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.

Characterization of single non-inactivating potassium channels in primary neuronal cultures of Drosophila

1989 ◽  
Vol 145 (1) ◽  
pp. 173-184
Author(s):  
D. Yamamoto ◽  
N. Suzuki
Keyword(s):  
Time Distribution ◽  
Single Channel ◽  
K Channel ◽  
Delayed Rectifier ◽  
Neuronal Cultures ◽  
Patch Clamp Technique ◽  
Primary Neuronal Cultures ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Cytoplasmic Side

Permeability and gating properties of single, non-inactivating, K+ channel currents in cultured Drosophila neurons were studied using the gigaohm-seal patch-clamp technique. The non-inactivating K+ currents were activated by depolarizing the membrane to −30 mV or to more positive potentials. The slope conductance of the channel was estimated to be 17.6 +/− 3.70 pS when the cytoplasmic side of the inside-out membrane patch was perfused with solutions containing 145 mmoll-1 K+. The single-channel conductance was temperature-sensitive, with a Q10 of 1.44 between 10 and 20 degrees C. Single-channel currents could be recorded when the cytoplasmic K+ was replaced with NH4+, Rb+ or Na+, but not with Cs+. The conductance ratio of the channel for these cations was: K+ (1) greater than NH4+(0.53) greater than Rb+ (0.47) greater than Na+ (0.44). Tetraethylammonium (TEA+) ions applied at a concentration of 10 mmoll-1 to the cytoplasmic side of the membrane increased the frequency of ‘blank’ traces which contained no channel openings during repetitive depolarization. In addition, single-channel amplitude was reduced by about 20%. The open-time distribution was fitted by a single exponential function, whereas the closed-time distribution required a three-exponential fit. Permeability and gating properties of single, non-inactivating K+ channel currents in neurons of eag, a mutant which has defects in the delayed rectifier K+ channel, were indistinguishable from those recorded from wild-type neurons.

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 Stretch-activated whole cell currents in adult rat cardiac myocytes

2000 ◽  
Vol 278 (2) ◽  
pp. H548-H557 ◽  
Author(s):  
Tao Zeng ◽  
Glenna C. L. Bett ◽  
Frederick Sachs
Keyword(s):  
Single Channel ◽  
Ventricular Myocytes ◽  
Reversal Potential ◽  
Cell Voltage ◽  
Cellular Level ◽  
Whole Cell ◽  
Adult Rat ◽  
Longitudinal Stretch ◽  
Single Channel Currents ◽  
Channel Currents

Mechanoelectric transduction can initiate cardiac arrhythmias. To examine the origins of this effect at the cellular level, we made whole cell voltage-clamp recordings from acutely isolated rat ventricular myocytes under controlled strain. Longitudinal stretch elicited noninactivating inward cationic currents that increased the action potential duration. These stretch-activated currents could be blocked by 100 μM Gd3+ but not by octanol. The current-voltage relationship was nearly linear, with a reversal potential of approximately −6 mV in normal Tyrode solution. Current density varied with sarcomere length (SL) according to I (pA/pF) = 8.3 − 5.0SL (μm). Repeated attempts to record single channel currents from stretch-activated ion channels failed, in accord with the absence of such data from the literature. The inability to record single channel currents may be a result of channels being located on internal membranes such as the T tubules or, possibly, inactivation of the channels by the mechanics of patch formation.

scholarly journals Kinetic properties of single sodium channels in rat heart and rat brain.

1989 ◽  
Vol 93 (1) ◽  
pp. 85-99 ◽  
Author(s):  
G E Kirsch ◽  
A M Brown
Keyword(s):  
Cortical Neurons ◽  
Single Channel ◽  
Ventricular Myocytes ◽  
Na Channel ◽  
Kinetic Properties ◽  
Open Time ◽  
Excised Patches ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Near Threshold

Single Na channel currents were compared in ventricular myocytes and cortical neurons of neonatal rats using the gigaseal patch-clamp method to determine whether tissue-specific differences in gating can be detected at the single-channel level. Single-channel currents were recorded in cell-attached and excised membrane patches at test potentials of -70 to -20 mV and at 9-11 degrees C. In both cell-attached and excised patches brain Na channel mean open time progressively increased from less than 1 ms at -70 mV to approximately 2 ms at -20 mV. Near threshold, single openings with dispersed latencies were observed. By contrast, in cell-attached patches, heart Na channel mean open time peaked near -50 mV, was three times brain Na channel mean open time, and declined continuously to approximately 2 ms at -20 mV. Near threshold, openings occurred frequently usually as brief bursts lasting several milliseconds and rarely as prolonged bursts lasting tens of milliseconds. Unlike what occurs in brain tissue where excision did not change gating, in excised heart patches both the frequency of prolonged bursting and the mean open time of single units increased markedly. Brain and cardiac Na channels can therefore be distinguished on the basis of their mean open times and bursting characteristics.

scholarly journals The nature and origin of spontaneous noise in G protein-gated ion channels.

1991 ◽  
Vol 97 (6) ◽  
pp. 1279-1293 ◽  
Author(s):  
K Okabe ◽  
A Yatani ◽  
A M Brown
Keyword(s):  
Ion Channels ◽  
G Protein ◽  
Single Channel ◽  
Coupled Systems ◽  
Dependent Manner ◽  
Protein Receptor ◽  
Spontaneous Noise ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Inside Out

Arrival of agonist is generally thought to initiate the signal transduction process in G protein-receptor coupled systems. However, the muscarinic atrial K+ (K+[ACh]) channel opens spontaneously in the absence of applied agonist, giving a noisy appearance to the current records. We investigated the nature and origin of the noise by measuring single channel currents in cell-attached or excised, inside-out membrane patches. Guanosine triphosphate (GTP) produced identical single channel currents in a concentration- and Mg(2+)-dependent manner in the presence or absence of carbachol, but the requirements for GTP were greater in the absence of agonist. Hence the agonist-independent currents appeared to be produced by an endogenous G protein, Gk. This prediction was confirmed when an affinity-purified, sequence-specific Gi-3 alpha antibody or pertussis toxin (PTX) blocked the agonist-independent currents. Candidate endogenous agonists were ruled out by the lack of effect of their corresponding antagonists. Thus agonist-independent currents had the same nature as agonist-dependent K+[ACh] currents and seemed to originate in the same way. We have developed a hypothesis in which agonist-free, empty receptors prime Gk with GTP and Gk activates atrial K+ [ACh] channels producing basal currents or noise. Agonist-independent activation by G proteins of effectors including ion channels appears to be a common occurrence.

scholarly journals Ion channels activated by light in Limulus ventral photoreceptors.

1986 ◽  
Vol 87 (1) ◽  
pp. 73-89 ◽  
Author(s):  
J Bacigalupo ◽  
K Chinn ◽  
J E Lisman
Keyword(s):  
Light Adaptation ◽  
Voltage Dependence ◽  
Single Channel ◽  
Patch Clamp Technique ◽  
Channel Activation ◽  
Voltage Range ◽  
Secondary Result ◽  
Channel Open Time ◽  
Single Channel Currents ◽  
Channel Currents

The light-activated conductance of Limulus ventral photoreceptors was studied using the patch-clamp technique. Channels (40 pS) were observed whose probability of opening was greatly increased by light. In some cells the latency of channel activation was nearly the same as that of the macroscopic response, while in other cells the channel latency was much greater. Like the macroscopic conductance, channel activity was reduced by light adaptation but enhanced by the intracellular injection of the calcium chelator EGTA. The latter observation indicates that channel activation was not a secondary result of the light-induced rise in intracellular calcium. A two-microelectrode voltage-clamp method was used to measure the voltage dependence of the light-activated macroscopic conductance. It was found that this conductance is constant over a wide voltage range more negative than zero, but it increases markedly at positive voltages. The single channel currents measured over this same voltage range show that the single channel conductance is independent of voltage, but that channel gating properties are dependent on voltage. Both the mean channel open time and the opening rate increase at positive voltages. These properties change in a manner consistent with the voltage dependence of the macroscopic conductance. The broad range of similarities between the macroscopic and single channel currents supports the conclusion that the 40-pS channel that we have observed is the principal channel underlying the response to light in these photoreceptors.

scholarly journals Effect of N-bromoacetamide on single sodium channel currents in excised membrane patches.

1982 ◽  
Vol 79 (3) ◽  
pp. 333-351 ◽  
Author(s):  
J Patlak ◽  
R Horn
Keyword(s):  
Sodium Channel ◽  
Open Channel ◽  
Single Channel ◽  
Voltage Range ◽  
Cytoplasmic Face ◽  
Before And After ◽  
Excised Patches ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Single Sodium Channel

We have studied the effect of N-bromoacetamide (NBA) on the behavior of single sodium channel currents in excised patches of rat myotube membrane at 10 degree C. Inward sodium currents were activated by voltage steps from holding potentials of about -100 mV to test potentials of -40 mV. The cytoplasmic-face solution was isotonic CsF. Application of NBA or pronase to the cytoplasmic face of the membrane irreversibly removed sodium channel inactivation, as determined by averaged single-channel records. Teh lifetime of the open channel at -40 mV was increased about 10-fold by NBA treatment without affecting the amplitude of single-channel currents. A binomial analysis was used both before and after treatment to determine the number of channels within the excised patch. NBA was shown to have little effect on activation kinetics, as determined by an examination of both the rising phase of averaged currents and measurements f the delay between the start of the pulse and the first channel opening. Our data support a kinetic model of sodium channel activation in which the rate constant leading back from the open state to the last closed state is slower than expected from a strict Hodgkin-Huxley model. The data also suggest that the normal open-channel lifetime is primarily determined by the inactivation process in the voltage range we have examined.

scholarly journals Low pH Potentiates Both Capsaicin Binding and Channel Gating of VR1 Receptors

2003 ◽  
Vol 122 (1) ◽  
pp. 45-61 ◽  
Author(s):  
Sujung Ryu ◽  
Beiying Liu ◽  
Feng Qin
Keyword(s):  
Single Channel ◽  
Channel Gating ◽  
General Mechanism ◽  
Low Concentrations ◽  
Excised Patches ◽  
High Concentrations ◽  
Activation Pathways ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Important Basis

Capsaicin ion channels are highly expressed in peripheral nervous terminals and involved in pain and thermal sensations. One characteristic of the cloned VR1 receptor is its multimodal responses to various types of noxious stimuli. The channel is independently activated by capsaicin and related vanilloids at submicromolar range, by heat above 40°C, and by protons at pH below 6.5. Furthermore, simultaneous applications of two or more stimuli lead to cross sensitization of the receptor, with an apparent increase in the sensitivity to any individual stimulus when applied alone. We studied here the mechanism underlying such cross-sensitization; in particular, between capsaicin and pH, two prototypical stimuli for the channel. By analyzing single-channel currents recorded from excised-patches expressing single recombinant VR1 receptors, we examined the effect of pH on burst properties of capsaicin activation at low concentrations and the effect on gating kinetics at high concentrations. Our results indicate that pH has dual effects on both capsaicin binding and channel gating. Lowering pH enhances the apparent binding affinity of capsaicin, promotes the occurrences of long openings and short closures, and stabilizes at least one of the open conformations of the channel. Our data also demonstrate that capsaicin binding and protonation of the receptor interact allosterically, where the effect of one can be offset by the effect of the other. These results provide important basis to further understand the nature of the activation pathways of the channel evoked by different stimuli as well as the general mechanism underling the cross-sensitization of pain.

Sign in / Sign up

Export Citation Format

Share Document