scholarly journals Development of an automated system to measure ion channel currents using a surface-modified gold probe

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Minako Hirano ◽  
Masahisa Tomita ◽  
Chikako Takahashi ◽  
Nobuyuki Kawashima ◽  
Toru Ide
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
Lipid Bilayer ◽  
Measurement System ◽  
Automated System ◽  
Channel Activity ◽  
Channel Current ◽  
Gold Probe ◽  
Measurement Efficiency ◽  
Channel Currents

AbstractArtificial lipid bilayer single-channel recording technique has been employed to determine the biophysical and pharmacological properties of various ion channels. However, its measurement efficiency is very low, as it requires two time-consuming processes: preparation of lipid bilayer membranes and incorporation of ion channels into the membranes. In order to address these problems, we previously developed a technique based on hydrophilically modified gold probes on which are immobilized ion channels that can be promptly incorporated into the bilayer membrane at the same time as the membrane is formed on the probes’ hydrophilic area. Here, we improved further this technique by optimizing the gold probe and developed an automated channel current measurement system. We found that use of probes with rounded tips enhanced the efficiency of channel current measurements, and introducing a hydrophobic area on the probe surface, beside the hydrophilic one, further increased measurement efficiency by boosting membrane stability. Moreover, we developed an automated measurement system using the optimized probes; it enabled us to automatically measure channel currents and analyze the effects of a blocker on channel activity. Our study will contribute to the development of high-throughput devices to identify drug candidates affecting ion channel activity.

scholarly journals TOK Homologue in Neurospora crassa: First Cloning and Functional Characterization of an Ion Channel in a Filamentous Fungus

2003 ◽  
Vol 2 (1) ◽  
pp. 181-190 ◽  
Author(s):  
Stephen K. Roberts
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
Neurospora Crassa ◽  
Filamentous Fungus ◽  
Single Channel ◽  
Functional Characterization ◽  
Reversal Potential ◽  
Channel Activity ◽  
Biophysical Properties ◽  
Patch Clamp Technique

ABSTRACT In contrast to animal and plant cells, very little is known of ion channel function in fungal physiology. The life cycle of most fungi depends on the “filamentous” polarized growth of hyphal cells; however, no ion channels have been cloned from filamentous fungi and comparatively few preliminary recordings of ion channel activity have been made. In an attempt to gain an insight into the role of ion channels in fungal hyphal physiology, a homolog of the yeast K+ channel (ScTOK1) was cloned from the filamentous fungus, Neurospora crassa. The patch clamp technique was used to investigate the biophysical properties of the N. crassa K+ channel (NcTOKA) after heterologous expression of NcTOKA in yeast. NcTOKA mediated mainly time-dependent outward whole-cell currents, and the reversal potential of these currents indicated that it conducted K+ efflux. NcTOKA channel gating was sensitive to extracellular K+ such that channel activation was dependent on the reversal potential for K+. However, expression of NcTOKA was able to overcome the K+ auxotrophy of a yeast mutant missing the K+ uptake transporters TRK1 and TRK2, suggesting that NcTOKA also mediated K+ influx. Consistent with this, close inspection of NcTOKA-mediated currents revealed small inward K+ currents at potentials negative of EK. NcTOKA single-channel activity was characterized by rapid flickering between the open and closed states with a unitary conductance of 16 pS. NcTOKA was effectively blocked by extracellular Ca2+, verapamil, quinine, and TEA+ but was insensitive to Cs+, 4-aminopyridine, and glibenclamide. The physiological significance of NcTOKA is discussed in the context of its biophysical properties.

Nuclear ion channel activity is regulated by actin filaments

1996 ◽  
Vol 270 (5) ◽  
pp. C1532-C1543 ◽  
Author(s):  
A. G. Prat ◽  
H. F. Cantiello
Keyword(s):  
Ion Channels ◽  
Actin Cytoskeleton ◽  
Nuclear Envelope ◽  
Ion Channel ◽  
Actin Filaments ◽  
Actin Binding ◽  
Bathing Solution ◽  
Channel Activity ◽  
Nuclear Ion Channels ◽  
Inside Out

Actin filaments are novel second messengers involved in ion channel regulation. Because cytoskeletal components interact with the nuclear envelope, the actin cytoskeleton may also control nuclear membrane function. In this report, the patch-clamp technique was applied to isolated nuclei from amphibian A6 epithelial cells to assess the role of actin filaments on nuclear ion channel activity under nucleus-attached or -excised conditions. The most prevalent spontaneous nuclear ion channel species, 76% (n = 46), was cation selective and had a maximal single-channel conductance of approximately 420 pS. Nuclear ion channels also displayed multiple subconductance states, including channel activity of 26 pS that was frequently observed. Nuclear ion channel activity on otherwise quiescent patches was induced by either addition of the actin cytoskeleton disrupter cytochalasin D (CD; 5 micrograms/ml, 60%, 3 of 5 patches) or actin (10-1,000 micrograms/ml) to the bathing solution of nucleus-attached patches (59%, 13 of 22 patches). Actin also induced ion channel activity in quiescent excised inside-out patches from the nuclear envelope (80%, 4 of 5 patches). In contrast, addition of bovine serum albumin (10-1,000 micrograms/ml) to the bathing solution of nucleus-attached patches was without effect on nuclear ion channel activity (5 of 5 patches). The monoclonal antibody MAb414, specific for nuclear pore complex proteins, completely prevented either spontaneous or cytosolic actin-induced nuclear ion channels under nucleus-attached conditions (4 of 4 patches) but not intranuclear actin-induced nuclear ion channels under excised inside-out conditions (3 of 3 patches). In nucleus-attached patches, channel activity was readily activated by addition of the G-actin-binding protein deoxyribonuclease I to nucleus-attached patches (56%, 5 of 9 patches) or further addition of the actin-cross-linker filamin in the presence of actin (57%, 4 of 7 patches). The data indicate that dynamic changes in actin filament organization may represent a novel mechanism to control nuclear function.

Ion channel regulation by G proteins

1995 ◽  
Vol 75 (4) ◽  
pp. 865-885 ◽  
Author(s):  
K. Wickman ◽  
D. E. Clapham
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
G Protein ◽  
G Proteins ◽  
Second Messengers ◽  
Channel Activity ◽  
Ion Channel Regulation ◽  
Protein Subunits ◽  
Channel Regulation ◽  
Intracellular Cascades

Ion channels are poised uniquely to initiate, mediate, or regulate such distinct cellular activities as action potential propagation, secretion, and gene transcription. In retrospect, it is not surprising that studies of ion channels have revealed considerable diversities in their primary structures, regulation, and expression. From a functional standpoint, the various mechanisms coopted by cells to regulate channel activity are particularly fascinating. Extracellular ligands, membrane potential, phosphorylation, ions themselves, and diffusible second messengers are all well-established regulators of ion channel activity. Heterotrimeric GTP-binding proteins (G proteins) mediate many of these types of ion channel regulation by stimulating or inhibiting phosphorylation pathways, initiating intracellular cascades leading to elevation of cytosolic Ca2+ or adenosine 3',5'-cyclic monophosphate levels, or by generating various lipid-derived compounds. In some cases, it seems that activated G protein subunits can interact directly with ion channels to elicit regulation. Although there is currently little direct biochemical evidence to support such a mechanism, it is the working hypothesis for the most-studied G protein-regulated ion channels.

ATP-sensitive K + -channel run-down is Mg 2+ dependent

1990 ◽  
Vol 240 (1298) ◽  
pp. 397-410 ◽  
Keyword(s):  
State Probability ◽  
K Channel ◽  
Channel Activity ◽  
Channel Current ◽  
Phosphatase Inhibitor ◽  
Voltage Dependent ◽  
Insulin Secreting Cells ◽  
Channel Currents

ATP-sensitive K + -channel currents were recorded from isolated mem­brane patches and voltage-clamped CRI-G1 insulin-secreting cells. Inter­nal Mg 2+ ions inhibited ATP-K + channels by a voltage-dependent block of the channel current and decrease of open-state probability. The run­ down of ATP-K + channel activity was also shown to be [Mg 2+ ] i depen­dent, being almost abolished in Mg 2+ -free conditions. Substitution of Mn 2+ for Mg 2+ did not prevent run-down, nor did the presence of phos­phate-donating nucleotides, a protease or phosphatase inhibitor or replacement of Cl by gluconate.

scholarly journals Fluorescence Microscopy of Piezo1 in Droplet Hydrogel Bilayers

2018 ◽  
Author(s):  
Oskar B. Jaggers ◽  
Pietro Ridone ◽  
Boris Martinac ◽  
Matthew A. B. Baker
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
Simultaneous Recording ◽  
Mechanical Stimuli ◽  
Channel Activity ◽  
Channel Proteins ◽  
Mechanosensitive Ion Channel ◽  
Lymphatic Dysplasia ◽  
Hydrogel Bilayer

AbstractMechanosensitive ion channels are membrane gated pores which are activated by mechanical stimuli. The focus of this study is on Piezo1, a newly discovered, large, mammalian, mechanosensitive ion channel, which has been linked to diseases such as dehydrated hereditary stomatocytosis (Xerocytosis) and lymphatic dysplasia. Here we utilize an established in-vitro artificial bilayer system to interrogate single Piezo1 channel activity. The droplet-hydrogel bilayer (DHB) system uniquely allows the simultaneous recording of electrical activity and fluorescence imaging of labelled protein. We successfully reconstituted fluorescently labelled Piezo1 ion channels in DHBs and verified activity using electrophysiology in the same system. We demonstrate successful insertion and activation of hPiezo1-GFP in bilayers of varying composition. Furthermore, we compare the Piezo1 bilayer reconstitution with measurements of insertion and activation of KcsA channels to reproduce the channel conductances reported in the literature. Together, our results showcase the use of DHBs for future experiments allowing simultaneous measurements of ion channel gating while visualising the channel proteins using fluorescence.

Dependence of Ca2+ channel currents on endogenous and exogenous sources of ATP in portal vein smooth muscle

1997 ◽  
Vol 272 (2) ◽  
pp. H987-H994 ◽  
Author(s):  
J. N. Lorenz ◽  
R. J. Paul
Keyword(s):  
Portal Vein ◽  
Smooth Muscles ◽  
Channel Activity ◽  
Vascular Smooth Muscles ◽  
Vascular Contractility ◽  
Channel Current ◽  
Metabolic Substrates ◽  
Oxidative Processes ◽  
Myosin Interaction ◽  
Channel Currents

Metabolic pathways in vascular smooth muscles (VSM) appear to be functionally compartmentalized such that glycolysis fuels membrane-related processes, whereas oxidative processes fuel actin-myosin interaction. Because ATP influences Ca2+ channel activity, we examined the effects of ATP and metabolic substrates on Ca2+ channel activity with patch-clamp techniques in VSM cells isolated from rat portal vein. The peak magnitude of the Ca2+ channel currents was found to depend on the ATP concentration in the patch pipette. Cells perfused with 1, 3, and 5 mMATP had mean peak currents of 4.7 +/- 0.6, 12.2 +/- 1.9, and 17.6 +/- 2.0 pA/pF, respectively, and all currents showed substantial rundown. In separate experiments performed in the absence of intracellular ATP, provision of glycolytic but not oxidative substrates was able to maintain Ca2+ channel currents at levels comparable with those seen in the presence of 1 mM ATP. In the presence of 5 mM ATP, provision of glycolytic substrates resulted in a high peak current amplitude that was also very stable. Finally, metabolic inhibition with cyanide and iodoacetate caused a significant increase in the rate of current rundown, even in the presence of 5 mM ATP. These findings indicate that Ca2+ channel current is strongly dependent on ATP and that the source of ATP can also be an important factor. Compared with exogenous provision of ATP, endogenous metabolism preferentially maintained Ca2+ channel currents, consistent with the hypothesis of a functionally separate subsarcolemmal compartment. This provides an effective pathway for linking E-C coupling and vascular contractility to the metabolic state of the vascular cell.

Ion Channel Sensor on a Silicon Support

2004 ◽  
Vol 820 ◽  
Author(s):  
Michael Goryll ◽  
Seth Wilk ◽  
Gerard M. Laws ◽  
Stephen M. Goodnick ◽  
Trevor J. Thornton ◽  
...  
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Chemical Vapor ◽  
Fast Method ◽  
Fluorocarbon Films ◽  
Novel Approach ◽  
Voltage Dependent ◽  
Assembled Monolayers

AbstractWe are building a biosensor based on ion channels inserted into lipid bilayers that are suspended across an aperture in silicon. The process flow only involves conventional optical lithography and deep Si reactive ion etching to create micromachined apertures in a silicon wafer. In order to provide surface properties for lipid bilayer attachment that are similar to those of the fluorocarbon films that are currently used, we coated the silicon surface with a fluoropolymer using plasma-assisted chemical vapor deposition. When compared with the surface treatment methods using self-assembled monolayers of fluorocarbon chemicals, this novel approach towards modifying the wettability of a silicon dioxide surface provides an easy and fast method for subsequent lipid bilayer formation. Current-Voltage measurements on OmpF ion channels incorporated into these membranes show the voltage dependent gating action expected from a working porin ion channel.

Ion channels and disease

2020 ◽  
pp. 246-255
Author(s):  
Frances Ashcroft ◽  
Paolo Tammaro
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
Single Channel ◽  
Cell Types ◽  
Channel Structure ◽  
Channel Proteins ◽  
Channel Function ◽  
Ion Channel Proteins ◽  
Single Channel Currents ◽  
Channel Currents

Ion channels are membrane proteins that act as gated pathways for the movement of ions across cell membranes. They are found in both surface and intracellular membranes and play essential roles in the physiology of all cell types. An ever-increasing number of human diseases are now known to be caused by defects in ion channel function. To understand how ion channel defects give rise to disease, it is helpful to understand how the ion channel proteins work. This chapter therefore considers what is known of ion channel structure, explains the properties of the single ion channel, and shows how single-channel currents give rise to action potentials and synaptic potentials.

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