Calcium-activated chloride channels in cultured embryonic Xenopus spinal neurons

1992 ◽  
Vol 68 (6) ◽  
pp. 2042-2050 ◽  
Author(s):  
N. Hussy
Keyword(s):  
Single Channel ◽  
Developmental Change ◽  
Reversal Potential ◽  
Equilibrium Potential ◽  
Ionophore A23187 ◽  
Spinal Neurons ◽  
Patch Clamp Technique ◽  
Cl Channels ◽  
Single Channel Currents

1. Single-channel currents were recorded from Xenopus spinal neurons developing in vitro using the patch-clamp technique, to identify the channels underlying the large and small macroscopic Ca(2+)-activated Cl- currents (ICl(Ca)) present in these cells. 2. Channels of large (maxi-channels; 310 pS) and smaller conductance (mini-channels; 50-60 pS) are activated by elevation of cytoplasmic Ca2+ concentration. Channel activity is not altered by subsequent removal of Ca2+ from the bath, arguing against a direct ligand-type Ca2+ dependence. The much higher incidence of channel activation in cell-attached patches from cells permeabilized with the Ca2+ ionophore A23187 than in excised patches also suggests the involvement of some unidentified intracellular factor. 3. The reversal potential of maxi-Cl- channels is not altered by changes in Na+ concentration, but is shifted in the negative direction by the substitution of Cl- by methanesulfonate on the intracellular side of the patch, indicating their anionic selectivity. 4. Maxi-Cl- channels exhibited the presence of multiple probable subconductance states and showed marked voltage-dependent inactivation above and below +/- 20 mV. 5. Examination of maxi-Cl- channels at early times in culture (6-9 h) and 24 h later did not reveal any developmental change in the characteristics described above. However, the mean open duration of the channel was found to increase twofold during this period of time. 6. The simultaneous presence of maxi- and mini-Cl- channels prevented detailed characterization of the latter. The anionic selectivity of mini-Cl- channels is suggested by their reversal potential that lies close to the Cl- equilibrium potential.(ABSTRACT TRUNCATED AT 250 WORDS)

Nonselective cation and Cl channels in luminal membrane of the marginal cell

1993 ◽  
Vol 265 (1) ◽  
pp. C72-C78 ◽  
Author(s):  
H. Sunose ◽  
K. Ikeda ◽  
Y. Saito ◽  
A. Nishiyama ◽  
T. Takasaka
Keyword(s):  
Single Channel ◽  
Patch Clamp Technique ◽  
Cl Channel ◽  
Luminal Membrane ◽  
Cl Channels ◽  
Cytoplasmic Acidification ◽  
Marginal Cells ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Linear Conductance

Single-channel currents of the luminal membrane of marginal cells dissected from the guinea pig cochlea were investigated using the patch-clamp technique. Nonselective cation channels having a linear conductance of 27 pS were activated by depolarization, cytoplasmic Ca2+, and cytoplasmic acidification. Cytoplasmic ATP inactivated the channel. A mixture of 3-isobutyl-1-methylxanthine and forskolin activated a small-conductance Cl channel in the cell-attached mode. On excision in the inside-out mode, the Cl channel was inactivated, but it was reactivated by a cytoplasmic catalytic subunit of protein kinase A with ATP. This Cl channel had a linear conductance of 12 pS, and its activity was little affected by voltage. The sequence of permeation by anions was Br- > Cl > I-. The Cl channel blocker diphenylamine-2-carboxylic acid (3 mM) completely blocked the channel, but 5-nitro-2-(3-phenylpropylamino)-benzoic acid (50 microM) blocked it only partially. The above-mentioned characteristics are similar to those of the well-demonstrated Cl- channel, cystic fibrosis transmembrane regulator.

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.

scholarly journals Heterogeneity of chloride channels in the apical membrane of isolated mitochondria-rich cells from toad skin.

1996 ◽  
Vol 108 (5) ◽  
pp. 421-433 ◽  
Author(s):  
J B Sørensen ◽  
E H Larsen
Keyword(s):  
Chloride Channels ◽  
Single Channel ◽  
Apical Membrane ◽  
Single Cells ◽  
Membrane Depolarization ◽  
Equilibrium Potential ◽  
Toad Skin ◽  
Patch Clamp Technique ◽  
Cl Channels ◽  
Inside Out

The isolated epithelium of toad skin was disintegrated into single cells by treatment with collagenase and trypsine. Chloride channels of cell-attached and excised inside-out apical membrane-patches of mitochondria-rich cells were studied by the patch-clamp technique. The major population of Cl- channels constituted small 7-pS linear channels in symmetrical solutions (125 mM Cl-). In cell-attached and inside-out patches the single channel i/V-relationship could be described by electrodiffusion of Cl- with a Goldmann-Hodgkin-Katz permeability of, PCl = 1.2 x 10(-14) - 2.6 x 10(-14) cm3. s-1. The channel exhibited voltage-independent activity and could be activated by cAMP. This channel is a likely candidate for mediating the well known cAMP-induced transepithelial Cl- conductance of the amphibian skin epithelium. Another population of Cl- channels exhibited large, highly variable conductances (upper limit conductances, 150-550 pS) and could be activated by membrane depolarization. A group of intermediate-sized Cl(-)-channels included: (a) channels (mean conductance, 30 pS) with linear or slightly outwardly rectifying i/V-relationships and activity occurring in distinct "bursts," (b) channels (conductance-range, 10-27 pS) with marked depolarization-induced activity, and (c) channels with unresolvable kinetics. The variance of current fluctuations of such "noisy" patches exhibited a minimum close to the equilibrium-potential for Cl-. With channels occurring in only 38% of sealed patches and an even lower frequency of voltage-activated channels, the chloride conductance of the apical membrane of mitochondria-rich cells did not match quantitatively that previously estimated from macroscopic Ussing-chamber experiments. From a qualitative point of view, however, we have succeeded in demonstrating the existence of Cl-channels in the apical membrane with features comparable to macroscopic predictions, i.e., activation of channel gating by cAMP and, in a few patches, also by membrane depolarization.

Anion channels for amino acids in MDCK cells

1992 ◽  
Vol 263 (6) ◽  
pp. C1200-C1207 ◽  
Author(s):  
U. Banderali ◽  
G. Roy
Keyword(s):  
Amino Acids ◽  
Single Channel ◽  
Mdck Cells ◽  
Reversal Potential ◽  
Patch Clamp Technique ◽  
Excised Patches ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Inside Out ◽  
Cytoplasmic Side

Large losses of amino acids by diffusion were previously observed in Madin-Darby canine kidney (MDCK) cells during volume regulation. Also, an outward rectifying anion channel was activated. Because this channel was not selective among anions, it was suggested that it could be permeable to amino acids. Its permeability to aspartate, glutamate, and taurine was studied using the patch-clamp technique in the inside-out configuration. Solutions containing 500 mM aspartate or glutamate were used on the cytoplasmic side of excised patches to detect single-channel currents carried by these anions. Permeability ratios were estimated in two different ways: 1) from the shift in reversal potential of current-voltage curves after anion replacement in the bath solution and 2) from comparisons of amplitudes of single-channel currents carried by tested anions and chloride, respectively. The values of aspartate-to-chloride and glutamate-to-chloride permeability ratios obtained with both methods were quite consistent and were of the order of 0.2 for both amino acids. Taurine in solutions at physiological pH 7.3 is a zwitterionic molecule and bears no net charge. To detect single-channel currents carried by taurine, solutions containing 500 mM taurine at pH 8.2 were used in inside-out experiments. Under these conditions 120 mM of negatively charged taurine was present in the solutions bathing the cytoplasmic side of excised patches. The permeability ratio estimated from the shift in reversal potential was 0.75. These results showed that some of the organic compounds released by cells during regulatory volume decrease could diffuse through this outwardly rectifying anionic channel.

Glutamate-gated chloride channel with glutamate-transporter-like properties in cone photoreceptors of the tiger salamander

1995 ◽  
Vol 74 (4) ◽  
pp. 1760-1771 ◽  
Author(s):  
S. A. Picaud ◽  
H. P. Larsson ◽  
G. B. Grant ◽  
H. Lecar ◽  
F. S. Werblin
Keyword(s):  
Single Channel ◽  
Noise Analysis ◽  
Glutamate Transporter ◽  
Reversal Potential ◽  
Equilibrium Potential ◽  
Channel Blockers ◽  
Cone Photoreceptors ◽  
Patch Clamp Technique ◽  
Cl Channel ◽  
Open Time

1. Using the patch-clamp technique, we investigated whether the glutamate-elicited current in mechanically isolated cone photoreceptors from the salamander retina is generated by a Cl- channel or a glutamate transporter. 2. The current reversed near the equilibrium potential for Cl-, was decreased by three Cl- channel blockers, 5-nitro-2-(3-phenyl-propylamino) benzoic acid, 4,4'-diisothiocyanostilbene-2,2'-disulfonate, and diphenylamine 2,2'-dicarboxylic acid, and was eliminated when gluconate was substituted for both internal and external Cl-, features consistent with the current being mediated by a Cl- channel. 3. The single-channel conductance of the Cl- channel was estimated by noise analysis of the glutamate-elicited current fluctuations to be 0.7 pS with an open time of 2 ms. 4. The magnitude of the current was dependent on both internal and external Na+ and K+, features consistent with the current being related to the activation of a glutamate transporter. Yet changes in their concentrations did not affect the reversal potential of the current. 5. Taken together with earlier reports on this current showing that it has a glutamate-transporter-like pharmacology, our results suggest that the glutamate-elicited current is carried by a Cl- channel but gated by a glutamate receptor whose pharmacology and ionic requirement resemble those previously described for glutamate transporters.

scholarly journals A whole-cell and single-channel study of the voltage-dependent outward potassium current in avian hepatocytes.

1988 ◽  
Vol 91 (2) ◽  
pp. 255-274 ◽  
Author(s):  
C Marchetti ◽  
R T Premont ◽  
A M Brown
Keyword(s):  
Single Channel ◽  
Reversal Potential ◽  
Outward Current ◽  
Single Type ◽  
K Channel ◽  
Delayed Rectifier ◽  
Patch Clamp Technique ◽  
Voltage Dependent ◽  
Single Channel Currents ◽  
K Currents

Voltage-dependent membrane currents were studied in dissociated hepatocytes from chick, using the patch-clamp technique. All cells had voltage-dependent outward K+ currents; in 10% of the cells, a fast, transient, tetrodotoxin-sensitive Na+ current was identified. None of the cells had voltage-dependent inward Ca2+ currents. The K+ current activated at a membrane potential of about -10 mV, had a sigmoidal time course, and did not inactivate in 500 ms. The maximum outward conductance was 6.6 +/- 2.4 nS in 18 cells. The reversal potential, estimated from tail current measurements, shifted by 50 mV per 10-fold increase in the external K+ concentration. The current traces were fitted by n2 kinetics with voltage-dependent time constants. Omitting Ca2+ from the external bath or buffering the internal Ca2+ with EGTA did not alter the outward current, which shows that Ca2+-activated K+ currents were not present. 1-5 mM 4-aminopyridine, 0.5-2 mM BaCl2, and 0.1-1 mM CdCl2 reversibly inhibited the current. The block caused by Ba was voltage dependent. Single-channel currents were recorded in cell-attached and outside-out patches. The mean unitary conductance was 7 pS, and the channels displayed bursting kinetics. Thus, avian hepatocytes have a single type of K+ channel belonging to the delayed rectifier class of K+ channels.

Single voltage-dependent potassium channels in cultured rat hippocampal neurons

1986 ◽  
Vol 56 (2) ◽  
pp. 481-493 ◽  
Author(s):  
M. A. Rogawski
Keyword(s):  
Hippocampal Neurons ◽  
Single Channel ◽  
Reversal Potential ◽  
Resting Potential ◽  
Moderate Degree ◽  
Patch Clamp Technique ◽  
Membrane Pore ◽  
Channel Opening ◽  
Voltage Dependent ◽  
Single Channel Currents

Single-channel recordings using the gigohm seal patch-clamp technique were carried out on the somatic membranes of dissociated embryonic rat hippocampal neurons grown in cell culture. The recording medium contained tetrodotoxin to block the voltage-dependent Na+ conductance and Cd2+ to block Ca2+ and Ca2+-activated conductances. In the cell-attached configuration, depolarizing voltage steps activated outward directed single-channel currents with conductance 15-20 pS. The channel openings exhibited a moderate degree of flickering. The mean burst lifetimes ranged from 5 to 13 ms with a tendency to increase slightly at more depolarized potentials (T = 21-25 degrees C). Reversal potential measurements using excised membrane patches indicated that the channels behaved as expected of a K+-selective membrane pore. Channel opening occurred in Ca2+-free EGTA-containing solutions but was never observed in the presence of tetraethylammonium (TEA; 20 mM). The frequency of channel opening increased as the membrane was depolarized by up to 50 mV from resting potential; the fraction of time spent in the open state during the first 300 ms following a step depolarization increased e-fold for a 8-25 mV change in potential. First-latency histograms and simulations of the macroscopic current based on channel data obtained during repeated depolarizing voltage steps indicated that the probability of the channel being in the open state increases gradually with time after a step depolarization. During repeated depolarizing steps the channels appeared to randomly enter and exit a long-lived inactive state. It is concluded that these channels may underly the slowly activating, very slowly inactivating, TEA-sensitive voltage-dependent K+ current (IK) in cultured hippocampal neurons.

Permeation and gating properties of a cloned renal K+ channel

1995 ◽  
Vol 268 (2) ◽  
pp. C389-C401 ◽  
Author(s):  
S. Chepilko ◽  
H. Zhou ◽  
H. Sackin ◽  
L. G. Palmer
Keyword(s):  
Single Channel ◽  
Reversal Potential ◽  
Cation Binding ◽  
K Channel ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Membrane Hyperpolarization ◽  
Channel Current ◽  
Inward Currents ◽  
Kinetics Of

The renal K+ channel (ROMK2) was expressed in Xenopus oocytes, and the patch-clamp technique was used to assess its conducting and gating properties. In cell-attached patches with 110 mM K+ in the bath and pipette, the reversal potential was near zero and the inward conductance (36 pS) was larger than the outward conductance (17 pS). In excised inside-out patches the channels showed rectification in the presence of 5 mM Mg2+ on the cytoplasmic side but not in Mg(2+)-free solution. Inward currents were also observed when K+ was replaced in the pipette by Rb+, NH4+, or thallium (Tl+). The reversal potentials under these conditions yielded a selectivity sequence of Tl+ > K+ > Rb+ > NH4+. On the other hand, the slope conductances for inward current gave a selectivity sequence of K+ = NH4+ > Tl+ > Rb+. The differences in the two sequences can be explained by the presence of cation binding sites within the channel, which interact with Rb+ and Tl+ more strongly and with NH4+ less strongly than with K+. Two other ions, Ba2+ and Cs+, blocked the channel from the outside. The effect of Ba2+ (1 mM) was to reduce the open probability of the channels, whereas Cs+ (10 mM) reduced the apparent single-channel current. The effects of both blockers are enhanced by membrane hyperpolarization. The kinetics of the channel were also studied in cell-attached patches. With K+ in the pipette the distribution of open times could be described by a single exponential (tau 0 = 25 ms), whereas two exponentials (tau 1 = 1 ms, tau 2 = 30 ms) were required to describe the closed-time distribution. Hyperpolarization of the oocyte membrane decreased the open probability and tau 0, and increased tau 1, tau 2, and the number of long closures. The presence of Tl+ in the pipette significantly altered the kinetics, reducing tau 0 and eliminating the long-lived closures. These results suggest that the gating of the channel may depend on the nature of the ion in the pore.

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.

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