A novel voltage clamp technique for mapping ionic currents from cultured skeletal myotubes.

The biophysical properties and cellular distribution of ion channels largely determine the input/output relationships of electrically excitable cells. A variety of patch pipette voltage clamp techniques are available to characterize ionic currents. However, when used by themselves, such techniques are not well suited to the task of mapping low-density channel distributions. We describe here a new voltage clamp method (the whole cell loose patch (WCLP) method) that combines whole-cell recording through a tight-seal pipette with focal extracellular stimulation through a loose-seal pipette. By moving the stimulation pipette across the cell surface and using a stationary whole-cell pipette to record the evoked patch currents, this method should be suitable for mapping channel distributions, even on large cells possessing low channel densities. When we applied this method to the study of currents in cultured chick myotubes, we found that the cell cable properties and the series resistance of the recording pipette caused significant filtering of the membrane currents, and that the filter characteristics depended in part upon the distance between the stimulating and recording pipettes. We describe here how we determined the filter impulse response for each loose-seal pipette placement and subsequently recovered accurate estimates of patch membrane current through deconvolution.     

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.     

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.     

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.Here, we employ the human cardiac sodium channel (hNa_V1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.The properties of fast activating ion channels, such as hNa_V1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest¹. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.     

MEC-2 and MEC-6 in the Caenorhabditis elegans sensory mechanotransduction complex: auxiliary subunits that enable channel activity

The ion channel formed by the homologous proteins MEC-4 and MEC-10 forms the core of a sensory mechanotransduction channel in Caenorhabditis elegans. Although the products of other mec genes are key players in the biophysics of transduction, the mechanism by which they contribute to the properties of the channel is unknown. Here, we investigate the role of two auxiliary channel subunits, MEC-2 (stomatin-like) and MEC-6 (paraoxonase-like), by coexpressing them with constitutively active MEC-4/MEC-10 heteromeric channels in Xenopus oocytes. This work extends prior work demonstrating that MEC-2 and MEC-6 synergistically increase macroscopic current. We use single-channel recordings and biochemistry to show that these auxiliary subunits alter function by increasing the number of channels in an active state rather than by dramatically affecting either single-channel properties or surface expression. We also use two-electrode voltage clamp and outside-out macropatch recording to examine the effects of divalent cations and proteases, known regulators of channel family members. Finally, we examine the role of cholesterol binding in the mechanism of MEC-2 action by measuring whole-cell and single-channel currents in MEC-2 mutants deficient in cholesterol binding. We suggest that MEC-2 and MEC-6 play essential roles in modulating both the local membrane environment of MEC-4/MEC-10 channels and the availability of such channels to be gated by force in vivo.     

Intramembrane Charge Movement Associated with Endogenous K+ Channel Activity in HEK-293 Cells

1. The use of molecular biology in combination with electrophysiology in the HEK-293 cell line has given fascinating insights into neuronal ion channel function. Nevertheless, to fully understand the properties of channels exogenously expressed in these cells, a detailed evaluation of endogenous channels is indispensable. 2. Previous studies have shown the expression of endogenous voltage-gated K+, Ca2+, and Cl- channels and this predicts that changes in membrane potential will cause intramembrane charge movement, though this gating charge translocation remain undefined. Here, we confirm this prediction by performing patch-clamp experiments to record ionic and gating currents. Our data show that HEK-293 cells express at least two types of K+-selective endogenous channels which sustain the majority of the ionic current, and exclude a significant contribution from Ca2+ and Cl- channels to the whole-cell current. 3. Gating currents were unambiguously resolved after ionic current blockade enabling this first report of intramembrane charge movement in HEK-293 cells arising entirely from endogenous K+ channel activity, and providing valuable information concerning the activation mechanism of voltage-gated K+ channels in these cells.     

A novel membrane potential-sensitive fluorescent dye improves cell-based assays for ion channels

The study of ion channel-mediated changes in membrane potential using the conventional bisoxonol fluorescent dye DiBAC(4)(3) has several limitations, including a slow onset of response and multistep preparation, that limit both the fidelity of the results and the throughput of membrane potential assays. Here, we report the characterization of the FLIPR Membrane Potential Assay Kit (FMP) in cells expressing voltage- and ligand-gated ion channels. The steady-state and kinetics fluorescence properties of FMP were compared with those of DiBAC(4)(3), using both FLIPR and whole-cell patch-clamp recording. Our experiments with the voltage-gated K(+) channel, hElk-1, revealed that FMP was 14-fold faster than DiBAC(4)(3) in response to depolarization. On addition of 60 mM KCl, the kinetics of fluorescence changes of FMP using FLIPR were identical to those observed in the electrophysiological studies using whole-cell current clamp. In addition, KCl concentration-dependent increases in FMP fluorescence correlated with the changes of membrane potential recorded in whole-cell patch clamp. In studies examining vanilloid receptor-1, a ligand-gated nonselective cation channel, FMP was superior to DiBAC(4)(3) with respect to both kinetics and amplitude of capsaicin-induced fluorescence changes. FMP has also been used to measure the activation of K(ATP) and hERG. Thus this novel membrane potential dye represents a powerful tool for developing high-throughput screening assays for ion channels.     

Variable ratio of permeability to gating charge of rBIIA sodium channels and sodium influx in Xenopus oocytes

Whole-cell gating current recording from rat brain IIA sodium channels in Xenopus oocytes was achieved using a high-expression system and a newly developed high-speed two-electrode voltage-clamp. The resulting ionic currents were increased by an order of magnitude. Surprisingly, the measured corresponding gating currents were approximately 5-10 times larger than expected from ionic permeability. This prompted us to minimize uncertainties about clamp asymmetries and to quantify the ratio of sodium permeability to gating charge, which initially would be expected to be constant for a homogeneous channel population. The systematic study, however, showed a 10- to 20-fold variation of this ratio in different experiments, and even in the same cell during an experiment. The ratio of P(Na)/Q was found to correlate with substantial changes observed for the sodium reversal potential. The data suggest that a cytoplasmic sodium load in Xenopus oocytes or the energy consumption required to regulate the increase in cytoplasmic sodium represents a condition where most of the expressed sodium channels keep their pore closed due to yet unknown mechanisms. In contrast, the movements of the voltage sensors remain undisturbed, producing gating current with normal properties.     

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。     

Expression and characterization of delayed rectifying K+ channels in anterior rat taste buds

Delayed rectifying K⁺ (DRK) channels in taste cells have been implicated in the regulation of cell excitability and as potential targets for direct and indirect modulation by taste stimuli. In the present study, we have used patch-clamp recording to determine the biophysical properties and pharmacological sensitivity of DRK channels in isolated rat fungiform taste buds. Molecular biological assays at the taste bud and single-cell levels are consistent with the interpretation that taste cells express a variety of DRK channels, including members from each of the three major subfamilies: KCNA, KCNB, and KCNC. Real-time PCR assays were used to quantify expression of the nine DRK channel subtypes. While taste cells express a number of DRK channels, the electrophysiological and molecular biological assays indicate that the Shaker Kv1.5 channel (KCNA5) is the major functional DRK channel expressed in the anterior rat tongue. transduction     

Membrane currents in the oocyte of the toad Bufo arenarum

The amphibian oocyte cell model is widely used for heterologous expression of ionic channels and receptors. Little is known, however, about the physiology of oocyte cell models other than Xenopus laevis. In this study, the two-electrode voltage clamp technique was used to assess the most common electrical patterns of oocytes of the South American toad Bufo arenarum. Basal membrane resistance, resting potential, and ionic currents were determined in this cell model. The oocyte transmembrane resistance was 0.35 M(Omega), and the resting potential in normal saline was about -33 mV with a range between -20 mV and -50 mV. This is, to our knowledge, the first attempt to begin an understanding of the ion transport mechanisms of Bufo arenarum oocytes. This cell model may provide a viable alternative to the expression of ion channels, in particular those endogenously observed in Xenopus laevis oocytes.     

The normalized slope conductance as a tool for quantitative analysis of current-voltage relations

The patch-clamp method, which was awarded the Nobel Prize in 1991, is a well-established and indispensable method to study ion channels in living cells and to biophysically characterize non-voltage-gated ion channels, which comprise about 70% of all ion channels in the human genome. To investigate the biophysical properties of non-voltage-gated ion channels, whole-cell measurements with application of continuous voltage ramps are routinely conducted to obtain current-voltage (IV) relationships. However, adequate tools for detailed and quantitative analysis of IV curves are still missing. We use the example of the transient receptor potential classical (TRPC) channel family to elucidate whether the normalized slope conductance (NSC) is an appropriate tool for reliable discrimination of the IV curves of diverse TRPC channels that differ in their individual curve progression. We provide a robust calculation method for the NSC, and, by applying this method, we find that TRPC channel activators and modulators can evoke different NSC progressions independent from their expression levels, which points to distinguishable active channel states. TRPC6 mutations in patients with focal segmental glomerulosclerosis resulted in distinct NSC progressions, suggesting that the NSC is suitable for investigating structure-function relations and might help unravel the unknown pathomechanisms leading to focal segmental glomerulosclerosis. The NSC is an effective algorithm for extended biophysical characterization of non-voltage-gated ion channels.     

Independent and joint modulation of rat Nav1.6 voltage-gated sodium channels by coexpression with the auxiliary β1 and β2 subunits

The Na_v1.6 voltage-gated sodium channel α subunit isoform is the most abundant isoform in the brain and is implicated in the transmission of high frequency action potentials. Purification and immunocytochemical studies imply that Na_v1.6 exist predominantly as Na_v1.6 + β1 + β2 heterotrimeric complexes. We assessed the independent and joint effects of the rat β1 and β2 subunits on the gating and kinetic properties of rat Na_v1.6 channels by recording whole-cell currents in the two-electrode voltage clamp configuration following transient expression in Xenopus oocytes. The β1 subunit accelerated fast inactivation of sodium currents but had no effect on the voltage dependence of their activation and steady-state inactivation and also prevented the decline of currents following trains of high-frequency depolarizing prepulses. The β2 subunit selectively retarded the fast phase of fast inactivation and shifted the voltage dependence of activation towards depolarization without affecting other gating properties and had no effect on the decline of currents following repeated depolarization. The β1 and β2 subunits expressed together accelerated both kinetic phases of fast inactivation, shifted the voltage dependence of activation towards hyperpolarization, and gave currents with a persistent component typical of those recorded from neurons expressing Na_v1.6 sodium channels. These results identify unique effects of the β1 and β2 subunits and demonstrate that joint modulation by both auxiliary subunits gives channel properties that are not predicted by the effects of individual subunits.     

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.     

Biophysical properties of zebrafish ether-à-go-go related gene potassium channels

The zebrafish is increasingly recognized as an animal model for the analysis of hERG-related diseases. However, functional properties of the zebrafish orthologue of hERG have not been analyzed yet. We heterologously expressed cloned ERG channels in Xenopus oocytes and analyzed biophysical properties using the voltage clamp technique. zERG channels conduct rapidly activating and inactivating potassium currents. However, compared to hERG, the half-maximal activation voltage of zERG current is shifted towards more positive potentials and the half maximal steady-state inactivation voltage is shifted towards more negative potentials. zERG channel activation is delayed and channel deactivation is accelerated significantly. However, time course of zERG conducted current under action potential clamp is highly similar to the human orthologue. In summary, we show that ERG channels in zebrafish exhibit biophysical properties similar to the human orthologue. Considering the conserved channel function, the zebrafish represents a valuable model to investigate human ERG channel related diseases.     

Endogenous KCNE subunits govern Kv2.1 K+ channel activation kinetics in Xenopus oocyte studies

Kv2.1 is a voltage-gated potassium (Kv) channel that generates delayed rectifier currents in mammalian heart and brain. The biophysical properties of Kv2.1 and other ion channels have been characterized by functional expression in heterologous systems, and most commonly in Xenopus laevis oocytes. A number of previous oocyte-based studies of mammalian potassium channels have revealed expression-level-dependent changes in channel properties, leading to the suggestion that endogenous oocyte factors regulate channel gating. Here, we show that endogenous oocyte potassium channel KCNE ancillary subunits xMinK and xMiRP2 slow the activation of oocyte-expressed mammalian Kv2.1 channels two-to-fourfold. This produces a sigmoidal relationship between Kv2.1 current density and activation rate in oocyte-based two-electrode voltage clamp studies. The effect of endogenous xMiRP2 and xMinK on Kv2.1 activation is diluted at high Kv2.1 expression levels, or by RNAi knockdown of either endogenous subunit. RNAi knockdown of both xMiRP2 and xMinK eliminates the correlation between Kv2.1 expression level and activation kinetics. The data demonstrate a molecular basis for expression-level-dependent changes in Kv channel gating observed in heterologous expression studies.     

Free energy analysis of conductivity and charge selectivity of M2GlyR-derived synthetic channels

Significant progresses have been made in the design, synthesis, modeling and in vitro testing of channel-forming peptides derived from the second transmembrane domain of the α-subunit of the glycine receptor (GlyR). The latest designs, including p22 (KKKKP ARVGL GITTV LTMTT QS), are highly soluble in water with minimal aggregation propensity and insert efficiently into cell membranes to form highly conductive ion channels. The last obstacle to a potential lead sequence for channel replacement treatment of CF patients is achieving adequate chloride selectivity. We have performed free energy simulation to analyze the conductance and charge selectivity of M2GlyR-derived synthetic channels. The results reveal that the pentameric p22 pore is non-selective. Moderate barriers for permeation of both K⁺ and Cl⁻ are dominated by the desolvation cost. Despite previous evidence suggesting a potential role of threonine side chains in anion selectivity, the hydroxyl group is not a good surrogate of water for coordinating these ions. We have also tested initial ideas of introducing additional rings of positive changes to various positions along the pore to increase anion selectivity. The results support the feasibility of achieving anion selectivity by modifying the electrostatic properties of the pore, but at the same time suggest that the peptide assembly and pore topology may also be dramatically modified, which could abolish the effects of modified electrostatics on anion selectivity. This was confirmed by subsequent two-electrode voltage clamp measurements showing that none of the tested mono-, di- and tri-Dap substituted sequences was selective. The current study thus highlights the importance of controlling channel topology besides modifying pore electrostatics for achieving anion selectivity. Several strategies are now being explored in our continued efforts to design an anion selective peptide channel with suitable biophysical, physiological and pharmacological properties as a potential treatment modality for channel replacement therapy. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

Development of a novel automated ion channel recording method using "inside-out" whole-cell membranes

Efforts to develop novel methods for recording from ion channels have been receiving increased attention in recent years. In this study, the authors report a unique "inside-out" whole-cell configuration of patch-clamp recording that has been developed. This method entails adding cells into a standard patch pipette and, with positive pressure, obtaining a gigaseal recording from a cell at the inside tip of the electrode. In this configuration, the cell may be moved through the air, first rupturing part of the cellular membrane and enabling bath access to the intracellular side of the membrane, and then into a series of wells containing differing solutions, enabling robotic control of all the steps in an experiment. The robotic system developed here fully automates the electrophysiological experiments, including gigaseal formation, obtaining whole-cell configuration, data acquisition, and drug application. Proof-of-principle experiments consisting of application of intracellularly acting potassium channel blockers to K+ channel cell lines resulted in a very rapid block, as well as block reversal, of the current. This technique allows compound application directly to the intracellular side of ion channels and enables the dissociation of compound in activities due to cellular barrier limitations. This technique should allow for parallel implementation of recording pipettes and the future development of larger array-based screening methods.