Manual Patch Research Articles

Fluorescence-Based HTS Assays for Ion Channel Modulation in Drug Discovery Pipelines.

Ion channels represent a druggable family of transmembrane pore-forming proteins with important (patho)physiological functions. While electrophysiological measurement (manual patch clamp) remains the only direct method for detection of ion currents, it is a labor-intensive technique. Although automated patch clamp instruments have become available to date, their high costs limit their use to large pharma companies or commercial screening facilities. Therefore, fluorescence-based assays are particularly important for initial screening of compound libraries. Despite their numerous disadvantages, they are highly amenable to high-throughput screening and in many cases, no sophisticated instrumentation or materials are required. These features predispose them for implementation in early phases of drug discovery pipelines (hit identification), even in an academic environment. This review summarizes the advantages and pitfalls of individual methodological approaches for identification of ion channel modulators employing fluorescent probes (i. e., membrane potential and ion flux assays) with emphasis on practical aspects of their adaptation to high-throughput format.

Digital sand patch: Using laser scanning and discrete element simulation for rapider pavement texture depth measurement

The sand patch method is one of the most basic methods for measuring pavement texture depth (TD), which is an important reference index for pavement anti-skid performance evaluation and road maintenance. However, manual sand patch method heavily relies on the experience of the operators. The emergence of laser 3D scanning and simulation has made it possible to conduct digital sand patch. This study introduces a digital sand patch framework based on 3D scanning and discrete element simulation for precise TD measurement. We developed a 3D scanner with an accuracy of 0.04 mm to obtain the pavement texture data, along with algorithms for outlier removal and slope correction in point cloud data. The method of 3D reconstruction of triangular meshes was adopted to transform discrete point clouds into continuous 3D models to build 3D pavement models. In addition, the virtual process of rotation-spreading-measurement of the digital sand patch method was constructed by discrete element simulation with reference to the physical principles of the manual sand patch method. At the same time, an adaptive dynamic feedback control logic was proposed to accurately determine the termination conditions of the sand patching process. To validate our method, we collected 126 samples from over 20 km of pavement surfaces with various gradations. The results demonstrated an average relative error of 2.92 % compared to the manual sand patch method, and a correlation coefficient of 0.9926 with the laser scanning Mean Profile Depth (MPD) algorithm. Stability and accuracy tests, including scanning downsampling and volatility assessments, confirmed the robustness of our method. This innovative technique for rapid and precise TD measurement not only offers immediate practical benefits but also sets the stage for future research on pavement skid resistance.

Single-cell ionic current phenotyping elucidates non-canonical features and predictive potential of cardiomyocytes during automated drug experiments.

All new drugs must go through preclinical screening tests to determine their proarrhythmic potential. While these assays effectively filter out dangerous drugs, they are too conservative, often misclassifying safe compounds as proarrhythmic. In this study, we attempt to address this shortcoming with a novel, medium-throughput drug-screening approach: we use an automated patch-clamp system to acquire optimized voltage clamp (VC) and action potential (AP) data from human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) at several drug concentrations (baseline, 3×, 10× and 20× the effective free plasma concentrations). With our novel method, we show correlations between INa block and upstroke slowing after treatment with flecainide or quinine. Additionally, after quinine treatment, we identify significant reductions in current during voltage steps designed to isolate If and IKs. However, we do not detect any IKr block by either drug, and upon further investigation, do not see any IKr present in the iPSC-CMs when prepared for automated patch experiments (i.e. in suspension) - this is in contrast to similar experiments we have conducted with these cells using the manual patch setup. In this study, we: (1) present a proof-of-concept demonstration of a single-cell medium-throughput drug study, and (2) characterize the non-canonical electrophysiology of iPSC-CMs when prepared for experiments in a medium-throughput setting. KEY POINTS: Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) offer potential as an in vitro model to study the proarrhythmic potential of drugs, but insights from these cells are often limited by the low throughput of manual patch-clamp. In this study, we use a medium-throughput automated patch-clamp system to acquire action potential (AP) and complex voltage clamp (VC) data from single iPSC-CMs at multiple drug concentrations. A correlation between AP upstroke and INa transients was identified and drug-induced changes in ionic currents found. We also characterize the substantially altered physiology of iPSC-CMs when patched in an automated system, suggesting the need to investigate differences between manual and automated patch experiments.

How to differentiate induced pluripotent stem cells into sensory neurons for disease modelling: a functional assessment.

Human induced pluripotent stem cell (iPSC)-derived peripheral sensory neurons present a valuable tool to model human diseases and are a source for applications in drug discovery and regenerative medicine. Clinically, peripheral sensory neuropathies can result in maladies ranging from a complete loss of pain to severe painful neuropathic disorders. Sensory neurons are located in the dorsal root ganglion and are comprised of functionally diverse neuronal types. Low efficiency, reproducibility concerns, variations arising due to genetic factors and time needed to generate functionally mature neuronal populations from iPSCs remain key challenges to study human nociception in vitro. Here, we report a detailed functional characterization of iPSC-derived sensory neurons with an accelerated differentiation protocol ("Anatomic" protocol) compared to the most commonly used small molecule approach ("Chambers" protocol). Anatomic's commercially available RealDRG™ were further characterized for both functional and expression phenotyping of key nociceptor markers. Multiple iPSC clones derived from different reprogramming methods, genetics, age, and somatic cell sources were used to generate sensory neurons. Manual patch clamp was used to functionally characterize both control and patient-derived neurons. High throughput techniques were further used to demonstrate that RealDRGs™ derived from the Anatomic protocol are amenable to high throughput technologies for disease modelling. The Anatomic protocol rendered a purer culture without the use of mitomycin C to suppress non-neuronal outgrowth, while Chambers differentiations yielded a mix of cell types. Chambers protocol results in predominantly tonic firing when compared to Anatomic protocol. Patient-derived nociceptors displayed higher frequency firing compared to control subject with both, Chambers and Anatomic differentiation approaches, underlining their potential use for clinical phenotyping as a disease-in-a-dish model. RealDRG™ sensory neurons show heterogeneity of nociceptive markers indicating that the cells may be useful as a humanized model system for translational studies. We validated the efficiency of two differentiation protocols and their potential application for functional assessment and thus understanding the disease mechanisms from patients suffering from pain disorders. We propose that both differentiation methods can be further exploited for understanding mechanisms and development of novel treatments in pain disorders.

Development of automated patch clamp assays to overcome the burden of variants of uncertain significance in inheritable arrhythmia syndromes.

Advances in next-generation sequencing have been exceptionally valuable for identifying variants in medically actionable genes. However, for most missense variants there is insufficient evidence to permit definitive classification of variants as benign or pathogenic. To overcome the deluge of Variants of Uncertain Significance, there is an urgent need for high throughput functional assays to assist with the classification of variants. Advances in parallel planar patch clamp technologies has enabled the development of automated high throughput platforms capable of increasing throughput 10- to 100-fold compared to manual patch clamp methods. Automated patch clamp electrophysiology is poised to revolutionize the field of functional genomics for inheritable cardiac ion channelopathies. In this review, we outline i) the evolution of patch clamping, ii) the development of high-throughput automated patch clamp assays to assess cardiac ion channel variants, iii) clinical application of these assays and iv) where the field is heading.

Epilepsy-associated SCN2A (NaV1.2) variants exhibit diverse and complex functional properties.

Pathogenic variants in voltage-gated sodium (NaV) channel genes including SCN2A, encoding NaV1.2, are discovered frequently in neurodevelopmental disorders with or without epilepsy. SCN2A is also a high-confidence risk gene for autism spectrum disorder (ASD) and nonsyndromic intellectual disability (ID). Previous work to determine the functional consequences of SCN2A variants yielded a paradigm in which predominantly gain-of-function variants cause neonatal-onset epilepsy, whereas loss-of-function variants are associated with ASD and ID. However, this framework was derived from a limited number of studies conducted under heterogeneous experimental conditions, whereas most disease-associated SCN2A variants have not been functionally annotated. We determined the functional properties of SCN2A variants using automated patch-clamp recording to demonstrate the validity of this method and to examine whether a binary classification of variant dysfunction is evident in a larger cohort studied under uniform conditions. We studied 28 disease-associated variants and 4 common variants using two alternatively spliced isoforms of NaV1.2 expressed in HEK293T cells. Automated patch-clamp recording provided a valid high throughput method to ascertain detailed functional properties of NaV1.2 variants with concordant findings for variants that were previously studied using manual patch clamp. Many epilepsy-associated variants in our study exhibited complex patterns of gain- and loss-of-functions that are difficult to classify by a simple binary scheme. The higher throughput achievable with automated patch clamp enables study of variants with greater standardization of recording conditions, freedom from operator bias, and enhanced experimental rigor. This approach offers an enhanced ability to discern relationships between channel dysfunction and neurodevelopmental disorders.

Electrical impedance spectroscopy for drug screening on ligand-gated ion channels.

Ligand-gated ion channels (LGICs) are involved in many pathophysiological processes and represent a relevant, although challenging, target for drug discovery. The gold standards for studying the ligand-induced ion channel currents and their modulations by drugs are manual patch clamp or two-electrode voltage clamp (TEVC) electrophysiology, but the throughput is low. Higher throughput has been achieved with automated patch clamp instruments, but these have immense acquisition and significant operating costs. In a proof-of-concept study, we developed a reliable, easy to use and multiplexable microfluidic setup to screen the action of drugs at LGICs heterologously expressed in X. laevis oocytes by measuring the electrical impedance changes across the plasma membrane induced by ligand pulses. We validated our setup by simultaneous TEVC electrophysiology and tested the effects of the reference drugs suramin, TNP-ATP, PPADS or diazepam on oocytes expressing P2X2 or GABAAreceptors, respectively. The drugs modulate ATP-induced and GABA-induced currents of P2X2 and GABAA receptors, respectively. The results obtained with TEVC are similar in terms of drug efficacy to those obtained with simultaneously recorded membrane impedances, which are determined by electrical impedance changes. Thus, the study shows the reliability of our setup as a faster (higher throughput) and more cost-effective method for drug screening compared to conventional electrophysiological techniques. After multiplexing of the system, the setup may facilitate commercial and academic drug screening of ligand gated ion channels that are receiving less attention due to limitations in current assays. [Funded under the Excellence Strategy of the Federal Government and the Länder - OPSF671]

Abstract 12145: Cell Surface Trafficking and Functional Deep Mutational Scans of the Potassium Channel Gene KCNE1

Introduction: Loss of function variants in KCNE1 cause type 5 Long QT Syndrome. Deep mutational scanning is a highly multiplexed method to assay the function of thousands of genetic variants. Hypothesis: A deep mutational scan of KCNE1 will identify variants that disrupt KCNE1 function and may lead to Long QT Syndrome. Methods: A comprehensive library of nearly all possible KCNE1 variants was generated by inverse PCR. The library was integrated in a one-variant-per-cell format into HEK293 landing pad cells expressing the KCNE1 partner KCNQ1 . To assess surface trafficking, cells expressing an HA-tagged KCNE1 variant library were stained with an anti-HA antibody, FACS sorted into 4 equal bins, and deep sequenced. For each variant, a weighted average of variant counts across the 4 bins was calculated. To assess channel function, the KCNE1 library was introduced into landing pad cells expressing a gain of function S140G KCNQ1 variant, which causes KCNE1-dependent K+ efflux and cell death, for 12 days. Functional scores for each variant were calculated as a normalized ratio of initial and day 12 counts. Results: The KCNE1 library had 88% of 2,451 possible missense variants. The results of the 2 assays indicate that the KCNE1 C-terminus is dispensable for trafficking but not function. Missense variant trafficking scores (n=1,886) were normally distributed (0.93+/-0.37, mean+/-SD); 277 loss- and 183 gain-of-trafficking variants were identified. Missense variant functional scores were bimodally distributed. The functional assay identified loss of function variants with trafficking-deficient or trafficking-normal scores (likely gating variants). The assays identified structurally important regions, such as an N-terminal salt bridge, a transmembrane helix at residues 47-59 resistant to hydrophilic variants, and a cytoplasmic amphipathic helix at residues 90-114 resistant to hydrophobic variants. Manual patch clamp results for 4 previously studied and 4 novel variants correlated with deep mutational scanning scores. Conclusions: Two deep mutational scans of KCNE1 identified variants that disrupt channel or function. This dataset elucidates structure-function relationships and may help predict LQT5 variant pathogenicity.

Reclassification of a likely pathogenic Dutch founder variant in KCNH2; implications of reduced penetrance.

Variants in KCNH2, encoding the human ether a-go-go (hERG) channel that is responsible for the rapid component of the cardiac delayed rectifier K+ current (IKr), are causal to long QT syndrome type 2 (LQTS2). We identified eight index patients with a new variant of unknown significance (VUS), KCNH2:c.2717C > T:p.(Ser906Leu). We aimed to elucidate the biophysiological effect of this variant, to enable reclassification and consequent clinical decision-making. A genotype-phenotype overview of the patients and relatives was created. The biophysiological effects were assessed independently by manual-, and automated calibrated patch clamp. HEK293a cells expressing (i) wild-type (WT) KCNH2, (ii) KCNH2-p.S906L alone (homozygous, Hm) or (iii) KCNH2-p.S906L in combination with WT (1:1) (heterozygous, Hz) were used for manual patching. Automated patch clamp measured the variants function against known benign and pathogenic variants, using Flp-In T-rex HEK293 KCNH2-variant cell lines. Incomplete penetrance of LQTS2 in KCNH2:p.(Ser906Leu) carriers was observed. In addition, some patients were heterozygous for other VUSs in CACNA1C, PKP2, RYR2 or AKAP9. The phenotype of carriers of KCNH2:p.(Ser906Leu) ranged from asymptomatic to life-threatening arrhythmic events. Manual patch clamp showed a reduced current density by 69.8 and 60.4% in KCNH2-p.S906L-Hm and KCNH2-p.S906L-Hz, respectively. The time constant of activation was significantly increased with 80.1% in KCNH2-p.S906L-Hm compared with KCNH2-WT. Assessment of KCNH2-p.S906L-Hz by calibrated automatic patch clamp assay showed a reduction in current density by 35.6%. The reduced current density in the KCNH2-p.S906L-Hz indicates a moderate loss-of-function. Combined with the reduced penetrance and variable phenotype, we conclude that KCNH2:p.(Ser906Leu) is a low penetrant likely pathogenic variant for LQTS2.

Development of ASIC1a ligand-gated ion channel drug screening assays across multiple automated patch clamp platforms.

Human acid-sensing ion channels (ASIC) are ligand-gated ionotropic receptors expressed widely in peripheral tissues as well as sensory and central neurons and implicated in detection of inflammation, tissue injury, and hypoxia-induced acidosis. This makes ASIC channels promising targets for drug discovery in oncology, pain and ischemia, and several modulators have progressed into clinical trials. We describe the use of hASIC1a as a case study for the development and validation of low, medium and high throughput automated patch clamp (APC) assays suitable for the screening and mechanistic profiling of new ligands for this important class of ligand-gated ion channel. Initial efforts to expand on previous manual patch work describing an endogenous hASIC1a response in HEK cells were thwarted by low current expression and unusual pharmacology, so subsequent work utilized stable hASIC1a CHO cell lines. Ligand-gated application protocols and screening assays on the Patchliner, QPatch 48, and SyncroPatch 384 were optimized and validated based on pH activation and nM-μM potency of reference antagonists (e.g., Amiloride, Benzamil, Memantine, Mambalgin-3, A-317567, PcTx1). By optimizing single and stacked pipette tip applications available on each APC platform, stable pH-evoked currents during multiple ligand applications enabled cumulative EC50 and IC50 determinations with minimized receptor desensitization. Finally, we successfully demonstrated for the first time on an APC platform the ability to use current clamp to implement the historical technique of input resistance tracking to measure ligand-gated changes in membrane conductance on the Patchliner platform.

There is no F in APC: Using physiological fluoride-free solutions for high throughput automated patch clamp experiments.

Fluoride has been used in the internal recording solution for manual and automated patch clamp experiments for decades because it helps to improve the seal resistance and promotes longer lasting recordings. In manual patch clamp, fluoride has been used to record voltage-gated Na (NaV) channels where seal resistance and access resistance are critical for good voltage control. In automated patch clamp, suction is applied from underneath the patch clamp chip to attract a cell to the hole and obtain a good seal. Since the patch clamp aperture cannot be moved to improve the seal like the patch clamp pipette in manual patch clamp, automated patch clamp manufacturers use internal fluoride to improve the success rate for obtaining GΩ seals. However, internal fluoride can affect voltage-dependence of activation and inactivation, as well as affecting internal second messenger systems and therefore, it is desirable to have the option to perform experiments using physiological, fluoride-free internal solution. We have developed an approach for high throughput fluoride-free recordings on a 384-well based automated patch clamp system with success rates >40% for GΩ seals. We demonstrate this method using hERG expressed in HEK cells, as well as NaV1.5, NaV1.7, and KCa3.1 expressed in CHO cells. We describe the advantages and disadvantages of using fluoride and provide examples of where fluoride can be used, where caution should be exerted and where fluoride-free solutions provide an advantage over fluoride-containing solutions.

Biophysical characterization of light-gated ion channels using planar automated patch clamp.

Channelrhodopsins (ChRs) are proteins that guide phototaxis in protists and exhibit light-gated channel conductance when their genes are heterologously expressed in mammalian cells. ChRs are widely used as molecular tools to control neurons and cardiomyocytes with light (optogenetics). Cation- and anion-selective ChRs (CCRs and ACRs, respectively) enable stimulation and inhibition of neuronal activity by depolarization and hyperpolarization of the membrane, respectively. More than 400 natural ChR variants have been identified so far, and high-throughput polynucleotide sequencing projects add many more each year. However, electrophysiological characterization of new ChRs lags behind because it is mostly done by time-consuming manual patch clamp (MPC). Here we report using a high-throughput automated patch clamp (APC) platform, SyncroPatch 384i from Nanion Technologies, for ChR research. We find that this instrument can be used for determination of the light intensity dependence and current-voltage relationships in ChRs and discuss its advantages and limitations.

HERG block potencies for 5 positive control drugs obtained per ICH E14/S7B Q&As best practices: Impact of recording temperature and drug loss

According to the ICH S7B guideline, drug candidates are screened for hERG block prior to first-in-human testing to predict the likelihood of delayed repolarization associated with a rare, but life-threatening, ventricular tachyarrhythmia. The new ICH E14 Q&As guideline allows hERG results to be used in later clinical development for decision-making (Q&As 5.1 and 6.1). To pursue this path, the hERG assay should be conducted following the new ICH S7B Q&A 2.1 guideline, which calls for best practice considerations of the recording temperature, voltage protocol, stimulation frequency, recording/data quality, and concentration verification. This study investigated hERG block by cisapride, dofetilide, terfenadine, sotalol, and E-4031 – positive controls commonly used to demonstrate assay sensitivity – using the manual whole cell patch clamp method and an action potential-like voltage protocol presented at 0.2 Hz. Recordings were conducted at room and near physiological temperature. Drug concentrations were measured using samples collected during real patch clamp experiments and satellite experiments. Results showed temperature effects for E-4031, terfenadine, and sotalol, but not cisapride and dofetilide. Cisapride and terfenadine showed substantial concentration losses, largely due to nonspecific binding to the perfusion apparatus. Using concentrations measured from the real and satellite experiments to assess block potencies yielded comparable results, indicating that satellite sample collection may be viable for drugs with nonspecific binding concerns only. In summary, this study provides block potencies for 5 hERG positive controls, and serves as a case study for hERG assays conducted, and results illustrated in accordance with the new ICH E14/S7B Q&As.

High throughput measurement of hERG drug block kinetics using the CiPA dynamic protocol

The Comprehensive in vitro Proarrhythmic Assay (CiPA) has promoted use of in silico models of drug effects on cardiac repolarization to improve proarrhythmic risk prediction. These models contain a pharmacodynamic component describing drug binding to hERG channels that required in vitro data for kinetics of block, in addition to potency, to constrain them. To date, development and validation has been undertaken using data from manual patch-clamp. The application of this approach at scale requires the development of a high-throughput, automated patch-clamp (APC) implementation. Here, we present a comprehensive analysis of the implementation of the Milnes, or CiPA dynamic protocol, on an APC platform, including quality control and data analysis. Kinetics and potency of block were assessed for bepridil, cisapride, terfenadine and verapamil with data retention/QC pass rate of 21.8% overall, or as high as 50.4% when only appropriate sweep lengths were considered for drugs with faster kinetics. The variability in IC50 and kinetics between manual and APC was comparable to that seen between sites/platforms in previous APC studies of potency. Whilst the experimental success is less than observed in screens of potency alone, it is still significantly greater than manual patch. With the modifications to protocol design, including sweep length, number of repetitions, and leak correction recommended in this study, this protocol can be applied on APC to acquire data comparable to manual patch clamp.

An efficient and scalable data analysis solution for automated electrophysiology platforms.

Ion channels are drug targets for neurologic, cardiac, and immunologic diseases. Many disease-associated mutations and drugs modulate voltage-gated ion channel activation and inactivation, suggesting that characterizing state-dependent effects of test compounds at an early stage of drug development can be of great benefit. Historically, the effects of compounds on ion channel biophysical properties and voltage-dependent activation/inactivation could only be assessed by using low-throughput, manual patch clamp recording techniques. In recent years, automated patch clamp (APC) platforms have drastically increased in throughput. In contrast to their broad utilization in compound screening, APC platforms have rarely been used for mechanism of action studies, in large part due to the lack of sophisticated, scalable analysis methods for processing the large amount of data generated by APC platforms. In the current study, we developed a highly efficient and scalable software workflow to overcome this challenge. This method, to our knowledge the first of its kind, enables automated curve fitting and complex analysis of compound effects. Using voltage-gated sodium channels as an example, we were able to immediately assess the effects of test compounds on a spectrum of biophysical properties, including peak current, voltage-dependent steady state activation/inactivation, and time constants of activation and fast inactivation. Overall, this automated data analysis method provides a novel solution for in-depth analysis of large-scale APC data, and thus will significantly impact ion channel research and drug discovery.

Determining the role of uremic toxins on cardiac electrophysiology and pro-arrhythmia during chronic kidney disease

Abstract Funding Acknowledgements Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Young Talent Programme, CVON Introduction Chronic kidney disease (CKD) is represented by a diminished filtration capacity of the kidneys. End stage renal disease patients need dialysis treatment to remove waste and toxins from the circulation. However, endogenously produced uremic toxins (UTs) cannot always be filtered during dialysis. UTs are one of the CKD-related factors already linked to maladaptive and pathophysiological remodeling of the heart. Importantly, 50% of the deaths in dialysis patients is cardiovascular related, with sudden cardiac death predominating. However, the mechanisms responsible still remain poorly understood. Purpose To assess the potentially increased pro-arrhythmic risk of pre-identified UTs at clinically relevant concentrations: 1) acutely in isolated adult dog ventricular cardiomyocytes (ADCMs) and human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs), 2) chronically in iPSC-CMs and 3) both conditions in human embryonic kidney (HEK) cells transfected with constructs that generate different ion channels, using optical and manual electrophysiological in vitro approaches. Methods and results Using the voltage sensitive membrane dye FluoVolt and manual patch clamp, action potential duration (APD) prolongation, considered as a pro-arrhythmic parameter, was assessed in ADCMs and iPSC-CMs. Acute exposure of indoxyl sulfate (IS), kynurenine (KYN) or kynurenic acid (KYNA) induced no alterations in APD in ADCMs and iPSC-CMs. iPSC-CMs, although having rather immature electrophysiological characteristics, showed significant APD prolongation when exposed chronically (48h) to those UTs. Manual patch clamp on HEK293 cells was performed to investigate individual ion currents to reveal underlying mechanisms. The repolarizing current IKr, often most sensitive and responsible for APD alterations, was not acutely affected by IS, KYN, or KYNA. However, chronic exposure (48h) to IS or KYNA significantly decreased IKr at clinically relevant concentrations. Finally, the protein level of the ion channel conducting IKr was determined in HEK293 cells, to further unveil the mechanism behind the effects of UTs on channels conducting IKr. Chronic exposure (48-72h) of the three UTs decreased total ion channel expression at pathological concentrations, which could be responsible for the decreased IKr and prolonged APD. Conclusion The results show the proof of concept to identify potentially pro-arrhythmogenic UTs and their mode of action, allowing the provision of a clinically relevant overview of (patho)physiological concentrations of UTs in both acute and chronic exposure.

Stiripentol inhibits spike-and-wave discharges in animal models of absence seizures: A new mechanism of action involving T-type calcium channels.

SummaryObjectiveStiripentol (STP; Diacomit®) is an antiepileptic drug indicated for Dravet syndrome that has been identified as a γ‐aminobutyric acid (GABAergic) positive allosteric modulator. Dravet syndrome is characterized by multiple seizure types: generalized tonic–clonic, focal, myoclonic, and absence seizures. In addition to its antiepileptic effects on tonic–clonic seizures, STP has also been reported to reduce the frequency of atypical absence seizures in patients. Our study focused on STP potential effects on absence seizures, to better characterize its full spectrum of mechanisms of action.MethodsSTP effects on absence seizures were quantified by electroencephalographic recording in two animal models: rats treated with a low dose of pentylenetetrazol (20 mg/kg ip) and rats from the WAG/Rij strain. In addition, we characterized STP effects on T‐type calcium channel activity. Peak currents were recorded with manual patch clamp on cells transfected with cDNA encoding for the human isoform for Cav3.1, Cav3.2, and Cav3.3.ResultsSTP administered before pentylenetetrazol almost completely abolished the generation of spike‐and‐wave discharges (SWDs) at the dose of 300 mg/kg. At this dose, STP also statistically significantly decreased SWD cumulated duration and number in WAG/Rij rats. Its antiepileptic effect was maintained in WAG/Rij rats, whose seizures were aggravated by the GABA agonist THIP (gaboxadol hydrochloride). Furthermore, electrophysiological recordings showed that STP inhibits T‐type calcium channel peak activity, with a higher specificity for the Cav3.3 subtype.SignificanceIn addition to its previously characterized anticonvulsive properties, these data highlight a new mechanism of action of STP on abnormal thalamocortical activity. This strong antiabsence effect on seizures is correlated with an inhibition of T‐type calcium channels. This new mechanism of action could be implicated in the specificity of STP therapeutic effects in Dravet syndrome.

Pharmacological characterization of a primary nicotinic receptor from the neuromuscular junction

Nicotinic receptors at the neuromuscular junction are important targets in the study of autoimmune disorders such as myasthenia gravis. Historic efforts targeting nAChR drug discovery focused on fluorescence based calcium flux assays along with low throughput manual patch clamp techniques due to its dependence on complete liquid exchange. With the advent of next generation automated patch champ instruments with integrated solution control and rapid solution change, extensive pharmacological characterization of the neuromuscular nAChR ((α1)2β1γδ) is now possible.

Crack Group Effect Analysis of the Stress-Release Boot of Solid Rocket Motor Based on Numerical Simulation

Due to the complex process of the insulation layer patch in solid rocket motor (SRM), only manual patch could be used. Sometimes weak bonding or debonding in each joint surface was inevitable. This study is aimed at determining the crack group effect of insulation and interfacial debonded crack in the wide-temperature SRM. The crack group appeared in the front area of the ahead stress-release boot and was induced by low temperature, axial overload, or interface bonding failure. Based on the viscoelastic finite element method, singular crack elements and singular interfacial crack elements at the tips of crack group were established to calculateJ-integral. Varying according to the length and position of cracks, theJ-integral of crack tips was, respectively, calculated to prejudge their stability and the crack group effect. The results showed that collinear crack group appeared in the front stress-release boot layer, and the crack group had a certain shielding effect on the main crack when the SRM was launched at low temperature. When noncollinear crack group appeared in the front stress-release boot layer, the crack group effect changed with the length of the main crack. The crack group first had a shielding effect on the main crack and then had a strong strengthening effect. The experimental test of the simulated specimen revealed that numerical simulation results matched the experimental test results.

A nonlinear and time-dependent leak current in the presence of calcium fluoride patch-clamp seal enhancer.

Automated patch-clamp platforms are widely used and vital tools in both academia and industry to enable high-throughput studies such as drug screening. A leak current to ground occurs whenever the seal between a pipette and cell (or internal solution and cell in high-throughput machines) is not perfectly insulated from the bath (extracellular) solution. Over 1 GΩ seal resistance between pipette and bath solutions is commonly used as a quality standard for manual patch work. With automated platforms it can be difficult to obtain such a high seal resistance between the intra- and extra-cellular solutions. One suggested method to alleviate this problem is using an F - containing internal solution together with a Ca 2+ containing external solution - so that a CaF 2 crystal forms when the two solutions meet which 'plugs the holes' to enhance the seal resistance. However, we observed an unexpected nonlinear-in-voltage and time-dependent current using these solutions on an automated patch-clamp platform. We performed manual patch-clamp experiments with the automated patch-clamp solutions, but no biological cell, and observed the same nonlinear time-dependent leak current. The current could be completely removed by washing out F - ions to leave a conventional leak current that was linear and not time-dependent. We therefore conclude fluoride ions interacting with the CaF 2 crystal are the origin of the nonlinear time-dependent leak current. The consequences of such a nonlinear and time-dependent leak current polluting measurements should be considered carefully if it cannot be isolated and subtracted.