Changes In Ionic Currents Research Articles

Engineering a ClyA nanopore for monitoring enzymatic reactions at the single molecule level

Using electric field to load and hold individual intact proteins within a nanopore makes it possible to monitor biochemical reactions in real time and at a single molecule level. Such monitoring, however, requires the conformational transition induced by the chemical reaction in the protein to be large enough to alter the nanopore ionic current. Previous experiments reported small yet detectable ionic current changes when a folded protein (substrate binding domain, SBD) was trapped inside a ClyA nanopore and exposed to glutamate, a small molecule known to produce conformational transition in SBD.

Discerning Tyrosine Phosphorylation from Multiple Phosphorylations Using a Nanofluidic Logic Platform.

Discerning tyrosine phosphorylation (pTyr) catalyzed by Tyr kinase is central to the revelation of oncogenic mechanisms and the development of targeted anticancer drugs. Despite some techniques, this goal remains challenging, especially when faced with the interference of multiple phosphorylation events, including serine (pSer) and threonine phosphorylation (pThr). We describe here a functional polymer-modified artificial ion nanochannel, which enables the sensitive and selective recognition of phosphotyrosine (pY) peptide by the distinct ionic current change. Such a recognition effect allows for the nanochannel to work in a complex protein digest condition. Further, the implementation of nanofluidic logic functions with the addition of Ca2+ dramatically improves the selectivity of the nanochannel to pY peptide and thus can discern pTyr by the Tyr kinase from pSer by the Ser/Thr kinase through simultaneously monitoring multisite phosphorylation at the same or different peptide substrates in one-pot. This logic sensing platform displays the potential in differentiating Tyr kinase and Ser/Thr kinase and assessing multi-kinase activities in multi-targeted drug design.

Single-Walled Carbon Nanotube-Based Field-Effect Transistors Functionalized with Odorant Receptors for Biosensing Applications

Bioelectronic detection with functionalized carbon nanotube transistor devices, known as CNT-FET, offers signal levels comparable with voltage clamp, multiplexing potential similar to microarray and amenability for miniaturization. The feasibility of CNT-FET biosensors was demonstrated in the detection of nucleic acids and various protein biomarkers and they are considered the next generation bioelectronic-based biosensors. Detection of volatiles and specifically volatile organic compounds (VOC) with CNT-FETs is less common, let alone devices that were biofunctionalized for specificity. The detection of VOCs has been demonstrated in a wide range of applications, with early reports focusing on environmental exogenous VOCs due to their adverse effects on human health; bacterial VOC signatures used as disease biomarkers, etc.. Methods that rely on separation techniques coupled with mass spectrometry are considered the ’gold standard’ in this field. These methods require expensive equipment, labor-intensive preparation steps and trained personnel, and are not suitable for field use. A method enabling high-throughput screening of VOCs such as insect behavior-modifying drugs, disease biomarkers, etc. is highly desirable.Bioelectronic sensors are ideally suited for the detection of pathogenic volatile markers (VOC). In order to confer ultrahigh specificity on an electronic sensor, a bio-recognition element needs to be incorporated with the transducer. In the case of VOC sensing, one such promising element is the natural insect odorant receptor (OR). Insect OR exhibit remarkable sensitivities and the ability to selectively detect a vast number of VOCs. These heteromeric transmembrane proteins transduce signals by acting as ligand-gated ion channels. We have recently developed OR-functionalized carbon nanotube field-effect transistor (FET) devices. These FET devices integrate an isolated single-walled carbon nanotube (SWCNT) as the conducting channel material.Indole and skatoles are microbial volatile organic compounds (VOCs). Depending on their concentrations, these odorants indicate environmental contaminants, metabolic disorders, food spoilage, human presence and malodorants.The indolergic receptor (IndolOR) named OR9 is the most sensitive odorant receptor discovered so far. We have developed a bioelectronic assay platform in the form of OR9-functionalized CNT-FET arrays. Our unique functionalization method is based on directing the attachment of a native OR9-containing nanovesicles (which we have exogenously expressed and functionally characterized) to the CNT-FET point defect. Target binding-induced ionic current and conformational changes of OR9 affect an electric field that modulates the device conductance. Figure 1

Deficiency of CXXC finger protein 1 leads to small changes in heart rate but moderate epigenetic alterations and significant protein downregulation of hyperpolarization-activated cyclic nucleotide-gated 4 (HCN4) ion channels in mice.

The normal cardiac rhythm is generated in the sinoatrial node (SAN). Changes in ionic currents of the SAN may cause sinus arrhythmia. CXXC finger protein 1 (Cfp1) is an epigenetic regulator that is involved in transcriptional regulation of multiple genes. The purpose of this study was to explore whether Cfp1 controls SAN function through regulation of ion channel-related genes. Electrophysiological study, patch clamp recording, reverse transcriptase polymerase chain reaction, optical mapping, chromatin immunoprecipitation, and immunofluorescence staining were performed to evaluate the function of SAN and underlying mechanism on Cfp1 heterozygous knockout (Cfp1+/-) mice. Heart rate was slower slightly and SAN recovery time was longer in Cfp1+/- mice than controls. Whole-cell patch-clamp recording showed that the firing rate of action potentials was reduced in Cfp1+/- mice. The density of If current was reduced by 66% in SAN cells of Cfp1+/- mice but the densities of ICa, ICa-L, and ICa-T were not changed. The hyperpolarization-activated cyclic nucleotide-gated 4 (HCN4) mRNA level in SAN tissue of Cfp1+/- mice was reduced. The HCN4 protein was significantly decreased in SAN cells and tissues after heterozygous deletion of Cfp1. Chromatin immunoprecipitation assay on cultured HL-1 cells demonstrated that Cfp1 was enriched in the promoter regions of HCN4. Knockdown of Cfp1 reduced H3K4 trimethylation, H3K9 acetylation, and H3K27 acetylation of HCN4 promoter region. Deficiency of Cfp1 leads to small changes in heart rate by moderate epigenetic modification alterations and significant protein downregulation of HCN4 ion channels in mice.

High sensitivity of deoxyribonucleic acid detection via graphene nanohole/silicon micro-nanopore structure fabricated by focused ion beam

Detection via nanohole is a simple sensing tool to analyze various biopolymers with high sensitivity. This paper reports the detection of deoxyribonucleic acid (DNA) biomolecules using an integration of 30 nm circular-shaped graphene nanohole and conical-shaped silicon (Si) micro-nanopore fabricated by focused ion beam technique. The ionic current changes at different DNA volume measured via a single and an array of nanoholes seem to confirm the ionic current blockade phenomena where the current decreases in stable linearity with the increase of volume. Regardless of the number of nanoholes, the sensitivity of around 110 nA/µL for both devices shows relatively much higher value as compared to the sensitivity reported in the other literatures. Such significant ionic current blockade detection suggests that a proposed combination of conical-shaped Si micro-nanopore and graphene nanohole are able to act as a promising fluidic channel and sensing membrane, respectively, towards a realization of high sensitive DNA biosensors.

Comodulation of h- and Na+/K+ Pump Currents Expands the Range of Functional Bursting in a Central Pattern Generator by Navigating between Dysfunctional Regimes.

Central pattern generators (CPGs), specialized oscillatory neuronal networks controlling rhythmic motor behaviors such as breathing and locomotion, must adjust their patterns of activity to a variable environment and changing behavioral goals. Neuromodulation adjusts these patterns by orchestrating changes in multiple ionic currents. In the medicinal leech, the endogenous neuromodulator myomodulin speeds up the heartbeat CPG by reducing the electrogenic Na+/K+ pump current and increasing h-current in pairs of mutually inhibitory leech heart interneurons (HNs), which form half-center oscillators (HN HCOs). Here we investigate whether the comodulation of two currents could have advantages over a single current in the control of functional bursting patterns of a CPG. We use a conductance-based biophysical model of an HN HCO to explain the experimental effects of myomodulin. We demonstrate that, in the model, comodulation of the Na+/K+ pump current and h-current expands the range of functional bursting activity by avoiding transitions into nonfunctional regimes, such as asymmetric bursting and plateau-containing seizure-like activity. We validate the model by finding parameters that reproduce temporal bursting characteristics matching experimental recordings from HN HCOs under control, three different myomodulin concentrations, and Cs+ treated conditions. The matching cases are located along the border of an asymmetric regime away from the border with more dangerous seizure-like activity. We found a simple comodulation mechanism with an inverse relation between the pump and h-currents makes a good fit of the matching cases and comprises a general mechanism for the robust and flexible control of oscillatory neuronal networks.SIGNIFICANCE STATEMENT Rhythm-generating neuronal circuits adjust their oscillatory patterns to accommodate a changing environment through neuromodulation. In different species, chemical messengers participating in such processes may target two or more membrane currents. In medicinal leeches, the neuromodulator myomodulin speeds up the heartbeat central pattern generator by reducing Na+/K+ pump current and increasing h-current. In a computational model, we show that this comodulation expands the range of central pattern generator's functional activity by navigating the circuit between dysfunctional regimes resulting in a much wider range of cycle period. This control would not be attainable by modulating only one current, emphasizing the synergy of combined effects. Given the prevalence of h-current and Na+/K+ pump current in neurons, similar comodulation mechanisms may exist across species.

Post myocardial infarction ionic remodelling promotes repolarisation dispersions and abnormalities in acute and chronic stages

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Wellcome Trust Background Sudden cardiac death after a myocardial infarction (MI) was reported in both the acute stage and the chronic stage with the development of heart failure (HF). Around 50% of these deaths were due to ventricular tachyarrhythmia which is related to the temporal dispersion of repolarisation caused by the post-MI remodelling of ionic currents. While variable extent of ionic current changes was observed in multiple animal species in different post-MI phases, the proarrhythmic effects of such remodelling in post infarction human hearts is not fully explored. Purpose The goal of this study is to explore the proarrhythmic effects of post-MI ionic remodelling in human ventricular myocyte models of electrophysiology for both the acute and the chronic stages. Methods The ToR-ORd human ventricular cellular model was used as the baseline model, and a new formulation of the calcium activated potassium current was implemented. We constructed and calibrated a population of 253 human ventricular cell models (POM) to represent electrophysiological variability of normal zone (NZ) myocytes. For the acute phase, three types of border zone (BZ) remodelling (Acute BZ1-3) were introduced based on dog experimental data collected within one week after infarction. For the chronic phase with the development of HF, BZ (Chronic BZ) and one type of remote zone (RZ) remodelling (Chronic RZ1) were introduced based on minipig data collected 5 months post-MI. Another type of RZ remodelling (Chronic RZ2) was introduced based on experimental data from failing human hearts. The cellular models were paced at cycle lengths of 1000ms, 500ms, 400ms and 300ms for 500 beats before the analysis of repolarization abnormalities. Results For both the acute and the chronic stages post-MI, the extent of repolarisation dispersion between BZ and the corresponding NZ and RZ varied from 2ms up to 120ms, and largely depended on the level of potassium current remodelling. A weaker calcium transient was always observed in the BZ. Early after depolarisations (EADs) and repolarisation failure (RF) were observed in the chronic stages, caused by the reactivation of L-type calcium current due to the augmentation of late sodium current and the inhibition of hERG current. Beat-to-beat repolarization alternans was observed in both the acute and the chronic stages, with the key ionic mechanisms being the inhibition of SERCA activity and the slower calcium release due to enhanced CaMKII. Conclusions Post-MI ionic remodelling of the acute and chronic stages can cause a substantial dispersion of repolarisation in human hearts by inducing the prolongation of action potential durations, EADs, RFs, and alternans, providing substrates for the generation and maintenance of lethal reentrant waves. Abstract Figure. BZ and RZ ionic current remodelling

Recent advances in ionic current rectification based nanopore sensing: a mini-review

In recent years, the intensive studies have been devoted to nanopores due to their unique charge transport properties including ion permselectivity, ion concentration polarization, and ionic current rectification (ICR). Based on ICR, the analytical applications of nanopore can be realized by monitoring ionic current change when the analyte molecule recognition events occur inside nanopore. To better understand ICR based nanopore sensing, its recent advances are reviewed and categorized in two main sensing strategies including nanopore inner surface charge variation and nanopore inner molecular conformation change. Moreover, the nanopore regeneration strategies are also reviewed. The future challenges and opportunities in ICR based nanopore sensing are further discussed. This review is expected to expedite the research and development of nanopore analytical applications.

Direct and Accurate Detection of DNA Methylation Positions using Biological Nanopore

We describe a method of detecting 5-methylcytosine (5mC) positions using nanopore measurement. The determination of 5mC in the genome is promising to be used as epigenetic detection. In the general method of the 5mC determination, bisulfite conversion, it requires multiple procedures and the incomplete conversion sometimes occurs to reduce the reliability of the epigenetic analysis. In the previous study, Wang et al. detected the 5mC existence using nanopore measurement with tetramethylammonium salt1. However, this method is hard to detect the position of 5mC. Here, we report a simple method of determining a 5mC position using α-hemolysin (αHL) nanopore without bisulfite conversion, chemical modification, or enzyme amplification. αHL reconstituted in the lipid bilayers made a nanopore for capturing the target molecules under an applied voltage. A probe DNA hybridizes to a DNA which has a 5mC, and then the complementary DNA is captured and passed through in the nanopore with unzipping strands electrophoretically. The αHL-filling electrolyte record the ion current change in a single-molecular level triggered by unzipping the double-stranded (dsDNA). To determine the 5mC-position information, we analyzed the unzipping current data including the unzipping time and blocking ratio using the bootstrap method2. The bootstrap method is based on the resampling of the original random sample drawn from a population. The bootstrapped data offered the estimation of the unknown distribution, which was shown by plotting the blocking ratio against unzipping time. As a result, the scatter plot graph showed the possibility to distinguish between the 5mC positions at the single nucleotide level using nanopore sensing technology. Our method offers a simple and rapid tool for a 5mC-position determination as a nanopore epigenetic detection. Reference 1. Wang, Y. et al., Scientific Reports (2017). 2. Liu, P. et al., Small Methods (2020).

Ultra-Sensitive and Selective Electrochemical Bio-Fluid Biopsy for Oral Cancer Screening.

The early diagnosis of recurrence and metastasis is critically important for decreasing the morbidity and mortality associated with oral cancers. Although liquid biopsy methods hold great promise that provide a successive "time-slice" profile of primary and metastatic oral cancer, the development of non-invasive, rapid, simple, and cost-effective liquid biopsy techniques remains challenging. In this study, an ultrasensitive and selective electrochemical liquid biopsy is developed for oral cancer screening based on tracking trace amounts of cancer biomarker by functionalized asymmetric nano-channels. Detection via antigen-antibody reactions is assayed by evaluating changes in ionic current. Upon the recognition of cancer biomarker antigens in bio-fluids, the inner wall of nano-channel immobilized with the corresponding antibodies undergoes molecular conformation transformation and surface physicochemical changes, which significantly regulate the ion transport through the nano-channel and help achieve sensitivity with a detection limit of 10-12 g mL-1 . Furthermore, owing to the specificity of the monoclonal antibody for the antigen, the nano-channel exhibits high selectivity for the biomarker than for structurally similar biological molecules present in bio-fluids. The effectiveness of this technique is confirmed through the diagnosis of clinical cases of oral squamous cell carcinoma. This study presents a novel diagnostic tool for oral cancer detection in bio-fluids.

Interfacial‐Potential‐Gradient Induced a Significant Enhancement of Photoelectric Conversion: Thiophene Polyelectrolyte (PTE‐BS) and Bipyridine Ruthenium (N3) Cooperative Regulated Biomimetic Nanochannels

AbstractInspired by photosynthesis, the ion transport‐based artificial light harvesting system shows unprecedented superiority in photoelectric conversion. However, how to high‐efficiently utilize solar energy, just like photosystem I and photosystem II working together in the thylakoid membrane, remains a great challenge. Here, a facile strategy for patterning two photosensitive molecules is demonstrated, that is, thiophene polyelectrolyte (PTE‐BS) and bipyridine ruthenium (N3), onto the two segments of symmetric/asymmetric hourglass‐shaped alumina nanochannels. Owing to the different energy levels, an interfacial‐potential‐gradient is established in the tip junction of the nanochannels, wherein photoinduced excited electrons transfer from PTE‐BS to N3, resulting in the efficient separation of electron–holes. Simultaneously, the increasing surface‐charge‐density enhances transmembrane ion transport performance. Thus, the photo‐induced ionic current change ratio increases up to 100% to realize a significant photoelectric conversion, which is superior to all of the N3 or PTE‐BS individually modified nanochannel systems. By changing the channel geometry from symmetric to asymmetric, the biomimetic nanochannels also exhibit a diode‐like ion transport behavior. This work may provide guidance for the development of high‐performance photoelectric conversion nanochannel systems.

Mechanisms underlying pro-arrhythmic abnormalities arising from Pitx2-induced electrical remodelling: an in silico intersubject variability study.

BackgroundElectrical remodelling as a result of the homeodomain transcription factor 2 (Pitx2)-dependent gene regulation induces atrial fibrillation (AF) with different mechanisms. The purpose of this study was to identify Pitx2-induced changes in ionic currents that cause action potential (AP) shortening and lead to triggered activity.MethodsPopulations of computational atrial AP models were developed based on AP recordings from sinus rhythm (SR) and AF patients. Models in the AF population were divided into triggered and untriggered AP groups to evaluate the relationship between each ion current regulated by Pitx2 and triggered APs. Untriggered AP models were then divided into shortened and unshortened AP groups to determine which Pitx2-dependent ion currents contribute to AP shortening.ResultsAccording to the physiological range of AP biomarkers measured experimentally, populations of 2,885 SR and 4,781 AF models out of the initial pool of 30,000 models were selected. Models in the AF population predicted AP shortening and triggered activity observed in experiments in Pitx2-induced remodelling conditions. The AF models included 925 triggered AP models, 1,412 shortened AP models and 2,444 unshortened AP models. Intersubject variability in IKs and ICaL primarily modulated variability in AP duration (APD) in all shortened and unshortened AP models, whereas intersubject variability in IK1 and SERCA mainly contributed to the variability in AP morphology in all triggered and untriggered AP models. The incidence of shortened AP was positively correlated with IKs and IK1 and was negatively correlated with INa, ICaL and SERCA, whereas the incidence of triggered AP was negatively correlated with IKs and IK1 and was positively correlated with INa, ICaL and SERCA.ConclusionsElectrical remodelling due to Pitx2 upregulation may increase the incidence of shortened AP, whereas electrical remodelling arising from Pitx2 downregulation may favor to the genesis of triggered AP.

Gauging surface charge distribution of live cell membrane by ionic current change using scanning ion conductance microscopy.

The distribution of surface charge and potential of cell membrane plays an indispensable role in cellular activities. However, probing surface charge of live cells under physiological conditions, until recently, remains an arduous challenge owing to the lack of effective methods. Scanning ion conductance microscopy (SICM) is an emerging imaging technique for imaging a live cell membrane in its native state. Here, we introduce a simple SICM based imaging technique to effectively map the surface charge contrast distribution of soft substrates including cell membranes by utilizing the higher surface charge sensitivity of the ionic current when the nanopipette tip is close to the substrate with a relatively high current change. This technique was assessed on charged model substrates made of polydimethylsiloxane, and the surface charge sensitivity of ionic current change was supported by finite element method simulations. With this method, we can distinguish the surface charge difference between the cell membrane and the supporting collagen matrix. We also observed the surface charge change induced by the small membrane damage after 1% dimethyl sulfoxide (DMSO) treatment. This new SICM technique provides opportunities to study interfacial and cell membrane processes with high spatial resolution.

Micrometer-scale transient ion transport for real-time pH assay in living rat brains.

Ion transport has been widely used for various applications such as sensing, desalination and energy conversion; however, nearly all applications are based on steady-state ion transport. Herein, we for the first time demonstrate the capability of transient ion transport for in vivo sensing with both high spatial (∼μm) and temporal (∼ms) resolution by using pH as the model target. Transient ion transport behavior (i.e., time-dependent ion current change) was observed by applying high-frequency pulse potential. Importantly, we proposed the ion distribution transient model for this time-dependent ion transport behavior. With this model, the temporal resolution of the as-developed pH microsensor based on ion current was improved to the ms level, thus satisfying the requirement of neurochemical recording. Moreover, our microsensor features good reproducibility, selectivity, and reversibility, and can thus real-time monitor the pH change in living rat brains. This study demonstrates the first example of in vivo sensing based on ion transport, opening a new way to neurochemical monitoring with ultrahigh spatiotemporal resolution. This study is also helpful to understand the transient process of asymmetric ion transport.

Coordination of Ligand-Protected Metal Nanoclusters and Glass Nanopipettes: Conversion of a Liquid-Phase Fluorometric Assay into an Enhanced Nanopore Analysis.

We propose a unique concept for transforming the liquid-phase fluorometric assay into an enhanced nanopore analysis, which is based on the analyte binding-mediated changes in the surface chemistry of noble metal nanostructures in a confined space. In a proof-of-concept trial, the bovine serum albumin-protected gold nanoclusters (BSA-Au NCs) were designed as the sensing unit for biothiol determination. Through the specific interaction between biothiols and BSA-Au NCs, the validation system not only performed well in aqueous fluorescent detection but also can be developed into a more selective and sensitive nanopore sensor. In the confined space of the nanopore, the BSA-Au NC film with high density formed, and the addition of biothiols triggered the fluorescence enhancement as well as the ionic current response, hence leading to the construction of the dual-signal-output (fluorescence/ion current signal) system. The fluorescence signal proved that the ionic current change corresponded to the specific recognition process, improving the reliability of our nanopore method. Moreover, the ionic current response from the BSA-Au NC film can be used to quantify cysteine in a broad dynamic range of 0.001-1 pM with a detection limit as low as 1 fM. Such a strategy can be used to detect biothiols in complex biological fluids such as human serum. Therefore, the present work provided a new design strategy for a glass nanopipette sensor inspired by the principles of numerous and diverse fluorometric assays. It also sheds light on how the coupling of electrical and optical signals improves the accuracy, sensitivity, and selectivity of the glass nanopipette platform.

Lysenin Channels as Sensors for Ions and Molecules.

Lysenin is a pore-forming protein extracted from the earthworm Eisenia fetida, which inserts large conductance pores in artificial and natural lipid membranes containing sphingomyelin. Its cytolytic and hemolytic activity is rather indicative of a pore-forming toxin; however, lysenin channels present intricate regulatory features manifested as a reduction in conductance upon exposure to multivalent ions. Lysenin pores also present a large unobstructed channel, which enables the translocation of analytes, such as short DNA and peptide molecules, driven by electrochemical gradients. These important features of lysenin channels provide opportunities for using them as sensors for a large variety of applications. In this respect, this literature review is focused on investigations aimed at the potential use of lysenin channels as analytical tools. The described explorations include interactions with multivalent inorganic and organic cations, analyses on the reversibility of such interactions, insights into the regulation mechanisms of lysenin channels, interactions with purines, stochastic sensing of peptides and DNA molecules, and evidence of molecular translocation. Lysenin channels present themselves as versatile sensing platforms that exploit either intrinsic regulatory features or the changes in ionic currents elicited when molecules thread the conducting pathway, which may be further developed into analytical tools of high specificity and sensitivity or exploited for other scientific biotechnological applications.

Genetic basis and molecular biology of cardiac arrhythmias in cardiomyopathies.

Cardiac arrhythmias are common, often the first, and sometimes the life-threatening manifestations of hereditary cardiomyopathies. Pathogenic variants in several genes known to cause hereditary cardiac arrhythmias have also been identified in the sporadic cases and small families with cardiomyopathies. These findings suggest a shared genetic aetiology of a subset of hereditary cardiomyopathies and cardiac arrhythmias. The concept of a shared genetic aetiology is in accord with the complex and exquisite interplays that exist between the ion currents and cardiac mechanical function. However, neither the causal role of cardiac arrhythmias genes in cardiomyopathies is well established nor the causal role of cardiomyopathy genes in arrhythmias. On the contrary, secondary changes in ion currents, such as post-translational modifications, are common and contributors to the pathogenesis of arrhythmias in cardiomyopathies through altering biophysical and functional properties of the ion channels. Moreover, structural changes, such as cardiac hypertrophy, dilatation, and fibrosis provide a pro-arrhythmic substrate in hereditary cardiomyopathies. Genetic basis and molecular biology of cardiac arrhythmias in hereditary cardiomyopathies are discussed.

Co‐modulation of the Na+/K+ pump and hyperpolarization‐activated currents as a mechanism for robust neuromodulation

Specialized oscillatory circuits, central pattern generators (CPGs), control rhythmic motor behaviors such as locomotion and breathing. To accommodate a motor pattern to environmental changes and behavioral goals, neuromodulators adjust the dynamics of CPGs by orchestrating changes in various ionic currents in a wide range of their biophysical parameters to expand temporal characteristics of the functional pattern. Recent studies provide evidence that the Na+/K+ pump contributes to the dynamics of CPGs and is controlled by neuromodulation [1– 4]. In the leech heartbeat CPG, the neuropeptide myomodulin reduces the period of bursting activity by increasing the hyperpolarization‐activated (h)‐current and decreasing the Na+/K+ pump current [4]. The application of myomodulin reduces the period of oscillatory activity by 17%. Application of Cs+, which is an h‐current blocker, increases the period of bursting by 24% relative to control. The application of myomodulin along with Cs+ decreases the period by 12% relative to treatment with Cs+ [4]. Here we investigate how the period of a bursting can be controlled in a wide range while functional bursting is maintained with a focus on the role of the Na+/K+ pump. We also investigate the risk associated with multistability in neuronal dynamics.We optimized a model of the leech heart interneuron (HN), which includes the Na+/K+ pump current and intracellular Na+ dynamics [1] and investigated the activity regimes of pairs of mutually inhibitory coupled HNs forming half‐center oscillators (HCOs). HCOs form the kernel of the leech heartbeat CPG. We investigated eight model variants representing combinations of three experimental treatments: the blockade of chemical synapses representing the application of bicuculline, the blockade of h‐current representing the application of Cs+, and the enhancement of the h‐current and inhibition of the Na+/K+ pump current representing the application of myomodulin. The model captures the qualitative trends in change of cycle period observed in experiments with myomodulin and Cs+. We found ranges of parameters where neurons showed functional bursting. The coordinated changes of maximal conductance of h‐current (Gh) and maximal pump activity (IPumpMax) increases the range of period of functional bursting as well as the range of parameters Gh and IPumpMax. We hypothesize that myomodulin co‐modulates h‐ and pump currents to expand the domain of the functional activity.Support or Funding InformationSupported by NINDS 1 R01 NS085006 to RLC and 1 R21 NS111355 to RLC and GSC.

Influence of humidity on conduction processes in gas‐insulated devices

Humidity has been considered as one of the main influencing factors that determine the conduction processes and electric strength of gas-insulated systems. Whereas in the past, various studies focused on the change in the partial discharge inception voltages, breakdown strength of homogeneous and inhomogeneous field arrangements, and insulator flashover voltage, recent studies have investigated the changes in ion currents measured through different gas gaps. In the framework of this contribution, a highly precise humidity control circuit has been developed to analyse the significance of humidity in the range from −25 to −5°C frost-point, which is fully applicable to operating gas-insulated devices. Using sulphur hexafluoride (SF6) as the insulation gas at 0.45 MPa, Al2O3-filled epoxy resin insulators, and technically rough electrodes, the humidity was found to significantly influence the intensity of microdischarges at interfaces. Charge generation from microdischarges at the interfaces substantially increased with increasing humidity. For an electric field of 5 kV/mm that were applicable to the dimensioning of gas-insulated devices, humidity strongly influences the charge provision from technically rough interfaces and potentially contributes to the surface-charge accumulation at insulator surfaces. On the other hand, for low-field conduction phenomena, no increase in the ion currents from natural ionisation or electrophoretic conduction was observed. For the investigated range of parameters, humidity is expected to be highly relevant for the design of gas-insulated devices.

Electrophysiological Examination of the Effects of Classical Pharmacological Agent Verapamil in Plant Based Model System

Homologs of human two pore channels (TPC) were documented in plants thus the examination of the effects of medicines against TPC-related diseases could be performed in plant model systems. Examination of electrical signalling properties - generation of Action Potentials (AP) - in Characeaen macroalgae Nitellopsis obtusa is an established method offering insights into effects of ion channel modulators in vivo. Standardized current clamp and voltage clamp approaches have been used for this purpose for decades. Verapamil is a well-known L-type Ca2+ channel blocker, also it was demonstrated to inhibit Kv channels in a variety of tissues and cells. Investigation of changes in ion currents during AP generation after verapamil treatment was carried out by the application of the C-C, V-C and patch clamp techniques in Nitellopsis obtusa model system. High discretization frequency allowed high temporal resolution analysis of real-time processes such as changes in excitation threshold, AP amplitude and velocity of repolarization phase along with activation/inactivation dynamics of ion channels. These parameters reflected modulation of activity of ion channels involved in AP generation. The effect of verapamil was found to be concentration dependent and manifested as a depolarization of membrane resting potential and excitation threshold, also as an increase of AP duration as well as a prolongation of inactivation times of transient currents during excitation. Our investigation demonstrate direct and rapid effect of verapamil on electrical signalling in plants on single cell level. The research was funded by DAINA – Polish-Lithuanian joint project S-LL-18-1.