Abstract 950: Direct Evidence for an Important Role of Agonist-independent Constitutive I K,ACh Channel Activity in Atrial Tachycardia Remodeling

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Niels Voigt ◽  
Ange Maguy ◽  
Yung-Hsin Yeh ◽  
Xiao-Yan Qi ◽  
Ursula Ravens ◽  
...  
Keyword(s):  
Atrial Tachycardia ◽  
Single Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Direct Demonstration ◽  
Open Time ◽  
Therapy Target ◽  
Channel Properties ◽  
Constitutively Active

Background: Although atrial tachycardia (AT) appears to promote agonist-independent constitutively active I K,ACh that increases susceptibility to AF, direct demonstration of dysregulated I K,ACh channel function is lacking. We studied AT effects on single I K,ACh channel activity in dog atria. Methods: I K,ACh channel activity was recorded with cell-attached patch clamp in isolated atrial myocytes of control (CTL) and AT (7 days, 400 min −1 ) dogs. Results : AT prolonged inducible AF duration from 44±22 to 413±167 s; N=9 dogs/gp, P<0.001. In the absence of cholinergic stimulation, single-channel openings with typical I K,ACh conductance and rectification were observed in CTL and AT (Figure ). AT produced prominent agonist-independent I K,ACh activity due to 7-fold increased opening frequency (f o ) and 10-fold increased open probability (P o ) vs CTL (P<0.01 for each), but unaltered open time and single channel conductance. With maximum I K,ACh activation (10 μm carbachol, CCh), f o was 38% lower, open time constant 25% higher, and P o and unitary conductance unchanged for AT vs CTL. The selective Kir3 blocker tertiapin (100 nM) reduced f o and P o by 48% and 51% (P<0.05 each) without altering other channel properties, confirming the identity of I K,ACh. Conclusions : AT produces prominent agonist-independent constitutive single-channel I K,ACh activity, providing a molecular basis for previously-observed AT-enhanced macroscopic I K,ACh , as well as associated AP-shortening and tertiapin-suppressible AF promotion. These results suggest an important role for constitutively active I K,ACh channels in AT-remodeling and support their interest as a potential novel AF-therapy target.

Chloride efflux inhibits single calcium channel open probability in vertebrate photoreceptors: Chloride imaging and cell-attached patch-clamp recordings

2000 ◽  
Vol 17 (2) ◽  
pp. 197-206 ◽  
Author(s):  
WALLACE B. THORESON ◽  
RON NITZAN ◽  
ROBERT F. MILLER
Keyword(s):  
Time Distribution ◽  
Single Channel ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Open Probability ◽  
Pipette Solution ◽  
Open Time ◽  
The Mean ◽  
Channel Properties ◽  
Distribution Fit

The present study uses cell-attached patch-recording techniques to study the single-channel properties of Ca2+ channels in isolated salamander photoreceptors and investigate their sensitivity to reductions in intracellular Cl−. The results show that photoreceptor Ca2+ channels possess properties similar to L-type Ca2+ channels in other preparations, including (1) enhancement of openings by the dihydropyridine agonist, (−)BayK8644; (2) suppression by a dihydropyridine antagonist, nisoldipine; (3) single-channel conductance of 22 pS with 82 mM Ba2+ as the charge carrier; (4) mean open probability of 0.1; (5) open-time distribution fit with a single exponential (τ0 = 1.1 ms) consistent with a single open state; and (6) closed time distribution fit with two exponentials (τc1 = 0.7 ms, τc2 = 25.4 ms) consistent with at least two closed states. Using a Cl−-sensitive dye to measure intracellular [Cl−], it was found that perfusion with gluconate-containing, low Cl− medium depleted intracellular [Cl−]. It was therefore possible to reduce intracellular [Cl−] by perfusion with a low Cl− solution while maintaining the extracellular channel surface in high Cl− pipette solution. Under these conditions, the single-channel conductance was unchanged, but the mean open probability fell to 0.03. This reduction can account for the 66% reduction in whole-cell Ca2+ currents produced by perfusion with low Cl− solutions. Examination of the open and closed time distributions suggests that the reduction in open probability arises from increases in closed-state dwell times. Changes in intracellular [Cl−] may thus modulate photoreceptor Ca2+ channels.

Ethanol enhances caffeine-induced Ca2+-release channel activation in skeletal muscle sarcoplasmic reticulum

1997 ◽  
Vol 272 (2) ◽  
pp. C622-C627 ◽  
Author(s):  
T. Oba ◽  
M. Koshita ◽  
M. Yamaguchi
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Lipid Bilayers ◽  
Skeletal Muscles ◽  
Single Channel ◽  
Channel Activity ◽  
Electrical Parameters ◽  
Open Probability ◽  
Release Channel ◽  
Channel Current ◽  
Open Time

When sarcoplasmic reticulum (SR) vesicles prepared from frog skeletal muscles were actively loaded with Ca2+, pretreatment of the SR with 2.2 mM (0.01%) ethanol for 30 s significantly potentiated 5 mM caffeine-induced release of Ca2+ from 16.7 +/- 3.7 nmol/mg protein in control without ethanol to 28.0 +/- 2.6 nmol/mg (P < 0.05, n = 5). Ethanol alone caused no release of Ca2+ from the SR. Exposure of the Ca2+-release channel, incorporated into planar lipid bilayers, to 2 mM caffeine significantly increased open probability (Po) and mean open time, but unitary conductance was not affected. Ethanol (2.2 mM) enhanced caffeine-induced Ca2+-release channel activity, with Po reaching 3.02-fold and mean open time 2.85-fold the values in the absence of ethanol. However, ethanol alone did not affect electrical parameters of single-channel current, over a concentration range of 2.2 mM (0.01%) to 217 mM (1%). The synergistic action of ethanol and caffeine on the channel activity could be attributable to enhancement of caffeine-induced release of Ca2+ from the SR vesicles in the presence of ethanol.

scholarly journals Single-channel Properties of Human NaV1.1 and Mechanism of Channel Dysfunction in SCN1A-associated Epilepsy

2005 ◽  
Vol 127 (1) ◽  
pp. 1-14 ◽  
Author(s):  
Carlos G. Vanoye ◽  
Christoph Lossin ◽  
Thomas H. Rhodes ◽  
Alfred L. George
Keyword(s):  
Sodium Channel ◽  
Single Channel ◽  
Sodium Current ◽  
Febrile Seizures ◽  
Persistent Sodium Current ◽  
Open Probability ◽  
Α Subunit ◽  
Whole Cell ◽  
Open Time ◽  
Channel Properties

Mutations in genes encoding neuronal voltage-gated sodium channel subunits have been linked to inherited forms of epilepsy. The majority of mutations (&gt;100) associated with generalized epilepsy with febrile seizures plus (GEFS+) and severe myoclonic epilepsy of infancy (SMEI) occur in SCN1A encoding the NaV1.1 neuronal sodium channel α-subunit. Previous studies demonstrated functional heterogeneity among mutant SCN1A channels, revealing a complex relationship between clinical and biophysical phenotypes. To further understand the mechanisms responsible for mutant SCN1A behavior, we performed a comprehensive analysis of the single-channel properties of heterologously expressed recombinant WT-SCN1A channels. Based on these data, we then determined the mechanisms for dysfunction of two GEFS+-associated mutations (R1648H, R1657C) both affecting the S4 segment of domain 4. WT-SCN1A has a slope conductance (17 pS) similar to channels found in native mammalian neurons. The mean open time is ∼0.3 ms in the −30 to −10 mV range. The R1648H mutant, previously shown to display persistent sodium current in whole-cell recordings, exhibited similar slope conductance but had an increased probability of late reopening and a subfraction of channels with prolonged open times. We did not observe bursting behavior and found no evidence for a gating mode shift to explain the increased persistent current caused by R1648H. Cells expressing R1657C exhibited conductance, open probability, mean open time, and latency to first opening similar to WT channels but reduced whole-cell current density, suggesting decreased number of functional channels at the plasma membrane. In summary, our findings define single-channel properties for WT-SCN1A, detail the functional phenotypes for two human epilepsy-associated sodium channel mutants, and clarify the mechanism for increased persistent sodium current induced by the R1648H allele.

Acetylcholine-sensitive potassium channels in human atrial myocytes

1990 ◽  
Vol 259 (6) ◽  
pp. H1730-H1735 ◽  
Author(s):  
R. Sato ◽  
I. Hisatome ◽  
J. A. Wasserstrom ◽  
C. E. Arentzen ◽  
D. H. Singer
Keyword(s):  
Single Channel ◽  
K Channel ◽  
Channel Activity ◽  
Atrial Myocytes ◽  
Open Probability ◽  
Human Atrial Myocytes ◽  
Open Time ◽  
Patch Pipette ◽  
Single Channel Activity ◽  
Gamma S

Single channel recording techniques were used to study acetylcholine (ACh)-sensitive K+ channel activity in human atrial myocytes isolated from specimens obtained during corrective cardiac surgery. Under conditions of cell-attached patch, the presence of ACh in the patch pipette activated K+ channels. Single channel activity occurred in periodic bursts. The channels exhibited a slope conductance of 46 +/- 2 pS inwardly (means +/- SD, n = 4). During a burst, both open and closed time histograms were fitted by a single exponential curve, suggesting the existence of one open and one closed state during a burst. Open probability increased directly with ACh concentration without affecting open time. The channel could be activated by GTP and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) (in the presence and absence of ACh in the pipette, respectively). Slope conductance, the response to GTP and GTP gamma S, and the independence of activation from Ca2+ were similar to those for other species. In contrast, sensitivity to ACh appeared diminished compared with frog atrial myocytes.

Single-channel properties of high- and low-voltage-activated calcium channels in rat pituitary melanotropic cells

1994 ◽  
Vol 71 (3) ◽  
pp. 840-855 ◽  
Author(s):  
J. A. Keja ◽  
K. S. Kits
Keyword(s):  
Calcium Channels ◽  
Kinetic Analysis ◽  
Calcium Current ◽  
Time Course ◽  
Single Channel ◽  
Low Voltage ◽  
Channel Activity ◽  
Open Probability ◽  
Test Pulse ◽  
Channel Properties

1. Single-channel properties of voltage-dependent calcium channels were investigated in rat melanotropes in short-term primary culture. Unitary currents were resolved using the cell-attached configuration. 2. Depolarizations higher than -50 mV activated a population of 8.1-pS calcium channels [low-voltage activated (LVA)]. The LVA channel ensembles displayed a monoexponential time course of inactivation and a sigmoidal time course of activation fitted best by an m2h Hodgkin-Huxley-type model. Microscopic kinetic analysis suggested that at least one open state, two closed states, and one inactivated state are involved in channel gating. 3. At potentials positive to -20 mV a second class of calcium channels was activated with a conductance of 24.7 pS [high-voltage activated (HVA)]. HVA channels display different gating modes. Gating with high open probability (mode 2) and low open probability (mode 1) as well as blank traces (mode 0) are observed. The HVA channels were heterogeneous with respect to their inactivation properties. Ensembles that decayed entirely during a 300-ms test pulse as well as nondecaying ensembles were observed. Both HVA channel subtypes displayed sigmoidal activation, which was fitted by an m2 model. Microscopic kinetic analysis suggested that at least one open state and two closed states are involved in mode two gating of both HVA channel subtypes. 4. Depolarizing prepulses did not recruit or facilitate calcium channel activity in response to a test pulse, but inactivating HVA channel activity was strongly reduced. Depolarizing prepulses (+50 mV) did not affect the probability of opening of the noninactivating HVA channel. 5. The voltage dependence and kinetics of the LVA as well as both HVA channels are in good agreement with previously published data on the properties of the various calcium current components derived from whole-cell recordings of rat melanotropes. The data suggest that a T-type as well as two L-type channels (an inactivating and noninactivating channel) underlie the calcium current in these cells.

scholarly journals Role of constitutively active acetylcholine-mediated potassium current in atrial contractile dysfunction caused by atrial tachycardia remodelling

EP Europace ◽  
2010 ◽  
Vol 12 (10) ◽  
pp. 1490-1497 ◽  
Author(s):  
S.-H. Koo ◽  
R. Wakili ◽  
J.-H. Heo ◽  
D. Chartier ◽  
H.-S. Kim ◽  
...  
Keyword(s):  
Potassium Current ◽  
Atrial Tachycardia ◽  
Contractile Dysfunction ◽  
Constitutively Active

scholarly journals Membrane-delimited Inhibition of Maxi-K Channel Activity by the Intermediate Conductance Ca2+-activated K Channel

2006 ◽  
Vol 127 (2) ◽  
pp. 159-169 ◽  
Author(s):  
Jill Thompson ◽  
Ted Begenisich
Keyword(s):  
Single Channel ◽  
Expression System ◽  
Chemical Activation ◽  
K Channel ◽  
Channel Activity ◽  
Single Family ◽  
Open Probability ◽  
K Channels ◽  
Channel Proteins ◽  
K Activity

The complexity of mammalian physiology requires a diverse array of ion channel proteins. This diversity extends even to a single family of channels. For example, the family of Ca2+-activated K channels contains three structural subfamilies characterized by small, intermediate, and large single channel conductances. Many cells and tissues, including neurons, vascular smooth muscle, endothelial cells, macrophages, and salivary glands express more than a single class of these channels, raising questions about their specific physiological roles. We demonstrate here a novel interaction between two types of Ca2+-activated K channels: maxi-K channels, encoded by the KCa1.1 gene, and IK1 channels (KCa3.1). In both native parotid acinar cells and in a heterologous expression system, activation of IK1 channels inhibits maxi-K activity. This interaction was independent of the mode of activation of the IK1 channels: direct application of Ca2+, muscarinic receptor stimulation, or by direct chemical activation of the IK1 channels. The IK1-induced inhibition of maxi-K activity occurred in small, cell-free membrane patches and was due to a reduction in the maxi-K channel open probability and not to a change in the single channel current level. These data suggest that IK1 channels inhibit maxi-K channel activity via a direct, membrane-delimited interaction between the channel proteins. A quantitative analysis indicates that each maxi-K channel may be surrounded by four IK1 channels and will be inhibited if any one of these IK1 channels opens. This novel, regulated inhibition of maxi-K channels by activation of IK1 adds to the complexity of the properties of these Ca2+-activated K channels and likely contributes to the diversity of their functional roles.

Regulation of high-conductance anion channels by G proteins and 5-HT1A receptors in CHO cells

1993 ◽  
Vol 264 (3) ◽  
pp. F490-F495 ◽  
Author(s):  
A. W. Mangel ◽  
J. R. Raymond ◽  
J. G. Fitz
Keyword(s):  
G Proteins ◽  
Pertussis Toxin ◽  
Cho Cells ◽  
Single Channel ◽  
Chinese Hamster ◽  
Channel Activity ◽  
Anion Channels ◽  
Open Probability ◽  
Single Channel Currents ◽  
High Conductance

This study addresses the mechanisms responsible for regulation of high-conductance anion channels by GTP binding proteins in Chinese hamster ovary (CHO) cells. Single-channel currents were measured in inside-out membrane patches using patch-clamp techniques. Anion-selective channels with a unitary conductance of 381 +/- 8 pS activated spontaneously in 48% of excised patches. In patches with no spontaneous channel activity, addition of GppNHp, a nonhydrolyzable analogue of GTP, activated channels in 8 of 12 studies, and in patches with spontaneous channel activity, GppNHp increased open probability in 4 of 4 experiments. In contrast, GDP beta S, a nonhydrolyzable GDP analogue, inhibited both spontaneous and GppNHp-induced channel activity. In patches without spontaneous channel activity, addition of cholera toxin activated channels in five of eight studies. Interestingly, pertussis toxin had a similar effect, activating channels in five of seven previously quiescent patches. To further evaluate the possible role of inhibitory G proteins in channel regulation, activity was measured in cell-attached patches in cells transfected with the serotonin 5-HT1A receptor, which is coupled to effector mechanisms through a pertussis toxin-sensitive G protein. Stimulation of 5-HT1A-transfected cells with the receptor agonist (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin caused a transient decrease in open probability in either standard or high-potassium solutions. In aggregate, these findings suggest that both cholera and pertussis toxin-sensitive G proteins contribute to regulation of high-conductance anion channels in CHO cells.

Intracellular ADP-ribose inhibits ATP-sensitive K+ channels in rat ventricular myocytes

1996 ◽  
Vol 271 (2) ◽  
pp. C464-C468 ◽  
Author(s):  
Y. G. Kwak ◽  
S. K. Park ◽  
U. H. Kim ◽  
M. K. Han ◽  
J. S. Eun ◽  
...  
Keyword(s):  
Single Channel ◽  
Ventricular Myocytes ◽  
Physiological Role ◽  
Hill Coefficient ◽  
K Channel ◽  
Channel Activity ◽  
Rat Ventricular Myocytes ◽  
Cyclic Adp Ribose ◽  
The Hill

Cyclic ADP-ribose (cADPR), an NAD metabolite, has been shown to be a messenger for Ca2+ mobilization from intracellular Ca2+ stores. However, the physiological role of ADP-ribose (ADPR), another metabolite of NAD, is not known. We examined the effects of cADPR and ADPR on the ATP-sensitive K+ channel (KATP) activity in rat ventricular myocytes by use of the inside-out patch-clamp configuration. ADPR, but not cADPR, inhibited the channel activity at micromolar range with an inhibitor constant (Ki) of 38.4 microM. The Hill coefficient was 0.9. ATP inhibited the K+ channel with a Ki of 77.8 microM, and the Hill coefficient was 1.8. Single-channel conductance was not affected by ADPR. These findings strongly suggest that ADPR may act as a regulator of KATP channel activity.

Modulation of ATP-sensitive K+ channels by internal acidification in insulin-secreting cells

1994 ◽  
Vol 267 (4) ◽  
pp. C1036-C1044 ◽  
Author(s):  
Z. Fan ◽  
Y. Tokuyama ◽  
J. C. Makielski
Keyword(s):  
Insulin Secretion ◽  
Cell Line ◽  
Single Channel ◽  
Inhibitory Effect ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Activation Effect ◽  
Insulin Secreting Cells ◽  
Single Channel Currents

The effect of intracellular acidification (low pHi) on open probability of the ATP-sensitive K+ (KATP) channel was examined in insulin-secretion cells using an inside-out configuration of the patch-clamp technique. In an insulin-secreting cell line beta-TC3, KATP single-channel currents (IKATP) were readily recorded in the absence of internal ATP. ATP (50 microM and 0.5 mM) dramatically decreased the channel activity. A step decrease of intracellular pH (pHi) from 7.4 to 6.7 or 6.3 in the presence of ATP gradually increased the channel activity. In addition, low pHi in the presence of ATP could partially restore channel activity lost in a process called "rundown." Kinetic analysis revealed a change in channel gating at low pHi with ATP. The bursting durations of IKATP at pHi 6.3 in the presence of ATP were significantly longer than those at pHi 7.4 in the absence of ATP. These results suggest that the increased channel activity at low pHi might have resulted from a mechanism involving an alteration of channel conformation. We also observed an inhibitory effect of low pHi on channel activity. However, the inhibitory effect was much more apparent at pHi 5.7 and was only partially reversible. The activation effect of low pHi on IKATP in the presence of ATP was also observed in acutely isolated rat islet cells and in another insulin-secretion cell line RINm5F, although the effect was weaker and was variable among experiments. We conclude that, as in frog skeletal muscle and cardiac muscle, an increase in channel activity at low pHi is one of the mechanisms underlying proton modulation of IKATP in insulin-secreting cells.

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