functionally relevant in human CaV1.3. Imitating phosphorylation states at S1475 alters current density and inactivation in a calmodulin-dependent manner. In wildtype CaV1.3 but not in the phosphorylation-resistant variant S1475A, CaMKII activation elicits effects similar to constitutively mimicking phosphorylation at S1475. Our findings provide novel insights on the interplay of modulatory mechanisms of human CaV1.3 channels, and present a possible target for CaV1.3-specific gating modulation in physiological and pathophysiological conditions.

Biophysical essentials – A full stack open-source software framework for conserved and advanced analysis of patch-clamp recordings

Background and ObjectivesPatch-Clamp recordings allow for in depth electrophysiological characterization of single cells, their general biophysical properties as well as characteristics of voltage- and ligand-gated ionic currents. Different acquisition modes, such as whole-cell patch-clamp recordings in the current or voltage clamp configuration, capacitance measurements or single channel recordings from cultured cells as well as acute brain slices are routinely performed for these purposes. Nevertheless, multipurpose transparent and adaptable software tools to perform reproducible state-of-the-art analysis of multiple experiment types and to manage larger sets of experimental data are currently unavailable. MethodsBiophysical Essentials (BPE) was developed as an open-source full stack python software for transparent and reproducible analysis of electrophysiological recordings. For validation, BPE results were compared with manually analyzed single-cell patch-clamp data acquired from a human in vitro nociceptor-model and mouse dorsal root ganglia neurons. ResultsWhile initially designed to improve time consuming and repetitive analysis steps, BPE was further optimized as a technical software solution for entire workflow processing including data acquisition, data preprocessing, normalization and visualization and of single recordings up to stacked calculations and statistics of multiple experiments. BPE can operate with different file formats from different amplifier systems and producers. An in-process database logs all analysis steps reproducible review and serves as a central storage point for recordings. Statistical testing as well as advanced analysis functions like Boltzmann-fitting and dimensional reduction methods further support the researchers' needs in projects involving electrophysiology techniques. ConclusionsBPE extends beyond available patch-clamp specific, open source – and commercial analysis tools in particular because of reproducible and sharable analysis workflows. BPE enables full analysis from raw data acquisition to publication ready result visualizations – all within one single program. Thereby, BPE significantly enhances transparency in the analytical process of patch-clamp data analysis. BPEs function scope is completely accessible through an easy-to-use graphical user interface eliminating the need for programing language proficiency as required by many community patch-clamp analysis frameworks and algorithms.

The structural remodeling of the mitochondrial Ca2+ uniporter complex underlies its functional impairment during metabolic cardiomyopathy

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): ANR Introduction Calcium (Ca2+) uptake by the mitochondrial Ca2+ uniporter complex (MCUC) controls key steps of oxidative metabolism to adapt energy production to cardiac demand. To ensure this critical function, MCUC assembly and maturation is a tightly regulated process involving the AAA proteases. We recently demonstrated that mitochondrial Ca2+ uptake is impaired during the early stages of metabolic cardiomyopathy in the atria. Purpose Our aim was to investigate mitochondrial Ca2+ handling in the left ventricle during the early stages of metabolic cardiomyopathy and to characterize the mechanisms underlying the functional remodeling of the MCUC. Methods Male C57Bl6/J mice were fed a high-fat sucrose diet (HFS) for two weeks to induce metabolic cardiomyopathy. Mitochondrial Ca2+ uptake was then studied (1) in isolated ventricular cardiomyocytes using whole-cell patch-clamp coupled with confocal microscopy and (2) in isolated mitochondria using Ca2+ Green-5N. The biophysical properties of MCUC were recorded in a planar lipid bilayer. MCUC structure and AAA-proteases expression were assessed by Western blot and co-immunoprecipitation. Control mice were treated with AAV9 harboring shRNA to downregulate the expression of MAIP1. Results Metabolic cardiomyopathy was associated with reduced beat-to-beat mitochondrial Ca2+ uptake in ventricular cardiomyocytes, which was further confirmed in isolated mitochondria. Single-channel recordings of the native cardiac MCUC embedded in a planar lipid bilayer revealed a reduction in ionic conductance and open probability, demonstrating functional impairment of the MCUC. Biochemical analysis of the MCUC revealed an accumulation of the unprocessed form of the EMRE subunit and a decreased interaction of the EMRE-MICU1 subcomplex with the pore-forming MCU subunits, indicating a structural remodeling of the MCUC. The expression of the AAA proteases YME1L and AFG3L2/SPG7, which are responsible for EMRE maturation and insertion into the MCUC, was unchanged. However, the expression of m-AAA protease-interacting protein 1 (MAIP1), which orchestrates EMRE processing, was reduced. In control mice, shRNA-mediated downregulation of MAIP1 recapitulated the structural and functional changes of the MCUC. Conclusions The structural remodeling of the MCUC integrity by downregulation of its assembly affects mitochondrial Ca2+ homeostasis in the early stages of metabolic cardiomyopathy. MAIP1 is a key regulator of MCUC assembly and its downregulation underlies the functional remodeling of the MCUC. These findings provide a novel post-translational paradigm of MCUC regulation in pathophysiology and suggest novel therapeutic targets for the treatment of the metabolic cardiomyopathy.

One-shot design elevates functional expression levels of a voltage-gated potassium channel.

Membrane proteins play critical physiological roles as receptors, channels, pumps, and transporters. Despite their importance, however, low expression levels often hamper the experimental characterization of membrane proteins. We present an automated and web-accessible design algorithm called mPROSS (https://mPROSS.weizmann.ac.il), which uses phylogenetic analysis and an atomistic potential, including an empirical lipophilicity scale, to improve native-state energy. As a stringent test, we apply mPROSS to the Kv1.2-Kv2.1 paddle chimera voltage-gated potassium channel. Four designs, encoding 9-26 mutations relative to the parental channel, were functional and maintained potassium-selective permeation and voltage dependence in Xenopus oocytes with up to 14-fold increase in whole-cell current densities. Additionally, single-channel recordings reveal no significant change in the channel-opening probability nor in unitary conductance, indicating that functional expression levels increase without impacting the activity profile of individual channels. Our results suggest that the expression levels of other dynamic channels and receptors may be enhanced through one-shot design calculations.

Neuroprotective amyloid β N-terminal peptides differentially alter human α7- and α7β2-nicotinic acetylcholine (nACh) receptor single-channel properties.

Oligomeric amyloid β 1-42 (oAβ1-42) exhibits agonist-like action at human α7- and α7β2-containing nicotinic receptors. The N-terminal amyloid β1-15 fragment (N-Aβ fragment) modulates presynaptic calcium and enhances hippocampal-based synaptic plasticity via α7-containing nicotinic receptors. Further, the N-Aβ fragment and its core sequence, the N-amyloid-beta core hexapeptide (N-Aβcore), protect against oAβ1-42-associated synapto- and neurotoxicity. Here, we investigated how oAβ1-42, the N-Aβ fragment, and the N-Aβcore regulate the single-channel properties of α7- and α7β2-nicotinic receptors. Single-channel recordings measured the impact of acetylcholine, oAβ1-42, the N-Aβ fragment, and the N-Aβcore on the unitary properties of human α7- and α7β2-containing nicotinic receptors expressed in nicotinic-null SH-EP1 cells. Molecular dynamics simulations identified potential sites of interaction between the N-Aβ fragment and orthosteric α7+/α7- and α7+/β2- nicotinic receptor binding interfaces. The N-Aβ fragment and N-Aβcore induced α7- and α7β2-nicotinic receptor single-channel openings. Relative to acetylcholine, oAβ1-42 preferentially enhanced α7β2-nicotinic receptor single-channel open probability and open-dwell times. Co-application with the N-Aβcore neutralized these effects. Further, administration of the N-Aβ fragment alone, or in combination with acetylcholine or oAβ1-42, selectively enhanced α7-nicotinic receptor open probability and open-dwell times (compared to acetylcholine or oAβ1-42). Amyloid-beta peptides demonstrate functional diversity in regulating α7- and α7β2-nicotinic receptor function, with implications for a wide range of nicotinic receptor-mediated functions in Alzheimer's disease. The effects of these peptides on α7- and/or α7β2-nicotinic receptors revealed complex interactions with these subtypes, providing novel insights into the neuroprotective actions of amyloid β-derived fragments against the toxic effects of oAβ1-42.

Activation of BKα channel provented uremic cardiomyopathy by attenuating oxidative stress

Background: Uremic cardiomyopathy and renal fibrosis contribute to chronic kidney disease (CKD)-induced morbidity and mortality. Our previous study found that activation of the large conductance calcium-activated potassium channels (BK channels) attenuated renal fibrosis in mice (Wang et al, Kid Int, 2021). We hypothesized that upregulation of BK channels would suppress cardiac fibrosis in CKD. Methods: CKD mice were induced by 5/6 nephrectomy. To upregulation of BKa channels, BMS-191011 (10 mg/kg BW) was given by IP injection every day for 6-8 weeks. Single channel recordings were used to analyze the activation of BKa channel. Blood pressure was measured by non-invasive photoplethysmography using the tail-cuff method. Echocardiogram was used to assess cardiac function. Cultured H9C2 cardiac myoblasts were used to detect the impact of BK opener (NS1916 20mM) on fibrosis markers and oxidative stress. The mRNA expression of BKa in heart was assayed by qPCR. The Hydrogen peroxide (H2O2) was tested by Amplex Red Hydrogen Peroxide Kit. DHE (Dihydroethidium) was assayed to detect superoxide. Superoxide Dismutase (SOD), a defense of oxidative enzyme, was measured using colorimetric activity kit. Results: The expression of BKa mRNA and protein were decreased and cardiac fibrosis was increased in the heart of CKD mice. Single channel recordings showed that human uremic serum (User) attenuated the activation of BKa channel. Upregulation of BKa channel by BSM-191011 decreased the CKD-induced increase in systolic and diastolic blood pressure. Echocardiogram showed that LV end-diastolic dimension increased in 5/6Nx mice, but back to normal after BK opener treatment. Ejection fraction also improved by this treatment. Fibronectin and vimentin were also significantly increased in CKD heart, whereas BMS treatment attenuated the amount of both proteins. In cultured cardiac myoblasts, User and TGFβ increased fibronectin and collagen I, which were in a dose-dependent manner. BKa openers (NS1916) limited the User and TGF-induced upregulation of both proteins in H9C2 cells. User also significantly increased H2O2 and superoxide production in cultured H9C2 cells. Activation of BKa significantly abolished this upregulation. In addition, User and TGFβ decrease SOD production, NS1916 blocked this decline in a dose dependent manner. Conclusions: Activation of BK channel in CKD animals attenuates cardiac fibrosis likely through reduction of uremia-induced oxidative stress. This study could provide new approaches for developing therapeutic strategies for treating uremic cardiomyopathy in chronic kidney diseases. the Department of Veteran Affairs MERIT Award 5I01BX000994 to HC. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Real-time Single-Channel EOG removal based on Empirical Mode Decomposition

In recent years, single-channel physiological recordings have gained popularity in portable health devices and research settings due to their convenience. However, the presence of electrooculogram (EOG) artifacts can significantly degrade the quality of the recorded data, impacting the accuracy of essential signal features. Consequently, artifact removal from physiological signals is a crucial step in signal processing pipelines. Current techniques often employ Independent Component Analysis (ICA) to efficiently separate signal and artifact sources in multichannel recordings. However, limitations arise when dealing with single or a few channel measurements in minimal instrumentation or portable devices, restricting the utility of ICA. To address this challenge, this paper introduces an innovative artifact removal algorithm utilizing enhanced empirical mode decomposition to extract the intrinsic mode functions (IMFs). Subsequently, the algorithm targets the removal of segments related to EOG by isolating them within these IMFs. The proposed method is compared with existing single-channel EEG artifact removal algorithms, demonstrating superior performance. The findings demonstrate the effectiveness of our approach in isolating artifact components, resulting in a reconstructed signal characterized by a strong correlation and a power spectrum closely resembling the ground-truth EEG signal. This outperforms the existing methods in terms of artifact removal. Additionally, the proposed algorithm exhibits significantly reduced execution time, enabling real-time online analysis.

PCA-Based Preprocessing for Clustering-Based Fetal Heart Rate Extraction in Non-Invasive Fetal Electrocardiograms

Non-invasive fetal electrocardiography (NI-ECG) is based on the acquisition of signals from electrodes on the mother’s abdominal surface. This abdominal ECG (aECG) signal consists of the maternal ECG (mECG) along with the fetal ECG (fECG) and other noises and artifacts. These records allow the acquisition of valuable and reliable information that helps ensure fetal well-being during pregnancy. This paper proposes a procedure based on principal component analysis (PCA) to obtain a single-channel master abdominal ECG record that can be used as input to fetal heart rate extraction techniques. The new procedure requires three main processing stages: PCA-based analysis for fECG-component extraction, polarity test, and curve fitting. To show the advantages of the proposal, this PCA-based method has been used as the feeding stage to a previously developed clustering-based method for single-channel aECG fetal heart rate monitoring. The results obtained for a set of real abdominal ECG recordings from annotated public aECG databases, the Abdominal and Direct Fetal ECG Database and the Challenge 2013 Training Set A, show improved efficiency in fetal heart rate extraction and illustrate the benefits derived from the use of such a master abdominal ECG channel. This allows us to achieve proper fetal heart rate monitoring without the need for manual inspection and selection of channels to be processed, while also allowing us to analyze records that would have been discarded otherwise.

Exploring the Neural Correlates of Flow Experience with Multifaceted Tasks and a Single-Channel Prefrontal EEG Recording.

Flow experience, characterized by deep immersion and complete engagement in a task, is highly recognized for its positive psychological impacts. However, previous studies have been restricted to using a single type of task, and the exploration of its neural correlates has been limited. This study aimed to explore the neural correlates of flow experience with the employment of multifaceted flow-induction tasks. Six tasks spanning mindfulness, artistic tasks, free recall, and varying levels of Tetris complexity (easy, flow, and hard conditions) were employed to have relatively complete coverage of the known flow-induction tasks for a better induction of individualized flow experience. Twenty-eight participants were recruited to perform these six tasks with a single-channel prefrontal EEG recording. Significant positive correlations were observed between the subjective flow scores of the individual's best-flow-experience task and the EEG activities at the delta, gamma, and theta bands, peaking at latencies around 2 min after task onset. The outcomes of regression analysis yield a maximum R2 of 0.163. Our findings report the EEG correlates of flow experience in naturalistic settings and highlight the potential of portable and unobtrusive EEG technology for an objective measurement of flow experience.

A novel, patient-derived RyR1 mutation impairs muscle function and calcium homeostasis in mice.

RYR1 is the most commonly mutated gene associated with congenital myopathies, a group of early-onset neuromuscular conditions of variable severity. The functional effects of a number of dominant RYR1 mutations have been established; however, for recessive mutations, these effects may depend on multiple factors, such as the formation of a hypomorphic allele, or on whether they are homozygous or compound heterozygous. Here, we functionally characterize a new transgenic mouse model knocked-in for mutations identified in a severely affected child born preterm and presenting limited limb movement. The child carried the homozygous c.14928C>G RYR1 mutation, resulting in the p.F4976L substitution. In vivo and ex vivo assays revealed that homozygous mice fatigued sooner and their muscles generated significantly less force compared with their WT or heterozygous littermates. Electron microscopy, biochemical, and physiological analyses showed that muscles from RyR1 p.F4976L homozygous mice have the following properties: (1) contain fewer calcium release units and show areas of myofibrillar degeneration, (2) contain less RyR1 protein, (3) fibers show smaller electrically evoked calcium transients, and (4) their SR has smaller calcium stores. In addition, single-channel recordings indicate that RyR1 p.F4976L exhibits higher Po in the presence of 100 μM [Ca2+]. Our mouse model partly recapitulates the clinical picture of the homozygous human patient and provides significant insight into the functional impact of this mutation. These results will help understand the pathology of patients with similar RYR1 mutations.