Ion Channel Recordings Research Articles

Lipid Bilayers at Gel/Gel Interface for Ion Channel Recordings

We have developed a practical method to produce durable artificial lipid bilayers using a hydrogel-hydrogel interface for ion channel measurements. Bilayers were formed by forcing a hydrogel-bead into contact with the hydrogel layer (hydrogel plate) in a lipid solution. The immediate formation of a bilayer was observed (<1 s). This allows channel recordings to be repeated more easily and quickly as compared to conventional methods. Currents of various types of channel such as gramicidin, hemolysin and BK-channel have been recorded. Our channel property results mirrored those of other techniques and were reproducible. Hydrogel solutions containing gramicidin were extremely stable and could be used months after preparation for bilayer experiments. [DOI: 10.1380/ejssnt.2008.130]

Single-channel recording of ligand-gated ion channels

Electrophysiological recording of single-channel currents is the most direct method available for obtaining detailed and precise information about the kinetic behavior of ion channels. A wide variety of cell types can be used for single-channel recording, but to obtain the highest resolution of the briefest channel opening and closing events, low-noise recordings, coupled with a minimal filtering frequency, are required. Here, we present a protocol designed to help those with some electrophysiological expertise who wish to explore the properties of native and recombinant single ligand-gated ion channels. We have focused on the practical aspects of recording single GABA channels from cell-attached and outside-out patches and also introduced some of the preliminary considerations that are necessary for the analysis of single-channel data, including an introduction to single-channel analysis software.

A bead-based kinase competition assay | A multiplexed, high-throughput disease diagnostic system | Labeling proteins with interchangeable fluorophores | Nanowire electrodes detect PSA activity | Control over DNA movement through nanopores | Detection of amino acids from complex mixtures | Glass nanopores for ion-channel recordings | Magnetic microposts apply mechanical forces on cells

A bead-based kinase competition assay | A multiplexed, high-throughput disease diagnostic system | Labeling proteins with interchangeable fluorophores | Nanowire electrodes detect PSA activity | Control over DNA movement through nanopores | Detection of amino acids from complex mixtures | Glass nanopores for ion-channel recordings | Magnetic microposts apply mechanical forces on cells

Single Ion-Channel Recordings Using Glass Nanopore Membranes

Protein ion-channel recordings using a glass nanopore (GNP) membrane as the support structure for lipid bilayer membranes are presented. The GNP membrane is composed of a single conical-shaped nanopore embedded in a approximately 50 microm-thick glass membrane chemically modified with a 3-cyanopropyldimethylchlorosilane monolayer to produce a surface of intermediate hydrophobicity. This surface modification results in lipid monolayer formation on the glass surface and a lipid bilayer suspended across the small orifice (100-400 nm-radius) of the GNP membrane, while allowing aqueous solutions to fully wet the glass nanopore. The GNP membrane/bilayer structures, which exhibit ohmic seal resistances of approximately 70 GOmega and electrical breakdown voltages of approximately 0.8 V, are exceptionally stable to mechanical disturbances and have lifetimes of at least 2 weeks. These favorable characteristics result from the very small area of bilayer (10(-10)-10(-8) cm(2)) that is suspended across the GNP membrane orifice. Fluorescence microscopy and vibrational sum frequency spectroscopy demonstrate that a lipid monolayer forms on the 3-cyanopropyl-dimethylchlorosilane modified glass surface with the lipid tails oriented toward the glass. The GNP membrane/bilayer structure is well suited for single ion-channel recordings. Reproducible insertion of the protein ion channel, wild-type alpha-hemolysin (WTalphaHL), and stochastic detection of a small molecule, heptakis(6-O-sulfo)-beta-cyclodextrin, are demonstrated. In addition, the insertion and removal of WTalphaHL channels are reproducibly controlled by applying small pressures (-100 to 350 mmHg) across the lipid bilayer. The electrical and mechanical stability of the bilayer, the ease of which bilayer formation is achieved, and the ability to control ion-channel insertion, coupled with the small bilayer capacitance of the GNP membrane-based system, provide a new and nearly optimal system for single ion-channel recordings.

Laser ablation of micropores for formation of artificial planar lipid bilayers

Artificial lipid bilayers are a powerful tool for studying synthetic or reconstituted ion channels. Key to forming these lipid bilayers is having a small aperture in a septum separating two solution chambers. Traditional methods of aperture generation involve manually punching the aperture into the septum. While these techniques work, they are difficult to implement reliably and do not produce consistently sized apertures. Presented here is a method of using a UV excimer laser with a nanosecond scale pulse width to laser ablate apertures from 4 to 105 microm in 20 microm thick polycarbonate films for use in artificial lipid bilayer experiments. The data demonstrate that the apertures produced by laser ablation are highly reproducible and can support both the formation of stable, long-lasting lipid bilayers as well as the recording of ion channels incorporated into the bilayers.

Isolation of protoplasts and ion channel recording of plasma membrane patches and whole-cell recordings of the marine alga, Ulva pertusa (Chlorophyta)

Whole-cell patch-clamp techniques were used to study ion channels of a marine alga. High quality protoplasts suitable for electrophysiological studies were isolated from the green marine alga, Ulva pertusa, using enzyme mixtures consisting of cellulase and abalone power and identified by calcofluor fluorescence. The vitality of protoplasts varied depending on the alga growth stage, and those isolated from younger tissue in March maintained a high vitality with high sealing success rate compared with protoplasts isolated from mature or non-growing plants in August or November. In the whole-cell configuration, large inward currents were elicited by negative voltage pulses. The voltage-dependent component was predominantly carried by Cl−, as confirmed by the use of the Cl− channel inhibitor DIDS and reversal potential of current-voltage plots. This evidence suggests that hyperpolarization-activated Cl− permeable channels are responsible for the influx of Cl− into U. pertusa cells. Voltage-dependent outward currents were also recorded in several protoplasts, and their properties need further investigation.

CLIC4 (p64H1) and its putative transmembrane domain form poorly selective, redox-regulated ion channels

Despite being synthesized in the cytosol without a leader sequence, the soluble 253-residue mammalian protein CLIC4 (Chloride Intracellular Channel 4, or p64H1), a structural homologue of Omega-type glutathione-S-transferase, autoinserts into membranes to form an integral membrane protein with ion channel activity. A predicted transmembrane domain (TMD) near the N-terminus of CLIC4 could mediate membrane insertion, and contribute to oligomeric pores, with minimal reorganization of the soluble protein structure. We tested this idea by reconstituting recombinant CLIC4 in planar bilayers containing phosphatidyethanolamine, phosphatidylserine and cholesterol, recording ion channels with a maximum conductance of approximately 15 pS in KCl under both oxidizing and reducing conditions. The channels discriminated poorly between anions and cations, incompatible with the current "CLIC" nomenclature, and their conductance was modified by the trans (external or luminal) redox potential, as previously observed for CLIC1. We then reconstituted a truncated version of the protein, limited to the first 61 residues containing the predicted TMD. This included a single trans cysteine residue in the putative pore-forming subunits, at the external entrance to the pore. The truncated protein formed non-selective channels with a reduced conductance, but they retained their trans-redox sensitivity, and could still be blocked or inactivated by trans (not cis) thiol-reative dithiobisnitrobenzoic acid. We suggest that oligomers containing the putative TMD are essential components of the CLIC4 pore. However, the pore is inherently non-selective, and any ionic selectivity in CLIC4 (and other membrane CLICs) may be attributable to other regions of the protein, including the channel vestibules.

2P242 並列イオンチャネル計測のための平面脂質二重膜アレイ(生体膜・人工膜(構造・物性),ポスター発表,第45回日本生物物理学会年会)

2P242 並列イオンチャネル計測のための平面脂質二重膜アレイ(生体膜・人工膜(構造・物性),ポスター発表,第45回日本生物物理学会年会)

Ginsenoside-Rd from panax notoginseng blocks Ca 2+ influx through receptor- and store-operated Ca 2+ channels in vascular smooth muscle cells

Previously, it was found that total saponins from panax notoginseng inhibited Ca 2+ influx coupling to activation of α 1-adrenoceptor. This study was designed to investigate the effects of ginsenoside-Rd from total saponins of panax notoginseng on receptor-operated (ROCC) and store-operated (SOCC) Ca 2+ channels in vascular smooth muscle cells using fura-2 fluorescence, whole cell patch clamp ion channel recording, radio-ligand-receptor binding, 45Ca 2+ radio-trace and organ bath techniques. It was found that ginsenoside-Rd reduced phenylephrine-induced contractile responses and Ca 2+ influx in normal media without significant effect on these responses in Ca 2+-free media. Ginsenoside-Rd also decreased phenylephrine- and thapsigargin-induced inward Ca 2+ currents, and attenuated thapsigargin- and 1-oleoy-2-acetyl- sn-glycerol (OAG)-induced cation entries that are coupled to ROCC and SOCC respectively. Ginsenoside-Rd failed to inhibit KCl-induced contraction of rat aortal rings and Ca 2+ influx, and did not alter voltage-dependent inward Ca 2+ current (VDCC) which was blocked by nifedipine. Also, ginsenoside-Rd did not change binding site and affinity of [ 3H]-prazosin for α 1-adrenoceptor in the vascular plasma membrane. These results suggest that ginsenoside-Rd, as an inhibitor, remarkably inhibits Ca 2+ entry through ROCC and SOCC without effects on VDCC and Ca 2+ release in vascular smooth muscle cells.

Electrophysiological recordings of single ion channels in planar lipid bilayers using a polymethyl methacrylate microfluidic chip

Planar lipid bilayers are used for functional studies of ion channel proteins using electrophysiological techniques. We have been developing a plastic micro-fluidic device for the reconstitution of planar lipid bilayers and electrophysiological recordings toward a "membrane protein chip" for high-throughput screening. In the previous report [Suzuki, H., Tabata, K.V., Noji, H., Takeuchi, S., 2006. Highly reproducible method of planar lipid bilayer reconstitution in polymethyl methacrylate microfluidic chip. Langmuir 22 (4), 1937-1942], we presented the method and device in which the reproducibility of planar lipid bilayers reached 90%, and multiple bilayers were formed simultaneously. In this communication, we show that our device has excellent electric properties suitable for ion channel analysis down to single molecular level. Additional aspects on the optical accessibility and controllability on lipid bilayer formation are also presented.

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.

Transgenic Mouse Overexpressing Syntaxin-1A as a Diabetes Model

Soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) protein syntaxin-1A (STX-1A) plays a role not only in exocytosis, but also binds and regulates Ca(2+) and K(+) (voltage-gated K(+) and ATP-sensitive K(+) channels) to influence the sequence of events leading to secretion. Islet levels of STX-1A and cognate SNARE proteins are reduced in type 2 diabetic rodents, suggesting their role in dysregulated insulin secretion contributing to the abnormal glucose homeostasis. We investigated the specific role of STX-1A in pancreatic beta-cells by generating transgenic mice, which express a moderately increased level ( approximately 30% higher) of STX-1A in pancreatic islets (hereafter called STX-1A mice). The STX-1A mice displayed fasting hyperglycemia and a more sustained elevation of plasma glucose levels after an intraperitoneal glucose tolerance test, with correspondingly reduced plasma insulin levels. Surprisingly, beta-cells from the STX-1A male mice also exhibited abnormal insulin tolerance. To unequivocally determine the beta-cell secretory defects, we used single-cell analyses of exocytosis by patch clamp membrane capacitance measurements and ion channel recordings. Depolarization-evoked membrane capacitance increases were reduced in the STX-1A mouse islet beta-cells. The STX-1A mouse also exhibited reduced currents through the Ca(2+) channels but little change in the voltage-gated K(+) channel or ATP-sensitive K(+) channel. These results suggest that fluctuation of islet STX-1A levels in diabetes could influence the pathological and differential regulation of beta-cell ion channels and the exocytotic machinery, collectively contributing to the impaired insulin secretion.

Development of a lung slice preparation for recording ion channel activity in alveolar epithelial type I cells

BackgroundLung fluid balance in the healthy lung is dependent upon finely regulated vectorial transport of ions across the alveolar epithelium. Classically, the cellular locus of the major ion transport processes has been widely accepted to be the alveolar type II cell. Although evidence is now emerging to suggest that the alveolar type I cell might significantly contribute to the overall ion and fluid homeostasis of the lung, direct assessment of functional ion channels in type I cells has remained elusive.MethodsHere we describe a development of a lung slice preparation that has allowed positive identification of alveolar type I cells within an intact and viable alveolar epithelium using living cell immunohistochemistry.ResultsThis technique has allowed, for the first time, single ion channels of identified alveolar type I cells to be recorded using the cell-attached configuration of the patch-clamp technique.ConclusionThis exciting new development should facilitate the ascription of function to alveolar type I cells and allow us to integrate this cell type into the general model of alveolar ion and fluid balance in health and disease.

A human congenital myasthenia‐causing mutation (ɛL78P) of the muscle nicotinic acetylcholine receptor with unusual single channel properties

A mutation in the epsilon subunit of the human nicotinic acetylcholine receptor (epsilonL78P) is known to cause a congenital slow channel myasthenic syndrome. We have investigated the changes in receptor function that result in the mutant receptor producing prolonged endplate currents, and consequent muscle damage. The rate constants for channel gating and for the binding and dissociation of acetylcholine were investigated by analysis of single ion channel recordings. A conventional mechanism with two non-equivalent binding sites, and variations upon this mechanism, were fitted to data using a maximum likelihood method that uses the Hawkes-Jalali-Colquhoun (HJC) treatment of missed brief events. The mutant receptor produced prolonged activations, bursts of openings that cause a slow decay of simulated synaptic currents. The main reason for the longer bursts of openings seen with mutant receptor was a decrease in the rate of ACh dissociation from diliganded receptors, though the lifetime of individual openings was somewhat increased too. As well as producing long bursts, the mutant receptor also produced many very short openings, though these carry little current. The burst structure for the mutant receptor at low ACh concentration is unusual in that most long bursts appear to start in a very brief monoliganded open state that then usually binds another ACh molecule to produce a long diliganded activation. The first opening is so short that it will usually be missed (together with the shut time that follows it), so the true burst length is likely to be underestimated.

Model selection and parameter estimation for ion channel recordings with an application to the K+ outward-rectifier in barley leaf.

We present a statistical method, and its accompanying algorithms, for the selection of a mathematical model of the gating mechanism of an ion channel and for the estimation of the parameters of this model. The method assumes a hidden Markov model that incorporates filtering, colored noise and state-dependent white excess noise for the recorded data. The model selection and parameter estimation are performed via a Bayesian approach using Markov chain Monte Carlo. The method is illustrated by its application to single-channel recordings of the K(+) outward-rectifier in barley leaf.

Characterization of a MEMS BioChip for planar patch-clamp recording

We describe a planar MEMS silicon structure to record ion channel currents in biological cells. The conventional method of performing an electrophysiological experiment, `patch-clamping', employs a glass micropipette. The micropipette tip is a source of thermal noise because of its inherent, tapered, conical structure, giving rise to a large pipette resistance. This pipette resistance, when coupled with the biological cell capacitance, limits the available bandwidth of single ion channel recording. In this work, we propose a current transport model to characterize the series resistance and capacitance of a planar pipette fabricated on a silicon BioChip. Our model provides a deeper insight into how currents injected into a micropore are quantitatively partitioned into the individual ion transports, and goes beyond just describing the solute and solvent kinetics inside pores of microscale dimensions. The device topology and fabrication sequence of the planar patch-clamp setup are also discussed. The theoretical predictions by the model are in close agreement with the experimental results.

Microfabricated Teflon Membranes for Low-Noise Recordings of Ion Channels in Planar Lipid Bilayers

We present a straightforward, accessible method for the fabrication of micropores with diameters from 2 to 800μm in films of amorphous Teflon (Teflon AF). Pores with diameters ≤40μm made it possible to record ion fluxes through ion channels in planar bilayers with excellent signal characteristics. These pores afforded: i), stable measurements at transmembrane voltages up to 460mV; ii), recordings at low noise levels (0.4pA rms at 4.3kHz bandwidth); iii), recordings at high effective bandwidth (10.7kHz); and iv), formation of multiple planar lipid bilayers in parallel. Microfabricated pores in films of Teflon AF made it possible to examine, experimentally and theoretically, the influence of the pore diameter on the current noise in planar bilayer recordings. Reducing the pore diameter below 40μm mainly increased the stability of the planar bilayers, but had only a small effect on the level of the current noise. The low-noise properties of bilayer recordings on micropores in Teflon AF films were exploited to record the smallest conductance state of alamethicin (24 pS) at an unprecedentedly high bandwidth of 10.7kHz.

Simultaneous Optical and Electrical Recording of Single Gramicidin Channels

We report here an approach for simultaneous fluorescence imaging and electrical recording of single ion channels in planar bilayer membranes. As a test case, fluorescently labeled (Cy3 and Cy5) gramicidin derivatives were imaged at the single-molecule level using far-field illumination and cooled CCD camera detection. Gramicidin monomers were observed to diffuse in the plane of the membrane with a diffusion coefficient of 3.3 × 10 −8 cm 2s −1. Simultaneous electrical recording detected gramicidin homodimer (Cy3/Cy3, Cy5/Cy5) and heterodimer (Cy3/Cy5) channels. Heterodimer formation was observed optically by the appearance of a fluorescence resonance energy transfer (FRET) signal (irradiation of Cy3, detection of Cy5). The number of FRET signals was significantly smaller than the number of Cy3 signals (Cy3 monomers plus Cy3 homodimers) as expected. The number of FRET signals increased with increasing channel activity. In numerous cases the appearance of a FRET signal was observed to correlate with a channel opening event detected electrically. The heterodimers also diffused in the plane of the membrane with a diffusion coefficient of 3.0 × 10 −8 cm 2s −1. These experiments demonstrate the feasibility of simultaneous optical and electrical detection of structural changes in single ion channels as well as suggesting strategies for improving the reliability of such measurements.

Ion Channels in Small Cells and Subcellular Structures Can Be Studied with a Smart Patch-Clamp System

We have developed a scanning patch-clamp technique that facilitates single-channel recording from small cells and submicron cellular structures that are inaccessible by conventional methods. The scanning patch-clamp technique combines scanning ion conductance microscopy and patch-clamp recording through a single glass nanopipette probe. In this method the nanopipette is first scanned over a cell surface, using current feedback, to obtain a high-resolution topographic image. This same pipette is then used to make the patch-clamp recording. Because image information is obtained via the patch electrode it can be used to position the pipette onto a cell with nanometer precision. The utility of this technique is demonstrated by obtaining ion channel recordings from the top of epithelial microvilli and openings of cardiomyocyte T-tubules. Furthermore, for the first time we have demonstrated that it is possible to record ion channels from very small cells, such as sperm cells, under physiological conditions as well as record from cellular microstructures such as submicron neuronal processes.

High-conductance chloride channels generate pacemaker currents in interstitial cells of Cajal

Background & Aims: Interstitial cells of Cajal (ICCs) are responsible for slow, wave-driven, rhythmic, peristaltic motor patterns in the gastrointestinal tract. The aim was to identify and characterize the ion channels that generate the underlying pacemaker activity. Methods: Single ion channel recordings were obtained from nonenzymatically isolated ICCs and studied by using the cell attached and inside-out configurations of the patch clamp technique. Results: A high-conductance chloride channel was observed in ICCs that was spontaneously and rhythmically active at the same frequency as the rhythmic inward currents defining ICC pacemaker activity, 20–30 cycles/min at room temperature. Main conductance levels occurred between 122–144 pS and between 185–216 pS. Periodicity in the channel opening coincided with periodicity in membrane potential change, hence, at the single channel level, chloride channels were seen to be associated with the generation of rhythmic changes in membrane potential. Conclusions: ICCs harbor high-conductance chloride channels that participate in the generation of pacemaker activity and may become a target for pharmacologic treatment of gut motor disorders.GASTROENTEROLOGY 2002;123:1627-1636