Studying mechanosensitive ion channels with an automated patch clamp

Patch clamp electrophysiology is the main technique to study mechanosensitive ion channels (MSCs), however, conventional patch clamping is laborious and success and output depends on the skills of the operator. Even though automated patch systems solve these problems for other ion channels, they could not be applied to MSCs. Here, we report on activation and single channel analysis of a bacterial mechanosensitive ion channel using an automated patch clamp system. With the automated system, we could patch not only giant unilamellar liposomes but also giant Escherichia coli (E. coli) spheroplasts. The tension sensitivity and channel kinetics data obtained in the automated system were in good agreement with that obtained from the conventional patch clamp. The findings will pave the way to high throughput fundamental and drug screening studies on mechanosensitive ion channels.     

Ion channel electrophysiology via integrated planar patch-clamp chip with on-demand drug exchange

Planar patch clamp has revolutionized characterization of ion channel behavior in drug discovery primarily via advancement in high throughput. Lab use of planar technology, however, addresses different requirements and suffers from inflexibility to enable wide range of interrogation via a single cell. This work presents integration of planar patch clamp with microfluidics, achieving multiple solution exchanges for tailor-specific measurement and allowing rapid replacement of the cell-contacting aperture. Studies via endogenously expressed ion channels in HEK 293T cells were commenced to characterize the device. Results reveal the microfluidic concentration generator produces distinct solution/drug combination/concentrations on-demand. Volume-regulated chloride channel and voltage-gated potassium channels in HEK 293T cells immersed in generated solutions under various osmolarities or drug concentrations show unique channel signature under specific condition. Excitation and blockage of ion channels in a single cell was demonstrated via serial solution exchange. Robustness of the reversible bonding and ease of glass substrate replacement were proven via repeated usage of the integrated device. The present approach reveals the capability and flexibility of integrated microfluidic planar patch-clamp system for ion channel assays.     

Population patch clamp improves data consistency and success rates in the measurement of ionic currents

Present whole-cell patch-clamp methodology has only moderate consistency and throughput, rendering impractical functional measurements on large numbers of ion channel ligands or on large numbers of unknown or mutant channel genes. In the population patch clamp (PPC) described herein, a single voltage-clamp amplifier sums the whole-cell currents from multiple cells at once, each sealed to a separate aperture in a planar substrate well. The resulting ensemble currents are more consistent from well to well, and the success rate for each recording attempt is >95%. The PPC was implemented by modifying the PatchPlate substrate and amplifiers in the IonWorks patch-clamp instrument. The increased data consistency and likelihood of a successful recording in each well, combined with 384-well measurements in parallel, allow the direct electrophysiological recording of thousands of ensemble ionic currents per day. Therapeutic groups in drug discovery programs require this order of throughput to screen directed compound libraries against ion channel targets. The potential for studying the function of large numbers of ion channel mutants may be realized with the technique. The procedure incorporates subtraction methods that correct for expected distortions and also reliably produces data that agree with previous patch-clamp studies.     

Novel screening techniques for ion channel targeting drugs

Ion channels are integral membrane proteins that regulate the flux of ions across the cell membrane. They are involved in nearly all physiological processes, and malfunction of ion channels has been linked to many diseases. Until recently, high-throughput screening of ion channels was limited to indirect, e.g. fluorescence-based, readout technologies. In the past years, direct label-free biophysical readout technologies by means of electrophysiology have been developed. Planar patch-clamp electrophysiology provides a direct functional label-free readout of ion channel function in medium to high throughput. Further electrophysiology features, including temperature control and higher-throughput instruments, are continually being developed. Electrophysiological screening in a 384-well format has recently become possible. Advances in chip and microfluidic design, as well as in cell preparation and handling, have allowed challenging cell types to be studied by automated patch clamp. Assays measuring action potentials in stem cell-derived cardiomyocytes, relevant for cardiac safety screening, and neuronal cells, as well as a large number of different ion channels, including fast ligand-gated ion channels, have successfully been established by automated patch clamp. Impedance and multi-electrode array measurements are particularly suitable for studying cardiomyocytes and neuronal cells within their physiological network, and to address more complex physiological questions. This article discusses recent advances in electrophysiological technologies available for screening ion channel function and regulation.     

Novel 384-well population patch clamp electrophysiology assays for Ca2+-activated K+ channels

Planar array electrophysiology techniques were applied to assays for modulators of recombinant hIK and hSK3 Ca2+-activated K+ channels. In CHO-hIK-expressing cells, under asymmetric K+ gradients, small-molecule channel activators evoked time- and voltage-independent currents characteristic of those previously described by classical patch clamp electrophysiology methods. In single-hole (cell) experiments, the large cell-to-cell heterogeneity in channel expression rendered it difficult to generate activator concentration-response curves. However, in population patch clamp mode, in which signals are averaged from up to 64 cells, well-to-well variation was substantially reduced such that concentration-response curves could be easily constructed. The absolute EC50 values and rank order of potency for a range of activators, including 1-EBIO and DC-EBIO, corresponded well with conventional patch clamp data. Activator responses of hIK and hSK3 channels could be fully and specifically blocked by the selective inhibitors TRAM-34 and apamin, with IC50 values of 0.31 microM and 3 nM, respectively. To demonstrate assay precision and robustness, a test set of 704 compounds was screened in a 384-well format of the hIK assay. All plates had Z' values greater than 0.6, and the statistical cutoff for activity was 8%. Eleven hits (1.6%) were identified from this set, in addition to the randomly spiked wells with known activators. Overall, our findings demonstrate that population patch clamp is a powerful and enabling method for screening Ca2+-activated K+ channels and provides significant advantages over single-cell electrophysiology (IonWorks(HT)) and other previously published approaches. Moreover, this work demonstrates for the 1st time the utility of population patch clamp for ion channel activator assays and for non-voltage-gated ion channels.     

膜片钳技术的新进展及其在药物高通量筛选中的应用

作为先进的细胞电生理技术,膜片钳一直被奉为研究离子通道的“金标准”。应用膜片钳技术可以证实细胞膜上离子通道的存在并能对其电生理特性、分子结构、药物作用机制等进行深入的研究。基因组学、蛋白质组学研究表明,以离子通道为靶标的药物研究在未来具有很大的发展空间。为了突破由于筛选技术所造成的针对离子通道为靶标的药物研发的瓶颈,近年来,对膜片钳技术进行了改进以适合药物高通量筛选的需求,由此产生了一些新的技术。本文就最近几年膜片钳技术的新进展及其在药物高通量筛选中的应用进行了综述。     

膜片钳阵列技术及其应用于药物筛选的研究进展

离子通道是一类重要的药物作用耙点。膜片钳技术是目前进行离子通道研究和影响离子通道药物研究的最好方法。但膜片钳技术通量低,成为应用该法进行药物筛选的最大障碍。膜片钳阵列技术是在普通膜片钳技术基础上发展起来的高通量技术,包括平面膜片钳阵列技术和微管自动化膜片钳技术,已经在药物筛选中得到应用。本文仅就这2种方法当前的研究进展及其在药物筛选中的应用做简单的介绍。     

Ionworks HT: a new high-throughput electrophysiology measurement platform

To address the throughput restrictions of classical patch clamp electrophysiology, Essen Instruments has developed a plate-based electrophysiology measurement platform. The instrument is an integrated platform that consists of computer-controlled fluid handling, recording electronics, and processing tools capable of voltage clamp whole-cell recordings from thousands of individual cells per day. To establish a recording, the system uses a planar, multiwell substrate (a PatchPlate). The system effectively positions 1 cell into a hole separating 2 fluid compartments in each well of the substrate. Voltage control and current recordings from the cell membrane are made subsequent to gaining access to the cell interior by applying a permeabilizing agent to the intracellular side. Based on the multiwell design of the PatchPlate, voltage clamp recordings of up to 384 individual cells can be made in minutes and are comparable to measurements made using traditional electrophysiology techniques. An integrated pipetting system allows for up to 2 additions of modulation agents. Typical throughput, measurement fidelity, stability, and comparative pharmacology of a recombinant voltage-dependent sodium channel (hNav1.3) and a voltage-gated potassium channel (hKv1.5) exogenously expressed in CHO cells are presented. The IonWorks HT device can be used in biophysical and pharmacological profiling of ion channels in an environment compatible with high-capacity screening.     

Trends in ion channel drug discovery: advances in screening technologies

Ion channels mediate and regulate crucial electrical functions throughout the body. They are therapeutic drug targets for a variety of disorders and, in some cases, the direct cause of unwanted side-effects. Advances in medical genetics have increased our knowledge of ion channel structure–function relationships and identified disease-causing mutations in ion channel genes. The recognized importance of these proteins in health and disease has led to an active search for ion channel targets in the multi-billion-dollar worldwide drug discovery market. Trends in ion channel screening technologies have focused on increasing throughput and enhancing information content of assays through electrophysiological approaches. The ability to study ion channels by voltage clamp and their time-, voltage- and state-dependent drug interactions with enhanced throughput will ultimately play a key role in the development of novel, safe ion channel-targeted drugs.     

Identification of Specific Sensory Neuron Populations for Study of Expressed Ion Channels

Sensory neurons transmit signals from various parts of the body to the central nervous system. The soma for these neurons are located in the dorsal root ganglia that line the spinal column. Understanding the receptors and channels expressed by these sensory afferent neurons could lead to novel therapies for disease. The initial step is to identify the specific subset of sensory neurons of interest. Here we describe a method to identify afferent neurons innervating the muscles by retrograde labeling using a fluorescent dye DiI (1,1''-dioctadecyl-3,3,3'',3''-tetramethylindocarbocyanine perchlorate). Understanding the contribution of ion channels to excitation of muscle afferents could help to better control excessive excitability induced by certain disease states such as peripheral vascular disease or heart failure. We used two approaches to identify the voltage dependent ion channels expressed by these neurons, patch clamp electrophysiology and immunocytochemistry. While electrophysiology plus pharmacological blockers can identify functional ion channel types, we used immunocytochemistry to identify channels for which specific blockers were unavailable and to better understand the ion channel distribution pattern in the cell population. These techniques can be applied to other areas of the nervous system to study specific neuronal groups.     

膜片钳技术及其在抗菌肽作用机制研究中的应用

抗菌肽是广泛存在于自然界生物体内的一类具有抗微生物、抗肿瘤等活性的多肽,有关抗菌肽作用机理的研究是近年来的热点之一。膜片钳技术自发明以来演化出适合不同研究需要的多种记录模式,并成为现代膜生物学和电生理学研究的重要手段。利用该技术对跨膜离子电流的记录分析,可以对细胞膜离子通道、膜选择性通透以及通道调节机制等方面进行深入的研究。本文介绍了抗菌肽的分类、组成及理化性质,阐述了膜片钳技术在抗菌肽对细菌细胞膜作用机制研究中的应用及最新研究进展。     

Advances in patch clamp technique: towards higher quality and quantity

The patch clamp technique, developed in late 1970s, started a new period of experimental cardiac electrophysiology enabling measurement of ionic currents on isolated cardiomyocytes down to the level of single channels. Since that time, the technique has been substantially improved by development of several upgraded modifications providing so far unavailable data (e.g. action potential clamp, dynamic clamp, high-resolution scanning patch clamp), or facilitating the patch clamp technique by increasing its efficiency (planar patch clamp, automated patch clamp). The current review summarizes the leading new patch clamp based techniques used in cardiac cellular electrophysiology, their principles and prominent related papers.     

Predict potential drug targets from the ion channel proteins based on SVM

The identification of molecular targets is a critical step in the drug discovery and development process. Ion channel proteins represent highly attractive drug targets implicated in a diverse range of disorders, in particular in the cardiovascular and central nervous systems. Due to the limits of experimental technique and low-throughput nature of patch-clamp electrophysiology, they remain a target class waiting to be exploited. In our study, we combined three types of protein features, primary sequence, secondary structure and subcellular localization to predict potential drug targets from ion channel proteins applying classical support vector machine (SVM) method. In addition, our prediction comprised two stages. In stage 1, we predicted ion channel target proteins based on whole-genome target protein characteristics. Firstly, we performed feature selection by Mann-Whitney U test, then made predictions to identify potential ion channel targets by SVM and designed a new evaluating indicator Q to prioritize results. In stage 2, we made a prediction based on known ion channel target protein characteristics. Genetic algorithm was used to select features and SVM was used to predict ion channel targets. Then, we integrated results of two stages, and found that five ion channel proteins appeared in both prediction results including CGMP-gated cation channel beta subunit and Gamma-aminobutyric acid receptor subunit alpha-5, etc., and four of which were relative to some nerve diseases. It suggests that these five proteins are potential targets for drug discovery and our prediction strategies are effective.     

2-Aminoethyl diphenyl borinate (2-APB) inhibits TRPM7 channels through an intracellular acidification mechanism

2-APB is a widely used compound in ion channel research. It affects numerous channels including inositol 1,4,5-trisphosphate receptors, store-operated calcium channels and TRP channels, TRPV3 and TRPM7 among them. A characteristic property of TRPM7 channels is their sensitivity to intracellular Mg²⁺ and pH. Using patch clamp electrophysiology we find that in Jurkat T lymphocytes, 100–300 µM extracellular 2-APB reversibly inhibits TRPM7 channels when internal HEPES concentration is low (1 mM). Increasing the concentration to 140 mM abolishes the 2-APB effect. Using single-cell fluorescence pH video imaging, we show that at concentrations of 100 µM and higher, 2-APB potently acidifies the cytoplasm. We conclude that TRPM7 sensitivity to 2-APB is not direct but rather, can be explained by cytoplasmic acidification and a resulting channel inhibition.     

High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology

Incorporation of noncanonical amino acids (ncAAs) can endow proteins with novel functionalities, such as crosslinking or fluorescence. In ion channels, the function of these variants can be studied with great precision using standard electrophysiology, but this approach is typically labor intensive and low throughput. Here, we establish a high-throughput protocol to conduct functional and pharmacological investigations of ncAA-containing human acid-sensing ion channel 1a (hASIC1a) variants in transiently transfected mammalian cells. We introduce 3 different photocrosslinking ncAAs into 103 positions and assess the function of the resulting 309 variants with automated patch clamp (APC). We demonstrate that the approach is efficient and versatile, as it is amenable to assessing even complex pharmacological modulation by peptides. The data show that the acidic pocket is a major determinant for current decay, and live-cell crosslinking provides insight into the hASIC1a–psalmotoxin 1 (PcTx1) interaction. Further, we provide evidence that the protocol can be applied to other ion channels, such as P2X2 and GluA2 receptors. We therefore anticipate the approach to enable future APC-based studies of ncAA-containing ion channels in mammalian cells.

This study describes a method to rapidly screen hundreds of ion channel variants containing non-canonical amino acids. A proof-of-principle introducing photocrosslinking non-canonical amino acids into the human ion channel hASIC1a shows how this approach can provide insights into function and pharmacology.     

2-Aminoethyl diphenyl borinate (2-APB) inhibits TRPM7 channels through an intracellular acidification mechanism

2-APB is a widely used compound in ion channel research. It affects numerous channels including inositol 1,4,5-trisphosphate receptors, store-operated calcium channels and TRP channels, TRPV3 and TRPM7 among them. A characteristic property of TRPM7 channels is their sensitivity to intracellular Mg²⁺ and pH. Using patch clamp electrophysiology we find that in Jurkat T lymphocytes, 100–300 µM extracellular 2-APB reversibly inhibits TRPM7 channels when internal HEPES concentration is low (1 mM). Increasing the concentration to 140 mM abolishes the 2-APB effect. Using single-cell fluorescence pH video imaging, we show that at concentrations of 100 µM and higher, 2-APB potently acidifies the cytoplasm. We conclude that TRPM7 sensitivity to 2-APB is not direct but rather, can be explained by cytoplasmic acidification and a resulting channel inhibition.     

Different Ligands of the TRPV3 Cation Channel Cause Distinct Conformational Changes as Revealed by Intrinsic Tryptophan Fluorescence Quenching

TRPV3 is a thermosensitive ion channel primarily expressed in epithelial tissues of the skin, nose, and tongue. The channel has been implicated in environmental thermosensation, hyperalgesia in inflamed tissues, skin sensitization, and hair growth. Although transient receptor potential (TRP) channel research has vastly increased our understanding of the physiological mechanisms of nociception and thermosensation, the molecular mechanics of these ion channels are still largely elusive. In order to better comprehend the functional properties and the mechanism of action in TRP channels, high-resolution three-dimensional structures are indispensable, because they will yield the necessary insights into architectural intimacies at the atomic level. However, structural studies of membrane proteins are currently hampered by difficulties in protein purification and in establishing suitable crystallization conditions. In this report, we present a novel protocol for the purification of membrane proteins, which takes advantage of a C-terminal GFP fusion. Using this protocol, we purified human TRPV3. We show that the purified protein is a fully functional ion channel with properties akin to the native channel using planar patch clamp on reconstituted channels and intrinsic tryptophan fluorescence spectroscopy. Using intrinsic tryptophan fluorescence spectroscopy, we reveal clear distinctions in the molecular interaction of different ligands with the channel. Altogether, this study provides powerful tools to broaden our understanding of ligand interaction with TRPV channels, and the availability of purified human TRPV3 opens up perspectives for further structural and functional studies.     

Whole cell patch clamp recording performed on a planar glass chip

The state of the art technology for the study of ion channels is the patch clamp technique. Ion channels mediate electrical current flow, have crucial roles in cellular physiology, and are important drug targets. The most popular (whole cell) variant of the technique detects the ensemble current over the entire cell membrane. Patch clamping is still a laborious process, requiring a skilled experimenter to micromanipulate a glass pipette under a microscope to record from one cell at a time. Here we report on a planar, microstructured quartz chip for whole cell patch clamp measurements without micromanipulation or visual control. A quartz substrate of 200 microm thickness is perforated by wet etching techniques resulting in apertures with diameters of approximately 1 microm. The apertures replace the tip of glass pipettes commonly used for patch clamp recording. Cells are positioned onto the apertures from suspension by application of suction. Whole cell recordings from different cell types (CHO, N1E-115 neuroblastoma) are performed with microstructured chips studying K(+) channels and voltage gated Ca(2+) channels.     

Characterization of voltage-gated sodium-channel blockers by electrical stimulation and fluorescence detection of membrane potential

Voltage-gated ion channels regulate many physiological functions and are targets for a number of drugs. Patch-clamp electrophysiology is the standard method for measuring channel activity because it fulfils the requirements for voltage control, repetitive stimulation and high temporal resolution, but it is laborious and costly. Here we report an electro-optical technology and automated instrument, called the electrical stimulation voltage ion probe reader (E-VIPR), that measures the activity of voltage-gated ion channels using extracellular electrical field stimulation and voltage-sensitive fluorescent probes. We demonstrate that E-VIPR can sensitively detect drug potency and mechanism of block on the neuronal human type III voltage-gated sodium channel expressed in human embryonic kidney cells. Results are compared with voltage-clamp and show that E-VIPR provides sensitive and information-rich compound blocking activity. Furthermore, we screened approximately 400 drugs and observed sodium channel-blocking activity for approximately 25% of them, including the antidepressants sertraline (Zoloft) and paroxetine (Paxil).