Single Ion Channel Research Articles

TMEM175 plays a crucial role in osteoblast differentiation by regulating lysosomal function and autophagy

Bone provides structural support, enables movement, protects internal organs, regulates calcium and phosphorus levels, and contains bone marrow essential for hematopoiesis. Osteoblasts are specialized cells responsible for bone formation through the secretion of extracellular matrix components. Transmembrane protein 175 (TMEM175), which functions as an endosomal/lysosomal K+ channel and a lysosomal H+ channel, regulates lysosomal function and autophagy. Despite the recognized importance of lysosomes and autophagy in osteoblast differentiation, the specific role of TMEM175 in osteoblast differentiation has not been revealed. In this study, we investigated whether TMEM175 is associated with human bone mineral densityand fracture and examined the role of TMEM175 in osteoblast differentiation. In analyses of single nucleotide polymorphismsof pore ion channel genes using the mouse2human database, a significant correlation between TMEM175 single nucleotide polymorphisms and human bone mineral density and fracture was identified. TMEM175 expression levels were found to increase during osteoblast differentiation from bone chip-derived mesenchymal stem cells (BMSCs). Knockdown of TMEM175 in BMSCs suppressed osteoblast differentiation, as evidenced by decreased matrix mineralization and lower expression levels of osteoblast marker genes. Further analysis indicated that TMEM175 deficiency leads to lysosomal dysfunction and partially impairs autophagic clearance during osteoblast differentiation. Moreover, the TMEM175 inhibitor 4-aminopyridinedecreased osteoblast differentiation of BMSCs. Taken together, this study reveals that TMEM175 plays an important role in osteoblast differentiation by regulating lysosomal function and autophagic clearance.

Osteoarthritis year in review 2024: Biology

Osteoarthritis (OA) research is a fast-growing and extremely wide field, in which a substantial increase in knowledge has been achieved over the last year. It covers many different topics, however, a PubMed search using the terms 'osteoarthritis' and 'biology' resulted in only a limited number of studies that were published between April 2023 and April 2024. In order to identify OA-relevant studies that focus on mechanistic studies of biological processes at the tissue, cellular, and molecular level, the following keywords were included as search terms: tissue interactions, single cell sequencing, transcriptomics, extracellular matrix, signaling, ion channels, and pain. The final selection of publications presented in this 'year in review' was influenced by the personal preferences of the authors, and eventually three larger key themes emerged: 1) Joint tissue interactions covering meniscus, subchondral bone, fat tissue, synovium, and synovial fluid. 2) Degeneration of the cartilage extracellular matrix and generation of bioactive fragments. 3) Receptors, ion channels, signaling pathways, and cellular metabolism. Many of the studies summarized here identified novel potential targets for OA treatment, and promising results were already obtained addressing these targets in different animal models. It will be exciting to see which findings can be translated into future clinical studies and eventually lead to novel treatment approaches for human OA.

Building predictive Markov models of ion channel permeation from molecular dynamics simulations

Molecular dynamics (MD) simulation of biological processes has always been a challenging task due to the long timescales of the processes involved and the large amount of output data to handle. Markov state models (MSMs) have been introduced as a powerful tool in this area of research, as they provide a mechanistically comprehensible synthesis of the large amount of MD data and, at the same time, can be used to rapidly estimate experimental properties of biological processes. Herein, we propose a method for building MSMs of ion channel permeation from MD trajectories, which directly evaluates the current flowing through the channel from the model’s transition matrix (T), which is crucial for comparing simulations and experimental data. This is achieved by including in the model a flux matrix that summarizes information on the charge moving across the channel between each pair of states of the MSM and can be used in conjunction with T to predict the ion current. A procedure to drastically reduce the number of states in the MSM while preserving the estimated ion current is also proposed. Applying the method to the KcsA channel returned an MSM with five states with significant equilibrium occupancy, capable of accurately reproducing the single-channel ion current from microsecond MD trajectories.

Novel therapies for cancer-induced bone pain

Cancer pain is a growing problem, especially with the substantial increase in cancer survival. Reports indicate that bone metastasis, whose primary symptom is bone pain, occurs in 65–75% of patients with advanced breast or prostate cancer. We optimized a preclinical in vivo model of cancer-induced bone pain (CIBP) involving the injection of Lewis Lung Carcinoma cells into the intramedullary space of the femur of C57BL/6 mice or transgenic mice on a C57BL/6 background. Mice gradually reduce the use of the affected limb, leading to altered weight bearing. Symptoms of secondary cutaneous heat sensitivity also manifest themselves. Following optimization, three potential analgesic treatments were assessed; 1) single ion channel targets (targeting the voltage-gated sodium channels NaV1.7, NaV1.8, or acid-sensing ion channels), 2) silencing µ-opioid receptor-expressing neurons by modified botulinum compounds, and 3) targeting two inflammatory mediators simultaneously (nerve growth factor (NGF) and tumor necrosis factor (TNF)). Unlike global NaV1.8 knockout mice which do not show any reduction in CIBP-related behavior, embryonic conditional NaV1.7 knockout mice in sensory neurons exhibit a mild reduction in CIBP-linked behavior. Modified botulinum compounds also failed to cause a detectable analgesic effect. In contrast, inhibition of NGF and/or TNF resulted in a significant reduction in CIBP-driven weight-bearing alterations and prevented the development of secondary cutaneous heat hyperalgesia. Our results support the inhibition of these inflammatory mediators, and more strongly their dual inhibition to treat CIBP, given the superiority of combination therapies in extending the time needed to reach limb use score zero in our CIBP model.

Neher and Sakmann: Single Ion Channels.

Most biological functions have their basis in a rapid change in cell membrane permeability. Hodgkin and Huxley showed regulation of the flow of molecules and ions between the nerve cell and its environment (Nobel Prize 1963), by recording macroscopic currents. Two Germans Neher and Sakmann showed that specific ion channels actually exist, and specific membrane proteins act as gates or agents for active transport regulating in physiological and pathological processes.

A modelling study to dissect the potential role of voltage-gated ion channels in activity-dependent conduction velocity changes as identified in small fiber neuropathy patients.

Patients with small fiber neuropathy (SFN) suffer from neuropathic pain, which is still a therapeutic problem. Changed activation patterns of mechano-insensitive peripheral nerve fibers (CMi) could cause neuropathic pain. However, there is sparse knowledge about mechanisms leading to CMi dysfunction since it is difficult to dissect specific molecular mechanisms in humans. We used an in-silico model to elucidate molecular causes of CMi dysfunction as observed in single nerve fiber recordings (microneurography) of SFN patients. We analyzed microneurography data from 97 CMi-fibers from healthy individuals and 34 of SFN patients to identify activity-dependent changes in conduction velocity. Using the NEURON environment, we adapted a biophysical realistic preexisting CMi-fiber model with ion channels described by Hodgkin-Huxley dynamics for identifying molecular mechanisms leading to those changes. Via a grid search optimization, we assessed the interplay between different ion channels, Na-K-pump, and resting membrane potential. Changing a single ion channel conductance, Na-K-pump or membrane potential individually is not sufficient to reproduce in-silico CMi-fiber dysfunction of unchanged activity-dependent conduction velocity slowing and quicker normalization of conduction velocity after stimulation as observed in microneurography. We identified the best combination of mechanisms: increased conductance of potassium delayed-rectifier and decreased conductance of Na-K-pump and depolarized membrane potential. When the membrane potential is unchanged, opposite changes in Na-K-pump and ion channels generate the same effect. Our study suggests that not one single mechanism accounts for pain-relevant changes in CMi-fibers, but a combination of mechanisms. A depolarized membrane potential, as previously observed in patients with neuropathic pain, leads to changes in the contribution of ion channels and the Na-K-pump. Thus, when searching for targets for the treatment of neuropathic pain, combinations of several molecules in interplay with the membrane potential should be regarded.

Contrast detection is enhanced by deterministic, high-frequency transcranial alternating current stimulation with triangle and sine waveform.

Stochastic resonance (SR) describes a phenomenon where an additive noise (stochastic carrier-wave) enhances the signal transmission in a nonlinear system. In the nervous system, nonlinear properties are present from the level of single ion channels all the way to perception and appear to support the emergence of SR. For example, SR has been repeatedly demonstrated for visual detection tasks, also by adding noise directly to cortical areas via transcranial random noise stimulation (tRNS). When dealing with nonlinear physical systems, it has been suggested that resonance can be induced not only by adding stochastic signals (i.e., noise) but also by adding a large class of signals that are not stochastic in nature that cause "deterministic amplitude resonance" (DAR). Here, we mathematically show that high-frequency, deterministic, periodic signals can yield resonance-like effects with linear transfer and infinite signal-to-noise ratio at the output. We tested this prediction empirically and investigated whether nonrandom, high-frequency, transcranial alternating current stimulation (tACS) applied to the visual cortex could induce resonance-like effects and enhance the performance of a visual detection task. We demonstrated in 28 participants that applying 80-Hz triangular-waves or sine-waves with tACS reduced the visual contrast detection threshold for optimal brain stimulation intensities. The influence of tACS on contrast sensitivity was equally effective to tRNS-induced modulation, demonstrating that both tACS and tRNS can reduce contrast detection thresholds. Our findings suggest that a resonance-like mechanism can also emerge when deterministic electrical waveforms are applied via tACS.NEW & NOTEWORTHY Our findings extend our understanding of neuromodulation induced by noninvasive electrical stimulation. We provide the first evidence showing acute online benefits of transcranial alternating current stimulation (tACS)triangle and tACSsine targeting the primary visual cortex (V1) on visual contrast detection in accordance with the resonance-like phenomenon. The "deterministic" tACS and "stochastic" high-frequency-transcranial random noise stimulation (tRNS) are equally effective in enhancing visual contrast detection.

A Poisson-Nernst-Planck single ion channel model and its effective finite element solver

A single ion channel is a membrane protein with an ion selectivity filter that allows only a single species of ions (such as potassium ions) to pass through in the “open” state. Its selectivity filter also naturally separates a solvent domain into an intracellular domain and an extracellular domain. Such biological and geometrical characteristics of a single ion channel are novelly adopted in the construction of a new kind of dielectric continuum ion channel model, called the Poisson-Nernst-Planck single ion channel (PNPSIC) model, in this paper. An effective PNPSIC finite element solver is then developed and implemented as a software package workable for a single ion channel with a three-dimensional X-ray crystallographic molecular structure and a mixture of multiple ionic species. Numerical results for a potassium channel confirm the convergence and efficiency of the PNPSIC finite element solver and demonstrate the high performance of the software package. Moreover, the PNPSIC model is applied to the calculation of electric current and validated by biophysical experimental data.

Simultaneous single photon counting and ion channel recording from free-standing lipid bilayers on a microstructured array.

Fluorescence microscopy and, in particular, single molecule optical spectroscopy is of great potential value in characterizing the structural dynamics of membranes and membrane proteins. A particular challenge is to combine such high-resolution optical measurements with high-resolution voltage clamp electrical recordings that can provide direct information on single ion channel gating a block by drugs and analytes. Here, we report on the use of a novel chip-based, 2 × 2 arrangement of microelectrode cavities with borosilicate glass optical windows which facilitates optical access on an inverted microscope with water or oil-immersion objectives of high numerical aperture to horizontal free-standing lipid membranes, while controlling membrane voltage and recording currents using individual micropatterned, ring-shaped Ag/AgCl-electrodes to perform time-resolved single photon counting on free-standing membranes spanning sub-nanoliter cavities. Single channel activity induced by the fluorescently labelled pore-forming peptide ceratotoxin A was simultaneously acquired. This device allows for rapid formation of four membranes that are simultaneously monitored electrically using a four-channel amplifier and can be sequentially optically addressed, greatly reducing the time needed for successful experimentation. During our experiments, we noted autofluorescence of the borosilicate to be a limiting factor for wide-field low-intensity fluorescence recording, but this can likely be circumvented by using quartz glass instead. In summary, the novel device increases the likelihood of realizing the long standing ambition to correlate structural and functional dynamics of single membrane proteins on the single molecule level.

Predicting human cardiac QT alterations and pro-arrhythmic effects of compounds with a 3D beating heart-on-chip platform.

Determining the potential cardiotoxicity and pro-arrhythmic effects of drug candidates remains one of the most relevant issues in the drug development pipeline (DDP). New methods enabling to perform more representative preclinical in vitro studies by exploiting induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) are under investigation to increase the translational power of the outcomes. Here we present a pharmacological campaign conducted to evaluate the drug-induced QT alterations and arrhythmic events on uHeart, a 3D miniaturized in vitro model of human myocardium encompassing iPSC-CM and dermal fibroblasts embedded in fibrin. uHeart was mechanically trained resulting in synchronously beating cardiac microtissues in 1 week, characterized by a clear field potential (FP) signal that was recorded by means of an integrated electrical system. A drug screening protocol compliant with the new International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines was established and uHeart was employed for testing the effect of 11 compounds acting on single or multiple cardiac ion channels and well-known to elicit QT prolongation or arrhythmic events in clinics. The alterations of uHeart's electrophysiological parameters such as the beating period, the FP duration, the FP amplitude, and the detection of arrhythmic events prior and after drug administration at incremental doses were effectively analyzed through a custom-developed algorithm. Results demonstrated the ability of uHeart to successfully anticipate clinical outcome and to predict the QT prolongation with a sensitivity of 83.3%, a specificity of 100% and an accuracy of 91.6%. Cardiotoxic concentrations of drugs were notably detected in the range of the clinical highest blood drug concentration (Cmax), qualifying uHeart as a fit-to-purpose preclinical tool for cardiotoxicity studies.

Single ion channel recording in 3D culture of stem cells using patch-clamp technique

Tri-dimensional (3D) cell aggregates or spheroids are considered to be closer to physiological conditions than traditional 2D cell culture. Mesenchymal stem cells (MSCs) assembling in spheroids have increased the survival of transplanted cells. The organization of stem cells in 3D culture affects cell microenvironment and their mechanical properties. The regulation of the biological processes that maintain crucial physiological reactions of MSCs is closely related to the functioning of ion channels. The pattern of expression, role and regulatory mechanisms of ion channels could be significantly different in 3D compared to 2D culture, and, thus, needed to be properly analyzed on the level of ionic currents. Electrophysiological data on the features of ion channels functioning in 3D cell culture models are currently very limited in the literature. This gap of knowledge may be associated with technical difficulties that exist when researchers try to apply the standard patch clamp method for the registration of ion channels in cells aggregated in spheroids. In this regard, our study focuses on solving emerging technical difficulties and presents an example of their successful solution. Here, we developed a specific approach and have recorded the activity of mechanosensitive stretch-activated ion channels (SACs) in endometrial MSCs (eMSCs) assembled in spheroids. Moreover, we observed functional interplay of SACs with potassium channels of big conductance (BK) in the plasma membrane of eMSC spheroids consistently to revealed earlier in routine 2D cultured cells. Additionally, we observed a significant decrease in the frequency of SACs activation in spheroids that may indicate the differences in the level of functional expression of channels in 3D culture comparing to 2D culture of eMSCs.

Single Artificial Ion Channels with Tunable Ion Transport Based on the Surface Modification of pH-Responsive Polymers.

Artificial ion channels with tunable ionic transport control and intelligent iontronic functions at the nanoscale have a wide application in logic computing and biosensing. Although some artificial ion channels with smart ion transport characteristics have been developed, most of them are constructed on porous membranes with undefined channel numbers. It is still challenging to achieve multiple ion transport features in single nanochannels and to control the ion flow more accurately with excellent repeatability. In this paper, a design strategy is presented to fabricate pH-responsive ion channels with various ion transport features based on a single polydimethylsiloxane (PDMS) nanochannel. The single-ion nanochannel developed by this approach can be further integrated into electronic systems on a chip. Three types of artificial ion channels are demonstrated and investigated systematically in this work. With symmetric or asymmetric pH stimuli, these ion channels can alternatively change their working states among an opened state, a closed state, and an ionic diode state. Four different ion transport features can be realized in a triple-gated ion channel system. With these advantages of the design, it is promising to build smart nanofluidic iontronic devices with widespread applicability in energy conversions, active ion transport control, and biological analysis.

DeepGANnel: Synthesis of fully annotated single molecule patch-clamp data using generative adversarial networks

Development of automated analysis tools for “single ion channel” recording is hampered by the lack of available training data. For machine learning based tools, very large training sets are necessary with sample-by-sample point labelled data (e.g., 1 sample point every 100microsecond). In an experimental context, such data are labelled with human supervision, and whilst this is feasible for simple experimental analysis, it is infeasible to generate the enormous datasets that would be necessary for a big data approach using hand crafting. In this work we aimed to develop methods to generate simulated ion channel data that is free from assumptions and prior knowledge of noise and underlying hidden Markov models. We successfully leverage generative adversarial networks (GANs) to build an end-to-end pipeline for generating an unlimited amount of labelled training data from a small, annotated ion channel “seed” record, and this needs no prior knowledge of theoretical dynamical ion channel properties. Our method utilises 2D CNNs to maintain the synchronised temporal relationship between the raw and idealised record. We demonstrate the applicability of the method with 5 different data sources and show authenticity with t-SNE and UMAP projection comparisons between real and synthetic data. The model would be easily extendable to other time series data requiring parallel labelling, such as labelled ECG signals or raw nanopore sequencing data.

The neurosciences at the Max Planck Institute for Biophysical Chemistry in Göttingen

ABSTRACT The Max Planck Institute (MPI) for Biophysical Chemistry (Karl-Friedrich Bonhoeffer Institute) was founded in 1971 in Göttingen. Two of the 11 departments at the institute had a neuroscientific focus. Otto D. Creutzfeldt (1927–1992) and Victor P. Whittaker (1919–2016) were directors of the Neurobiological and Neurochemical Departments, respectively. Creutzfeldt’s department researched the structure and function of the cerebral cortex, and Whittaker’s department concentrated on the biochemical analysis of synapses and synaptic vesicles. Creutzfeldt and Whittaker were already internationally respected scientists when they were appointed to Göttingen. The next generation of departmental directors, Erwin Neher and Bert Sakmann, were “home-grown” researchers from the institute and, during their time as junior group leaders, they developed the so-called patch clamp technique, with which they were able to measure single ion channels in nerve cells. This technique revolutionized neurophysiology, and Neher and Sakmann were awarded the 1991 Nobel Prize in Physiology or Medicine for their work in this area. Neher was appointed director of the Membrane Biophysics Department in 1983 and, since then, his department has mainly examined the role of Ca2+ in the release of neurotransmitters at synapses and in the secretion of catecholamines from chromaffin cells. From 1985, Sakmann was director of the Cell Physiology Department, and his laboratory concentrated on the molecular and physiological characterization of transmitter receptors in postsynaptic membranes. In 1989, he was appointed to the MPI for Medical Research in Heidelberg. Reinhard Jahn became director of the Neurobiology Department in 1997, researching the molecular mechanisms of the release of neurotransmitters from the presynaptic terminals, and he discovered several proteins associated with the synaptic vesicles. With their work, Neher, Sakmann, and Jahn have made the MPI for Biophysical Chemistry one of the world’s leading research centers for the transmission of signals at synapses.

Arrhythmia Assessment in Heterotypic Human Cardiac Myocyte-Fibroblast Microtissues.

Risk assessment assays for chemically induced arrhythmia are critical, but significant limitations exist with current cardiotoxicity testing, including a focus on single select ion channels, the use of non-human species in vitro and in vivo, and limited direct physiological translation. To be predictive of actual adverse clinical arrhythmic risk, arrhythmia assessment models for chemicals and drugs should be fit-for-purpose and suited for evaluating compounds in which the mechanism of action may not be entirely known. Here, we describe methods for efficient and reliable screening for arrhythmogenic cardiotoxicity with a 3D human cardiac microtissue model using purified human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and human cardiac fibroblasts. Applying optical mapping of voltage and calcium-sensitive dyes-an established approach to evaluate cardiac action potentials and calcium transients-to 3D heterotypic cardiac myocyte-fibroblast tissues allows for the generation and functional analysis of a large number of individual microtissues to provide greater throughput and high statistical power in analyses. Hundreds of microtissues in standard cell culture plates can be produced with low variability beat-to-beat, microtissue-to-microtissue, and across hiPSC-cardiomyocyte differentiation batches, reducing the number of microtissues required per condition for predictive outputs. The platform described here can be used as a sensitive, efficient, and predictive preclinical model validated for the purpose of assessing human pro-arrhythmic risk.

A Poisson-Nernst-Planck Single Ion Channel Model and its Effective Finite Element Solver

A Poisson-Nernst-Planck Single Ion Channel Model and its Effective Finite Element Solver

A chip-based array for high-resolution fluorescence characterization of free-standing horizontal lipid membranes under voltage clamp.

Optical techniques, such as fluorescence microscopy, are of great value in characterizing the structural dynamics of membranes and membrane proteins. A particular challenge is to combine high-resolution optical measurements with high-resolution voltage clamp electrical recordings providing direct information on e.g. single ion channel gating and/or membrane capacitance. Here, we report on a novel chip-based array device which facilitates optical access with water or oil-immersion objectives of high numerical aperture to horizontal free-standing lipid membranes while controlling membrane voltage and recording currents using individual micropatterned Ag/AgCl-electrodes. Wide-field and confocal imaging, as well as time-resolved single photon counting on free-standing membranes spanning sub-nanoliter cavities are demonstrated while electrical signals, including single channel activity, are simultaneously acquired. This optically addressable microelectrode cavity array will allow combined electrical-optical studies of membranes and membrane proteins to be performed as a routine experiment.

Tosoh reports on its consolidated results for 1H FY 2022: Speciality Group

Depletion of intracellular calcium ion stores initiates a rapid cascade of events culminating with the activation of the so-called Store-Operated Channels (SOC) at the plasma membrane. Calcium influx via SOC is essential in the initiation of calcium-dependent intracellular signaling and for the refilling of internal calcium stores, ensuring the regeneration of the signaling cascade. In spite of the significance of this evolutionary conserved mechanism, the molecular identity of SOC has been the center of a heated controversy spanning over the last 20 years. Initial studies positioned some members of the transient receptor potential canonical (TRPC) channel superfamily of channels (with the more robust evidence pointing to TRPC1) as a putative SOC. Recent evidence indicates that Stromal Interacting Molecule 1 (STIM1) activates some members from the TRPC family of channels. However, the exact subunit composition of TRPC channels remains undetermined to this date. To identify the subunit composition of STIM1-activated TRPC channels, we developed novel method, which combines single channel electrophysiological measurements based on the patch clamp technique with single molecule fluorescence imaging. We termed this method Single ion Channel Single Molecule Detection technique (SC-SMD). Using SC-SMD method, we have obtained direct evidence of the subunit composition of TRPC channels activated by STIM1. Furthermore, our electrophysiological-imaging SC-SMD method provides evidence at the molecular level of the mechanism by which STIM1 and calmodulin antagonize to modulate TRPC channel activity.

Toward a More General Understanding of Bohr's Complementarity: Insights from Modeling of Ion Channels.

Some contemporary theorists such as Mazzocchi, Theise and Kafatos are convinced that the reformed complementarity may redefine how we might exploit the complexity theory in 21st-century life sciences research. However, the motives behind the profound re-invention of "biological complementarity" need to be substantiated with concrete shreds of evidence about this principle's applicability in real-life science experimentation, which we found missing in the literature. This paper discusses such pieces of evidence by confronting Bohr's complementarity and ion channel modeling practice. We examine whether and to what extent this principle might assist in developing ion channel models incorporating both deterministic and stochastic solutions. According to the "mutual exclusiveness of experimental setups" version of Bohr's complementarity, this principle is needed when two mutually exclusive perspectives or approaches are right, necessary in a particular context, and are not contradictory as they arise in mutually exclusive conditions (mutually exclusive experimental or modeling setups).A detailed examination of the modeling practice reveals that both solutions are often used simultaneously in a single ion channel model, suggesting that the opposite conceptual frameworks can coexist in the same modeling setup. We concluded that Bohr's complementarity might find applications in these complex modeling setups but only through its realistic phenomenological interpretation that allows applying different modes of description regardless of the nature of the underlying ion channel opening process. Also, we propose the combined use of complementarity and Complex thinking in building the multifaceted ion channel models. Overall, this paper's results support the efforts to establish a more general form of complementarity to meet today's complexity theory-inspired life sciences modeling demands.

Anion-Responsive Manganese Porphyrin Facilitates Chloride Transport and Induces Immunogenic Cell Death

Anion-Responsive Manganese Porphyrin Facilitates Chloride Transport and Induces Immunogenic Cell Death