Voltage-clamp Protocols Research Articles

Mathematical models of C-type and N-type inactivating heteromeric voltage gated potassium channels.

Voltage gated potassium channels can be composed of either four identical, or different, pore-forming protein subunits. While the voltage gated channels with identical subunits have been extensively studied both physiologically and mathematically, those with multiple subunit types, termed heteromeric channels, have not been. Here we construct, and explore the predictive outputs of, mechanistic models for heteromeric voltage gated potassium channels that possess either N-type or C-type inactivation kinetics. For both types of inactivation, we first build Markov models of four identical pore-forming inactivating subunits. Combining this with previous results regarding non-inactivating heteromeric channels, we are able to define models for heteromeric channels containing both non-inactivating and inactivating subunits of any ratio. We simulate each model through three unique voltage clamp protocols to identify steady state properties. In doing so, we generate predictions about the impact of adding additional inactivating subunits on a total channel's kinetics. We show that while N-type inactivating subunits appear to have a non-linear impact on the level of inactivation the channel experiences, the effect of C-type inactivating subunits is almost linear. Finally, to combat the computational issues of working with a large number of state variables we define model reductions for both types of heteromeric channels. For the N-type heteromers we derive a quasi-steady-state approximation and indicate where the approximation is appropriate. With the C-type heteromers we are able to write an explicit model reduction bringing models of greater than 10 dimensions down to 2.

Experimentally validated modeling of dynamic drug-hERG channel interactions reproducing the binding mechanisms and its importance in action potential duration.

Background and ObjectiveAssessment of drug cardiotoxicity is critical in the development of new compounds and modeling of drug-binding dynamics to hERG can improve early cardiotoxicity assessment. We previously developed a methodology to generate Markovian models reproducing preferential state-dependent binding properties, trapping dynamics and the onset of IKr block using simple voltage clamp protocols. Here, we test this methodology with real IKr blockers and investigate the impact of drug dynamics on action potential prolongation. MethodsExperiments were performed on HEK cells stably transfected with hERG and using the Nanion SyncroPatch 384i. Three protocols, P-80, P0 and P 40, were applied to obtain the experimental data from the drugs and the Markovian models were generated using our pipeline. The corresponding static models were also generated and a modified version of the O´Hara-Rudy action potential model was used to simulate the action potential duration. ResultsThe experimental Hill plots and the onset of IKr block of ten compounds were obtained using our voltage clamp protocols and the models generated successfully mimicked these experimental data, unlike the CiPA dynamic models. Marked differences in APD prolongation were observed when drug effects were simulated using the dynamic models and the static models. ConclusionsThese new dynamic models of ten well-known IKr blockers constitute a validation of our methodology to model dynamic drug–hERG channel interactions and highlight the importance of state-dependent binding, trapping dynamics and the time-course of IKr block to assess drug effects even at the steady-state.

Quantifying the efficacy of voltage protocols in characterising ion channel kinetics: A novel information-theoretic approach.

Voltage-clamp experiments are commonly utilised to characterise cellular ion channel kinetics. In these experiments, cells are stimulated using a known time-varying voltage, referred to as the voltage protocol, and the resulting cellular response, typically in the form of current, is measured. Parameters of models that describe ion channel kinetics are then estimated by solving an inverse problem which aims to minimise the discrepancy between the predicted response of the model and the actual measured cell response. In this paper, a novel framework to evaluate the information content of voltage-clamp protocols in relation to ion channel model parameters is presented. Additional quantitative information metrics that allow for comparisons among various voltage protocols are proposed. These metrics offer a foundation for future optimal design frameworks to devise novel, information-rich protocols. The efficacy of the proposed framework is evidenced through the analysis of seven voltage protocols from the literature. By comparing known numerical results for inverse problems using these protocols with the information-theoretic metrics, the proposed approach is validated. The essential steps of the framework are: (i) generate random samples of the parameters from chosen prior distributions; (ii) run the model to generate model output (current) for all samples; (iii) construct reduced-dimensional representations of the time-varying current output using proper orthogonal decomposition (POD); (iv) estimate information-theoretic metrics such as mutual information, entropy equivalent variance, and conditional mutual information using non-parametric methods; (v) interpret the metrics; for example, a higher mutual information between a parameter and the current output suggests the protocol yields greater information about that parameter, resulting in improved identifiability; and (vi) integrate the information-theoretic metrics into a single quantitative criterion, encapsulating the protocol's efficacy in estimating model parameters.

A biophysically comprehensive model of urothelial afferent neurons: implications for sensory signalling in urinary bladder.

The urothelium is the innermost layer of the bladder wall; it plays a pivotal role in bladder sensory transduction by responding to chemical and mechanical stimuli. The urothelium also acts as a physical barrier between urine and the outer layers of the bladder wall. There is intricate sensory communication between the layers of the bladder wall and the neurons that supply the bladder, which eventually translates into the regulation of mechanical activity. In response to natural stimuli, urothelial cells release substances such as ATP, nitric oxide (NO), substance P, acetylcholine (ACh), and adenosine. These act on adjacent urothelial cells, myofibroblasts, and urothelial afferent neurons (UAN), controlling the contractile activity of the bladder. There is rising evidence on the importance of urothelial sensory signalling, yet a comprehensive understanding of the functioning of the urothelium-afferent neurons and the factors that govern it remains elusive to date. Until now, the biophysical studies done on UAN have been unable to provide adequate information on the ion channel composition of the neuron, which is paramount to understanding the electrical functioning of the UAN and, by extension, afferent signalling. To this end, we have attempted to model UAN to decipher the ionic mechanisms underlying the excitability of the UAN. In contrast to previous models, our model was built and validated using morphological and biophysical properties consistent with experimental findings for the UAN. The model included all the channels thus far known to be expressed in UAN, including; voltage-gated sodium and potassium channels, N, L, T, P/Q, R-type calcium channels, large-conductance calcium-dependent potassium (BK) channels, small conductance calcium-dependent (SK) channels, Hyperpolarisation activated cation (HCN) channels, transient receptor potential melastatin (TRPM8), transient receptor potential vanilloid (TRPV1) channel, calcium-activated chloride(CaCC) channels, and internal calcium dynamics. Our UAN model a) was constrained as far as possible by experimental data from the literature for the channels and the spiking activity, b) was validated by reproducing the experimental responses to current-clamp and voltage-clamp protocols c) was used as a base for modelling the non-urothelial afferent neurons (NUAN). Using our models, we also gained insights into the variations in ion channels between UAN and NUAN neurons.

Abstract 12942: The D801N Variant in ATP1A3 -Encoded Na/K-atpase Alpha 3 Shortens the Cardiac Myocyte Action Potential and Increases Risk for Calcium Mediated Arrhythmias

Background: Alternating hemiplegia of childhood (AHC) is a rare de novo syndrome that manifests with episodic hemiplegia, seizures, dystonia, and, notably, sudden unexplained death. Gene positive patients carry a pathogenic variant in the ATP1A3 -encoded Na/K ATPase alpha 3 isoform (ATP1A3), and the variant D801N is the most common pathogenic variant. Our group recently found that AHC patients with ATP1A3-D801N missense have short QTc and are at risk of ventricular fibrillation. Hypothesis: We hypothesized that the D801N variant results in reduced ATPase function leading to Ca 2+ overload resulting in shortened repolarization time and increased arrhythmogenic risk. Methods: We differentiated iPSCs into cardiomyocytes from an AHC patient hosting the ATP1A3-D801N variant (iPSC-CM D801N ), and an ostensibly healthy individual (iPSC-CM WT ). Patch clamp was conducted to determine APD90, and the ratio of cells with delayed after depolarization events (DADs). iPSC-CM WT were perfused with 10 -7 M ouabain, an ATP1A3 inhibitor, and APD90 and DADs were measured. SR store ([Ca 2+ ] SR ) was measured with Cal-520 dye under 10 mM caffeine. Resting [Ca 2+ ] i was measured with Fura-2 dye, and Na/Ca exchanger (NCX) peak current was measured as Ni 2+ sensitive current in a stepwise voltage-clamp protocol. Results: iPSC-CM D801N APD90 was shorter 40% than iPSC-CM WT . IPSC-CM WT treated with ouabain had 17% shorter APD90 than vehicle. iPSC-CM D801N had DADs (3/11 cells) when iPSC-CM WT (0/24) did not, and 6/12 ouabain treated iPSC-CM WT had DADs vs vehicle treated (0/18). Post caffeine, iPSC-CM D801N had higher [Ca 2+ ] SR than iPSC-CM WT . Finally, iPSC-CM D801N had higher resting [Ca 2+ ] i than iPSC-CM WT and iPSC-CM D801N had higher peak NCX current density at +80mV but no difference at RMP. Conclusions: Our results show that both iPSC-CM D801N and ouabain-treated iPSC-CM WT , have a shorter APD compared to WT controls and exhibit DADs. This is associated with higher NCX-mediated influx of Ca 2+ and increased intracellular Ca 2+ . Our findings highlight the emerging role of ATP1A3-D801N in creating a myocardium vulnerable to arrhythmias.

Prolonged Exposure to Remdesivir Inhibits the Human Ether-A-Go-Go-Related Gene Potassium Current.

Remdesivir, approved for the treatment of COVID-19, has been associated with heart-rate corrected QT interval (QTc) prolongation and torsade de pointes in case reports. However, data are conflicting regarding the ability of remdesivir to inhibit the human ether-a-go-go-related gene (hERG) -related current. The objective of this study was to investigate the effects remdesivir and its primary metabolite, GS-441524, on hERG-related currents. Human embryonic kidney 293 cells stably expressing hERG were treated with various concentrations of remdesivir and GS-441524. The effects of acute and prolonged exposure on hERG-related current were assessed using whole-cell configuration of voltage-clamp protocols. Acute exposure to remdesivir and GS-441524 had no effect on hERG currents and the half-activation voltage (V 1/2 ). Prolonged treatment with 100 nM and 1 µM remdesivir significantly reduced peak tail currents and hERG current density. The propensity for remdesivir to prolong QTc intervals and induce torsade de pointes in predisposed patients warrants further investigation.

G-protein tonic inhibition of calcium channels in pancreatic β-cells.

Voltage-gated calcium channels (CaV) conduct Ca2+ influx promoting neurotransmitters and hormone release. CaV are finely regulated by voltage-dependent and independent pathways either by G-protein-coupled receptors (GPCRs) or intramembrane lipids, respectively, in neurons and glands. Interestingly, pancreatic β-cells are abundantly innervated by both sympathetic and parasympathetic neurons, while a variety of high-voltage activated (HVA) Ca2+ channels are present in these cells. Thus, autonomic system seems to exert a tonic inhibition on HVA Ca2+ channels throughout GPCRs, constitutively preventing hormone secretion. Therefore, this work aimed to investigate noradrenergic and cholinergic inhibition of HVA Ca2+ channels in pancreatic β-cells. Experiments were conducted in pancreatic β-cells of rat by using patch-clamping methods, immunocytochemistry, pharmacological probes, and biochemical reagents. A voltage-clamp protocol with a strong depolarizing prepulse was used to unmask tonic inhibition. Herein, we consistently find a basal tonic inhibition of HVA Ca2+ channels according to a GPCRs regulation. Facilitation ratio is enhanced by noradrenaline (NA) according to a voltage-dependent regulation and a membrane-delimited mechanism, while no facilitation changes are observed with carbachol or phosphatidylinositol 4,5-bisphosphate (PIP2). Furthermore, carbachol or intramembrane lipids, such as PIP2, do not change facilitation ratio according to a voltage-independent regulation. Together, HVA Ca2+ channels of pancreatic β-cells are constitutively inhibited by GPCRs, suggesting a natural brake preventing cells from exhaustive insulin secretion.NEW & NOTEWORTHY Our results support the hypothesis that GPCRs tonically inhibit HVA Ca2+ channels in pancreatic β-cells. A voltage-clamp protocol with a strong depolarizing prepulse was used to unmask voltage-dependent inhibition of Ca2+ channels. The novelty of these results strengthens the critical role of Gβγ's in Ca2+ channel regulation, highlighting kinetic slowing and increased facilitation ratio. Together, HVA Ca2+ channels of pancreatic β-cells are constitutively inhibited by GPCRs underlying fine-tuning modulation of insulin secretion.

#2696 IN SILICO ELECTROPHYSIOLOGICAL STUDY REVEALS TAMSULOSIN MEDIATE URETER SMOOTH MUSCLE CONTRACTION BY INCREASING POTASSIUM CURRENT

Abstract Background and Aims Tamsulosin is a common α1-AR antagonist that is prescribed to patients with acute kidney stone episodes [Scotland et al., 2021]. According to accumulated experimental data, abnormal peristaltic contraction of the ureter smooth muscle (USM) causes acute kidney stone episodes [Lang et al., 2006]. Intracellular electrical activities like membrane depolarization and action potentials play important roles in modulating the USM contraction by releasing intracellular calcium from the sarcoplasmic reticulum. Therefore, an electrophysiological study will be helpful to assess the USM cell's electrical activities and in diagnosing abnormal USM contraction. Transient potassium channels (KA) are prevalent in different systems and hold immense importance for maintaining/performing selective electrophysiological functions. The present study investigates the altered electrophysiological characteristics of the USM cell because of the induction of Tamsulosin doses. Method The single isolated USM cell comprises several voltage-gated ion channels, such as two voltage-gated calcium (T-type, and L-type) channels, one voltage-gated fast potassium (KA) channel, one calcium-dependent large conductance potassium channel, and an HCN channel. The individual ion channel currents and the membrane potential are recorded by utilizing both the voltage clamp and current clamp protocols. A Tamsulosin drug (0.5 mg) model for the KA channel is introduced by multiplying the maximal conductance of the KA channel with a scaling factor between 0 and 1 to mimic the drug concentration. Statistical analysis protocols are evaluated for analytical purposes. Results In this in silico electrophysiological model, the resting membrane potential is maintained at −75 mV, as this value is cited by various experimental studies. The ionic concentrations and biophysical parameters for all ion channels are varied in the physiological ranges. Under the voltage clamp protocol, the voltage steps are increased from a holding potential of −80 mV to −10 mV with a 10 mV of step voltage. In Figure 1, the red and blue solid lines represent recorded outward KA current from the isolated USM cell under both control and applied Tamsulosin dose conditions. Figure 1 reveals that the KA current is elevated after the application of the Tamsulosin drug regarding all step voltages. As a result, the total outward current is elevated and the USM action potential duration is shortened. It is also observed that the steady-state activation curve of the KA channel is shifted to more negative after applying Tamsulosin. The USM cell is electrically less excitable with the Tamsulosin. Conclusion This first in-silico study reveals the KA ionic current, which plays a major role in coordinating ureteral contractions, is altered due to Tamsulosin. It implies that the pharmacological maneuver of Tamsulosin is substantially beneficial to treat dysfunctional peristalsis by reducing the symptoms and installation complexities from the ureteral stents procedure.

Calcium- and voltage-driven atrial alternans: Insight from [Ca]i and Vm asynchrony.

Cardiac alternans is defined as beat-to-beat alternations in contraction strength, action potential duration (APD), and Ca transient (CaT) amplitude. Cardiac excitation-contraction coupling relies on the activity of two bidirectionally coupled excitable systems, membrane voltage (Vm ) and Ca release. Alternans has been classified as Vm - or Ca-driven, depending whether a disturbance of Vm or [Ca]i regulation drives the alternans. We determined the primary driver of pacing induced alternans in rabbit atrial myocytes, using combined patch clamp and fluorescence [Ca]i and Vm measurements. APD and CaT alternans are typically synchronized; however, uncoupling between APD and CaT regulation can lead to CaT alternans in the absence of APD alternans, and APD alternans can fail to precipitate CaT alternans, suggesting a considerable degree of independence of CaT and APD alternans. Using alternans AP voltage clamp protocols with extra APs showed that most frequently the pre-existing CaT alternans pattern prevailed after the extra-beat, indicating that alternans is Ca-driven. In electrically coupled cell pairs, dyssynchrony of APD and CaT alternans points to autonomous regulation of CaT alternans. Thus, with three novel experimental protocols, we collected evidence for Ca-driven alternans; however, the intimately intertwined regulation of Vm and [Ca]i precludes entirely independent development of CaT and APD alternans.

The role of potassium current in the pulmonary response to environmental oxidative stress

Exposure of human lung epithelial cells (A549 cell line) to the oxidant pollutant ozone (O3) alters cell membrane currents inducing its decrease, when the cell undergoes to a voltage-clamp protocol ranging from −90 to +70mV. The membrane potential of these cells is mainly maintained by the interplay of potassium and chloride currents. Our previous studies indicated the ability of O3 to activate ORCC (Outward Rectifier Chloride Channel) and consequently increases the chloride current.In this paper our aim was to understand the response of potassium current to oxidative stress challenge and to identify the kind potassium channel involved in O3 induced current changes.After measuring the total membrane current using an intracellular solution with or without potassium ions, we obtained the contribution of potassium to the overall membrane current in control condition by a mathematical approach. Repeating these experiments after O3 treatment we observed a significant decrease of Ipotassium. Treatment of the cells with Iberiotoxin (IbTx), a specific inhibitor of BK channel, we were able to verify the presence and the functionality of BK channels. In addition, the administration of 4-Aminopyridine (an inhibitor of voltage dependent K channels but not BK channels) and Tetraethylammonium (TEA) before and after O3 treatment we observed the formation of BK oxidative post-translation modifications.Our data suggest that O3 is able to inhibit potassium current by targeting BK channel. Further studies are needed to better clarify the role of this BK channel and its interplay with the other membrane channels under oxidative stress conditions. These findings can contribute to identify the biomolecular pathway induced by O3 allowing a possible pharmacological intervention against oxidative stress damage in lung tissue.

Charge-voltage curves of Shaker potassium channel are not hysteretic at steady state.

Charge-voltage curves of many voltage-gated ion channels exhibit hysteresis but such curves are also a direct measure of free energy of channel gating and, hence, should be path-independent. Here, we identify conditions to measure steady-state charge-voltage curves and show that these are curves are not hysteretic. Charged residues in transmembrane segments of voltage-gated ion channels (VGICs) sense and respond to changes in the electric field. The movement of these gating charges underpins voltage-dependent activation and is also a direct metric of the net free-energy of channel activation. However, for most voltage-gated ion channels, the charge-voltage (Q-V) curves appear to be dependent on initial conditions. For instance, Q-V curves of Shaker potassium channel obtained by hyperpolarizing from 0 mV is left-shifted compared to those obtained by depolarizing from a holding potential of -80 mV. This hysteresis in Q-V curves is a common feature of channels in the VGIC superfamily and raises profound questions about channel energetics because the net free-energy of channel gating is a state function and should be path independent. Due to technical limitations, conventional gating current protocols are limited to test pulse durations of <500 ms, which raises the possibility that the dependence of Q-V on initial conditions reflects a lack of equilibration. Others have suggested that the hysteresis is fundamental thermodynamic property of voltage-gated ion channels and reflects energy dissipation due to measurements under non-equilibrium conditions inherent to rapid voltage jumps (Villalba-Galea. 2017. Channels. https://doi.org/10.1080/19336950.2016.1243190). Using an improved gating current and voltage-clamp fluorometry protocols, we show that the gating hysteresis arising from different initial conditions in Shaker potassium channel is eliminated with ultra-long (18-25 s) test pulses. Our study identifies a modified gating current recording protocol to obtain steady-state Q-V curves of a voltage-gated ion channel. Above all, these findings demonstrate that the gating hysteresis in Shaker channel is a kinetic phenomenon rather than a true thermodynamic property of the channel and the charge-voltage curve is a true measure of the net-free energy of channel gating.

Automatic modeling of dynamic drug-hERG channel interactions using three voltage protocols and machine learning techniques: A simulation study

BackgroundAssessment of drug cardiac safety is critical in the development of new compounds and is commonly addressed by evaluating the half-maximal blocking concentration of the potassium human ether-à-go-go related gene (hERG) channels. However, recent works have evidenced that the modelling of drug-binding dynamics to hERG can help to improve early cardiac safety assessment. Our goal is to develop a methodology to automatically generate Markovian models of the drug-hERG channel interactions. MethodsThe training and the test sets consisted of 20800 and 5200 virtual drugs, respectively, distributed into 104 groups with different affinities and kinetics to the conformational states of the channel. In our system, drugs may bind to any state (individually or simultaneously), with different degrees of preference for a conformational state and the change of the conformational state of the drug bound channels may be restricted or allowed. To model such a wide range of possibilities, 12 Markovian chains are considered. Our approach uses the response of the drugs to our three previously developed voltage clamp protocols, which enhance the differences in the probabilities of occupying a certain conformational state of the channel (open, closed and inactivated). The computing tool is comprised of a classifier and a parameter optimizer and uses linear interpolation, support vector machines and a simplex method for function minimization. ResultsWe propose a novel methodology that automatically generates dynamic drug models using Markov model formulations and that elucidates the states where the drug binds and unbinds and the preferential binding state using data obtained from simple voltage clamp protocols that captures the preferential state-dependent binding properties, the relative affinities, trapping and non-trapping dynamics and the onset of IKr block. Overall, the tool correctly predicted the class of 92.04% of the drugs and the model provided by the tool accurately fitted the response of the target compound, the mean accuracy being 97.53%. Moreover, generation of the dynamic model of an IKr blocker from its response to our voltage clamp protocols usually takes less than an hour on a common desktop computer. ConclusionOur methodology could be very useful to model and simulate dynamic drug–hERG channel interactions. It would contribute to the improvement of the preclinical assessment of the proarrhythmic risk of drugs that inhibit IKr and the efficacy of antiarrhythmic IKr blockers.

An in silico-in vitro pipeline for drug cardiotoxicity screening identifies ionic pro-arrhythmia mechanisms.

Before advancing to clinical trials, new drugs are screened for their pro-arrhythmic potential using a method that is overly conservative and provides limited mechanistic insight. The shortcomings of this approach can lead to the mis-classification of beneficial drugs as pro-arrhythmic. An in silico-in vitro pipeline was developed to circumvent these shortcomings. A computational human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model was used as part of a genetic algorithm to design experiments, specifically electrophysiological voltage clamp (VC) protocols, to identify which of several cardiac ion channels were blocked during in vitro drug studies. Such VC data, along with dynamically clamped action potentials (AP), were acquired from iPSC-CMs before and after treatment with a control solution or a low- (verapamil), intermediate- (cisapride or quinine) or high-risk (quinidine) drug. Significant AP prolongation (a pro-arrhythmia marker) was seen in response to quinidine and quinine. The VC protocol identified block of IKr (a source of arrhythmias) by all strong IKr blockers, including cisapride, quinidine and quinine. The protocol also detected block of ICaL by verapamil and Ito by quinidine. Further demonstrating the power of the approach, the VC data uncovered a previously unidentified If block by quinine, which was confirmed with experiments using a HEK-293 expression system and automated patch-clamp. We developed an in silico-in vitro pipeline that simultaneously identifies pro-arrhythmia risk and mechanism of ion channel-blocking drugs. The approach offers a new tool for evaluating cardiotoxicity during preclinical drug screening.

FC025: Physiological Role of KV Channel in Ureter Smooth Muscle Cell Investigated Quantitatively by Electrophysiological Modeling

Abstract BACKGROUND AND AIMS Spontaneous peristaltic contraction disorder of the ureter smooth muscle (USM) is an important etiology of congenital giant ureter, congenital ureteropelvic junction deformity and non-obstructive hydronephrosis [1]. The USM cell action potential (AP) plays a key role in commencing USM contraction by allowing influx of Ca2+ from the extra cellular space [2]. Therefore, an in-depth quantitative investigation of the USM cell AP generation will assist in developing better pharmacological therapies for the USM abnormal contraction. The purpose of this in-silico study is to unfold the physiological role of Kv channel in modulating the USM AP generation. METHOD The model consists voltage- dependent Na+ currents (INa), hyperpolarization-activated cyclic nucleotide- gated current (Ih), voltage- dependent Ca2+ currents (ICa), Ca2+ dependent potassium channel (IBKCa) and voltage-dependent potassium channels (IKv) as the components of active electrophysiological characteristics [3]. All ion channels are represented by the Hodgkin–Huxley formalism. The maximum conductance of the is varied to investigate the physiological role of the Kv channel in modulating the USM AP generation. RESULTS The whole cell IKv current is generated by applying the voltage clamp protocol, where the membrane potential is stepped from a holding potential of –80 mV–60 mV for a duration of 500 ms. The half activation potential, half inactivation potential, activation slope factor and inactivation slope factors are: 43 mV, –76.1 mV, 15.4 mV and 6.5 mV, respectively, to fit the Boltzmann function equation. The model reveals that fast activation and inactivation properties are the essential feature of IKv for the USM cell. The resting membrane potential (RMP) is set at –50 mV. The AP was induced in the whole cell model by applying an external stimulus current (10–30 pA), as a brief square pulse of 500 ms duration. It is demonstrated that IKv opposes the Ca2+ current during the first 50 ms of the AP duration and then inactivated. It doesn't contribute to the repolarizing phase of the AP. CONCLUSION The plateau phase of the USM AP shows that the total AP duration much longer than that in other smooth muscle cells. It strongly supports the phenomenon is due to the activation of Ca2+-dependent K+ channel and inactivation of L-type Ca2+ channel. In ureter cells, the IKv does not contribute significantly to the repolarization of the AP, but it may regulate membrane excitability by opposing Ca2+ current activated around the threshold of the AP. Therefore, the agonist and antagonist of the IKv might be useful partially as new pharmacological targets for the USM abnormal contraction.

An in silico-in vitro pipeline for drug cardiotoxicity screening identifies ionic proarrhythmia mechanisms

Before advancing to clinical trials, new drugs are screened for their proarrhythmic potential using a method that is overly conservative and provides limited mechanistic insight. The shortcomings of this approach can lead to the misclassification of beneficial drugs as proarrhythmic. In this study, we develop an in silico-in vitro pipeline using mathematical models and human induced stem cell-derived cardiomyocytes (iPSC-CMs) to circumvent this problem. A computational iPSC-CM model is used with a genetic algorithm to design experiments, specifically electrophysiological voltage-clamp (VC) protocols to identify which of seven cardiac ion channels (IKr, INa, ICaL, Ito, IK1, If, IKs) are blocked during in vitro drug studies.

Creating Ion Channel Kinetic Models Using Cloud Computing.

Computational modeling of ion channels provides key insight into experimental electrophysiology results and can be used to connect channel dynamics to emergent phenomena observed at the tissue and organ levels. However, creation of these models requires substantial mathematical and computational background. This tutorial seeks to lower the barrier to creating these models by providing an automated pipeline for creating Markov models of an ion channel kinetics dataset. We start by detailing how to encode sample voltage-clamp protocols and experimental data into the program and its implementation in a cloud computing environment. We guide the reader on how to build a containerized instance, push the machine image, and finally run the routine on cluster nodes. While providing open-source code has become more standard in computational studies, this tutorial provides unprecedented detail on the use of the program and the creation of channel models, starting from inputting the raw experimental data. © 2022 Wiley Periodicals LLC. Basic Protocol: Creation of ion channel kinetic models with a cloud computing environment Alternate Protocol: Instructions for use in a standard high-performance compute cluster.

Intrinsic mechanisms in the gating of resurgent Na+ currents.

The resurgent component of the voltage-gated sodium current (INaR) is a depolarizing conductance, revealed on membrane hyperpolarizations following brief depolarizing voltage steps, which has been shown to contribute to regulating the firing properties of numerous neuronal cell types throughout the central and peripheral nervous systems. Although mediated by the same voltage-gated sodium (Nav) channels that underlie the transient and persistent Nav current components, the gating mechanisms that contribute to the generation of INaR remain unclear. Here, we characterized Nav currents in mouse cerebellar Purkinje neurons, and used tailored voltage-clamp protocols to define how the voltage and the duration of the initial membrane depolarization affect the amplitudes and kinetics of INaR. Using the acquired voltage-clamp data, we developed a novel Markov kinetic state model with parallel (fast and slow) inactivation pathways and, we show that this model reproduces the properties of the resurgent, as well as the transient and persistent, Nav currents recorded in (mouse) cerebellar Purkinje neurons. Based on the acquired experimental data and the simulations, we propose that resurgent Na+ influx occurs as a result of fast inactivating Nav channels transitioning into an open/conducting state on membrane hyperpolarization, and that the decay of INaR reflects the slow accumulation of recovered/opened Nav channels into a second, alternative and more slowly populated, inactivated state. Additional simulations reveal that extrinsic factors that affect the kinetics of fast or slow Nav channel inactivation and/or impact the relative distribution of Nav channels in the fast- and slow-inactivated states, such as the accessory Navβ4 channel subunit, can modulate the amplitude of INaR.

Lack of pro-arrhythmic potential for the novel soluble guanylate cyclase stimulator vericiguat: results from preclinical studies

Abstract Introduction Vericiguat is an orally-administered soluble guanylate cyclase stimulator, developed for the treatment of symptomatic chronic heart failure (HF) in adult patients who have had a previous decompensation event. At the maximum therapeutic dose of 10 mg once daily in patients with HF, the protein-unbound plasma concentrations of vericiguat and its major pharmacologically inactive N-glucuronide metabolite M-1 are approximately 18 nmol/l and 43 nmol/l, respectively. As part of an integrated risk assessment, vericiguat and its M-1 metabolite were characterised electrophysiologically in vivo and in vitro. This was performed according to the International Council for Harmonisation standard S7B guideline and to recent related “best practice” revisions (draft ICH E14/S7B Q&amp;A), being adopted as a result of the Comprehensive In Vitro Proarrhythmia Assay (CIPA) initiative. Purpose To assess the potential for a proarrhythmic risk from vericiguat and its M-1 metabolite in a series of preclinical studies. Methods The potential for proarrhythmic risk was investigated in conscious telemetered dogs and in a series of in vitro electrophysiological studies, including mechanistic ion channel studies, using both generally accepted and CIPA voltage-clamp protocols under conditions simulating normal and diseased physiological states. The ion channels studied were hERG, hNav1.5, hCav1.2, hKvLQT1/minK and hKv4.3. Transfected human embryonic kidney cell lines were used for the hERG, hNav1.5 and hKvLQT1/minK studies; transfected Chinese hamster ovary cell lines were used for the hCav1.2 and hKv4.3 studies. Results In dogs, administration of vericiguat as single oral doses was associated with dose-dependent decreases in arterial blood pressure (consistent with its mode of action) and compensatory increases in heart rate (Table 1). Heart rate-corrected QT (QTc) intervals were not prolonged by vericiguat to a clinically meaningful extent. Neither vericiguat nor its M-1 metabolite inhibited cardiac ion channels (hERG, hNav1.5, hCav1.2, hKvLQT1/minK and hKv4.3) at exposure multiples of &amp;gt;150-fold (Table 2). Conclusion There was no preclinical evidence of proarrhythmic risk from the in vitro (simulating normal and diseased physiological states) and in vivo assessment of vericiguat or its major N-glucuronide metabolite M-1. This integrated risk assessment of non-clinical data supports the conclusion that administration of vericiguat 10 mg once daily in humans is not associated with meaningful QTc prolongation. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Funding for this research was provided by Bayer AG, Berlin, Germany and Merck Sharp &amp; Dohme Corp., a subsidiary of Merck &amp; Co., Inc., Kenilworth, NJ, USA.

Attenuation of Native Hyperpolarization-Activated, Cyclic Nucleotide-Gated Channel Function by the Volatile Anesthetic Sevoflurane in Mouse Thalamocortical Relay Neurons

As thalamocortical relay neurons are ascribed a crucial role in signal propagation and information processing, they have attracted considerable attention as potential targets for anesthetic modulation. In this study, we analyzed the effects of different concentrations of sevoflurane on the excitability of thalamocortical relay neurons and hyperpolarization-activated, cyclic-nucleotide gated (HCN) channels, which play a decisive role in regulating membrane properties and rhythmic oscillatory activity. The effects of sevoflurane on single-cell excitability and native HCN channels were investigated in acutely prepared brain slices from adult wild-type mice with the whole-cell patch-clamp technique, using voltage-clamp and current-clamp protocols. Sevoflurane dose-dependently depressed membrane biophysics and HCN-mediated parameters of neuronal excitability. Respective half-maximal inhibitory and effective concentrations ranged between 0.30 (95% CI, 0.18–0.50) mM and 0.88 (95% CI, 0.40–2.20) mM. We witnessed a pronounced reduction of HCN dependent Ih current amplitude starting at a concentration of 0.45 mM [relative change at −133 mV; 0.45 mM sevoflurane: 0.85 (interquartile range, 0.79–0.92), n = 12, p = 0.011; 1.47 mM sevoflurane: 0.37 (interquartile range, 0.34–0.62), n = 5, p < 0.001] with a half-maximal inhibitory concentration of 0.88 (95% CI, 0.40–2.20) mM. In contrast, effects on voltage-dependent channel gating were modest with significant changes only occurring at 1.47 mM [absolute change of half-maximal activation potential; 1.47 mM: −7.2 (interquartile range, −10.3 to −5.8) mV, n = 5, p = 0.020]. In this study, we demonstrate that sevoflurane inhibits the excitability of thalamocortical relay neurons in a concentration-dependent manner within a clinically relevant range. Especially concerning its effects on native HCN channel function, our findings indicate substance-specific differences in comparison to other anesthetic agents. Considering the importance of HCN channels, the observed effects might mechanistically contribute to the hypnotic properties of sevoflurane.

The devil is in the C-tail: a NBCe1-carbonic anhydrase bicarbonate transport metabolon?

The concept of metabolons is well established in biochemistry as structural–metabolic complexes in cells that facilitate substrate channelling through the complex and is best exemplified by the citric acid cycle enzymes. Similarly, an arrangement of enzymes was suggested to promote the transport of acid/base equivalents across the plasma membrane by the Reithmeier group. The concept was supported by direct evidence of physical interaction of an acid/base transporter and carbonic anhydrase (Vince & Reithmeier, 1998) and emboldened by the demonstration of a functional advantage for such an arrangement called a ‘transport metabolon’ (Sterling et al. 2001). The direct protein interaction was first reported for the C-terminal of anion exchanger AE1 (band 3) and the cytosolic ubiquitous carbonic anhydrase II (CAII). Since then, the field has evolved to other acid/base transporters of the SLC4 gene family of HCO3− transporters, the Na+/H+ exchangers (NHE) and monocarboxylate transporters interacting with soluble or membrane-bound CA isozymes in various expression systems and native tissues. Discrepancies have since evolved both regarding the direct physical interaction of acid/base transporters and CA enzymes, and regarding the functional interaction between the individual molecules of such complexes. This ‘Perspectives’ article only addresses the case of the functional interaction of the human renal form of the electrogenic Na+:HCO3− cotransporter NBCe1-A (kNBCe1) and CAII when expressed in Xenopus laevis oocytes, but may prove highly relevant for the metabolon field as such. In one study, the NBCe1 function determined as the current slope conductance (Gm INBC) was not affected by injection of CAII protein or the expression of either NBCe1-CAII or eGFP-NBCe1-CAII fusion proteins (Lu et al. 2006). No effect of the cell-permeant CA inhibitor ethoxyzolamide (EZA) on the Gm INBC was observed. In a separate and opposing study, NBCe1 function assessed as the changes in membrane current (Im), the Na+ flux (d[Na+]i/dt), as well as the membrane conductance (Gm) was augmented by bovine CA protein injection or CAII co-expression (Becker & Deitmer 2007). The induced changes in current and Na+ fluxes were EZA-sensitive in this study. Thus, although the two studies applied similar methodological set-ups at first glance, the results were contrasting. Each paper is compelling to the reader, and both studies were carefully and elegantly performed. Moss and Boron (2020) first carefully replicate the findings by Lu et al. (2006) and then elegantly rule out differences in the amount of injected cRNA, CAII protein and eGFP tag as contributing factors to conflicting results by the two groups. Furthermore, they show that prolonged EZA exposure or the high EZA concentration by Lu et al. were not causing the inconsistency between the previous studies. It also appears that the variations in voltage-clamping protocols cannot explain the contradicting results per se (Moss & Boron 2020). Remarkably, the authors are able to closely replicate the fundamental observations by Becker & Deitmer (2007) that injection of bovine CA in oocytes expressing untagged NBCe1 increases the Na+ flux (d[Na+]i/dt)max as well as the membrane current Im, effects that are sensitive to CA inhibition by EZA. This is apparently only possible 1) in the absence of eGFP tag, 2) by bovine CA injection instead of human CAII, and 3) applying the prolonged -40mV voltage clamp and 10 s step duration of I-V ramps. Surprisingly, Moss and Boron demonstrate that injection of either human CAII or bovine CA alone in oocytes induces an electroneutral process that increases Na+ uptake and acid extrusion upon CO2-induced acid loading. The authors ascribe the effects to the induction of endogenous NHE activity, as this process is inhibited by EIPA. Interestingly, EIPA also prevents the EZA-sensitive change in Im and (d[Na+]i/dt)max induced by injection of bovine CA using the prolonged -40 mV voltage clamp protocol. Thus, Moss and Boron explain the discrepancy between the previous studies (Lu et al. 2006 vs. Becker & Deitmer 2007) by the induction of native NHE activity by exogenous CA in Xenopus oocytes. With the study by Moss and Boron (2020), the specific controversy regarding enhanced NBCe1 activity by CA in oocytes has most likely been fully resolved. The authors accomplished a systematic comparison of experimental approaches resulting in a study with high internal consistency presented in a respectful and transparent manner. The take-home message is that even the most careful studies by world-renowned laboratories can reach conflicting conclusions and that unexpected sources of discrepancies can be identified by hard and meticulous work. The findings by Moss and Boron (2020) should impact the broader transport metabolon field; however, the conclusions can probably not be extrapolated to all other cases of acid/base transporters, CA forms and cell types for which transport metabolons have been suggested. Hopefully, time will tell. None. Sole author. None.