Biophysical properties and metabolic regulation of a TASK-like potassium channel in rat carotid body type 1 cells

2004 ◽  
Vol 286 (1) ◽  
pp. L221-L230 ◽  
Author(s):  
Beatrice A. Williams ◽  
Keith J. Buckler
Keyword(s):  
Potassium Channel ◽  
Carotid Body ◽  
Metabolic Regulation ◽  
Single Channel ◽  
Divalent Cations ◽  
Channel Activity ◽  
Body Type ◽  
Oxygen Sensitive ◽  
Inside Out

The single channel properties of TASK-like oxygen-sensitive potassium channels were studied in rat carotid body type 1 cells. We observed channels with rapid bursting kinetics, active at resting membrane potentials. These channels were highly potassium selective with a slope conductance of 14–16 pS, values similar to those reported for TASK-1. In the absence of extracellular divalent cations, however, single channel conductance increased to 28 pS in a manner similar to that reported for TASK-3. After patch excision, channel activity ran down rapidly. Channel activity in inside-out patches was markedly increased by 2 and 5 mM ATP and by 2 mM ADP but not by 100 μM ADP or 1 mM AMP. In cell-attached patches, both cyanide and 2,4-dinitrophenol strongly inhibited channel activity. We conclude that 1) whilst the properties of this channel are consistent with it being a TASK-like potassium channel they do not precisely conform to those of either TASK-1 or TASK-3, 2) channel activity is highly dependent on cytosolic factors including ATP, and 3) changes in energy metabolism may play a role in regulating the activity of these background K+ channels.

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.

Different properties of the atrial G protein-gated K+ channels activated by extracellular ATP and adenosine

1995 ◽  
Vol 269 (4) ◽  
pp. H1349-H1358 ◽  
Author(s):  
C. Fu ◽  
A. Pleumsamran ◽  
U. Oh ◽  
D. Kim
Keyword(s):  
G Protein ◽  
Single Channel ◽  
Extracellular Atp ◽  
K Channel ◽  
Affinity Constant ◽  
Channel Activity ◽  
K Channels ◽  
Atrial Cells ◽  
K Current ◽  
Inside Out

Extracellular ATP (ATPo) and adenosine activate G protein-gated inwardly rectifying K+ currents in atrial cells. Earlier studies have suggested that the two agonists may use separate pathways to activate the K+ current. Therefore, we examined whether the K+ channels activated by the two agonists have different properties under identical ionic conditions. In cell-attached patches, K+ channels activated by 100 microM ATP in the pipette had a single-channel conductance and mean open time of 32.0 +/- 0.2 pS and 0.5 +/- 0.1 ms, respectively, compared with 31.3 +/- 0.3 pS and 0.9 +/- 0.1 ms for the K+ channels activated by adenosine (140 mM KCl). With ATPo as the agonist, the K+ channel activity in cell-attached patches was approximately threefold lower than that in inside-out patches with 100 microM GTP in the bath. Applying ATP to the cytoplasmic side of the membrane (ATPi) produced a biphasic concentration-dependent effect on channel activity: an increase at low [mean affinity constant (K0.5) = 190 microM] and a decrease at high (K0.5 = 1.3 mM) concentrations. In contrast, with adenosine as the agonist, K+ channel activity in cell-attached patches was approximately fourfold greater than that in inside-out patches with 100 microM GTP in the bath. In inside-out patches, ATPi only augmented the K+ channel activity (K0.5 = 32 microM). These results show that although both ATPo and adenosine activate kinetically similar K+ channels in atrial cells, the channels are regulated differently by intracellular nucleotides.

scholarly journals Characterization of the calcium-activated chloride channel in isolated guinea-pig hepatocytes.

1994 ◽  
Vol 104 (2) ◽  
pp. 357-373 ◽  
Author(s):  
S Koumi ◽  
R Sato ◽  
T Aramaki
Keyword(s):  
Guinea Pig ◽  
Single Channel ◽  
Hill Coefficient ◽  
Disulfonic Acid ◽  
Dependent Manner ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Cl Channels ◽  
Inside Out

Macroscopic and unitary currents through Ca(2+)-activated Cl- channels were examined in enzymatically isolated guinea-pig hepatocytes using whole-cell, excised outside-out and inside-out configurations of the patch-clamp technique. When K+ conductances were blocked and the intracellular Ca2+ concentration ([Ca2+]i) was set at 1 microM (pCa = 6), membrane currents were observed under whole-cell voltage-clamp conditions. The reversal potential of the current shifted by approximately 60 mV per 10-fold change in the external Cl- concentration. In addition, the current did not appear when Cl- was omitted from the internal and external solutions, indicating that the current was Cl- selective. The current was activated by increasing [Ca2+]i and was inactivated in Ca(2+)-free, 5 mM EGTA internal solution (pCa > 9). The current was inhibited by bath application of 9-anthracenecarboxylic acid (9-AC) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) in a voltage-dependent manner. In single channel recordings from outside-out patches, unitary current activity was observed, whose averaged slope conductance was 7.4 +/- 0.5 pS (n = 18). The single channel activity responded to extracellular Cl- changes as expected for a Cl- channel current. The open time distribution was best described by a single exponential function with mean open lifetime of 97.6 +/- 10.4 ms (n = 11), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 21.5 +/- 2.8 ms (n = 11) and that for the slow component of 411.9 +/- 52.0 ms (n = 11). In excised inside-out patch recordings, channel open probability was sensitive to [Ca2+]i. The relationship between [Ca2+]i and channel activity was fitted by the Hill equation with a Hill coefficient of 3.4 and the half-maximal activation was 0.48 microM. These results suggest that guinea-pig hepatocytes possess Ca(2+)-activated Cl- channels.

scholarly journals Disease-Linked Super-Trafficking of a Mutant Potassium Channel

2020 ◽  
Author(s):  
Hui Huang ◽  
Laura M. Chamness ◽  
Carlos G. Vanoye ◽  
Georg Kuenze ◽  
Jens Meiler ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Potassium Channel ◽  
Single Channel ◽  
Channel Activity ◽  
Wild Type ◽  
Channel Activation ◽  
Voltage Gated ◽  
Disease Mutations ◽  
Single Channel Activity

ABSTRACTGain-of-function (GOF) mutations in the KCNQ1 voltage-gated potassium channel can induce cardiac arrhythmia. We tested whether any of the known GOF disease mutations in KCNQ1 act by increasing the amount of KCNQ1 that reaches the cell surface—“super-trafficking”. We found that levels of R231C KCNQ1 in the plasma membrane are 5-fold higher than wild type KCNQ1. This arises from both enhanced translocon-mediated membrane integration of the S4 voltage-sensor helix and an energetic linkage of C231 with the V129 and F166 side chains. Whole-cell electrophysiology recordings confirmed that R231C KCNQ1 in complex with KCNE1 is constitutively active, but also revealed the single channel activity of this mutant to be only 20% that of WT. The GOF phenotype associated with R231C therefore reflects the net effects of super-trafficking, reduced single channel activity, and constitutive channel activation. These investigations document membrane protein super-trafficking as a contributing mechanism to human disease.

IP3-activated Ca2+ channels in the plasma membrane of cultured vascular endothelial cells

1995 ◽  
Vol 269 (3) ◽  
pp. C733-C738 ◽  
Author(s):  
L. Vaca ◽  
D. L. Kunze
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Single Channel ◽  
Vascular Endothelial Cells ◽  
Channel Activity ◽  
Open Probability ◽  
Aortic Endothelial Cells ◽  
Control Value ◽  
Selective Channel ◽  
Inside Out

Although it is clear that D-myo-inositol 1,4,5-trisphosphate (IP3) plays an important role in the activation of Ca2+ influx, the mechanisms by which this occurs remain controversial. In an attempt to determine the role of IP3 in the activation of Ca2+ influx, patch-clamp single-channel experiments in the cell-attached, inside-out, and outside-out configurations were performed on cultured bovine aortic endothelial cells (BAEC). The results presented indicate that both IP3 and intracellular Ca2+ can modulate the activity of a Ca(2+)-selective channel found in the plasma membrane of these cells. Addition of 10 microM IP3 increased channel open probability (P(o)) from a control value of 0.12 +/- 0.05 to 0.7 +/- 0.13 at a constant intracellular Ca2+ of 1 nM in excised inside-out patches. D-Myo-inositol 1,3,4,5-tetrakisphosphate at 50 microM was ineffective in altering channel P(o). Channel activity declined after approximately 2 min in the continuous presence of IP3. Three to four minutes after addition of IP3, channel P(o) was reduced from 0.7 +/- 0.2 to 0.2 +/- 0.1, indicating that an additional regulator might be required to maintain channel activity in excised patches. The channel was reversibly blocked by application of 1 microgram/ml heparin to the intracellular side of inside-out patches. This Ca(2+)-selective channel is indistinguishable from the depletion-activated Ca2+ channel we have previously described in BAEC.

scholarly journals Characterization of ryanodine receptor type 1 single channel activity using “on-nucleus” patch clamp

Cell Calcium ◽  
2014 ◽  
Vol 56 (2) ◽  
pp. 96-107 ◽  
Author(s):  
Larry E. Wagner ◽  
Linda A. Groom ◽  
Robert T. Dirksen ◽  
David I. Yule
Keyword(s):  
Patch Clamp ◽  
Ryanodine Receptor ◽  
Single Channel ◽  
Receptor Type ◽  
Channel Activity ◽  
Single Channel Activity

scholarly journals Dequalinium

2002 ◽  
Vol 121 (1) ◽  
pp. 37-47 ◽  
Author(s):  
Tamara Rosenbaum ◽  
León D. Islas ◽  
Anne E. Carlson ◽  
Sharona E. Gordon
Keyword(s):  
Single Channel ◽  
Divalent Cations ◽  
K Channel ◽  
Kinase C ◽  
Conducting Pathway ◽  
Voltage Dependent ◽  
Excised Patches ◽  
Ion Conducting ◽  
Inside Out ◽  
Cnga1 Channels

Cyclic nucleotide–gated (CNG) channels have been shown to be blocked by diltiazem, tetracaine, polyamines, toxins, divalent cations, and other compounds. Dequalinium is an organic divalent cation which suppresses the rat small conductance Ca2+-activated K+ channel 2 (rSK2) and the activity of protein kinase C. In this study, we have tested the ability of dequalinium to block CNGA1 channels and heteromeric CNGA1+CNGB1 channels. When applied to the intracellular side of inside-out excised patches from Xenopus oocytes, dequalinium blocks CNGA1 channels with a K1/2 ≈ 190 nM and CNGA1+CNGB1 channels with a K1/2 ≈ 385 nM, at 0 mV. This block occurs in a state-independent fashion, and is voltage dependent with a zδ ≈ 1. Our data also demonstrate that dequalinium interacts with the permeant ion probably because it occupies a binding site in the ion conducting pathway. Dequalinium applied to the extracellular surface also produced block, but with a voltage dependence that suggests it crosses the membrane to block from the inside. We also show that at the single-channel level, dequalinium is a slow blocker that does not change the unitary conductance of CNGA1 channels. Thus, dequalinium should be a useful tool for studying permeation and gating properties of CNG channels.

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