Rapid depolarization and cytosolic calcium increase go hand-in-hand in mesophyll cells' ozone response.

Plant stress signalling involves bursts of reactive oxygen species (ROS), which can be mimicked by the application of acute pulses of ozone. Such ozone-pulses inhibit photosynthesis and trigger stomatal closure in a few minutes, but the signalling that underlies these responses remains largely unknown. We measured changes in Arabidopsis thaliana gas exchange after treatment with acute pulses of ozone and set up a system for simultaneous measurement of membrane potential and cytosolic calcium with the fluorescent reporter R-GECO1. We show that within 1 min, prior to stomatal closure, O3 triggered a drop in whole-plant CO2 uptake. Within this early phase, O3 pulses (200-1000 ppb) elicited simultaneous membrane depolarization and cytosolic calcium increase, whereas these pulses had no long-term effect on either stomatal conductance or photosynthesis. In contrast, pulses of 5000 ppb O3 induced cell death, systemic Ca2+ signals and an irreversible drop in stomatal conductance and photosynthetic capacity. We conclude that mesophyll cells respond to ozone in a few seconds by distinct pattern of plasma membrane depolarizations accompanied by an increase in the cytosolic calcium ion (Ca2+ ) level. These responses became systemic only at very high ozone concentrations. Thus, plants have rapid mechanism to sense and discriminate the strength of ozone signals.

A systematic strategy for estimating hERG block potency and its implications in a new cardiac safety paradigm.

INTRODUCTION: hERG block potency is widely used to calculate a drug's safety margin against its torsadogenic potential. Previous studies are confounded by use of different patch clamp electrophysiology protocols and a lack of statistical quantification of experimental variability. Since the new cardiac safety paradigm being discussed by the International Council for Harmonisation promotes a tighter integration of nonclinical and clinical data for torsadogenic risk assessment, a more systematic approach to estimate the hERG block potency and safety margin is needed. METHODS: A cross-industry study was performed to collect hERG data on 28 drugs with known torsadogenic risk using a standardized experimental protocol. A Bayesian hierarchical modeling (BHM) approach was used to assess the hERG block potency of these drugs by quantifying both the inter-site and intra-site variability. A modeling and simulation study was also done to evaluate protocol-dependent changes in hERG potency estimates. RESULTS: A systematic approach to estimate hERG block potency is established. The impact of choosing a safety margin threshold on torsadogenic risk evaluation is explored based on the posterior distributions of hERG potency estimated by this method. The modeling and simulation results suggest any potency estimate is specific to the protocol used. DISCUSSION: This methodology can estimate hERG block potency specific to a given voltage protocol. The relationship between safety margin thresholds and torsadogenic risk predictivity suggests the threshold should be tailored to each specific context of use, and safety margin evaluation may need to be integrated with other information to form a more comprehensive risk assessment.

Repolarization studies using human stem cell-derived cardiomyocytes: Validation studies and best practice recommendations.

Human stem cell-derived cardiomyocytes (hSC-CMs) hold great promise as in vitro models to study the electrophysiological effects of novel drug candidates on human ventricular repolarization. Two recent large validation studies have demonstrated the ability of hSC-CMs to detect drug-induced delayed repolarization and "cellrhythmias" (interrupted repolarization or irregular spontaneous beating of myocytes) linked to Torsade-de-Pointes proarrhythmic risk. These (and other) studies have also revealed variability of electrophysiological responses attributable to differences in experimental approaches and experimenter, protocols, technology platforms used, and pharmacologic sensitivity of different human-derived models. Thus, when evaluating drug-induced repolarization effects, there is a need to consider 1) the advantages and disadvantages of different approaches, 2) the need for robust functional characterization of hSC-CM preparations to define "fit for purpose" applications, and 3) adopting standardized best practices to guide future studies with evolving hSC-CM preparations. Examples provided and suggested best practices are instructional in defining consistent, reproducible, and interpretable "fit for purpose" hSC-CM-based applications. Implementation of best practices should enhance the clinical translation of hSC-CM-based cell and tissue preparations in drug safety evaluations and support their growing role in regulatory filings.

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

BACKGROUND AND OBJECTIVE: Assessment 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. METHODS: Experiments 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. RESULTS: The 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. CONCLUSIONS: These 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.

Chamber-specific contractile responses of atrial and ventricular hiPSC-cardiomyocytes to GPCR and ion channel targeting compounds: A microphysiological system for cardiac drug development.

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) have found utility for conducting in vitro drug screening and disease modelling to gain crucial insights into pharmacology or disease phenotype. However, diseases such as atrial fibrillation, affecting >33 M people worldwide, demonstrate the need for cardiac subtype-specific cells. Here, we sought to investigate the base characteristics and pharmacological differences between commercially available chamber-specific atrial or ventricular hiPSC-CMs seeded onto ultra-thin, flexible PDMS membranes to simultaneously measure contractility in a 96 multi-well format. We investigated the effects of GPCR agonists (acetylcholine and carbachol), a Ca2+ channel agonist (S-Bay K8644), an HCN channel antagonist (ivabradine) and K+ channel antagonists (4-AP and vernakalant). We observed differential effects between atrial and ventricular hiPSC-CMs on contractile properties including beat rate, beat duration, contractile force and evidence of arrhythmias at a range of concentrations. As an excerpt of the compound analysis, S-Bay K8644 treatment showed an induced concentration-dependent transient increase in beat duration of atrial hiPSC-CMs, whereas ventricular cells showed a physiological increase in beat rate over time. Carbachol treatment produced marked effects on atrial cells, such as increased beat duration alongside a decrease in beat rate over time, but only minimal effects on ventricular cardiomyocytes. In the context of this chamber-specific pharmacology, we not only add to contractile characterization of hiPSC-CMs but propose a multi-well platform for medium-throughput early compound screening. Overall, these insights illustrate the key pharmacological differences between chamber-specific cardiomyocytes and their application on a multi-well contractility platform to gain insights for in vitro cardiac liability studies and disease modelling.

Reliable identification of cardiac conduction abnormalities in drug discovery using automated patch clamp II: Best practices for Nav1.5 peak current in a high throughput screening environment.

INTRODUCTION: For reliable identification of cardiac safety risk, compounds should be screened for activity on cardiac ion channels in addition to hERG, including NaV1.5 and CaV1.2. We identified different parameters that might affect IC50s of compounds on NaV1.5 peak and late currents recorded using automated patch clamp (APC) and suggest outlines for best practices. METHODS: APC instruments SyncroPatch 384 and Patchliner were used to record NaV1.5 peak and late current. Up to 24 CiPA compounds were used to investigate effects of voltage protocol, holding potential (-80 mV or - 95 mV) and temperature (23 ± 1 °C or 36 ± 1 °C) on IC50 values on hNaV1.5 overexpressed in HEK or CHO cells either as frozen cells or running cultures. RESULTS: The IC50s of 18 compounds on the NaV1.5 peak current recorded on the SyncroPatch 384 using the CiPA step-ramp protocol correlated well with the literature. The use of frozen or cultured cells did not affect IC50s but voltage protocol and holding potential did cause differences in IC50 values. Temperature can affect Vhalf of inactivation and also compound potency. A compound incubation time of 5-6 min was sufficient for most compounds, however slow acting compounds such as terfenadine required longer to reach maximum effect. DISCUSSION: We conclude that holding potential, voltage protocol and temperature can affect IC50 values and recommend the use of the CiPA step-ramp protocol at physiological temperature to record NaV1.5 peak and late currents for cardiac safety. Further recommendations include: a minimum compound incubation time of 5 min, a replicate number of 4 and the use of positive and negative controls for reliable IC50s.

Integration of mechanical conditioning into a high throughput contractility assay for cardiac safety assessment.

INDUCTION: Despite increasing acceptance of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in safety pharmacology, controversy remains about the physiological relevance of existing in vitro models for their mechanical testing. We hypothesize that existing signs of immaturity of the cell models result from an improper mechanical environment. With the presented study, we aimed at validating the newly developed FLEXcyte96 technology with respect to physiological responses of hiPSC-CMs to pharmacological compounds with known inotropic and/or cardiotoxic effects. METHODS: hiPSC-CMs were cultured in a 96-well format on hyperelastic silicone membranes imitating their native mechanical environment. Cardiomyocyte contractility was measured contact-free by application of capacitive displacement sensing of the cell-membrane biohybrids. Acute effects of positive inotropic compounds with distinct mechanisms of action were examined. Additionally, cardiotoxic effects of tyrosine kinase inhibitors and anthracyclines were repetitively examined during repeated exposure to drug concentrations for up to 5 days. RESULTS: hiPSC-CMs grown on biomimetic membranes displayed increased contractility responses to isoproterenol, S-Bay K8644 and omecamtiv mecarbil without the need for additional stimulation. Tyrosine kinase inhibitor erlotinib, vandetanib, nilotinib, gefitinib, A-674563 as well as anthracycline idarubicin showed the expected cardiotoxic effects, including negative inotropy and induction of proarrhythmic events. DISCUSSION: We conclude that the FLEXcyte 96 system is a reliable high throughput tool for invitro cardiac contractility research, providing the user with data obtained under physiological conditions which resemble the native environment of human heart tissue. We showed that the results obtained for both acute and sub-chronic compound administration are consistent with the respective physiological responses in humans.

Reliable identification of cardiac liability in drug discovery using automated patch clamp: Benchmarking best practices and calibration standards for improved proarrhythmic assessment.

INTRODUCTION: Screening compounds for activity on the hERG channel using patch clamp is a crucial part of safety testing. Automated patch clamp (APC) is becoming widely accepted as an alternative to manual patch clamp in order to increase throughput whilst maintaining data quality. In order to standardize APC experiments, we have investigated the effects on IC50 values under different conditions using several devices across multiple sites. METHODS: APC instruments SyncroPatch 384i, SyncroPatch 384PE and Patchliner, were used to record hERG expressed in HEK or CHO cells. Up to 27 CiPA compounds were used to investigate effects of voltage protocol, incubation time, labware and time between compound preparation and experiment on IC50 values. RESULTS: All IC50 values of 21 compounds recorded on the SyncroPatch 384PE correlated well with IC50 values from the literature (Kramer et al., 2013) regardless of voltage protocol or labware, when compounds were used immediately after preparation, but potency of astemizole decreased if prepared in Teflon or polypropylene (PP) compound plates 2-3 h prior to experiments. Slow acting compounds such as dofetilide, astemizole, and terfenadine required extended incubation times of at least 6 min to reach steady state and therefore, stable IC50 values. DISCUSSION: Assessing the influence of different experimental conditions on hERG assay reliability, we conclude that either the step-ramp protocol recommended by CiPA or a standard 2-s step-pulse protocol can be used to record hERG; a minimum incubation time of 5 min should be used and although glass, Teflon, PP or polystyrene (PS) compound plates can be used for experiments, caution should be taken if using Teflon, PS or PP vessels as some adsorption can occur if experiments are not performed immediately after preparation. Our recommendations are not limited to the APC devices described in this report, but could also be extended to other APC devices.

Publisher Correction: Cross-site and cross-platform variability of automated patch clamp assessments of drug effects on human cardiac currents in recombinant cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cross-site and cross-platform variability of automated patch clamp assessments of drug effects on human cardiac currents in recombinant cells.

Automated patch clamp (APC) instruments enable efficient evaluation of electrophysiologic effects of drugs on human cardiac currents in heterologous expression systems. Differences in experimental protocols, instruments, and dissimilar site procedures affect the variability of IC50 values characterizing drug block potency. This impacts the utility of APC platforms for assessing a drug's cardiac safety margin. We determined variability of APC data from multiple sites that measured blocking potency of 12 blinded drugs (with different levels of proarrhythmic risk) against four human cardiac currents (hERG [IKr], hCav1.2 [L-Type ICa], peak hNav1.5, [Peak INa], late hNav1.5 [Late INa]) with recommended protocols (to minimize variance) using five APC platforms across 17 sites. IC50 variability (25/75 percentiles) differed for drugs and currents (e.g., 10.4-fold for dofetilide block of hERG current and 4-fold for mexiletine block of hNav1.5 current). Within-platform variance predominated for 4 of 12 hERG blocking drugs and 4 of 6 hNav1.5 blocking drugs. hERG and hNav1.5 block. Bland-Altman plots depicted varying agreement across APC platforms. A follow-up survey suggested multiple sources of experimental variability that could be further minimized by stricter adherence to standard protocols. Adoption of best practices would ensure less variable APC datasets and improved safety margins and proarrhythmic risk assessments.

A general procedure to select calibration drugs for lab-specific validation and calibration of proarrhythmia risk prediction models: An illustrative example using the CiPA model.

INTRODUCTION: In response to the ongoing shift of the regulatory cardiac safety paradigm, a recent White Paper proposed general principles for developing and implementing proarrhythmia risk prediction models. These principles included development strategies to validate models, and implementation strategies to ensure a model developed by one lab can be used by other labs in a consistent manner in the presence of lab-to-lab experimental variability. While the development strategies were illustrated through the validation of the model under the Comprehensive In vitro Proarrhythmia Assay (CiPA), the implementation strategies have not been adopted yet. METHODS: The proposed implementation strategies were applied to the CiPA model by performing a sensitivity analysis to identify a subset of calibration drugs that were most critical in determining the classification thresholds for proarrhythmia risk prediction. RESULTS: The selected calibration drugs were able to recapitulate classification thresholds close to those calculated from the full list of CiPA drugs. Using an illustrative dataset it was shown that a new lab could use these calibration drugs to establish its own classification thresholds (lab-specific calibration), and verify that the model prediction accuracy in the new lab is comparable to that in the original lab where the model was developed (lab-specific validation). DISCUSSION: This work used the CiPA model as an example to illustrate how to adopt the proposed model implementation strategies to select calibration drugs and perform lab-specific calibration and lab-specific validation. Generic in nature, these strategies could be generally applied to different proarrhythmia risk prediction models using various experimental systems under the new paradigm.

Cross - site comparison of excitation-contraction coupling using impedance and field potential recordings in hiPSC cardiomyocytes.

INTRODUCTION: Since 2005 the S7B and E14 guidances from ICH and FDA have been in place to assess a potential drug candidate's ability to cause long QT syndrome. To refine these guidelines, the FDA proposed the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative, where the assessment of drug effects on cardiac repolarization was one subject of investigation. Within the myocyte validation study, effects of pharmaceutical compounds on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were assessed and this article will focus on the evaluation of the proarrhythmic potential of 23 blinded drugs in four hiPSC-CM cell lines. METHODS: Experiments were performed on the CardioExcyte 96 at different sites. A combined readout of contractility (via impedance) and electrophysiology endpoints (field potentials) was performed. RESULTS: Our data demonstrates that hERG blockers such as dofetilide and further high risk categorized compounds prolong the field potential duration. Arrhythmia were detected in both impedance as well as field potential recordings. Intermediate risk compounds induced arrhythmia in almost all cases at the highest dose. In the case of low risk compounds, either a decrease in FPDmax was observed, or not a significant change from pre-addition control values. DISCUSSION: With exceptions, hiPSC-CMs are sensitive and exhibit at least 10% delayed or shortened repolarization from pre-addition values and arrhythmia after drug application and thus can provide predictive cardiac electrophysiology data. The baseline electrophysiological parameters vary between iPS cells from different sources, therefore positive and negative control recordings are recommended.

A Hybrid Model for Safety Pharmacology on an Automated Patch Clamp Platform: Using Dynamic Clamp to Join iPSC-Derived Cardiomyocytes and Simulations of Ik1 Ion Channels in Real-Time.

An important aspect of the Comprehensive In Vitro Proarrhythmia Assay (CiPA) proposal is the use of human stem cell-derived cardiomyocytes and the confirmation of their predictive power in drug safety assays. The benefits of this cell source are clear; drugs can be tested in vitro on human cardiomyocytes, with patient-specific genotypes if needed, and differentiation efficiencies are generally excellent, resulting in a virtually limitless supply of cardiomyocytes. There are, however, several challenges that will have to be surmounted before successful establishment of hSC-CMs as an all-round predictive model for drug safety assays. An important factor is the relative electrophysiological immaturity of hSC-CMs, which limits arrhythmic responses to unsafe drugs that are pro-arrhythmic in humans. Potentially, immaturity may be improved functionally by creation of hybrid models, in which the dynamic clamp technique joins simulations of lacking cardiac ion channels (e.g., IK1) with hSC-CMs in real-time during patch clamp experiments. This approach has been used successfully in manual patch clamp experiments, but throughput is low. In this study, we combined dynamic clamp with automated patch clamp of iPSC-CMs in current clamp mode, and demonstrate that IK1 conductance can be added to iPSC-CMs on an automated patch clamp platform, resulting in an improved electrophysiological maturity.

Mechanisms of acquired long QT syndrome in patients with propionic academia.

BACKGROUND: Propionic acidemia (PROP) is a rare metabolic disorder caused by deficiency of propionyl-CoA carboxylase. PROP patients demonstrate QT prolongations associated with ventricular tachycardia and syncopes. Mechanisms responsible for this acquired long QT syndrome (acqLQTS) are unknown. OBJECTIVE: The aim of the study was to investigate acute and chronic effects of metabolites accumulating in PROP patients on major repolarizing potassium currents (IKs and IKr) and their channel subunits. METHODS: Voltage clamp studies were performed in CHO-KCNQ1/KCNE1 or HEK-KCNH2 cells to determine effects of propionic acid (PA; 1-10 mM), propionylcarnitine (PC; 25 µM-10 mM), methylcitrate (MC; 25 µM-10 mM), 0.2 M phosphate buffer (PB), or patient serum on IKs and IKr currents. Metabolite effects on action potentials were recorded in current clamp mode in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). Protein expression of α- and ß-subunits of IKs (KCNQ1/KCNE1) and IKr (KCNH2) was evaluated with Western blots. RESULTS: Acute application of PA, PC, MC, and patient serum had no direct effect on net IKr densities (and KCNH2 expression), although it changed IKr gating kinetics. In contrast, PA, PC, MC, and patient serum all reduced IKs-tail (-67% ± 4.2%, -27% ± 6.7%, -16% ± 6.3%, -42.8% ± 5.15; P < .001) and IKs-end pulse currents. PA significantly prolonged action potential duration (APD) in hiPSC-CM and QT interval in wild-type but not in LQT1 rabbits lacking IKs. Moreover, PC and MC (1 mM) decreased KCNQ1 protein expression (relative density: 0.58 ± 0.08 and 0.16 ± 0.05; P < .01). Chronic exposure to 10 mM PA, in contrast, increased KCNQ1 5.4-fold (P < .001) owing to decreased protein degradation. CONCLUSION: Acute reduction of IKs by PROP metabolites may be responsible for APD prolongation and acqLQTS observed in PROP patients.

Identification of Drug-Drug Interactions In Vitro: A Case Study Evaluating the Effects of Sofosbuvir and Amiodarone on hiPSC-Derived Cardiomyocytes.

Drug-drug interactions pose a difficult drug safety problem, given the increasing number of individuals taking multiple medications and the relative complexity of assessing the potential for interactions. For example, sofosbuvir-based drug treatments have significantly advanced care for hepatitis C virus-infected patients, yet recent reports suggest interactions with amiodarone may cause severe symptomatic bradycardia and thus limit an otherwise extremely effective treatment. Here, we evaluated the ability of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) to recapitulate the interaction between sofosbuvir and amiodarone in vitro, and more generally assessed the feasibility of hiPSC-CMs as a model system for drug-drug interactions. Sofosbuvir alone had negligible effects on cardiomyocyte electrophysiology, whereas the sofosbuvir-amiodarone combination produced dose-dependent effects beyond that of amiodarone alone. By comparison, GS-331007, the primary circulating metabolite of sofosbuvir, had no effect alone or in combination with amiodarone. Further mechanistic studies revealed that the sofosbuvir-amiodarone combination disrupted intracellular calcium (Ca2+) handling and cellular electrophysiology at pharmacologically relevant concentrations, and mechanical activity at supra-pharmacological (30x Cmax) concentrations. These effects were independent of the common mechanisms of direct ion channel block and P-glycoprotein activity. These results support hiPSC-CMs as a comprehensive, yet scalable model system for the identification and evaluation of cardioactive pharmacodynamic drug-drug interactions.

New easy-to-use hybrid system for extracellular potential and impedance recordings.

The need for predictive, in vitro cardiac safety screening drives further development of automated, high-throughput-compatible drug evaluation based on cardiac cell preparations. Recently, pluripotent stem cells are evaluated as a new, more predictive model for cardiovascular risk assessment pertaining to in vitro assays. We present a new screening platform, the CardioExcyte 96, a hybrid instrument that combines impedance (cell contractility) with extracellular field potential (EFP) recordings. The electrophysiological measurements are noninvasive, label free and have a temporal resolution of 1 ms. This hybrid technology addresses the lack of easy-to-use high-throughput screening for in vitro assays and permits the reliable investigation of short- and long-term pharmacological effects. Several models of cardiomyocyte preparations were successfully validated for use with the CardioExcyte96. Furthermore, the pharmacological effects of a number of reference compounds were evaluated. Compound effects on cell monolayers of human-induced pluripotent stem cell-derived cardiomyocytes are evaluated using a quasi-simultaneous hybrid recording mode that combines impedance and EFP readouts. A specialized software package for rapid data handling and real-time analysis was developed, which allows for comprehensive investigation of the cellular beat signal. Combining impedance readouts of cell contractility and EFP (microelectrode array-like) recordings, the system opens up new possibilities in the field of in vitro cardiac safety assessment.

State-of-the-art automated patch clamp: heat activation, action potentials, and high throughput in ion channel screening.

A successful robotic approach of the patch clamp technique is based on planar patch clamp chips where a glass pipette, as used in conventional patch clamping, is replaced by a thin planar glass sheet with a small hole in the middle. Automated patch clamp (APC) systems utilizing this chip design offer higher throughput capabilities and ease of use and thus have become common in basic research, drug development, and safety screening. Further development of existing devices and introduction of new systems widen the range of possible experiments and increase throughput. Here, two features with different areas of applications that meet the needs of drug discovery researchers and basic researchers alike are described. The utilized system is a medium throughput APC device capable of recording up to eight cells simultaneously. The temperature control capability and the possibility to perform recordings not only in the voltage clamp but also in the current clamp mode are described in detail. Since eight recordings can be generated in parallel without compromising data quality, reliable and cost-effective and time-effective screening of compounds against ion channels using voltage clamp and current clamp electrophysiology can be performed.

Automated Patch Clamp Analysis of nAChα7 and Na(V)1.7 Channels.

Automated patch clamp devices are now commonly used for studying ion channels. A useful modification of this approach is the replacement of the glass pipet with a thin planar glass layer with a small hole in the middle. Planar patch clamp devices, such as the three described in this unit, are overtaking glass pipets in popularity because they increase throughput, are easier to use, provide for the acquisition of high-quality and information-rich data, and allow for rapid perfusion and temperature control. Covered in this unit are two challenging targets in drug discovery: voltage-gated sodium subtype 1.7 (Na(V)1.7) and nicotinic acetylcholine α7 receptors (nAChα7R). Provided herein are protocols for recording activation and inactivation kinetics of Na(V)1.7, and activation and allosteric modulation of nAChα7R.