Single K+-channel current measurements from brain synaptosomes in lipid bilayers

1983 ◽  
Vol 245 (1) ◽  
pp. C151-C156 ◽  
Author(s):  
M. T. Nelson ◽  
M. Roudna ◽  
E. Bamberg
Keyword(s):  
Ion Channels ◽  
Lipid Bilayers ◽  
Single Channel ◽  
Mammalian Brain ◽  
K Channel ◽  
Kinetic Properties ◽  
Nerve Terminals ◽  
Planar Lipid Bilayers ◽  
Presynaptic Nerve Terminals ◽  
Single Channel Currents

Ion channels from a rat brain preparation enriched in presynaptic nerve terminals (synaptosomes) were incorporated into planar lipid bilayers. Experiments examined macroscopic (channel-ensemble) currents as well as single-channel currents. Four single-channel conductances (ranging from 10 to 40 pS) were usually observed, each with distinct kinetic properties. All the observed channels selected for K+ over Cl-. These K+ channels may contribute to the resting K+ conductance of brain nerve terminals. Furthermore, this report demonstrates that the properties of ion channels from mammalian brain can be studied in planar lipid bilayers and suggests that this system can be readily extended to many additional investigations on the electrical properties of brain membranes.

K+ channel currents in basolateral membrane of distal convoluted tubule of rabbit kidney

1989 ◽  
Vol 256 (2) ◽  
pp. F246-F254 ◽  
Author(s):  
J. Taniguchi ◽  
K. Yoshitomi ◽  
M. Imai
Keyword(s):  
Single Channel ◽  
Basolateral Membrane ◽  
Distal Convoluted Tubule ◽  
Rabbit Kidney ◽  
K Channel ◽  
Kinetic Properties ◽  
Open Probability ◽  
K Channels ◽  
Single Channel Currents ◽  
Channel Currents

To examine the nature of ion-conductive pathways in the basolateral membrane of rabbit distal convoluted tubule (DCT), we recorded single-channel currents from the tubule segment isolated from collagenase-treated kidney. Using cell-attached patch pipettes filled with 130 mM KCl, 5.4 mM CaCl2, and 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.4), we observed K+ channels in the basolateral membrane of DCT, having two different single-channel conductances of 48.7 +/- 1.4 (n = 9) and 60.6 +/- 1.4 pS (n = 7). Both types of channels were completely blocked by 0.1 mM BaCl2. Both channels have same open probability of approximately 0.5 at the intrinsic basolateral membrane voltage and were recorded with similar incidence. Mean open and closed times were 31.5 +/- 5.2 and 41.3 +/- 16.0 ms for the smaller channel, and 31.5 +/- 5.1 and 36.7 +/- 8.7 ms for the larger channel, respectively. These kinetic properties did not show any clear voltage dependence in both channels. Because of apparent similarity of channel kinetics, it is possible that both activities might represent different states of the same channel. For definite conclusion, however, further investigations are necessary. In three recordings from 54 successful patches, we observed a flickering channel with rapid kinetics, which was insensitive to 1 meq/l Ba2+. The conductance of this channel was 76.6 pS (n = 2). The extrapolated zero current voltage was 76.0 mV (n = 2), indicating that this channel is permeable to K+. From these results, we suggest that K+ channels constitute conductive pathways for K+ in the basolateral membrane of rabbit DCT.

scholarly journals Incorporation of RyR2 and Other Ion Channels into Nanopore Based Planar Lipid Bilayers for Low Noise Single Channel Recordings

2010 ◽  
Vol 98 (3) ◽  
pp. 539a-540a
Author(s):  
Prithwish Pal ◽  
Geoffrey A. Barrall ◽  
Ariel L. Escobar ◽  
Melissa A. Poquette ◽  
Patricio Velez ◽  
...  
Keyword(s):  
Ion Channels ◽  
Lipid Bilayers ◽  
Single Channel ◽  
Low Noise ◽  
Planar Lipid Bilayers ◽  
Single Channel Recordings

scholarly journals Unitary Ca2+ Current through Cardiac Ryanodine Receptor Channels under Quasi-Physiological Ionic Conditions

1999 ◽  
Vol 113 (2) ◽  
pp. 177-186 ◽  
Author(s):  
Rafael Mejía-Alvarez ◽  
Claudia Kettlun ◽  
Eduardo Ríos ◽  
Michael Stern ◽  
Michael Fill
Keyword(s):  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Single Channel ◽  
Monovalent Cation ◽  
Planar Lipid Bilayers ◽  
Physiological Conditions ◽  
Single Channel Currents ◽  
The Relationship ◽  
Channel Currents ◽  
Cardiac Ryanodine Receptor

Single canine cardiac ryanodine receptor channels were incorporated into planar lipid bilayers. Single-channel currents were sampled at 1–5 kHz and filtered at 0.2–1.0 kHz. Channel incorporations were obtained in symmetrical solutions (20 mM HEPES-Tris, pH 7.4, and pCa 5). Unitary Ca2+ currents were monitored when 2–30 mM Ca2+ was added to the lumenal side of the channel. The relationship between the amplitude of unitary Ca2+ current (at 0 mV holding potential) and lumenal [Ca2+] was hyperbolic and saturated at ∼4 pA. This relationship was then defined in the presence of different symmetrical CsCH3SO3 concentrations (5, 50, and 150 mM). Under these conditions, unitary current amplitude was 1.2 ± 0.1, 0.65 ± 0.1, and 0.35 ± 0.1 pA in 2 mM lumenal Ca2+; and 3.3 ± 0.4, 2.4 ± 0.2, and 1.63 ± 0.2 pA in 10 mM lumenal Ca2+ (n > 6). Unitary Ca2+ current was also defined in the presence of symmetrical [Mg2+] (1 mM) and low [Cs+] (5 mM). Under these conditions, unitary Ca2+ current in 2 and 10 mM lumenal Ca2+ was 0.66 ± 0.1 and 1.52 ± 0.06 pA, respectively. In the presence of higher symmetrical [Cs+] (50 mM), Mg2+ (1 mM), and lumenal [Ca2+] (10 mM), unitary Ca2+ current exhibited an amplitude of 0.9 ± 0.2 pA (n = 3). This result indicates that the actions of Cs+ and Mg2+ on unitary Ca2+ current were additive. These data demonstrate that physiological levels of monovalent cation and Mg2+ effectively compete with Ca2+ as charge carrier in cardiac ryanodine receptor channels. If lumenal free Ca2+ is 2 mM, then our results indicate that unitary Ca2+ current under physiological conditions should be <0.6 pA.

scholarly journals Sodium channels in planar lipid bilayers. Channel gating kinetics of purified sodium channels modified by batrachotoxin.

1986 ◽  
Vol 88 (1) ◽  
pp. 1-23 ◽  
Author(s):  
B U Keller ◽  
R P Hartshorne ◽  
J A Talvenheimo ◽  
W A Catterall ◽  
M Montal
Keyword(s):  
Lipid Bilayers ◽  
Sodium Channels ◽  
Single Channel ◽  
Channel Gating ◽  
Neuroblastoma Cells ◽  
Planar Lipid Bilayers ◽  
Transition Rates ◽  
Biophysical Journal ◽  
Single Channel Currents ◽  
Kinetics Of

Single channel currents of sodium channels purified from rat brain and reconstituted into planar lipid bilayers were recorded. The kinetics of channel gating were investigated in the presence of batrachotoxin to eliminate inactivation and an analysis was conducted on membranes with a single active channel at any given time. Channel opening is favored by depolarization and is strongly voltage dependent. Probability density analysis of dwell times in the closed and open states of the channel indicates the occurrence of one open state and several distinct closed states in the voltage (V) range-120 mV less than or equal to V less than or equal to +120 mV. For V less than or equal to 0, the transition rates between stages are exponentially dependent on the applied voltage, as described in mouse neuroblastoma cells (Huang, L. M., N. Moran, and G. Ehrenstein. 1984. Biophysical Journal. 45:313-322). In contrast, for V greater than or equal to 0, the transition rates are virtually voltage independent. Autocorrelation analysis (Labarca, P., J. Rice, D. Fredkin, and M. Montal. 1985. Biophysical Journal. 47:469-478) shows that there is no correlation in the durations of successive open or closing events. Several kinetic schemes that are consistent with the experimental data are considered. This approach may provide information about the mechanism underlying the voltage dependence of channel activation.

scholarly journals Batrachotoxin-modified sodium channels in planar lipid bilayers. Ion permeation and block.

1987 ◽  
Vol 89 (6) ◽  
pp. 841-872 ◽  
Author(s):  
W N Green ◽  
L B Weiss ◽  
O S Andersen
Keyword(s):  
Lipid Bilayers ◽  
Sodium Channels ◽  
Single Channel ◽  
Divalent Cations ◽  
Cation Binding ◽  
Planar Lipid Bilayers ◽  
Surface Charges ◽  
High Concentrations ◽  
Conductance States ◽  
Single Channel Currents

Batrachotoxin-modified, voltage-dependent sodium channels from canine forebrain were incorporated into planar lipid bilayers. Single-channel conductances were studied for [Na+] ranging between 0.02 and 3.5 M. Typically, the single-channel currents exhibited a simple two-state behavior, with transitions between closed and fully open states. Two other conductance states were observed: a subconductance state, usually seen at [NaCl] greater than or equal to 0.5 M, and a flickery state, usually seen at [NaCl] less than or equal to 0.5 M. The flickery state became more frequent as [NaCl] was decreased below 0.5 M. The K+/Na+ permeability ratio was approximately 0.16 in 0.5 and 2.5 M salt, independent of the Na+ mole fraction, which indicates that there are no interactions among permeant ions in the channels. Impermeant and permeant blocking ions (tetraethylammonium, Ca++, Zn++, and K+) have different effects when added to the extracellular and intracellular solutions, which indicates that the channel is asymmetrical and has at least two cation-binding sites. The conductance vs. [Na+] relation saturated at high concentrations, but could not be described by a Langmuir isotherm, as the conductance at low [NaCl] is higher than predicted from the data at [NaCl] greater than or equal to 1.0 M. At low [NaCl] (less than or equal to 0.1 M), increasing the ionic strength by additions of impermeant monovalent and divalent cations reduced the conductance, as if the magnitude of negative electrostatic potentials at the channel entrances were reduced. The conductances were comparable for channels in bilayers that carry a net negative charge and bilayers that carry no net charge. Together, these results lead to the conclusion that negative charges on the channel protein near the channel entrances increase the conductance, while lipid surface charges are less important.

scholarly journals Activation by divalent cations of a Ca2+-activated K+ channel from skeletal muscle membrane.

1988 ◽  
Vol 92 (1) ◽  
pp. 67-86 ◽  
Author(s):  
A Oberhauser ◽  
O Alvarez ◽  
R Latorre
Keyword(s):  
Lipid Bilayers ◽  
Single Channel ◽  
Divalent Cations ◽  
Hill Coefficient ◽  
Muscle Membrane ◽  
K Channel ◽  
Single Channel Currents ◽  
The Hill ◽  
Channel Currents ◽  
Cytoplasmic Side

Several divalent cations were studied as agonists of a Ca2+-activated K+ channel obtained from rat muscle membranes and incorporated into planar lipid bilayers. The effect of these agonists on single-channel currents was tested in the absence and in the presence of Ca2+. Among the divalent cations that activate the channel, Ca2+ is the most effective, followed by Cd2+, Sr2+, Mn2+, Fe2+, and Co2+. Mg2+, Ni2+, Ba2+, Cu2+, Zn2+, Hg2+, and Sn2+ are ineffective. The voltage dependence of channel activation is the same for all the divalent cations. The time-averaged probability of the open state is a sigmoidal function of the divalent cation concentration. The sigmoidal curves are described by a dissociation constant K and a Hill coefficient N. The values of these parameters, measured at 80 mV are: N = 2.1, K = 4 X 10(-7) mMN for Ca2+; N = 3.0, K = 0.02 mMN for Cd2+; N = 1.45, K = 0.63 mMN for Sr2+; N = 1.7, K = 0.94 mMN for Mn2+; N = 1.1, K = 3.0 mMN for Fe2+; and N = 1.1 K = 4.35 mMN for Co2+. In the presence of Ca2+, the divalent cations Cd2+, Co2+, Mn2+, Ni2+, and Mg2+ are able to increase the apparent affinity of the channel for Ca2+ and they increase the Hill coefficient in a concentration-dependent fashion. These divalent cations are only effective when added to the cytoplasmic side of the channel. We suggest that these divalent cations can bind to the channel, unmasking new Ca2+ sites.

Ryanodine receptor dysfunction in porcine stunned myocardium

1997 ◽  
Vol 273 (2) ◽  
pp. H796-H804 ◽  
Author(s):  
C. Valdivia ◽  
J. O. Hegge ◽  
R. D. Lasley ◽  
H. H. Valdivia ◽  
R. Mentzer
Keyword(s):  
Coronary Artery ◽  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Myocardial Stunning ◽  
Single Channel ◽  
Stunned Myocardium ◽  
Wall Thickening ◽  
Planar Lipid Bilayers ◽  
Open Probability ◽  
Single Channel Recordings

We investigated the effects of myocardial stunning on the function of the two main Ca2+ transport proteins of the sarcoplasmic reticulum (SR), the Ca(2+)-adenosinetriphosphatase and the Ca(2+)-release channel or ryanodine receptor. Regional myocardial stunning was induced in open-chest pigs (n = 6) by a 10-min occlusion of the left anterior descending coronary artery (LAD) and 2 h reperfusion. SR vesicles isolated from the LAD-perfused region (stunned) and the normal left circumflex coronary artery (LC)-perfused region were used to assess the oxalate-supported 45Ca2+ uptake, [3H]ryanodine binding, and single-channel recordings of ryanodine-sensitive Ca(2+)-release channels in planar lipid bilayers. Myocardial stunning decreased LAD systolic wall thickening to 20% of preischemic values. The rate of SR 45Ca2+ uptake in the stunned LAD bed was reduced by 37% compared with that of the normal LC bed (P < 0.05). Stunning was also associated with a 38% reduction in the maximal density of high-affinity [3H]ryanodine binding sites (P < 0.05 vs. normal LC) but had no effect on the dissociation constant. The open probability of ryanodine-sensitive Ca(2+)-release channels determined by single channel recordings in planar lipid bilayers was 26 +/- 2% for control SR (n = 33 channels from 3 animals) and 14 +/- 2% for stunned SR (n = 21 channels; P < 0.05). This depressed activity of SR function observed in postischemic myocardium could be one of the mechanisms underlying myocardial stunning.

Single potassium channels blocked by lidocaine and quinidine in isolated turtle colon epithelial cells

1986 ◽  
Vol 251 (1) ◽  
pp. C85-C89 ◽  
Author(s):  
N. W. Richards ◽  
D. C. Dawson
Keyword(s):  
Epithelial Cells ◽  
Single Channel ◽  
Reversal Potential ◽  
Cell Swelling ◽  
K Channel ◽  
Patch Clamp Technique ◽  
Colon Cells ◽  
High K ◽  
A Cell ◽  
Single Channel Currents

The patch-clamp technique for recording single-channel currents across cell membranes was applied to single turtle colon epithelial cells isolated with hyaluronidase. With electrodes fabricated from Corning #7052 glass, high-resistance seals were consistently formed to these cells. In on-cell patches with low K (2.5 mM) in the pipette and high K (114.5 mM) in the bath, outward K currents were recorded that had a slope conductance of 17 pS and a reversal potential greater than -70 mV. Currents through this K channel were blocked by lidocaine, quinidine, and barium. These agents also block a cell swelling-induced K conductance identified by macroscopic current measurements in the basolateral membranes of the intact colonic epithelium, suggesting that the 17 pS K channel identified by single-channel recording in isolated turtle colon cells may be responsible for this macroscopically defined K conductance.

Regulation of ATP-sensitive K+ channels by ATP and nucleotide diphosphate in rabbit portal vein

1994 ◽  
Vol 266 (5) ◽  
pp. H1687-H1698 ◽  
Author(s):  
M. Kamouchi ◽  
K. Kitamura
Keyword(s):  
Portal Vein ◽  
Katp Channel ◽  
Single Channel ◽  
Katp Channels ◽  
K Channel ◽  
Channel Activity ◽  
Rabbit Portal Vein ◽  
Internal Side ◽  
The Right ◽  
Single Channel Currents

The modulation of ATP-sensitive K+ (KATP)-channel activity was investigated by recording single-channel currents in isolated smooth muscle cells from rabbit portal vein. K(+)-channel openers (KCOs; pinacidil, lemakalim, and nicorandil) induced burstlike openings of single KATP channels in the cell-attached configuration. After patch excision, KATP channels showed "run-down" phenomenon in the presence of KCOs, but subsequent application of Mg-ATP (1 mM) restored KATP-channel activity. Removal of Mg-ATP resulted in transient augmentation of KATP currents, which eventually decayed out. Nucleotide diphosphates (NDPs; GDP, ADP, UDP, IDP, and CDP) also induced channel reopening in the presence of KCOs, which was markedly enhanced by addition of Mg2+ in millimolar concentrations at the internal side of the membrane. The dose-response relation between ATP and the UDP-induced KATP-channel activity was shifted to the right in the presence of Mg2+ (2 mM). These results suggest that intracellular ATP, NDPs, and Mg2+ regulate the channel state of KATP channels (operative and inoperative states) and that KCOs open KATP channels only in the operative state.

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