Ion Channel Recordings Research Articles

Real-time quantitative characterization of ion channel activities for automated control of a lipid bilayer system

This paper describes a novel signal processing method to characterize the activity of ion channels on a lipid bilayer system in a real-time and quantitative manner. Lipid bilayer systems, which enable single-channel level recordings of ion channel activities against physiological stimuli in vitro, are gaining attention in various research fields. However, the characterization of ion channel activities has heavily relied on time-consuming analyses after recording, and the inability to return the quantitative results in real time has long been a bottleneck to incorporating the system into practical products. Herein, we report a lipid bilayer system that integrates real-time characterization of ion channel activities and real-time response based on the characterization result. Unlike conventional batch processing, an ion channel signal is divided into short segments and processed during the recording. After optimizing the system to maintain the same characterization accuracy as conventional operation, we demonstrated the usability of the system with two applications. One is quantitative control of a robot based on ion channel signals. The velocity of the robot was controlled every second, which was around tens of times faster than the conventional operation, in proportion to the stimulus intensity estimated from changes in ion channel activities. The other is the automation of data collection and characterization of ion channels. By constantly monitoring and maintaining the functionality of a lipid bilayer, our system enabled continuous recording of ion channels over 2 h without human intervention, and the time of manual labor has been reduced from conventional 3 h to 1 min at a minimum. We believe the accelerated characterization and response in the lipid bilayer systems presented in this work will facilitate the transformation of lipid bilayer technology toward a practical level, finally leading to its industrialization.

Adventures and Advances in Time Travel With Induced Pluripotent Stem Cells and Automated Patch Clamp.

In the Hollywood blockbuster “The Curious Case of Benjamin Button” a fantastical fable unfolds of a man’s life that travels through time reversing the aging process; as the tale progresses, the frail old man becomes a vigorous, vivacious young man, then man becomes boy and boy becomes baby. The reality of cellular time travel, however, is far more wondrous: we now have the ability to both reverse and then forward time on mature cells. Four proteins were found to rewind the molecular clock of adult cells back to their embryonic, “blank canvas” pluripotent stem cell state, allowing these pluripotent stem cells to then be differentiated to fast forward their molecular clocks to the desired adult specialist cell types. These four proteins – the “Yamanaka factors” – form critical elements of this cellular time travel, which deservedly won Shinya Yamanaka the Nobel Prize for his lab’s work discovering them. Human induced pluripotent stem cells (hiPSCs) hold much promise in our understanding of physiology and medicine. They encapsulate the signaling pathways of the desired cell types, such as cardiomyocytes or neurons, and thus act as model cells for defining the critical ion channel activity in healthy and disease states. Since hiPSCs can be derived from any patient, highly specific, personalized (or stratified) physiology, and/or pathophysiology can be defined, leading to exciting developments in personalized medicines and interventions. As such, hiPSC married with high throughput automated patch clamp (APC) ion channel recording platforms provide a foundation for significant physiological, medical and drug discovery advances. This review aims to summarize the current state of affairs of hiPSC and APC: the background and recent advances made; and the pros, cons and challenges of these technologies. Whilst the authors have yet to finalize a fully functional time traveling machine, they will endeavor to provide plausible future projections on where hiPSC and APC are likely to carry us. One future projection the authors are confident in making is the increasing necessity and adoption of these technologies in the discovery of the next blockbuster, this time a life-enhancing ion channel drug, not a fantastical movie.

Single ion channel recording in 3D culture of stem cells using patch-clamp technique

Tri-dimensional (3D) cell aggregates or spheroids are considered to be closer to physiological conditions than traditional 2D cell culture. Mesenchymal stem cells (MSCs) assembling in spheroids have increased the survival of transplanted cells. The organization of stem cells in 3D culture affects cell microenvironment and their mechanical properties. The regulation of the biological processes that maintain crucial physiological reactions of MSCs is closely related to the functioning of ion channels. The pattern of expression, role and regulatory mechanisms of ion channels could be significantly different in 3D compared to 2D culture, and, thus, needed to be properly analyzed on the level of ionic currents. Electrophysiological data on the features of ion channels functioning in 3D cell culture models are currently very limited in the literature. This gap of knowledge may be associated with technical difficulties that exist when researchers try to apply the standard patch clamp method for the registration of ion channels in cells aggregated in spheroids. In this regard, our study focuses on solving emerging technical difficulties and presents an example of their successful solution. Here, we developed a specific approach and have recorded the activity of mechanosensitive stretch-activated ion channels (SACs) in endometrial MSCs (eMSCs) assembled in spheroids. Moreover, we observed functional interplay of SACs with potassium channels of big conductance (BK) in the plasma membrane of eMSC spheroids consistently to revealed earlier in routine 2D cultured cells. Additionally, we observed a significant decrease in the frequency of SACs activation in spheroids that may indicate the differences in the level of functional expression of channels in 3D culture comparing to 2D culture of eMSCs.

A standardised hERG phenotyping pipeline to evaluate KCNH2 genetic variant pathogenicity.

Background and aimsMutations in KCNH2 cause long or short QT syndromes (LQTS or SQTS) predisposing to life‐threatening arrhythmias. Over 1000 hERG variants have been described by clinicians, but most remain to be characterised. The objective is to standardise and accelerate the phenotyping process to contribute to clinician diagnosis and patient counselling. In silico evaluation was also included to characterise the structural impact of the variants.MethodsWe selected 11 variants from known LQTS patients and two variants for which diagnosis was problematic. Using the Gibson assembly strategy, we efficiently introduced mutations in hERG cDNA despite GC‐rich sequences. A pH‐sensitive fluorescent tag was fused to hERG for efficient evaluation of channel trafficking. An optimised 35‐s patch‐clamp protocol was developed to evaluate hERG channel activity in transfected cells. R software was used to speed up analyses.ResultsIn the present work, we observed a good correlation between cell surface expression, assessed by the pH‐sensitive tag, and current densities. Also, we showed that the new biophysical protocol allows a significant gain of time in recording ion channel properties and provides extensive information on WT and variant channel biophysical parameters, that can all be recapitulated in a single parameter defined herein as the repolarisation power. The impacts of the variants on channel structure were also reported where structural information was available. These three readouts (trafficking, repolarisation power and structural impact) define three pathogenicity indexes that may help clinical diagnosis.ConclusionsFast‐track characterisation of KCNH2 genetic variants shows its relevance to discriminate mutants that affect hERG channel activity from variants with undetectable effects. It also helped the diagnosis of two new variants. This information is meant to fill a patient database, as a basis for personalised medicine. The next steps will be to further accelerate the process using an automated patch‐clamp system.

Fast Track hERG phenotyping to evaluate the pathogenicity of KCNH2 genetic variants

Mutations in KCNH2 (coding for hERG) cause long or short QT syndromes (LQTS or SQTS), predisposing to life-threatening arrhythmias. More than 1000 variations in hERG sequences have been described, most of them are to be characterized. The objective is to standardize and accelerate the entire process necessary to phenotype hERG variants. An in silico evaluation was also included to characterize the structural impact of the variants. We selected 12 variants from patients with LQTS, and 1 with SQTS. We optimized the protocol to efficiently introduce mutations in hERG cDNA despite GC-rich sequences, using the Gibson assembly strategy. A pH-sensitive fluorescent tag was fused to hERG for fast-track evaluation of hERG cell trafficking. An optimized patch-clamp protocol of 35 sec was developed to evaluate hERG channel activity in transfected cells. R software was used to speed up analyses. In the present work, we observed a good correlation between cell surface expression, assessed by the pH-sensitive tag, and current densities. Also, we showed that the new biophysical protocol allows a significant gain of time in recording ion channel properties and provides extensive information on WT and variant channel biophycical parameters, that can all be recapitulated in a single parameter defined herein as the repolarization power. The impact of the variants on channel structure were also reported where structural information was available. These three readouts (trafficking, repolarization power and structural impact) define 3 pathogenicity indexes that may help clinical diagnosis. Fast track characterization of KCNH2 genetic variants shows its relevance to discriminate mutants that affect hERG channel activity from variants with undetectable effects. This information is meant to fill a patient database, as a basis for personalized medicine. The next steps will be to further accelerate the process using an automated patch-clamp system.

Multiscale Quantile Segmentation

We introduce a new methodology for analyzing serial data by quantile regression assuming that the underlying quantile function consists of constant segments. The procedure does not rely on any distributional assumption besides serial independence. It is based on a multiscale statistic, which allows to control the (finite sample) probability for selecting the correct number of segments S at a given error level, which serves as a tuning parameter. For a proper choice of this parameter, this probability tends exponentially fast to one, as sample size increases. We further show that the location and size of segments are estimated at minimax optimal rate (compared to a Gaussian setting) up to a log-factor. Thereby, our approach leads to (asymptotically) uniform confidence bands for the entire quantile regression function in a fully nonparametric setup. The procedure is efficiently implemented using dynamic programming techniques with double heap structures, and software is provided. Simulations and data examples from genetic sequencing and ion channel recordings confirm the robustness of the proposed procedure, which at the same time reliably detects changes in quantiles from arbitrary distributions with precise statistical guarantees. Supplementary materials for this article are available online.

Advancing Ion Channel Research with Automated Patch Clamp (APC) Electrophysiology Platforms.

Since its development on the cusp of the new millennium, automated patch clamp (APC) technology has matured over the last two decades. The increased throughput it afforded promised a new paradigm in ion channel recordings: It offered the potential to overcome the time-consuming, low-throughput bottleneck arising from manual patch clamp (MPC) investigations. This chapter highlights the advances in technology, showing how APC platforms have 'democratised' ion channel recordings, lowering the technical bar whilst substantially raising throughput. It will describe the background of the seminal first-generation and updates on advances in second-generation platforms. Furthermore, the chapter summarises the advances APC has made in ion channel studies, including finding new tool compounds and medicines. New functionality and applications on APC platforms give ion channel researchers flexible tools to study ion channels with high quality and high throughput.

Electrophysiological Recordings of the Polycystin Complex in the Primary Cilium of Cultured Mouse IMCD-3 Cell Line.

PC-1 and PC-2 form an ion channel complex called the polycystin complex, which predominantly localizes to a small hair-like organelle called the primary cilium. The polycystin complex permeates cations, K+, Na+, and Ca2+, and has an unusual 1:3 stoichiometry that combines one PC-1 subunit with three PC-2 subunits. However, the small size and shape of primary cilia impose technical challenges to study the polycystin complex in its native environment. In this paper, we describe the methodology to directly record ion channel activity in primary cilia. This method will allow a detailed functional characterization of how mutations within the polycystin complex cause Autosomal Dominant Polycystic Kidney Disease (ADPKD), essential to develop novel therapeutics for this ciliopathy.

Heterogeneous Idealization of Ion Channel Recordings - Open Channel Noise.

We propose a new model-free segmentation method for idealizing ion channel recordings. This method is designed to deal with heterogeneity of measurement errors. This in particular applies to open channel noise which, in general, is particularly difficult to cope with for model-free approaches. Our methodology is able to deal with lowpass filtered data which provides a further computational challenge. To this end we propose a multiresolution testing approach, combined with local deconvolution to resolve the lowpass filter. Simulations and statistical theory confirm that the proposed idealization recovers the underlying signal very accurately at presence of heterogeneous noise, even when events are shorter than the filter length. The method is compared to existing approaches in computer experiments and on real data. We find that it is the only one which allows to identify openings of the PorB porine at two different temporal scales. An implementation is available as an R package.

A Lipid Bilayer Formed on a Hydrogel Bead for Single Ion Channel Recordings.

Ion channel proteins play important roles in various cell functions, making them attractive drug targets. Artificial lipid bilayer recording is a technique used to measure the ion transport activities of channel proteins with high sensitivity and accuracy. However, the measurement efficiency is low. In order to improve the efficiency, we developed a method that allows us to form bilayers on a hydrogel bead and record channel currents promptly. We tested our system by measuring the activities of various types of channels, including gramicidin, alamethicin, α-hemolysin, a voltage-dependent anion channel 1 (VDAC1), a voltage- and calcium-activated large conductance potassium channel (BK channel), and a potassium channel from Streptomyces lividans (KcsA channel). We confirmed the ability for enhanced measurement efficiency and measurement system miniaturizion.

Perforated Whole-Cell Recordings in Automated Patch Clamp Electrophysiology.

The automated patch clamp (APC) technology is used for increasing the data throughput of electrophysiological measurements, especially in safety pharmacology and drug discovery. Typically, electrical access to the cells are obtained using standard whole-cell formation by rupturing the membrane, thereby causing a rapid washout of cytosolic components. In contrast the perforated whole-cell configuration provides electrical access to the cell interior while limiting intracellular wash-out. This method allows for recordings of ion channels that are gated by intracellular modulators (e.g., ATP, cyclic nucleotides, or Ca2+), prevents channel current "run down," and maintains a physiological membrane potential for action potential recordings. Here we present some practical approaches to the use of perforated patch clamp for APC recordings. Our findings from these high-throughput, data-rich measurements (e.g., defining optimized concentrations and practical recommendations for four different perforating agents) can be more broadly applied to perforated patch clamp experiments in general (automated and manual), improving success rates, experimental conditions, and applications.

Shot noise sets the limit of quantification in electrochemical measurements

Abstract Detection of single molecules, particles, and rapid redox events is a challenge of electrochemical investigations and requires either an amplification strategy or significant averaging in order to boost the electrochemical current above the noise level. We consider the minimum number of electrons required to reach the limit of quantification in these electrochemical measurements. A survey of the literature indicates that the state-of-the-art limit in current detection for different types of measurements (e.g., voltammetry, single-molecule redox cycling, ion channel recordings of single molecules, metal nanoparticle collision, phase nucleation, etc.) is independent of the nature of the measurement and increases linearly with reciprocal response time, Δt-1, over ∼5 orders of magnitude (from ∼10 to ∼106 s-1). We demonstrate that the practical limit of quantification requires cumulative measurement of ∼2,100 electrons during Δt, and is determined by statistics of counting electrons, i.e., the shot noise in the current.

On hysteresis of ion channels

Ion channel proteins have many conformational (metastable) states and, for this reason, they exhibit hysteresis. This fact is responsible for the non-Markovian stochastic nature of single ion channel recordings. It is suggested in the paper that the stochastic single channel recordings can be modeled as the random outputs of rectangular hysteresis loops driven by stochastic processes. The latter problem can be mathematically treated as an exit problem for stochastic processes or by using the theory of stochastic processes on graphs. It is also demonstrated in the paper that the collective action of sodium and potassium channels responsible for the generation and propagation of action potentials exhibit hysteresis. This demonstration is accomplished by using the inverse problem approach to the nonlinear Hodgkin-Huxley diffusion equation.

Free-Standing Lipid Bilayers Based on Nanopore Array and Ion Channel Formation.

Integrated nanopores are novel and versatile single-molecule sensors for individual label-free biopolymer detection and characterization. However, their studies and application requires a stable lipid bilayer to maintain protein function. Herein, we describe a method for producing lipid bilayers across a nanopore array on a silicon nitride substrate. We used a painting technique commonly used with Teflon films to embed α-hemolysin (α-HL) into bilayer lipid membranes (BLMs) to form an ion channel. This was carried out in nanofluid developed in our lab. The membrane formation process, stability of BLMs and ion channel recordings were monitored by patch clamp in real-time. BLM formation was demonstrated by electrical recording (<10 pS conductance) of suspended lipid bilayers spanning a nanopore in the range of ±100 mV. Membrane resistance (Rm) and capacitance (Cm) of the device with the bilayer were assessed by membrane test as above 1.0 GΩ and ~20±2 pF, respectively. The silicon nitride surface and aperture edge were smooth at the nanometer lever leading to remarkable membrane stability. The membrane lifetime was 5-24 h. A single α-HL channel inserted in 30-60 min applied a potential of +100 mV. The α-HL channel currents were recorded at ~100±10 pA. Such integrated nanopores enable analysis of channel functions under various solution conditions from the same BLM. This will open up a variety of applications for ion channels including high-throughput medical screening and diagnosis.

Electrophysiological Recordings of Single-cell Ion Currents Under Well-defined Shear Stress.

Fluid shear stress is well known to play a major role in endothelial function. In most vascular beds, elevated shear stress from acute increases in blood flow triggers a signaling cascade resulting in vasodilation thereby alleviating mechanical stress on the vascular wall. The pattern of shear stress is also well known to be a critical factor in the development of atherosclerosis with laminar shear stress being atheroprotective and disturbed shear stress being pro-atherogenic. While we have a detailed understanding of the various intermediate cell signaling pathways, the receptors that first translate the mechanical stimulus into chemical mediators are not completely understood. Mechanosensitive ion channels are critical to the response to shear and regulate shear-induced cell signaling thereby controlling the production of vasoactive mediators. These channels are among the earliest activated signaling components to shear and have been linked to shear-induced vasodilation through promoting nitric oxide production (e.g., inwardly rectifying K+ [Kir] and transient receptor potential [TRP] channels) and endothelium hyperpolarizing factor (e.g., Kir and calcium-activated K+ [KCa] channels) and shear-induced vasoconstriction through an undetermined mechanism that involves piezo channels. Understanding the biophysical mechanism by which these channels are activated by shear forces (i.e., directly or through a primary mechano-receptor) could provide potential new targets to resolve the pathophysiology associated with endothelial dysfunction and atherogenesis. It is still a major challenge to record flow-induced activation of ion channels in real time using electrophysiology. The standard methods to expose cells to well-defined shear stress, such as the cone and plate rheometer and closed parallel plate flow chamber do not allow real time study of ion channel activation. The goal of this protocol is to describe a modified parallel plate flow chamber that allows real time electrophysiological recording of mechanosensitive ion channels under well-defined shear stress.

Spatially Resolved Chemical Detection with a Nanoneedle-Probe-Supported Biological Nanopore.

In this article, we describe the quantitative characterization of a gold nanoneedle ion channel probe and demonstrate the utility of this probe for spatially resolved detection of a small molecule using ion channel activity. Our report builds on recent reports of Ide and co-workers, who reported the use of an etched gold wire modified with a poly(ethylene) glycol monolayer as a support for a lipid bilayer and subsequent single ion channel recordings. Although this nanoneedle electrode approach was reported previously, in our report, we investigate the effects of several operational parameters on the performance of the ion channel measurement and electrochemical phenomenon that occur in the nanoconfined space between the supported bilayer and the gold electrode. More specifically, we address the effects of length of the supporting monolayer and the composition of the electrolyte baths on channel current measurements and provide a quantitative description of what carries current at the working electrode (double-layer charging). In addition, we demonstrate the ability to control the direction of protein insertion (tip side vs bath side) with freely diffusing protein, which has not been previously reported, with the former method (tip side) enabling single-molecule detection of β-cyclodextrin (βCD) using a reconstituted α-hemolysin channel. Finally, anticipating future use of a nanoneedle-based biological nanopore probe in a scanned-probe microscopy, we demonstrate the ability to quantify and spatially resolve the concentration of βCD molecules in a microfluidic channel. We believe, in the long term, the described nanoneedle-based biological nanopore probe can be employed in, for example, scanning ion conductance microscopy using ion channels.

Wavelet Denoising of High-Bandwidth Nanopore and Ion-Channel Signals.

Recent work has pushed the noise-limited bandwidths of solid-state nanopore conductance recordings to more than 5 MHz and of ion channel conductance recordings to more than 500 kHz through the use of integrated complementary metal-oxide-semiconductor (CMOS) integrated circuits. Despite the spectral spread of the pulse-like signals that characterize these recordings when a sinusoidal basis is employed, Bessel filters are commonly used to denoise these signals to acceptable signal-to-noise ratios (SNRs) at the cost of losing many of the faster temporal features. Here, we report improvements to the SNR that can be achieved using wavelet denoising instead of Bessel filtering. When combined with state-of-the-art high-bandwidth CMOS recording instrumentation, we can reduce baseline noise levels by over a factor of 4 compared to a 2.5 MHz Bessel filter while retaining transient properties in the signal comparable to this filter bandwidth. Similarly, for ion-channel recordings, we achieve a temporal response better than a 100 kHz Bessel filter with a noise level comparable to that achievable with a 25 kHz Bessel filter. Improvements in SNR can be used to achieve robust statistical analyses of these recordings, which may provide important insights into nanopore translocation dynamics and mechanisms of ion-channel function.

γ-Hemolysin Nanopore Is Sensitive to Guanine-to-Inosine Substitutions in Double-Stranded DNA at the Single-Molecule Level.

Biological nanopores provide a unique single-molecule sensing platform to detect target molecules based on their specific electrical signatures. The γ-hemolysin (γ-HL) protein produced by Staphylococcus aureus is able to assemble into an octamer nanopore with a ∼2.3 nm diameter β-barrel. Herein, we demonstrate the first application of γ-HL nanopore for DNA structural analysis. To optimize conditions for ion-channel recording, the properties of the γ-HL pore (e.g., conductance, voltage-dependent gating, and ion-selectivity) were characterized at different pH, temperature, and electrolyte concentrations. The optimal condition for DNA analysis using γ-HL corresponds to 3 M KCl, pH 5, and T = 20 °C. The γ-HL protein nanopore is able to translocate dsDNA at about ∼20 bp/ms, and the unique current-signature of captured dsDNA can directly distinguish guanine-to-inosine substitutions at the single-molecule level with ∼99% accuracy. The slow dsDNA threading and translocation processes indicate this wild-type γ-HL channel has potential to detect other base modifications in dsDNA.

TraceSpecks: A Software for Automated Idealization of Noisy Patch-Clamp and Imaging Data

Experimental records of single molecules or ion channels from fluorescence microscopy and patch-clamp electrophysiology often include high-frequency noise and baseline fluctuations that are not generated by the system under investigation and have to be removed. Moreover, multiple channels or conductance levels can be present at a time in the data that need to be quantified to accurately understand the behavior of the system. Manual procedures for removing these fluctuations and extracting conducting states or multiple channels are laborious, prone to subjective bias, and likely to hinder the processing of often very large data sets. We introduce a maximal likelihood formalism for separating signal from a noisy and drifting background such as fluorescence traces from imaging of elementary Ca2+ release events called puffs arising from clusters of channels, and patch-clamp recordings of ion channels. Parameters such as the number of open channels or conducting states, noise level, and background signal can all be optimized using the expectation-maximization algorithm. We implement our algorithm following the Baum-Welch approach to expectation-maximization in the portable Java language with a user-friendly graphical interface and test the algorithm on both synthetic and experimental data from the patch-clamp electrophysiology of Ca2+ channels and fluorescence microscopy of a cluster of Ca2+ channels and Ca2+ channels with multiple conductance levels. The resulting software is accurate, fast, and provides detailed information usually not available through manual analysis. Options for visual inspection of the raw and processed data with key parameters are provided, in addition to a range of statistics such as the mean open probabilities, mean open times, mean close times, dwell-time distributions for different number of channels open or conductance levels, amplitude distribution of all opening events, and number of transitions between different number of open channels or conducting levels in asci format with a single click.

Fully Automatic Multiresolution Idealization for Filtered Ion Channel Recordings: Flickering Event Detection.

We propose a new model-free segmentation method, JULES, which combines recent statistical multiresolution techniques with local deconvolution for idealization of ion channel recordings. The multiresolution criterion takes into account scales down to the sampling rate enabling the detection of flickering events, i.e., events on small temporal scales, even below the filter frequency. For such small scales the deconvolution step allows for a precise determination of dwell times and, in particular, of amplitude levels, a task which is not possible with common thresholding methods. This is confirmed theoretically and in a comprehensive simulation study. In addition, JULES can be applied as a preprocessing method for a refined hidden Markov analysis. Our new methodology allows us to show that gramicidin A flickering events have the same amplitude as the slow gating events. JULES is available as an R function jules in the package clampSeg.