RESUMEN
Ion channels have gained increased interest as therapeutic targets over recent years, since a growing number of human and animal diseases have been attributed to defects in ion channel function. Potassium channels are the largest and most diverse family of ion channels. Pharmaceutical agents such as Glibenclamide, an inhibitor of K(ATP) channel activity which promotes insulin release, have been successfully sold on the market for many years. So far, only a small group of the known ion channels have been addressed as potential drug targets. The functional testing of drugs on these ion channels has always been the bottleneck in the development of these types of pharmaceutical compounds.New generations of automated patch clamp screening platforms allow a higher throughput for drug testing and widen this bottleneck. Due to their planar chip design not only is a higher throughput achieved, but new applications have also become possible. One of the advantages of planar patch clamp is the possibility of perfusing the intracellular side of the membrane during a patch clamp experiment in the whole-cell configuration. Furthermore, the extracellular membrane remains accessible for compound application during the experiment.Internal perfusion can be used not only for patch clamp experiments with cell membranes, but also for those with artificial lipid bilayers. In this chapter we describe how internal perfusion can be applied to potassium channels expressed in Jurkat cells, and to Gramicidin channels reconstituted in a lipid bilayer.
Asunto(s)
Electrofisiología/métodos , Canales KATP/fisiología , Técnicas de Placa-Clamp , Células 3T3 , Animales , Automatización , Células CHO , Línea Celular , Cricetinae , Cricetulus , Eritrocitos/efectos de los fármacos , Eritrocitos/fisiología , Gramicidina/farmacología , Humanos , Canales Iónicos/efectos de los fármacos , Canales Iónicos/fisiología , Canales KATP/efectos de los fármacos , Riñón , Liposomas , Ratones , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Ratas , Linfocitos T/efectos de los fármacos , Linfocitos T/fisiologíaRESUMEN
The patch-clamp technique is the state-of-the-art technology for the study of a large class of membrane proteins called ion channels. Ion channels mediate electrical current flow, have crucial roles in cellular physiology, and are important drug targets. However, patch clamping is a laborious process requiring a skilled experimenter and is, therefore, not compatible with the high throughput needed in drug development. The solution for automated and parallel patch-clamp measurements that is provided by microchip technology is presented here.
Asunto(s)
Dispositivos Laboratorio en un Chip , Técnicas de Placa-Clamp/instrumentación , Animales , Humanos , Canales Iónicos/fisiologíaRESUMEN
INTRODUCTION: Chip-based automated patch clamp systems are widely used in drug development and safety pharmacology, allowing for high quality, high throughput screening at standardized experimental conditions. The merits of automation generally come at the cost of large amounts of cells needed, since cells are not targeted individually, but randomly positioned onto the chip aperture from cells in suspension. While cell usage is of little concern when using standard cell lines such as CHO or HEK cells, it becomes a crucial constraint with cells of limited availability, such as primary or otherwise rare and expensive cells, like induced pluripotent stem (IPS) cell-derived cardiomyocytes or neurons. METHODS: We established application protocols for CHO cells, IPS cell-derived neurons (iCell® Neurons, Cellular Dynamics International), cardiomyocytes (Cor.4U®, Axiogenesis) and pancreatic islet cells, minimizing cell usage for automated patch clamp recordings on Nanion's Patchliner. Use of 5 µl cell suspension per well for densities between 55,000 cells/ml and 400,000 cells/ml depending on cell type resulted in good cell capture. RESULTS: We present a new cell application procedure optimized for the Patchliner achieving>80% success rates for using as little as 300 to 2000 cells per well depending on cell type. We demonstrate that this protocol works for standard cell lines, as well as for stem cell-derived neurons and cardiomyocytes, and for primary pancreatic islet cells. We present recordings for these cell types, demonstrating that high data quality is not compromised by altered cell application. DISCUSSION: Our new cell application procedure achieves high success rates with unprecedentedly low cell numbers. Compared to other standard automated patch clamp systems we reduced the average amount of cells needed by more than 150 times. Reduced cell usage crucially improves cost efficiency for expensive cells and opens up automated patch clamp for primary cells of limited availability.
Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/citología , Neuronas/citología , Técnicas de Placa-Clamp/métodos , Animales , Automatización , Células CHO/citología , Cricetinae , Cricetulus , Humanos , Islotes Pancreáticos/citología , Ratones , Técnicas de Placa-Clamp/economíaRESUMEN
Neurons derived from human-induced pluripotent stem cells were characterized using manual and automated patch-clamp recordings. These cells expressed voltage-gated Na(+) (Na(v)), Ca(2+) (Ca(v)), and K(+) (K(v)) channels as expected from excitable cells. The Na(v) current was TTX sensitive, IC(50) = 12 ± 6 nM (n = 5). About 50% of the Ca(v) current was blocked by 10 µM of the L-type channel blocker nifedipine. Two populations of the K(v) channel were present in different proportions: an inactivating (A-type) and a noninactivating type. The A-type current was sensitive to 4-AP and TEA (IC(50) = 163 ± 93 µM; n = 3). Application of γ-aminobutyric acid (GABA) activated a current sensitive to the GABA(A) receptor antagonist bicuculline, IC(50) = 632 ± 149 nM (n = 5). In both devices, comparable action potentials were generated in the current clamp. With unbiased, automated patch clamp, about 40% of the cells expressed Na(v) currents, whereas visual guidance in manual patch clamp provided almost a 100% success rate of patching "excitable cells." These results show high potential for pluripotent stem cell-derived neurons as a useful model for drug discovery, in combination with automated patch-clamp recordings for high-throughput and high-quality drug assessments at human neuronal ion channels in their correct cellular background.
Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Activación del Canal Iónico/efectos de los fármacos , Canales Iónicos/metabolismo , Neuronas/metabolismo , Técnicas de Placa-Clamp/métodos , Bicuculina/farmacología , Canales de Calcio/metabolismo , Diferenciación Celular , Descubrimiento de Drogas/métodos , Humanos , Concentración 50 Inhibidora , Activación del Canal Iónico/fisiología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neuronas/efectos de los fármacos , Nifedipino/farmacología , Canales de Potasio con Entrada de Voltaje/metabolismo , Tetrodotoxina/farmacología , Canales de Sodio Activados por Voltaje/metabolismo , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Cardiovascular side effects are critical in drug development and have frequently led to late-stage project terminations or even drug withdrawal from the market. Physiologically relevant and predictive assays for cardiotoxicity are hence strongly demanded by the pharmaceutical industry. To identify a potential impact of test compounds on ventricular repolarization, typically a variety of ion channels in diverse heterologously expressing cells have to be investigated. Similar to primary cells, in vitro-generated stem cell-derived cardiomyocytes simultaneously express cardiac ion channels. Thus, they more accurately represent the native situation compared with cell lines overexpressing only a single type of ion channel. The aim of this study was to determine if stem cell-derived cardiomyocytes are suited for use in an automated patch clamp system. The authors show recordings of cardiac ion currents as well as action potential recordings in readily available stem cell-derived cardiomyocytes. Besides monitoring inhibitory effects of reference compounds on typical cardiac ion currents, the authors revealed for the first time drug-induced modulation of cardiac action potentials in an automated patch clamp system. The combination of an in vitro cardiac cell model with higher throughput patch clamp screening technology allows for a cost-effective cardiotoxicity prediction in a physiologically relevant cell system.
Asunto(s)
Productos Biológicos/efectos adversos , Evaluación Preclínica de Medicamentos/métodos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/metabolismo , Corazón/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Miocitos Cardíacos/efectos de los fármacos , Técnicas de Placa-Clamp , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Automatización de Laboratorios , Productos Biológicos/farmacología , Diferenciación Celular , Células Cultivadas , Humanos , Canales Iónicos/efectos de los fármacos , Canales Iónicos/metabolismo , Transporte Iónico/efectos de los fármacos , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Células Madre/citología , Células Madre/metabolismoRESUMEN
Ion channels are essential in a wide range of cellular functions and their malfunction underlies many disease states making them important targets in drug discovery. The availability of standardized cell lines expressing ion channels of interest lead to the development of diverse automated patch clamp (APC) systems with high-throughput capabilities. These systems are now available for drug screening, but there are limitations in the application range. However, further development of existing devices and introduction of new systems widen the range of possible experiments and increase throughput. The addition of well controlled and fast solution exchange, temperature control and the availability of the current clamp mode are required to analyze standard cell lines and excitable cells such as stem cell-derived cardiomyocytes in a more physiologically relevant environment. Here we describe two systems with different areas of applications that meet the needs of drug discovery researchers and basic researchers alike. The here utilized medium throughput APC device is a planar patch clamp system capable of recording up to eight cells simultaneously. Features such as temperature control and recordings in the current clamp mode are described here. Standard cell lines and excitable cells such as stem cell-derived cardiomyocytes have been used in the voltage clamp and current clamp modes with the view to finding new drug candidates and safety testing methods in a more physiologically relevant environment. The high-throughput system used here is a planar patch clamp screening platform capable of recording from 96 cells in parallel and offers a throughput of 5000 data points per day. Full dose response curves can be acquired from individual cells reducing the cost per data point. The data provided reveals the suitability and relevance of both APC platforms for drug discovery, ion channel research, and safety testing.
RESUMEN
Ion channel dysfunction is known to underlie several acute and chronic disorders and, therefore, ion channels have gained increased interest as drug targets. During the past decade, ion channel screening platforms have surfaced that enable high throughput drug screening from a more functional perspective. These two factors taken together have further inspired the development of more refined screening platforms, such as the automated patch clamp platforms described in this article. Approximately six years ago, Nanion introduced its entry level device for automated patch clamping - the Port-a-Patch. With this device, Nanion offers the world's smallest patch-clamp workstation, whilst greatly simplifying the experimental procedures. This makes the patch clamp technique accessible to researchers and technicians regardless of previous experience in electrophysiology. The same flexibility and high data quality is achieved in a fully automated manner with the Patchliner, Nanion's higher throughput patch clamp workstation. The system utilizes a robotic liquid handling environment for fully automated application of solutions, cells and compounds. The NPC-16 chips come in a sophisticated, yet simplistic, microfluidic cartridge, which allow for fast and precise perfusion. In this way, full concentration response curves are easily obtained. The Port-a-Patch and Patchliner workstations from Nanion are valuable tools for target validation, secondary screening and safety pharmacology (for example hERG and Nav1.5 safety screening). They are widely used in drug development efforts by biotechnological and pharmaceutical companies, as well as in basic and applied biophysical research within academia.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Electrofisiología/instrumentación , Canales Iónicos/efectos de los fármacos , Técnicas de Placa-Clamp/instrumentación , Evaluación Preclínica de Medicamentos/instrumentación , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , HumanosRESUMEN
Efficient high resolution techniques are required for screening efforts and research targeting ion channels. The conventional patch clamp technique, a high resolution but low efficiency technique, has been established for 25 years. Recent advances have opened up new possibilities for automated patch clamping. This new technology meets the need of drug developers for higher throughput and facilitates new experimental approaches in ion channel research. Specifically, Nanion's electrophysiology workstations, the Port-a-Patch and the Patchliner, have been successfully introduced as high-quality automated patch clamp platforms for industry as well as academic users. Both platforms give high quality patch clamp recordings, capable of true giga-seals and stable recordings, accessible to the user without the need for years of practical training. They also offer sophisticated experimental possibilities, such as accurate and fast ligand application, temperature control and internal solution exchange. This article describes the chip-based patch clamp technology and its usefulness in ion channel drug screening and academic research.
Asunto(s)
Canales Iónicos/metabolismo , Técnicas de Placa-Clamp/instrumentación , Técnicas de Placa-Clamp/métodos , Automatización/instrumentación , Automatización/métodos , Línea Celular , Electrofisiología/instrumentación , Electrofisiología/métodos , HumanosRESUMEN
Light is a central regulator of plant growth and development. Among the processes triggered by blue and UV-A light, phototropism, stomatal movement, and chloroplast orientation rely on the activation of blue-light receptors known as phototropins. So far, these photoreceptors constitute a class of light receptor kinases unique to the plant kingdom. In Arabidopsis thaliana, the two members phot1 and phot2 have been shown to display partially overlapping functions. Up to now little is known about the signaling cascade, which links these phototropins to the physiological responses downstream of blue-light perception. Here, we show that on illumination with blue light, but not red light, voltage-dependent and calcium-permeable channels activate in the plasma membrane of mesophyll cells. Blue-light stimulation in the presence of the photosynthetic electron transport inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, indicates that blue-light receptors rather than photosynthesis control channel activity. Sensitivity toward the protein kinase inhibitor K252a further pointed to the possible involvement of light receptor kinases. In support of this hypothesis, in the photoreceptor mutant phot1-5, blue-light induction of calcium currents was dramatically reduced and was eliminated in the double mutant phot1-5 phot2-1. By contrast, in cry1-304 cry2-1, an Arabidopsis mutant lacking another class of plant blue-light receptors, the channel remained sensitive to blue light. We thus conclude that blue light triggers calcium fluxes via the phototropin-activated calcium-permeable channel.