Flickery block of single CFTR chloride channels by intracellular anions and osmolytes

1996 ◽  
Vol 271 (2) ◽  
pp. C628-C634 ◽  
Author(s):  
P. Linsdell ◽  
J. W. Hanrahan
Keyword(s):  
Chloride Channels ◽  
Single Channel ◽  
Chinese Hamster Ovary Cells ◽  
Specific Interaction ◽  
Chinese Hamster ◽  
Chloride Concentration ◽  
Chloride Ion ◽  
Chloride Ions ◽  
Outward Rectification ◽  
Voltage Dependent

Cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation- and nucleotide-dependent chloride channel. Single CFTR currents recorded on cell show slight outward rectification, which has previously been suggested to be due to an asymmetrical chloride ion gradient or to a specific interaction between permeant intracellular anions and the channel. Using a single-channel recording from Chinese hamster ovary cells stably expressing CFTR, we have found that both the sparingly permeant anion glutamate and the impermeant anion gluconate cause a rapid, voltage-dependent block of CFTR channels when applied to the intracellular, but not the extracellular, face of excised patches. Both the affinity and the voltage dependence of block were affected by the extracellular chloride concentration in a manner consistent with chloride ions being able to repel these blocking ions from the pore. These results are discussed in terms of previous models of CFTR current outward rectification, and it is suggested that this rectification may result from a combination of asymmetrical chloride concentrations and voltage-dependent block of the channel by large cytoplasmic anions. In addition, we find that CFTR conductance is decreased by high concentrations of intracellular sucrose, sorbitol, and urea in a manner consistent with a rapid block of the channel by these molecules.

Small linear chloride channels are endogenous to nonepithelial cells

1992 ◽  
Vol 263 (3) ◽  
pp. C708-C713 ◽  
Author(s):  
S. E. Gabriel ◽  
E. M. Price ◽  
R. C. Boucher ◽  
M. J. Stutts
Keyword(s):  
Cell Lines ◽  
Chloride Channels ◽  
Single Channel ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Types ◽  
Ovary Cells ◽  
3T3 Fibroblasts ◽  
Whole Cell Patch Clamp ◽  
Cl Channels

We used both single-channel and whole cell patch-clamp techniques to characterize chloride channels and currents endogenous to Sf9 cells, 3T3 fibroblasts, and Chinese hamster ovary cells. In cell-attached patches from these cell types, anion channels were observed with low ohmic conductance (4-11 ps), linear current-voltage relationships, and little time- or voltage-dependent behavior. These channels are very similar to the Cl- channels reported to appear concomitant with the expression of cystic fibrosis transmembrane conductance regulator (CFTR) in these cell lines. The presence of such endogenous channels suggests either that low levels of CFTR are present in all of these cell lines prior to transfection or that an endogenous non-CFTR channel is present in these cell types. Our results suggest that at least some of the channel behaviors attributed to expressed, recombinant CFTR in previous studies may have been due to these endogenous Cl- channels.

scholarly journals Permeability of Wild-Type and Mutant Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels to Polyatomic Anions

1997 ◽  
Vol 110 (4) ◽  
pp. 355-364 ◽  
Author(s):  
Paul Linsdell ◽  
Joseph A. Tabcharani ◽  
Johanna M. Rommens ◽  
Yue-Xian Hou ◽  
Xiu-Bao Chang ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Chloride Channels ◽  
Single Channel ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Site Directed Mutagenesis ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Patch Clamp Technique ◽  
Cystic Fibrosis Transmembrane

Permeability of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel to polyatomic anions of known dimensions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. Biionic reversal potentials measured with external polyatomic anions gave the permeability ratio (PX/PCl) sequence NO3− > Cl− > HCO3− > formate > acetate. The same selectivity sequence but somewhat higher permeability ratios were obtained when anions were tested from the cytoplasmic side. Pyruvate, propanoate, methane sulfonate, ethane sulfonate, and gluconate were not measurably permeant (PX/PCl < 0.06) from either side of the membrane. The relationship between permeability ratios from the outside and ionic diameters suggests a minimum functional pore diameter of ∼5.3 Å. Permeability ratios also followed a lyotropic sequence, suggesting that permeability is dependent on ionic hydration energies. Site-directed mutagenesis of two adjacent threonines in TM6 to smaller, less polar alanines led to a significant (24%) increase in single channel conductance and elevated permeability to several large anions, suggesting that these residues do not strongly bind permeating anions, but may contribute to the narrowest part of the pore.

The Insecticide Avermectin Bla Activates A Chloride Channel in Crayfish Muscle Membrane

1989 ◽  
Vol 142 (1) ◽  
pp. 191-205
Author(s):  
F. ZUFALL ◽  
CH. FRANKE ◽  
H. HATT
Keyword(s):  
Chloride Channel ◽  
Chloride Channels ◽  
Single Channel ◽  
Chloride Concentration ◽  
Ibotenic Acid ◽  
Muscle Membrane ◽  
Stomach Muscle ◽  
Quisqualic Acid ◽  
Drug Actions ◽  
Inside Out

Effects of avermectin B1a (AVM) have been tested on excised outside-out or inside-out patches of crayfish stomach muscle membrane. Continuous supervision of AVM (0.1-1 pmoll−1) to the outside-out patches induced openings of channels (22 pS) which were similar in conductance and kinetics to the chloride channels activated by glutamate, quisqualic acid, ibotenic acid and nicotinic agonists, whereas GABA mainly activated a second, larger conductance state (44 pS). This effect was reversible. AVM did not activate the excitatory, glutamate-activated cation channel. Upon raising the AVM-concentration to 10 pmoll−1 and above, an enormous increase in the rate of openings of channels (22 pS) occurred. This effect could not be washed out during the lifetime of the patch. Using inside-out patches, it was shown that the single-channel current amplitude, for both the reversible and irreversible drug actions, strongly depended on intracellular chloride concentration. Applied to the sarcoplasmic side of inside-out patches, AVM did not activate any channel. The distribution of open times for 0.1 pmoll−1 AVM could be fitted by a single exponential (τ=3.3ms). For a higher AVM concentration (1 pmoll−1) two exponentials (τ1 = 0.5ms, τ2 = 2.4ms) were needed to fit the distribution. A similar effect was elicited by decreasing the extracellular Ca2+ concentration from 13.5 to 1 mmoll−1 during the application of 0.1 pmoll−1 AVM. Picrotoxin blocked the activation of chloride channels for both the reversible and irreversible effects of AVM. It is suggested that AVM activates the multitransmitter-gated chloride channel in this preparation. Binding sites for the drug are discussed.

Effect of cytoplasmic chloride concentration on open-state preference of glycine-activated chloride channels in cultured spinal cord cells

1993 ◽  
Vol 69 (3) ◽  
pp. 860-867 ◽  
Author(s):  
A. I. McNiven ◽  
A. R. Martin
Keyword(s):  
Spinal Cord ◽  
Chloride Channels ◽  
Single Channel ◽  
Chloride Concentration ◽  
Constant Field ◽  
Spinal Cord Neurons ◽  
Methane Sulphonate ◽  
State Preference ◽  
Lower Permeability ◽  
Open States

1. Glycine was bath-applied to outside-out patches taken from cultured mouse spinal cord neurons. Glycine-activated chloride channels had at least six open states with permeabilities of 40 (A), 68 (B), 100 (C), 145 (D), 180 (E), and 250 (F) al/s (1 attoliter = 10(-18) liter). Usually no more than two such states were seen in any given patch. The usual extracellular chloride concentration was 157 mM; chloride concentration in the electrode (on the cytoplasmic face of the patch) was varied between 7 and 170 mM, replaced at lower concentrations with either of the inert anions gluconate or methane sulphonate. 2. With normal (7 mM) chloride concentration in the electrode, openings to state D were the most probable, and the average permeability of all openings was 115 al/s. When the cytoplasmic chloride concentration was 20 mM, the most probable open state was the lower-permeability state (C), and the average permeability was 92 al/s. In symmetrical 170 mM chloride, the most probable open state was B and the mean permeability 77 al/s. 3. The glycine-activated channels had a vanishingly small permeability to fluoride. However, in experiments in which 150 mM gluconate in the pipette was replaced by fluoride, the most probable open state was B, as in the high-chloride solution. Thus the two halides had similar effects on open state preference. 4. With external and internal chloride concentrations of 7 and 157 mM, respectively, single-channel current-voltage relations could be fit with constant field relations appropriate to one or more of the permeability states.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Distinct subunit contributions to the activation of M-type potassium channels by PI(4,5)P2

2012 ◽  
Vol 140 (1) ◽  
pp. 41-53 ◽  
Author(s):  
Vsevolod Telezhkin ◽  
David A. Brown ◽  
Alasdair J. Gibb
Keyword(s):  
Potassium Channels ◽  
Single Channel ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Water Soluble ◽  
Activation Mechanism ◽  
Quantitative Relation ◽  
Open Probability ◽  
Activation Curve ◽  
M Channels

Low-threshold voltage-gated M-type potassium channels (M channels) are tetraheteromers, commonly of two Kv7.2 and two Kv7.3 subunits. Though gated by voltage, the channels have an absolute requirement for binding of the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) to open. We have investigated the quantitative relation between the concentration of a water-soluble PI(4,5)P2 analog, dioctanoyl-PI(4,5)P2 (DiC8-PI(4,5)P2), and channel open probability (Popen) by fast application of increasing concentrations of DiC8-PI(4,5)P2 to the inside face of membrane patches excised from Chinese hamster ovary cells expressing M channels as heteromeric Kv7.2/7.3 subunits. The rationale for the experiments is that this will mimic the effect of changes in membrane PI(4,5)P2 concentration. Single-channel conductances from channel current–voltage relations in cell-attached mode were 9.2 ± 0.1 pS with a 2.5-mM pipette [K+]. Plots of Popen against DiC8-PI(4,5)P2 concentration were best fitted using a two-component concentration–Popen relationship with high and low affinity, half-maximal effective concentration (EC50) values of 1.3 ± 0.14 and 75.5 ± 2.5 µM, respectively, and Hill slopes of 1.4 ± 0.06. In contrast, homomeric channels from cells expressing only Kv7.2 or Kv7.3 constructs yielded single-component curves with EC50 values of 76.2 ± 19.9 or 3.6 ± 1.0 µM, respectively. When wild-type (WT) Kv7.2 was coexpressed with a mutated Kv7.3 subunit with >100-fold reduced sensitivity to PI(4,5)P2, the high-affinity component of the activation curve was lost. Fitting the data for WT and mutant channels to an activation mechanism with independent PI(4,5)P2 binding to two Kv7.2 and two Kv7.3 subunits suggests that the two components of the M-channel activation curve correspond to the interaction of PI(4,5)P2 with the Kv7.3 and Kv7.2 subunits, respectively, that channels can open when only the two Kv7.3 subunits have bound DiC8-PI(4,5)P2, and that maximum channel opening requires binding to all four subunits.

Inositol trisphosphate activates a voltage-dependent calcium influx in Xenopus oocytes

1987 ◽  
Vol 231 (1262) ◽  
pp. 27-36 ◽  
Keyword(s):  
Chloride Channels ◽  
Calcium Influx ◽  
Inositol Trisphosphate ◽  
Ringer Solution ◽  
Chloride Ions ◽  
Bathing Solution ◽  
Calcium Dependent ◽  
Large Numbers ◽  
Voltage Dependent ◽  
Oocyte Membrane

Injection of inositol trisphosphate (IP 3 ) into oocytes of Xenopus laevis induces the appearance of a transient inward ( T in ) current on hyper­polarization of the membrane. This current is carried largely by chloride ions, but is shown to depend on extracellular calcium, because it is abolished by removal of calcium in the bathing fluid or by addition of manganese. Recordings with aequorin as an intracellular calcium indi­cator show that a calcium influx is activated by hyperpolarization after intracellular injection of IP 3 as well as after activation of neurotrans­mitter receptors thought to mediate a rise in IP 3 . Furthermore, by substituting barium for calcium in the bathing solution, inward barium currents can be recorded during hyperpolarization. We conclude that intracellular IP 3 modulates the activity of a class of calcium channels, so as to allow an influx of calcium on hyperpolarization. In normal Ringer solution this then leads to the generation of a chloride current, because of the large numbers of calcium-dependent chloride channels in the oocyte membrane.

scholarly journals The chloride effect in the human embryonic haemoglobins

1995 ◽  
Vol 309 (3) ◽  
pp. 959-962 ◽  
Author(s):  
O Hofmann ◽  
G Carrucan ◽  
N Robson ◽  
T Brittain
Keyword(s):  
Oxygen Affinity ◽  
Chloride Concentration ◽  
Chloride Ion ◽  
Chloride Ions ◽  
Ion Concentration ◽  
Chloride Binding ◽  
Independent Components ◽  
Binding Data ◽  
Chloride Effect ◽  
Allosteric Model

The interactions of the three human embryonic haemoglobins with chloride ions have been investigated. Each of the three embryonic haemoglobins exhibits a unique pattern of oxygen-affinity-dependence on chloride ion concentration. Human embryonic haemoglobin Portland (zeta 2 gamma 2) is found to be completely insensitive to chloride ion concentration. Haemoglobin Gower I (zeta 2 gamma 2) shows a small concentration dependence, whilst haemoglobin Gower II (alpha 2 epsilon 2) exhibits a dependence approaching that of the adult protein. The degree of co-operativity for each protein is essentially chloride concentration independent. The chloride-dependent and -independent components of the alkaline Bohr effects have been measured for each of the embryonic haemoglobins and compared with that of the adult protein. Both the chloride-binding data and the Bohr effect have been analysed in terms of the recently developed allosteric model proposed by Perutz [Perutz, Fermi, Poyart, Pagnier and Kister (1993) J. Mol. Biol. 233, 536-545].

scholarly journals Comparative Effects of Halogenated Inhaled Anesthetics on Voltage-gated Na+Channel Function

2009 ◽  
Vol 110 (3) ◽  
pp. 582-590 ◽  
Author(s):  
Wei Ouyang ◽  
Karl F. Herold ◽  
Hugh C. Hemmings
Keyword(s):  
Rank Order ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Na Channel ◽  
Dependent Manner ◽  
Fast Inactivation ◽  
Inhaled Anesthetics ◽  
Voltage Gated ◽  
Channel Inhibition ◽  
Voltage Dependent

Background Inhibition of voltage-gated Na channels (Na(v)) is implicated in the synaptic actions of volatile anesthetics. We studied the effects of the major halogenated inhaled anesthetics (halothane, isoflurane, sevoflurane, enflurane, and desflurane) on Na(v)1.4, a well-characterized pharmacological model for Na(v) effects. Methods Na currents (I(Na)) from rat Na(v)1.4 alpha-subunits heterologously expressed in Chinese hamster ovary cells were analyzed by whole cell voltage-clamp electrophysiological recording. Results Halogenated inhaled anesthetics reversibly inhibited Na(v)1.4 in a concentration- and voltage-dependent manner at clinical concentrations. At equianesthetic concentrations, peak I(Na) was inhibited with a rank order of desflurane > halothane approximately enflurane > isoflurane approximately sevoflurane from a physiologic holding potential (-80 mV). This suggests that the contribution of Na channel block to anesthesia might vary in an agent-specific manner. From a hyperpolarized holding potential that minimizes inactivation (-120 mV), peak I(Na) was inhibited with a rank order of potency for tonic inhibition of peak I(Na) of halothane > isoflurane approximately sevoflurane > enflurane > desflurane. Desflurane produced the largest negative shift in voltage-dependence of fast inactivation consistent with its more prominent voltage-dependent effects. A comparison between isoflurane and halothane showed that halothane produced greater facilitation of current decay, slowing of recovery from fast inactivation, and use-dependent block than isoflurane. Conclusions Five halogenated inhaled anesthetics all inhibit a voltage-gated Na channel by voltage- and use-dependent mechanisms. Agent-specific differences in efficacy for Na channel inhibition due to differential state-dependent mechanisms creates pharmacologic diversity that could underlie subtle differences in anesthetic and nonanesthetic actions.

scholarly journals One-Step Synthesis of Green Fluorescent Carbon Dots for Chloride Detecting and for Bioimaging

2021 ◽  
Vol 9 ◽  
Author(s):  
Juan Yue ◽  
Ling Yu ◽  
Li Li ◽  
Pai Liu ◽  
Qian Mei ◽  
...  
Keyword(s):  
Carbon Dots ◽  
Limit Of Detection ◽  
Chloride Concentration ◽  
Chloride Ion ◽  
Silver Ions ◽  
Chloride Ions ◽  
Cell Functions ◽  
Green Fluorescent ◽  
Fluorescent Carbon

The chloride ion is an essential ion in organisms, which plays an important role in maintaining normal cell functions. It is involved in many cell activities, such as cell proliferation, cell excitability regulation, immune response, and volume regulation. Accurate detection of the chloride ion can balance its concentration in vivo, which is of great significance. In this study, we developed a green fluorescent carbon quantum dot to detect chloride concentration through the “off–on” mechanism. First, the fluorescence of carbon dots is quenched by the complex of sulfhydryl and silver ions on the surface of carbon dots. Then, the addition of chloride ions pulls away the silver ions and restores the fluorescence. The fluorescence recovery is linearly related to the concentration of chloride ions, and the limit of detection is 2.817 μM, which is much lower than those of other reported chloride probes. Besides, cell and zebrafish experiments confirmed the biosafety and biocompatibility of the carbon dots, which provided a possibility for further applications in bioimaging in vivo.

scholarly journals Experimental Study on Chloride Ion Diffusion in Concrete under Uniaxial and Biaxial Sustained Stress

Materials ◽  
2020 ◽  
Vol 13 (24) ◽  
pp. 5717
Author(s):  
Xiaokang Cheng ◽  
Jianxin Peng ◽  
C.S. Cai ◽  
Jianren Zhang
Keyword(s):  
Diffusion Coefficient ◽  
Compressive Stress ◽  
Stress Level ◽  
Chloride Concentration ◽  
Chloride Ion ◽  
Chloride Ions ◽  
Chloride Diffusion ◽  
Chloride Diffusion Coefficient ◽  
Ion Diffusion ◽  
Chloride Ion Diffusion

The existence of axial and lateral compressive stress affect the diffusion of chloride ions in concrete will lead to the performance degradation of concrete structure. This paper experimentally studied the chloride diffusivity properties of uniaxial and biaxial sustained compressive stress under one-dimensional chloride solution erosion. The influence of different sustained compressive stress states on chloride ion diffusivity is evaluated by testing chloride concentration in concrete. The experiment results show that the existence of sustained compressive stress does not always inhibit the diffusion of chloride ions in concrete, and the numerical value of sustained compressive stress level can affect the diffusion law of chloride ions in concrete. It is found that the chloride concentration decreases most when the lateral compressive stress level is close to 0.15 times the compressive strength of concrete. In addition, the sustained compressive stress has a significant effect on chloride ion diffusion of concrete with high water/cement ratio. Then, the chloride diffusion coefficient model under uniaxial and biaxial sustained compressive stress is established based on the apparent chloride diffusion coefficient. Finally, the results demonstrate that the chloride diffusion coefficient model is reasonable and feasible by comparing the experimental data in the opening literature with the calculated values from the developed model.

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