scholarly journals Development of a Novel Automated Ion Channel Recording Method Using “Inside-Out” Whole-Cell Membranes

2005 ◽  
Vol 10 (8) ◽  
pp. 806-813 ◽  
Author(s):  
Dmitry V. Vasilyev ◽  
Thomas L. Merrill ◽  
Mark R. Bowlby
Keyword(s):  
Ion Channels ◽  
Parallel Implementation ◽  
Drug Application ◽  
K Channel ◽  
Positive Pressure ◽  
Screening Methods ◽  
Patch Clamp Recording ◽  
Whole Cell ◽  
Cell Configuration ◽  
Inside Out

Efforts to develop novelmethods 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 cellmay be moved through the air, first rupturing part of the cellularmembrane 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, aswell 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 inactivities 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.

Ion channel activities of cultured rat mesangial cells

1991 ◽  
Vol 261 (5) ◽  
pp. F808-F814 ◽  
Author(s):  
H. Matsunaga ◽  
N. Yamashita ◽  
Y. Miyajima ◽  
T. Okuda ◽  
H. Chang ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Ion Channels ◽  
Patch Clamp ◽  
Ion Channel ◽  
Arginine Vasopressin ◽  
Mesangial Cells ◽  
Cation Channel ◽  
K Channel ◽  
Patch Clamp Technique ◽  
Inside Out

We used the patch-clamp technique to clarify the nature of ion channels in renal mesangial cells in culture. In the cell-attached mode most patches were silent in the absence of agonists. In some patches a 25-pS nonselective channel was observed. This 25-pS cation channel was consistently observed in inside-out patches, and it was activated by intracellular Ca2+. Excised patch experiments also revealed the existence of a 40-pS K+ channel, which was activated by intracellular Ca2+. This 40-pS K+ channel was observed infrequently in the cell-attached mode. The activities of both channels were increased by arginine vasopressin or angiotensin II, resulting from an increase in intracellular Ca2+ concentration.

scholarly journals Calnexin revealed as an ether-a-go-go chaperone by getting mutant worms up and going

2018 ◽  
Vol 150 (8) ◽  
pp. 1059-1061
Author(s):  
Jonathan T. Pierce
Keyword(s):  
Ion Channels ◽  
Dynamic Control ◽  
Cell Types ◽  
K Channel ◽  
Membrane Domains ◽  
Excitable Cells ◽  
Patch Clamp Recording ◽  
Cell Excitability ◽  
Distinct Membrane

The role of ion channels in cell excitability was first revealed in a series of voltage clamp experiments by Hodgkin and Huxley in the 1950s. However, it was not until the 1970s that patch-clamp recording ushered in a revolution that allowed physiologists to witness how ion channels flicker open and closed at angstrom scale and with microsecond resolution. The unexpectedly tight seal made by the patch pipette in the whole-cell configuration later allowed molecular biologists to suck up the insides of identified cells to unveil their unique molecular contents. By refining these techniques, researchers have scrutinized the surface and contents of excitable cells in detail over the past few decades. However, these powerful approaches do not discern which molecules are responsible for the dynamic control of the genesis, abundance, and subcellular localization of ion channels. In this dark territory, teams of unknown and poorly understood molecules guide specific ion channels through translation, folding, and modification, and then they shuttle them toward and away from distinct membrane domains via different subcellular routes. A central challenge in understanding these processes is the likelihood that these diverse regulatory molecules may be specific to ion channel subtypes, cell types, and circumstance. In work described in this issue, Bai et al. (2018. J. Gen. Physiol. https://doi.org/10.1085/jgp.201812025) begin to shed light on the biogenesis of UNC-103, a K+ channel found in Caenorhabditis elegans.

A new oil-gate concentration jump technique applied to inside-out patch-clamp recording

1988 ◽  
Vol 255 (4) ◽  
pp. H980-H984 ◽  
Author(s):  
D. Y. Qin ◽  
A. Noma
Keyword(s):  
K Channel ◽  
Narrow Slit ◽  
Patch Clamp Recording ◽  
Concentration Jump ◽  
Guinea Pig Heart ◽  
Ventricular Cells ◽  
Pipette Tip ◽  
Time Courses ◽  
Inside Out ◽  
Membrane Patch

A new method was developed to instantaneously replace the solution on the inner side of an inside-out membrane patch in order to measure time courses with which active substances acted on single ionic channels. Inside-out membrane patches were isolated from single ventricular cells of the guinea pig heart. The recording bath consisted of two chambers separated by a partition having a narrow slit. Mixing of two test solutions through this slit was prevented by filling it with paraffin oil. The pipette tip with a tightly sealed inside-out membrane patch was moved through the oil from one solution to the other so that the pipette tip was instantaneously exposed to a new solution. When the pipette tip was jumped between different K+ concentrations, the leak current through the membrane patch increased or decreased with a half time of 6.3 +/- 3.0 ms (n = 15). The amplitude of single K+ channel currents changed to a new steady level within approximately 20 ms. These time courses were well explained by diffusion of K+ in the dead space between the pipette tip opening and the membrane patch. An application of this method to the ATP-regulated K+ channel revealed a latent period of 1-2 s before the channel started its activity after the instantaneous removal of ATP, whereas no obvious latency was observed in the rapid suppression of the channel, which was completed in 100-300 ms after reapplying ATP.

Calcium-dependent inactivation of inwardly rectifying K+ channel in a tumor mast cell line

1992 ◽  
Vol 262 (1) ◽  
pp. C84-C90 ◽  
Author(s):  
M. Mukai ◽  
I. Kyogoku ◽  
M. Kuno
Keyword(s):  
Mast Cell ◽  
Cell Line ◽  
Single Channel ◽  
K Channel ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Whole Cell ◽  
Mast Cell Line ◽  
Inwardly Rectifying ◽  
Inside Out

Antigenic stimulation of rat basophilic leukemia (RBL-2H3) cells, a tumor mast cell line, is associated with an increase in intracellular free Ca2+ concentrations ([Ca2+]i) and membrane polarization. We recorded whole cell and single-channel currents through the inwardly rectifying K+ channel, a major resting conductance of cells, using the patch-clamp technique, and we examined interactions between channel activity and [Ca2+]i. With 10 microM Ca2+ in the pipette, the amplitude of whole cell currents gradually declined within 5 min to 48 +/- 13% of that immediately after rupture of the patch membrane, in the presence of 1 mM ATP which minimized intrinsic rundown. In inside-out patches, activity of the channel was reduced by increasing the concentration of Ca2+ in the internal medium, both in the presence and absence of 1 mM ATP, with no apparent change in single-channel conductance. Time-averaged mean current activity in inside-out patches in the presence of 5 microM Ca2+ was less than 50% of that with Ca2+ of 100 nM or less. These results suggest that a rise in [Ca2+]i leads to a closure of the inwardly rectifying K+ channel. In some inside-out patches, inward currents characterized by burst composed of rapid transitions between open and closed states were observed (flickering currents). Single-channel properties of the flickering currents are similar to the inwardly rectifying K+ channel except for kinetics (single-channel conductance of 24.5 +/- 7.9 pS, inward rectification, and permeability to K+).(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Halothane Inhibits an Intermediate Conductance Ca2+-activated K+Channel by Acting at the Extracellular Side of the Ionic Pore

2003 ◽  
Vol 99 (6) ◽  
pp. 1340-1345 ◽  
Author(s):  
Mitsuko Hashiguchi-Ikeda ◽  
Tsunehisa Namba ◽  
Takahiro M. Ishii ◽  
Taizo Hisano ◽  
Kazuhiko Fukuda
Keyword(s):  
Ion Channels ◽  
K Channel ◽  
Xenopus Laevis Oocytes ◽  
Gamma Aminobutyric Acid ◽  
Time Constants ◽  
K Channels ◽  
Pore Domain ◽  
Inside Out ◽  
Gated Ion Channels ◽  
Ligand Gated Ion Channels

Background Actions of volatile anesthetics on ligand-gated ion channels, such as gamma-aminobutyric acid type A receptors, have been studied extensively. However, actions on other types of channels, such as K+ channels, are poorly understood. The authors previously showed that a Ca2+-activated K+ channel, IK, is sensitive to halothane, whereas SK1, another Ca2+-activated K+ channel, is insensitive. To explore how halothane acts on Ca2+-activated K+ channels, chimeras between IK and SK1 were constructed, and halothane sensitivity was analyzed. Methods IK, SK1, and chimera channels were expressed in Xenopus laevis oocytes. Currents of expressed channels were measured in the presence of 10 microm Ca2+ by excised patch clamp analysis. Time constants of inhibition by halothane were compared between inside-out and outside-out patch configurations. Results Currents from chimera channels possessing the pore domain derived from IK were inhibited by halothane, whereas those possessing the SK1 pore domain were insensitive. Time constants of inhibition by halothane were significantly smaller in the outside-out patches than in the inside-out patches of both wild-type IK and a chimera with pore domain of IK. Conclusions It is suggested that halothane interacts with the extracellular part of the ionic pore of IK. Whether this type of interaction is involved in the mechanism of anesthetic actions on ligand-gated ion channels warrants further investigation.

scholarly journals Apical K+ channels in Necturus taste cells. Modulation by intracellular factors and taste stimuli.

1992 ◽  
Vol 99 (4) ◽  
pp. 591-613 ◽  
Author(s):  
T A Cummings ◽  
S C Kinnamon
Keyword(s):  
Apical Membrane ◽  
Basolateral Membrane ◽  
Taste Receptor ◽  
K Channel ◽  
Patch Clamp Recording ◽  
Open Probability ◽  
K Channels ◽  
Voltage Dependent ◽  
Whole Cell Recordings ◽  
Inside Out

The apically restricted, voltage-dependent K+ conductance of Necturus taste receptor cells was studied using cell-attached, inside-out and outside-out configurations of the patch-clamp recording technique. Patches from the apical membrane typically contained many channels with unitary conductances ranging from 30 to 175 pS in symmetrical K+ solutions. Channel density was so high that unitary currents could be resolved only at negative voltages; at positive voltages patch recordings resembled whole-cell recordings. These multi-channel patches had a small but significant resting conductance that was strongly activated by depolarization. Patch current was highly K+ selective, with a PK/PNa ratio of 28. Patches containing single K+ channels were obtained by allowing the apical membrane to redistribute into the basolateral membrane with time. Two types of K+ channels were observed in isolation. Ca(2+)-dependent channels of large conductance (135-175 pS) were activated in cell-attached patches by strong depolarization, with a half-activation voltage of approximately -10 mV. An ATP-blocked K+ channel of 100 pS was activated in cell-attached patches by weak depolarization, with a half-activation voltage of approximately -47 mV. All apical K+ channels were blocked by the sour taste stimulus citric acid directly applied to outside-out and perfused cell-attached patches. The bitter stimulus quinine also blocked all channels when applied directly by altering channel gating to reduce the open probability. When quinine was applied extracellularly only to the membrane outside the patch pipette and also to inside-out patches, it produced a flickery block. Thus, sour and bitter taste stimuli appear to block the same apical K+ channels via different mechanisms to produce depolarizing receptor potentials.

scholarly journals Block of cardiac ATP-sensitive K+ channels by external divalent cations is modulated by intracellular ATP. Evidence for allosteric regulation of the channel protein.

1993 ◽  
Vol 102 (4) ◽  
pp. 693-712 ◽  
Author(s):  
W M Kwok ◽  
R S Kass
Keyword(s):  
Single Channel ◽  
Divalent Cations ◽  
Cation Binding ◽  
Patch Clamp Recording ◽  
Whole Cell ◽  
Channel Protein ◽  
Intracellular Atp ◽  
Ventricular Cells ◽  
Patch Configuration ◽  
Inside Out

We have investigated the interactions between extracellular divalent cations and the ATP-sensitive potassium channel in single guinea pig ventricular cells and found that, under whole-cell patch clamp recording conditions, extracellularly applied Co2+, Cd2+, and Zn2+ block current through the ATP-sensitive K channel (IKATP). The respective Kd's for block of IKATP by Cd2+ and Zn2+ are 28 and 0.46 microM. The Kd for Co2+ is > 200 microM. Extracellular Ca2+ and Mg2+ appear to have no effect at concentrations up to 1 and 2 mM, respectively. Block of IKATP by extracellular cations is not voltage dependent, and both onset and recovery from block occur within seconds. Single-channel experiments using the inside-out patch configuration show that internally applied Cd2+ and Zn2+ are not effective blockers of IKATP. Experiments in the outside-out patch configuration confirm that the divalent cations interact directly with IKATP channel activity. Our study also shows that this block of IKATP is dependent on intracellular ATP concentrations. Under whole-cell conditions, when cells are dialyzed with [ATP]pipette = 0, the degree of cation block is reduced. This dependence on intracellular ATP was confirmed at the single-channel level by experiments in excised, inside-out patch configurations. Our results show that some, but not all, divalent cations inhibit current through IKATP channels by binding to sites that are not within the transmembrane electric field, but are on the extracellular membrane surface. The interdependence of internal ATP and external divalent cation binding is consistent with an allosteric interaction between two binding sites and is highly suggestive of a modulatory mechanism involving conformational change of the channel protein.

scholarly journals A Novel Membrane Potential-Sensitive Fluorescent Dye Improves Cell-Based Assays for Ion Channels

2002 ◽  
Vol 7 (1) ◽  
pp. 79-85 ◽  
Author(s):  
Deborah F. Baxter ◽  
Martin Kirk ◽  
Amy F. Garcia ◽  
Alejandra Raimondi ◽  
Mats H. Holmqvist ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Ion Channels ◽  
Patch Clamp ◽  
High Throughput Screening ◽  
Fluorescent Dye ◽  
Patch Clamp Recording ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp ◽  
Electrophysiological Studies

The study of ion channel-mediated changes in membrane potential using the conventional bisoxonol fluorescent dye DiBAC4(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 DiBAC4(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 DiBAC4(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 DiBAC4(3) with respect to both kinetics and amplitude of capsaicin-induced fluorescence changes. FMP has also been used to measure the activation of KATP1 and hERG.2 Thus this novel membrane potential dye represents a powerful tool for developing high-throughput screening assays for ion channels.

scholarly journals Characterization of ion channels and O2 sensitivity in gill neuroepithelial cells of the anoxia-tolerant goldfish (Carassius auratus)

2017 ◽  
Vol 118 (6) ◽  
pp. 3014-3023 ◽  
Author(s):  
Peter C. Zachar ◽  
Wen Pan ◽  
Michael G. Jonz
Keyword(s):  
Ion Channels ◽  
Patch Clamp ◽  
Synaptic Vesicle ◽  
Resting Membrane Potential ◽  
Patch Clamp Recording ◽  
Whole Cell ◽  
Neuroepithelial Cells ◽  
Voltage Dependent ◽  
Intracellular Ca

The neuroepithelial cell (NEC) of the fish gill is an important model for O2 sensing in vertebrates; however, a complete picture of the chemosensory mechanisms in NECs is lacking, and O2 chemoreception in vertebrates that are tolerant to anoxia has not yet been explored. Using whole cell patch-clamp recording, we characterized four types of ion channels in NECs isolated from the anoxia-tolerant goldfish. A Ca2+-dependent K+ current ( IKCa) peaked at ~20 mV, was potentiated by increased intracellular Ca2+, and was reduced by 100 μM Cd2+. A voltage-dependent inward current in Ba2+ solution, with peak at 0 mV, confirmed the presence of Ca2+ channels. A voltage-dependent K+ current ( IKV) was inhibited by 20 mM tetraethylammonium and 5 mM 4-aminopyridine, revealing a background K+ current ( IKB) with open rectification. Mean resting membrane potential of −45.2 ± 11.6 mV did not change upon administration of hypoxia (Po2 = 11 mmHg), nor were any of the K+ currents sensitive to changes in Po2 during whole cell recording. By contrast, when the membrane and cytosol were left undisturbed during fura-2 or FM 1-43 imaging experiments, hypoxia increased intracellular Ca2+ concentration and initiated synaptic vesicle activity. 100 μM Cd2+ and 50 μM nifedipine eliminated uptake of FM 1-43. We conclude that Ca2+ influx via L-type Ca2+ channels is correlated with vesicular activity during hypoxic stimulation. In addition, we suggest that expression of IKCa in gill NECs is species specific and, in goldfish, may contribute to an attenuated response to acute hypoxia. NEW & NOTEWORTHY This study provides the first physiological characterization of oxygen chemoreceptors from an anoxia-tolerant vertebrate. Neuroepithelial cells (NECs) from the gills of goldfish displayed L-type Ca2+ channels and three types of K+ channels, one of which was dependent upon intracellular Ca2+. Although membrane currents were not inhibited by hypoxia during patch-clamp recording, this study is the first to show that NECs with an undisturbed cytosol responded to hypoxia with increased intracellular Ca2+ and synaptic vesicle activity.

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