Basolateral outward rectifier chloride channel in isolated crypts of mouse colon

2000 ◽  
Vol 279 (2) ◽  
pp. G277-G287 ◽  
Author(s):  
Olivier Mignen ◽  
Stéphane Egee ◽  
Martine Liberge ◽  
Brian J. Harvey
Keyword(s):  
Protein Kinase ◽  
Chloride Channels ◽  
Single Channel ◽  
Basolateral Membrane ◽  
Distal Colon ◽  
Open Probability ◽  
Voltage Dependent ◽  
Intracellular Ca ◽  
Dependent Protein ◽  
Inside Out

Single channel patch-clamp techniques were used to demonstrate the presence of outwardly rectifying chloride channels in the basolateral membrane of crypt cells from mouse distal colon. These channels were rarely observed in the cell-attached mode and, in the inside-out configuration, only became active after a delay and depolarizing voltage steps. Single channel conductance was 23.4 pS between −100 and −40 mV and increased to 90.2 pS between 40 and 100 mV. The channel permeability sequence for anions was: I− > SCN− > Br−> Cl− > NO3 − > F−≫ SO4 2− ≈ gluconate. In inside-out patches, the channel open probability was voltage dependent but insensitive to intracellular Ca2+ concentration. In cell-attached mode, forskolin, histamine, carbachol, A-23187, and activators of protein kinase C all failed to activate the channel, and activity could not be evoked in inside-out patches by exposure to the purified catalytic subunit of cAMP-dependent protein kinase A. The channel was inhibited by 5-nitro-2-(3-phenylpropylamino)benzoate, 9-anthracenecarboxylic acid, and DIDS. Stimulation of G proteins with guanosine 5′- O-(3-thiotriphosphate) decreased the channel open probability and conductance, whereas subsequent addition of guanosine 5′- O-(2-thiodiphosphate) reactivated the channel.

scholarly journals ATP mediates both activation and inhibition of K(ATP) channel activity via cAMP-dependent protein kinase in insulin-secreting cell lines.

1989 ◽  
Vol 94 (4) ◽  
pp. 693-717 ◽  
Author(s):  
B Ribalet ◽  
S Ciani ◽  
G T Eddlestone
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Single Channel ◽  
K Channel ◽  
Channel Activity ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Insulin Secreting Cells ◽  
Dependent Protein ◽  
Inside Out

The single-channel recording technique was employed to investigate the mechanism conferring ATP sensitivity to a metabolite-sensitive K channel in insulin-secreting cells. ATP stimulated channel activity in the 0-10 microM range, but depressed it at higher concentrations. In inside-out patches, addition of the cAMP-dependent protein kinase inhibitor (PKI) reduced channel activity, suggesting that the stimulatory effect of ATP occurs via cAMP-dependent protein kinase-mediated phosphorylation. Raising ATP between 10 and 500 microM in the presence of exogenous PKI progressively reduced the channel activity; it is proposed that this inactivation results from a reduction in kinase activity owing to an ATP-dependent binding of PKI or a protein with similar inhibitory properties to the kinase. A model describing the effects of ATP was developed, incorporating these two separate roles for the nucleotide. Assuming that the efficacy of ATP in controlling the channel activity depends upon the relative concentrations of inhibitor and catalytic subunit associated with the membrane, our model predicts that the channel sensitivity to ATP will vary when the ratio of these two modulators is altered. Based upon this, it is shown that the apparent discrepancy existing between the sensitivity of the channel to low ATP concentrations in the excised patch and the elevated intracellular level of ATP may be explained by postulating a change in the inhibitor/kinase ratio from 1:1 to 3:2 owing to the loss of protein kinase after patch excision. At a low concentration of ATP (10-20 microM), a nonhydrolyzable ATP analogue, AMP-PNP, enhanced the channel activity when present below 10 microM, whereas the analogue blocked the channel activity at higher concentrations. It is postulated that AMP-PNP inhibits the formation of the kinase-inhibitor complex in the former case, and prevents phosphate transfer in the latter. A similar mechanism would explain the interaction between ATP and ADP which is characterized by enhanced activity at low ADP concentrations and blocking at higher concentrations.

scholarly journals Spermine Block of the Strong Inward Rectifier Potassium Channel Kir2.1

2002 ◽  
Vol 120 (1) ◽  
pp. 53-66 ◽  
Author(s):  
Lai-Hua Xie ◽  
Scott A. John ◽  
James N. Weiss
Keyword(s):  
Single Channel ◽  
Quantitative Model ◽  
Inward Rectification ◽  
Channel Blockers ◽  
Surface Charges ◽  
Open Probability ◽  
Dependent Component ◽  
Voltage Dependent ◽  
Single Channel Currents ◽  
Inside Out

Inward rectification in strong inward rectifiers such as Kir2.1 is attributed to voltage-dependent block by intracellular polyamines and Mg2+. Block by the polyamine spermine has a complex voltage dependence with shallow and steep components and complex concentration dependence. To understand the mechanism, we measured macroscopic Kir2.1 currents in excised inside-out giant patches from Xenopus oocytes expressing Kir2.1, and single channel currents in the inside-out patches from COS7 cells transfected with Kir2.1. We found that as spermine concentration or voltage increased, the shallow voltage-dependent component of spermine block at more negative voltages was caused by progressive reduction in the single channel current amplitude, without a decrease in open probability. We attributed this effect to spermine screening negative surface charges involving E224 and E299 near the inner vestibule of the channel, thereby reducing K ion permeation rate. This idea was further supported by experiments in which increasing ionic strength also decreased Kir2.1 single channel amplitude, and by mutagenesis experiments showing that this component of spermine block decreased when E224 and E299, but not D172, were neutralized. The steep voltage-dependent component of block at more depolarized voltages was attributed to spermine migrating deeper into the pore and causing fast open channel block. A quantitative model incorporating both features showed excellent agreement with the steady-state and kinetic data. In addition, this model accounts for previously described substate behavior induced by a variety of Kir2.1 channel blockers.

Two types of voltage-dependent potassium channels in outer hair cells from the guinea pig cochlea

1999 ◽  
Vol 277 (5) ◽  
pp. C913-C925 ◽  
Author(s):  
Thierry van den Abbeele ◽  
Jacques Teulon ◽  
Patrice Tran Ba Huy
Keyword(s):  
Guinea Pig ◽  
Hair Cells ◽  
Basolateral Membrane ◽  
Outer Hair Cells ◽  
Catalytic Subunit ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Guinea Pig Cochlea ◽  
Voltage Dependent ◽  
Inside Out

Cell-attached and cell-free configurations of the patch-clamp technique were used to investigate the conductive properties and regulation of the major K+channels in the basolateral membrane of outer hair cells freshly isolated from the guinea pig cochlea. There were two major voltage-dependent K+ channels. A Ca2+-activated K+ channel with a high conductance (220 pS, P K/ P Na= 8) was found in almost 20% of the patches. The inside-out activity of the channel was increased by depolarizations above 0 mV and increasing the intracellular Ca2+concentration. External ATP or adenosine did not alter the cell-attached activity of the channel. The open probability of the excised channel remained stable for several minutes without rundown and was not altered by the catalytic subunit of protein kinase A (PKA) applied internally. The most frequent K+ channel had a low conductance and a small outward rectification in symmetrical K+ conditions (10 pS for inward currents and 20 pS for outward currents, P K/ P Na= 28). It was found significantly more frequently in cell-attached and inside-out patches when the pipette contained 100 μM acetylcholine. It was not sensitive to internal Ca2+, was inhibited by 4-aminopyridine, was activated by depolarization above −30 mV, and exhibited a rundown after excision. It also had a slow inactivation on ensemble-averaged sweeps in response to depolarizing pulses. The cell-attached activity of the channel was increased when adenosine was superfused outside the pipette. This effect also occurred with permeant analogs of cAMP and internally applied catalytic subunit of PKA. Both channels could control the cell membrane voltage of outer hair cells.

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.

scholarly journals Apical K+ channels in Necturus taste cells. Modulation by intracellular factors and taste stimuli.

1992 ◽  
Vol 99 (4) ◽  
pp. 591-613 ◽  
Author(s):  
T A Cummings ◽  
S C Kinnamon
Keyword(s):  
Apical Membrane ◽  
Basolateral Membrane ◽  
Taste Receptor ◽  
K Channel ◽  
Patch Clamp Recording ◽  
Open Probability ◽  
K Channels ◽  
Voltage Dependent ◽  
Whole Cell Recordings ◽  
Inside Out

The apically restricted, voltage-dependent K+ conductance of Necturus taste receptor cells was studied using cell-attached, inside-out and outside-out configurations of the patch-clamp recording technique. Patches from the apical membrane typically contained many channels with unitary conductances ranging from 30 to 175 pS in symmetrical K+ solutions. Channel density was so high that unitary currents could be resolved only at negative voltages; at positive voltages patch recordings resembled whole-cell recordings. These multi-channel patches had a small but significant resting conductance that was strongly activated by depolarization. Patch current was highly K+ selective, with a PK/PNa ratio of 28. Patches containing single K+ channels were obtained by allowing the apical membrane to redistribute into the basolateral membrane with time. Two types of K+ channels were observed in isolation. Ca(2+)-dependent channels of large conductance (135-175 pS) were activated in cell-attached patches by strong depolarization, with a half-activation voltage of approximately -10 mV. An ATP-blocked K+ channel of 100 pS was activated in cell-attached patches by weak depolarization, with a half-activation voltage of approximately -47 mV. All apical K+ channels were blocked by the sour taste stimulus citric acid directly applied to outside-out and perfused cell-attached patches. The bitter stimulus quinine also blocked all channels when applied directly by altering channel gating to reduce the open probability. When quinine was applied extracellularly only to the membrane outside the patch pipette and also to inside-out patches, it produced a flickery block. Thus, sour and bitter taste stimuli appear to block the same apical K+ channels via different mechanisms to produce depolarizing receptor potentials.

Basolateral membrane potassium channels in rabbit cortical thick ascending limb

1992 ◽  
Vol 263 (2) ◽  
pp. F262-F267 ◽  
Author(s):  
A. M. Hurst ◽  
M. Duplain ◽  
J. Y. Lapointe
Keyword(s):  
Patch Clamp ◽  
Single Channel ◽  
Basolateral Membrane ◽  
Fold Increase ◽  
Thick Ascending Limb ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Collagenase Treatment ◽  
Voltage Dependent ◽  
Selective Channel

The nature of K exit across the basolateral membrane of rabbit cortical thick ascending limb (CTAL) was investigated using the patch clamp technique. The basolateral membrane was exposed by mild collagenase treatment (0.1 U/ml), and a K-selective inwardly rectifying channel was identified. In cell-attached patches (140 mM K pipette) the inward conductance was 35.0 +/- 1.3 pS (n = 9) compared with an outward conductance of 7.0 +/- 0.9 pS (n = 5), and the current reversed at a pipette potential of -63.5 +/- 3.1 mV (n = 9). The channel is strongly voltage dependent, showing an e-fold increase in open probability per 18-mV depolarization. Barium blocked the channel, reducing both mean open probability and single-channel current amplitude; however, the channel was not Ca sensitive. On excision the channel exhibited rundown, which could not be prevented by 0.1 mM ATP or ATP plus 20 U/ml catalytic subunit of protein kinase A. A few excised patch recordings were possible, which confirmed the presence of a highly K-selective channel with a K-to-Na permeability ratio of 100. In conclusion, 1) it is possible to obtain patch clamp recordings from the rabbit CTAL basolateral membrane using a very mild collagenase treatment, and 2) the exit of K across the basolateral membrane is mediated at least in part by the presence of voltage-sensitive K channels.

Isoflurane-induced Facilitation of the Cardiac Sarcolemmal KATPChannel

2002 ◽  
Vol 97 (1) ◽  
pp. 57-65 ◽  
Author(s):  
Kazuhiro Fujimoto ◽  
Zeljko J. Bosnjak ◽  
Wai-Meng Kwok
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Single Channel ◽  
Intact Cell ◽  
Direct Interaction ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Whole Cell ◽  
Kinase C ◽  
Inside Out

Background Volatile anesthetics have cardioprotective effects that mimic ischemic preconditioning, including the involvement of adenosine triphosphate-sensitive potassium (K(ATP)) channels. However, evidence for a direct effect of volatile anesthetic on the K(ATP) channel is limited. In this study, the effects of isoflurane on the cardiac sarcolemmal K(ATP) channel were investigated. Methods Single ventricular myocytes were enzymatically isolated from guinea pig hearts. Whole cell and single-channel configurations, specifically the cell-attached and inside-out patch mode, of the patch clamp technique were used to monitor sarcolemmal K(ATP) channel current. Results In the cell-attached patch configuration, 2,4-dinitrophenol (150 microm) opened the sarcolemmal K(ATP) channel. Isoflurane (0.5 mm) further increased channel open probability and the number of active channels in the patch. In contrast, in the inside-out patch experiments, isoflurane had no significant effect on the K(ATP) channel activated by low ATP (0.2-0.5 mm). In addition, isoflurane had no effect on the K(ATP) channel when activated by adenosine diphosphate, adenosine + guanosine triphosphate, bimakalim, and 2,4-dinitrophenol under inside-out patch configurations. When K(ATP) current was monitored in the whole cell mode, isoflurane alone was unable to elicit channel opening. However, during sustained protein kinase C activation by 12,13-dibutyrate, isoflurane activated the K(ATP) current that was sensitive to glibenclamide. In contrast, isoflurane had no effect on the K(ATP) channel activated by 12,13-dibutyrate in a cell-free environment. Conclusions Isoflurane facilitated the opening of the sarcolemmal K(ATP) channel in the intact cell, but not in an excised, inside-out patch. The isoflurane effect was not due to a direct interaction with the K(ATP) channel protein, but required an intracellular component, likely including the translocation of specific protein kinase C isoforms. This suggests that the sarcolemmal K(ATP) channel may have a significant role in anesthetic-induced preconditioning.

scholarly journals Stimulation of unitary T-type Ca2+ channel currents by calmodulin-dependent protein kinase II

2000 ◽  
Vol 279 (6) ◽  
pp. C1694-C1703 ◽  
Author(s):  
Paula Q. Barrett ◽  
Hong-Kai Lu ◽  
Roger Colbran ◽  
Andrew Czernik ◽  
Joseph J. Pancrazio
Keyword(s):  
Protein Kinase ◽  
Single Channel ◽  
Low Voltage ◽  
Patch Clamp Technique ◽  
Dependent Protein Kinase ◽  
Protein Kinase Ii ◽  
Dependent Protein ◽  
Channel Open Time ◽  
Channel Currents ◽  
Inside Out

The effect of Ca2+/calmodulin-dependent protein kinase II (CaMKII) stimulation on unitary low voltage-activated (LVA) T-type Ca2+ channel currents in isolated bovine adrenal glomerulosa (AG) cells was measured using the patch-clamp technique. In cell-attached and inside-out patches, LVA channel activity was identified by voltage-dependent inactivation and a single-channel conductance of ∼9 pS in 110 mM BaCl2 or CaCl2. In the cell-attached patch, elevation of bath Ca2+ from 150 nM to 1 μM raised intracellular Ca2+ in K+-depolarized (140 mM) cells and evoked an increase in the LVA Ca2+ channel probability of opening ( NP o) by two- to sixfold. This augmentation was associated with an increase in the number of nonblank sweeps, a rise in the frequency of channel opening in nonblank sweeps, and a 30% reduction in first latency. No apparent changes in the single-channel open-time distribution, burst lengths, or openings/burst were apparent. Preincubation of AG cells with lipophilic or peptide inhibitors of CaMKII in the cell-attached or excised (inside-out) configurations prevented the rise in NP oelicited by elevated Ca2+ concentration. Furthermore, administration of a mutant recombinant CaMKIIα exhibiting cofactor-independent activity in the absence of elevated Ca2+ produced a threefold elevation in LVA channel NP o. These data indicate that CaMKII activity is both necessary and sufficient for LVA channel activation by Ca2+.

Calcium- and voltage-dependent ion channels in Saccharomyces cerevisiae

1992 ◽  
Vol 338 (1283) ◽  
pp. 63-72 ◽  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Ion Channels ◽  
Open Channel ◽  
Single Channel ◽  
Membrane Channel ◽  
Open Probability ◽  
Current Voltage ◽  
Voltage Dependent ◽  
Inside Out

Ion channels in both the tonoplast and the plasma membrane of Saccharomyces cerevisiae have been characterized at the single channel level by patch-clamp techniques. The predominant tonoplast channel is cation selective, has an open-channel conductance of 120 pS in 100 mM KCl, and conducts Na + or K + equally well, and Ca 2+ to a lesser extent. Its open probability (P„) is voltage-dependent, peaking at about — 80 mV (cytoplasm negative), and falling to near zero at + 8 0 mV. Elevated cytoplasmic Ca 2+ , alkaline cytoplasmic pH, and reducing agents activate the channel. The predominant plasma membrane channel is highly selective for K + over anions and other cations, and shows strong outward rectification of the time-averaged current-voltage curves in cell-attached experiments. In isolated inside-out patches with micromolar cytoplasmic Ca 2+ , this channel is activated by positive going membrane voltages: mean P o is zero at negative membrane voltages and near unity at 100 mV. At moderate positive membrane voltages (20-40 mV), elevating cytoplasmic Ca 2+ activates the channel to open in bursts of several hundred milliseconds duration. At higher positive membrane voltages, however, elevating cytoplasmic Ca 2+ blocks the channel in a voltage-dependent fashion for periods of 2-3 ms. The frequency of these blocking events depends on cytoplasmic Ca 2+ and membrane voltage according to second-order kinetics. Alternative cations, such as Mg 2+ of Na + , block the yeast plasma-membrane K + channel in a similar but less pronounced manner.

Chloride channels in the apical membrane of a distal nephron A6 cell line

1990 ◽  
Vol 258 (2) ◽  
pp. C352-C368 ◽  
Author(s):  
Y. Marunaka ◽  
D. C. Eaton
Keyword(s):  
Chloride Channels ◽  
Single Channel ◽  
Apical Membrane ◽  
The Other ◽  
Distal Nephron ◽  
Open Probability ◽  
Cl Channel ◽  
High Calcium ◽  
Voltage Dependent ◽  
Closing Rate

In this report, single-channel recording methods were used to determine whether there are Cl- conductive pathways in the apical membrane of cultured renal distal nephron cells (A6). Two different types of single Cl- channels were observed. In cell-attached patches, one had a unit conductance of 3 pS, whereas the unit conductance of the other was 8 pS. In cell-attached patches, the currents associated with the 3-pS Cl- channel outwardly rectified, whereas the current voltage relationship for the 8-pS Cl-channel was linear. The 3-pS Cl- channel has one open and one closed state; the 8-pS Cl- channel has one open and two closed states. The open probability of the 3-pS Cl- channel was voltage dependent (increasing with depolarization of the membrane) but even at very depolarized potentials (+140 mV) remained small (always less than 0.1). On the other hand, the open probability of the 8-pS Cl- channel was large (approximately 0.8) and voltage independent. The closing rate of the 3-pS Cl- channel was decreased when the patch membrane was depolarized, whereas the opening rate was increased. In contrast, the closing rate of the 8-pS Cl- channel decreased with depolarization, but the opening rates were voltage independent. The outward rectification of the 3-pS channel was markedly reduced in inside-out patches when high calcium concentrations (10-800 microM) were present on the intracellular surface. The open probability of the 3-pS Cl- channel is increased by membrane permeable analogues of adenosine 3',5'-cyclic monophosphate primarily by decreasing the mean closed time.

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