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.

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.

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.

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 Characterization of the calcium-activated chloride channel in isolated guinea-pig hepatocytes.

1994 ◽  
Vol 104 (2) ◽  
pp. 357-373 ◽  
Author(s):  
S Koumi ◽  
R Sato ◽  
T Aramaki
Keyword(s):  
Guinea Pig ◽  
Single Channel ◽  
Hill Coefficient ◽  
Disulfonic Acid ◽  
Dependent Manner ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Cl Channels ◽  
Inside Out

Macroscopic and unitary currents through Ca(2+)-activated Cl- channels were examined in enzymatically isolated guinea-pig hepatocytes using whole-cell, excised outside-out and inside-out configurations of the patch-clamp technique. When K+ conductances were blocked and the intracellular Ca2+ concentration ([Ca2+]i) was set at 1 microM (pCa = 6), membrane currents were observed under whole-cell voltage-clamp conditions. The reversal potential of the current shifted by approximately 60 mV per 10-fold change in the external Cl- concentration. In addition, the current did not appear when Cl- was omitted from the internal and external solutions, indicating that the current was Cl- selective. The current was activated by increasing [Ca2+]i and was inactivated in Ca(2+)-free, 5 mM EGTA internal solution (pCa > 9). The current was inhibited by bath application of 9-anthracenecarboxylic acid (9-AC) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) in a voltage-dependent manner. In single channel recordings from outside-out patches, unitary current activity was observed, whose averaged slope conductance was 7.4 +/- 0.5 pS (n = 18). The single channel activity responded to extracellular Cl- changes as expected for a Cl- channel current. The open time distribution was best described by a single exponential function with mean open lifetime of 97.6 +/- 10.4 ms (n = 11), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 21.5 +/- 2.8 ms (n = 11) and that for the slow component of 411.9 +/- 52.0 ms (n = 11). In excised inside-out patch recordings, channel open probability was sensitive to [Ca2+]i. The relationship between [Ca2+]i and channel activity was fitted by the Hill equation with a Hill coefficient of 3.4 and the half-maximal activation was 0.48 microM. These results suggest that guinea-pig hepatocytes possess Ca(2+)-activated Cl- channels.

scholarly journals Angiotensin II stimulates epithelial sodium channels in the cortical collecting duct of the rat kidney

2012 ◽  
Vol 302 (6) ◽  
pp. F679-F687 ◽  
Author(s):  
Peng Sun ◽  
Peng Yue ◽  
Wen-Hui Wang
Keyword(s):  
Angiotensin Ii ◽  
Single Channel ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Ang Ii ◽  
Cortical Collecting Duct ◽  
Patch Clamp Recording ◽  
Open Probability ◽  
Whole Cell ◽  
Na Currents

We examined the effect of angiotensin II (ANG II) on epithelial Na+channel (ENaC) in the rat cortical collecting duct (CCD) with single-channel and the perforated whole cell patch-clamp recording. Application of 50 nM ANG II increased ENaC activity, defined by NPo(a product of channel numbers and open probability), and the amiloride-sensitive whole cell Na currents by twofold. The stimulatory effect of ANG II on ENaC was absent in the presence of losartan, suggesting that the effect of ANG II on ENaC was mediated by ANG II type 1 receptor. Moreover, depletion of intracellular Ca2+with 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid (BAPTA)-AM failed to abolish the stimulatory effect of ANG II on ENaC but inhibiting protein kinase C (PKC) abolished the effect of ANG II, suggesting that the effect of ANG II was the result of stimulating Ca2+-independent PKC. This notion was also suggested by the experiments in which stimulation of PKC with phorbol ester derivative mimicked the effect of ANG II and increased amiloride-sensitive Na currents in the principal cell, an effect that was not abolished by treatment of the CCD with BAPTA-AM. Also, inhibition of NADPH oxidase (NOX) with diphenyleneiodonium chloride abolished the stimulatory effect of ANG II on ENaC and application of superoxide donors, pyrogallol or xanthine and xanthine oxidase, significantly increased ENaC activity. Moreover, addition of ANG II or H2O2diminished the arachidonic acid (AA)-induced inhibition of ENaC in the CCD. We conclude that ANG II stimulates ENaC in the CCD through a Ca2+-independent PKC pathway that activates NOX thereby increasing superoxide generation. The stimulatory effect of ANG II on ENaC may be partially the result of blocking AA-induced inhibition of ENaC.

Quinidine blocks adenosine 5'-triphosphate-sensitive potassium channels in heart

1990 ◽  
Vol 259 (5) ◽  
pp. H1609-H1612 ◽  
Author(s):  
A. I. Undrovinas ◽  
N. Burnashev ◽  
D. Eroshenko ◽  
I. Fleidervish ◽  
C. F. Starmer ◽  
...  
Keyword(s):  
Single Channel ◽  
Dependent Manner ◽  
Open Probability ◽  
Pipette Solution ◽  
Channel Current ◽  
Closed Intervals ◽  
Ventricular Cells ◽  
Voltage Dependent ◽  
Inside Out ◽  
Ischemic Arrhythmias

The ATP-sensitive potassium channel current (IK-ATP) was studied in excised inside-out patches from rat ventricular cells at 20-23 degrees C. The bath solution contained 140 mM KF, and the pipette solution contained 140 mM KCl and 1.2 mM MgCl2. ATP (0.5 mM) in the bath inhibited IK-ATP. In the absence of ATP, 10 microM quinidine decreased open probability 67 +/- 1% (n = 6) at -50 mV and 28 +/- 12% at -130 mV (n = 5) without affecting single channel conductance (48-52 pS). The block increased with 25 and 50 microM quinidine and could be reversed on washing quinidine for several minutes. Interburst (closed) intervals were increased by quinidine, whereas open and closed time distributions within bursts were not changed. We conclude that quinidine blocks IK-ATP in a "slow" and voltage-dependent manner in clinically relevant concentrations. Because of the postulated role for IK-ATP in cardiac ischemia, quinidine block of this channel may play a role in ischemic arrhythmias.

Biophysical properties and metabolic regulation of a TASK-like potassium channel in rat carotid body type 1 cells

2004 ◽  
Vol 286 (1) ◽  
pp. L221-L230 ◽  
Author(s):  
Beatrice A. Williams ◽  
Keith J. Buckler
Keyword(s):  
Potassium Channel ◽  
Carotid Body ◽  
Metabolic Regulation ◽  
Single Channel ◽  
Divalent Cations ◽  
Channel Activity ◽  
Body Type ◽  
Oxygen Sensitive ◽  
Inside Out

The single channel properties of TASK-like oxygen-sensitive potassium channels were studied in rat carotid body type 1 cells. We observed channels with rapid bursting kinetics, active at resting membrane potentials. These channels were highly potassium selective with a slope conductance of 14–16 pS, values similar to those reported for TASK-1. In the absence of extracellular divalent cations, however, single channel conductance increased to 28 pS in a manner similar to that reported for TASK-3. After patch excision, channel activity ran down rapidly. Channel activity in inside-out patches was markedly increased by 2 and 5 mM ATP and by 2 mM ADP but not by 100 μM ADP or 1 mM AMP. In cell-attached patches, both cyanide and 2,4-dinitrophenol strongly inhibited channel activity. We conclude that 1) whilst the properties of this channel are consistent with it being a TASK-like potassium channel they do not precisely conform to those of either TASK-1 or TASK-3, 2) channel activity is highly dependent on cytosolic factors including ATP, and 3) changes in energy metabolism may play a role in regulating the activity of these background K+ channels.

scholarly journals Dequalinium

2002 ◽  
Vol 121 (1) ◽  
pp. 37-47 ◽  
Author(s):  
Tamara Rosenbaum ◽  
León D. Islas ◽  
Anne E. Carlson ◽  
Sharona E. Gordon
Keyword(s):  
Single Channel ◽  
Divalent Cations ◽  
K Channel ◽  
Kinase C ◽  
Conducting Pathway ◽  
Voltage Dependent ◽  
Excised Patches ◽  
Ion Conducting ◽  
Inside Out ◽  
Cnga1 Channels

Cyclic nucleotide–gated (CNG) channels have been shown to be blocked by diltiazem, tetracaine, polyamines, toxins, divalent cations, and other compounds. Dequalinium is an organic divalent cation which suppresses the rat small conductance Ca2+-activated K+ channel 2 (rSK2) and the activity of protein kinase C. In this study, we have tested the ability of dequalinium to block CNGA1 channels and heteromeric CNGA1+CNGB1 channels. When applied to the intracellular side of inside-out excised patches from Xenopus oocytes, dequalinium blocks CNGA1 channels with a K1/2 ≈ 190 nM and CNGA1+CNGB1 channels with a K1/2 ≈ 385 nM, at 0 mV. This block occurs in a state-independent fashion, and is voltage dependent with a zδ ≈ 1. Our data also demonstrate that dequalinium interacts with the permeant ion probably because it occupies a binding site in the ion conducting pathway. Dequalinium applied to the extracellular surface also produced block, but with a voltage dependence that suggests it crosses the membrane to block from the inside. We also show that at the single-channel level, dequalinium is a slow blocker that does not change the unitary conductance of CNGA1 channels. Thus, dequalinium should be a useful tool for studying permeation and gating properties of CNG channels.

GABA-activated single channel currents in outside-out membrane patches from rat retinal ganglion cells

1989 ◽  
Vol 3 (3) ◽  
pp. 275-279 ◽  
Author(s):  
Stuart A. Lipton
Keyword(s):  
Retinal Ganglion Cells ◽  
Cortical Neurons ◽  
Single Channel ◽  
Ganglion Cells ◽  
Retinal Ganglion ◽  
Whole Cell ◽  
Cell Recording ◽  
Excised Patches ◽  
Patch Configuration ◽  
Single Channel Currents

Abstractγ-aminobutyric acid (GABA) evokes large whole-cell currents in solitary mammalian retinal ganglion cells studied by the patch-clamp method. This evidence suggests that GABA acts directly on the retinal ganglion cells as an inhibitory transmitter as it does elsewhere in the mammalian central nervous system. Here, single-channel recordings of the currents underlying the GABA-induced responses were studied in outside-out patches of cell membrane. In some other preparations, single GABAA channels recorded in the excised patch configuration have been shown to have altered properties in comparison to responses elicited during whole-cell recording. For example, in cortical neurons single GABA-activated channels in excised patches display accelerated desensitization kinetics as well as rapid rundown of the response. Therefore, in retinal ganglion cells, responses generated by GABA in cell-free patches were compared to whole-cell responses. After determining that the responses to GABA in acutely isolated outside-out patches were indeed similar to those of the whole-cell currents in retinal ganglion cells, the unitary conductances were studied. It was determined that these single-channel events resemble those reported in other nervous tissues with 4 elementary conductances of ~10 pS, 19–22 pS, 30–33 pS, and 45–50 pS at 33–35°C.

scholarly journals Application of Machine-Learning Methods to Recognize mitoBK Channels from Different Cell Types Based on the Experimental Patch-Clamp Results

2021 ◽  
Vol 22 (2) ◽  
pp. 840
Author(s):  
Monika Richter-Laskowska ◽  
Paulina Trybek ◽  
Piotr Bednarczyk ◽  
Agata Wawrzkiewicz-Jałowiecka
Keyword(s):  
Machine Learning ◽  
Patch Clamp ◽  
Single Channel ◽  
Conformational Dynamics ◽  
Cell Types ◽  
Inner Mitochondrial Membrane ◽  
Patch Clamp Recording ◽  
K Nearest Neighbors ◽  
Channel Protein ◽  
Different Cell Types

(1) Background: In this work, we focus on the activity of large-conductance voltage- and Ca2+-activated potassium channels (BK) from the inner mitochondrial membrane (mitoBK). The characteristic electrophysiological features of the mitoBK channels are relatively high single-channel conductance (ca. 300 pS) and types of activating and deactivating stimuli. Nevertheless, depending on the isoformal composition of mitoBK channels in a given membrane patch and the type of auxiliary regulatory subunits (which can be co-assembled to the mitoBK channel protein) the characteristics of conformational dynamics of the channel protein can be altered. Consequently, the individual features of experimental series describing single-channel activity obtained by patch-clamp method can also vary. (2) Methods: Artificial intelligence approaches (deep learning) were used to classify the patch-clamp outputs of mitoBK activity from different cell types. (3) Results: Application of the K-nearest neighbors algorithm (KNN) and the autoencoder neural network allowed to perform the classification of the electrophysiological signals with a very good accuracy, which indicates that the conformational dynamics of the analyzed mitoBK channels from different cell types significantly differs. (4) Conclusion: We displayed the utility of machine-learning methodology in the research of ion channel gating, even in cases when the behavior of very similar microbiosystems is analyzed. A short excerpt from the patch-clamp recording can serve as a “fingerprint” used to recognize the mitoBK gating dynamics in the patches of membrane from different cell types.

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