scholarly journals The long activations of α2 glycine channels can be described by a mechanism with reaction intermediates (“flip”)

2011 ◽  
Vol 137 (2) ◽  
pp. 197-216 ◽  
Author(s):  
Paraskevi Krashia ◽  
Remigijus Lape ◽  
Francesco Lodesani ◽  
David Colquhoun ◽  
Lucia G. Sivilotti
Keyword(s):  
Concentration Dependence ◽  
Binding Sites ◽  
Time Course ◽  
Single Channel ◽  
Glycine Receptor ◽  
Open Probability ◽  
Low Concentrations ◽  
Open Time ◽  
Single Channel Activity ◽  
Entire Sequence

The α2 glycine receptor (GlyR) subunit, abundant in embryonic neurons, is replaced by α1 in the adult nervous system. The single-channel activity of homomeric α2 channels differs from that of α1-containing GlyRs, as even at the lowest glycine concentration (20 µM), openings occurred in long (>300-ms) groups with high open probability (Popen; 0.96; cell-attached recordings, HEK-expressed channels). Shut-time intervals within groups of openings were dominated by short shuttings of 5–10 µs. The lack of concentration dependence in the groups of openings suggests that they represent single activations, separated by very long shut times at low concentrations. Several putative mechanisms were fitted by maximizing the likelihood of the entire sequence of open and shut times, with exact missed-events allowance (program hjcfit). Records obtained at several glycine concentrations were fitted simultaneously. The adequacy of the different schemes was judged by the accuracy with which they predicted not only single-channel data but also the time course and concentration dependence of macroscopic responses elicited by rapid glycine applications to outside-out patches. The data were adequately described only with schemes incorporating a reaction intermediate in the activation, and the best was a flip mechanism with two binding sites and one open state. Fits with this mechanism showed that for α2 channels, the opening rate constant is very fast, ∼130,000 s−1, much as for α1β GlyRs (the receptor in mature synapses), but the estimated true mean open time is 20 times longer (around 3 ms). The efficacy for the flipping step and the binding affinity were lower for α2 than for α1β channels, but the overall efficacies were similar. As we previously showed for α1 homomeric receptors, in α2 glycine channels, maximum Popen is achieved when fewer than all five of the putative binding sites in the pentamer are occupied by glycine.

Differential regulation of voltage-gated K+ channels by oxidized and reduced pyridine nucleotide coenzymes

2005 ◽  
Vol 288 (2) ◽  
pp. C366-C376 ◽  
Author(s):  
Srinivas M. Tipparaju ◽  
Nina Saxena ◽  
Si-Qi Liu ◽  
Rajiv Kumar ◽  
Aruni Bhatnagar
Keyword(s):  
Redox State ◽  
Redox Regulation ◽  
Single Channel ◽  
Pyridine Nucleotide ◽  
Pyridine Nucleotides ◽  
Open Probability ◽  
Kv Channels ◽  
Open Time ◽  
Dependent Inactivation ◽  
Single Channel Activity

The activity of the voltage-sensitive K+ (Kv) channels varies as a function of the intracellular redox state and metabolism, and several Kv channels act as oxygen sensors. However, the mechanisms underlying the metabolic and redox regulation of these channels remain unclear. In this study we investigated the regulation of Kv channels by pyridine nucleotides. Heterologous expression of Kvα1.5 in COS-7 cells led to the appearance of noninactivating currents. Inclusion of 0.1–1 mM NAD+ or 0.03–0.5 mM NADP+ in the internal solution of the patch pipette did not affect Kv currents. However, 0.5 and 1 mM NAD+ and 0.1 and 0.5 mM NADP+ prevented inactivation of Kv currents in cells transfected with Kvα1.5 and Kvβ1.3 and shifted the voltage dependence of activation to depolarized potentials. The Kvβ-dependent inactivation of Kvα currents was also decreased by internal pipette perfusion of the cell with 1 mM NAD+. The Kvα1.5-Kvβ1.3 currents were unaffected by the internal application of 0.1 mM NADPH or 0.1 or 1 mM NADH. Excised inside-out patches from cells expressing Kvα1.5-Kvβ1.3 showed transient single-channel activity. The mean open time and the open probability of these currents were increased by the inclusion of 1 mM NAD+ in the perfusate. These results suggest that NAD(P)+ prevents Kvβ-mediated inactivation of Kv currents and provide a novel mechanism by which pyridine nucleotides could regulate specific K+ currents as a function of the cellular redox state [NAD(P)H-to-NAD(P)+ ratio].

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.

Aldosterone alters the open probability of amiloride-blockable sodium channels in A6 epithelia

1992 ◽  
Vol 263 (4) ◽  
pp. C825-C837 ◽  
Author(s):  
A. E. Kemendy ◽  
T. R. Kleyman ◽  
D. C. Eaton
Keyword(s):  
Sodium Channels ◽  
Time Course ◽  
Single Channel ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Channel Number ◽  
Continuous Exposure ◽  
Open Probability ◽  
Similar Time ◽  
Open Time

We used patch-clamp methods to examine the effects of depletion and readdition of aldosterone on single, highly selective, amiloride-blockable sodium channels in the A6 cell line. Single-channel characteristics changed little before 24 h of continuous aldosterone depletion, although there was some reduction in short-circuit current. Thereafter, apical sodium permeability, measured as product of channel number per patch and individual channel open probability (NPo), was reduced between five- and sevenfold, primarily due to a large decrease in channel mean open time. With about the same time course, short-circuit current also decreased approximately fivefold. Readdition of aldosterone to depleted cells produced an increase in NPo within 2 h, primarily through an increase in mean open time. After readdition, channel number per patch increased twofold compared with cells not hormone deprived, with a return to control levels between 24 and 48 h after continuous exposure. The increase in short-circuit current followed a similar time course. The primary effect of aldosterone appears to be modulation of the open time of channels continuously present in the apical membrane, rather than promotion of the appearance or disappearance of channels from the membrane. In particular, it cannot be demonstrated statistically that aldosterone removal reduces the number of channels per patch, and there may actually be up to a twofold increase after a long period of aldosterone depletion.

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.

Gadolinium Reduces AMPA Receptor Desensitization and Deactivation in Hippocampal Neurons

2001 ◽  
Vol 86 (1) ◽  
pp. 173-182 ◽  
Author(s):  
Saobo Lei ◽  
John F. MacDonald
Keyword(s):  
Steady State ◽  
Ampa Receptor ◽  
Hippocampal Neurons ◽  
Time Course ◽  
Single Channel ◽  
Trivalent Cation ◽  
Receptor Desensitization ◽  
Whole Cell ◽  
Low Concentrations ◽  
Cultured Hippocampal Neurons

The actions of the trivalent cation Gd3+ on whole cell AMPA receptor-mediated currents were studied in isolated hippocampal neurons, in nucleated or outside-out patches taken from cultured hippocampal neurons, and on miniature excitatory postsynaptic currents (mEPSCs) recorded in cultured hippocampal neurons. Glutamate, AMPA, or kainate was employed to activate AMPA receptors. Applications of relatively low concentrations of Gd3+ (0.1–10 μM) substantially enhanced steady-state whole cell glutamate and kainate-evoked currents without altering peak currents, suggesting that desensitization was reduced. However, higher concentrations (>30 μM) depressed steady-state currents, indicating an underlying inhibition of channel activity. Lower concentrations of Gd3+also increased the potency of peak glutamate-evoked currents without altering that of steady-state currents. An ultrafast perfusion system and nucleated patches were then used to better resolve peak glutamate-evoked currents. Low concentrations of Gd3+ reduced peak currents, enhanced steady-state currents, and slowed the onset of desensitization, providing further evidence that this cation reduces desensitization. In the presence of cyclothiazide, a compound that blocks desensitization, a low concentration Gd3+ inhibited both peak and steady-state currents, indicating that Gd3+ both reduces desensitization and inhibits these currents. Gd3+ reduced the probability of channel opening at the peak of the currents but did not alter the single channel conductance calculated using nonstationary variance analysis. Recovery from desensitization was enhanced, and glutamate-evoked current activation and deactivation were slowed by Gd3+. The Gd3+-induced reduction in desensitization did not require the presence of the GluR2 subunit as this effect was seen in hippocampal neurons from GluR2 null-mutant mice. Gd3+ reduced the time course of decay of mEPSCs perhaps as a consequence of its slowing of AMPA receptor deactivation although an increase in the frequency of mEPSCs also suggested enhanced presynaptic release of transmitter. These results demonstrate that Gd3+ potently reduces AMPA receptor desensitization and mimics a number of the properties of the positive modulators of AMPA receptor desensitization such as cyclothiazide.

Characterization of RyR1-slow, a ryanodine receptor specific to slow-twitch skeletal muscle

2000 ◽  
Vol 279 (5) ◽  
pp. R1889-R1898 ◽  
Author(s):  
Jeffery Morrissette ◽  
Le Xu ◽  
Alexandra Nelson ◽  
Gerhard Meissner ◽  
Barbara A. Block
Keyword(s):  
Skeletal Muscle ◽  
Single Channel ◽  
Fiber Type ◽  
Ryanodine Receptors ◽  
Open Probability ◽  
Dependent Inhibition ◽  
Single Channel Activity ◽  
Intracellular Ca ◽  
Slow Twitch ◽  
Fast Twitch

Two distinct skeletal muscle ryanodine receptors (RyR1s) are expressed in a fiber type–specific manner in fish skeletal muscle (11). In this study, we compare [3H]ryanodine binding and single channel activity of RyR1-slow from fish slow-twitch skeletal muscle with RyR1-fast and RyR3 isolated from fast-twitch skeletal muscle. Scatchard plots indicate that RyR1-slow has a lower affinity for [3H]ryanodine when compared with RyR1-fast. In single channel recordings, RyR1-slow and RyR1-fast had similar slope conductances. However, the maximum open probability (Po) of RyR1-slow was threefold less than the maximum Po of RyR1-fast. Single channel studies also revealed the presence of two populations of RyRs in tuna fast-twitch muscle (RyR1-fast and RyR3). RyR3 had the highest Po of all the RyR channels and displayed less inhibition at millimolar Ca2+. The addition of 5 mM Mg-ATP or 2.5 mM β,γ-methyleneadenosine 5′-triphosphate (AMP-PCP) to the channels increased the Po and [3H]ryanodine binding of both RyR1s but also caused a shift in the Ca2+ dependency curve of RyR1-slow such that Ca2+-dependent inactivation was attenuated. [3H]ryanodine binding data also showed that Mg2+-dependent inhibition of RyR1-slow was reduced in the presence of AMP-PCP. These results indicate differences in the physiological properties of RyRs in fish slow- and fast-twitch skeletal muscle, which may contribute to differences in the way intracellular Ca2+ is regulated in these muscle types.

scholarly journals Mechanism of Tacrine Block at Adult Human Muscle Nicotinic Acetylcholine Receptors

2002 ◽  
Vol 120 (3) ◽  
pp. 369-393 ◽  
Author(s):  
Richard J. Prince ◽  
Richard A. Pennington ◽  
Steven M. Sine
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Binding Sites ◽  
Acetylcholine Receptors ◽  
Open Channel ◽  
Single Channel ◽  
Dependent Manner ◽  
Open Probability ◽  
Sequential Model ◽  
Hek 293 ◽  
Voltage Dependent

We used single-channel kinetic analysis to study the inhibitory effects of tacrine on human adult nicotinic receptors (nAChRs) transiently expressed in HEK 293 cells. Single channel recording from cell-attached patches revealed concentration- and voltage-dependent decreases in mean channel open probability produced by tacrine (IC50 4.6 μM at −70 mV, 1.6 μM at −150 mV). Two main effects of tacrine were apparent in the open- and closed-time distributions. First, the mean channel open time decreased with increasing tacrine concentration in a voltage-dependent manner, strongly suggesting that tacrine acts as an open-channel blocker. Second, tacrine produced a new class of closings whose duration increased with increasing tacrine concentration. Concentration dependence of closed-times is not predicted by sequential models of channel block, suggesting that tacrine blocks the nAChR by an unusual mechanism. To probe tacrine's mechanism of action we fitted a series of kinetic models to our data using maximum likelihood techniques. Models incorporating two tacrine binding sites in the open receptor channel gave dramatically improved fits to our data compared with the classic sequential model, which contains one site. Improved fits relative to the sequential model were also obtained with schemes incorporating a binding site in the closed channel, but only if it is assumed that the channel cannot gate with tacrine bound. Overall, the best description of our data was obtained with a model that combined two binding sites in the open channel with a single site in the closed state of the receptor.

scholarly journals Potassium channel types in arterial chemoreceptor cells and their selective modulation by oxygen.

1992 ◽  
Vol 100 (3) ◽  
pp. 401-426 ◽  
Author(s):  
M D Ganfornina ◽  
J López-Barneo
Keyword(s):  
Time Course ◽  
Single Channel ◽  
K Channel ◽  
Open Probability ◽  
K Channels ◽  
Glomus Cells ◽  
Control Value ◽  
Voltage Dependent ◽  
K Current ◽  
Chemoreceptor Cells

Single K+ channel currents were recorded in excised membrane patches from dispersed chemoreceptor cells of the rabbit carotid body under conditions that abolish current flow through Na+ and Ca2+ channels. We have found three classes of voltage-gated K+ channels that differ in their single-channel conductance (gamma), dependence on internal Ca2+ (Ca2+i), and sensitivity to changes in O2 tension (PO2). Ca(2+)-activated K+ channels (KCa channels) with gamma approximately 210 pS in symmetrical K+ solutions were observed when [Ca2+]i was greater than 0.1 microM. Small conductance channels with gamma = 16 pS were not affected by [Ca2+]i and they exhibited slow activation and inactivation time courses. In these two channel types open probability (P(open)) was unaffected when exposed to normoxic (PO2 = 140 mmHg) or hypoxic (PO2 approximately 5-10 mmHg) external solutions. A third channel type (referred to as KO2 channel), having an intermediate gamma(approximately 40 pS), was the most frequently recorded. KO2 channels are steeply voltage dependent and not affected by [Ca2+]i, they inactivate almost completely in less than 500 ms, and their P(open) reversibly decreases upon exposure to low PO2. The effect of low PO2 is voltage dependent, being more pronounced at moderately depolarized voltages. At 0 mV, for example, P(open) diminishes to approximately 40% of the control value. The time course of ensemble current averages of KO2 channels is remarkably similar to that of the O2-sensitive K+ current. In addition, ensemble average and macroscopic K+ currents are affected similarly by low PO2. These observations strongly suggest that KO2 channels are the main contributors to the macroscopic K+ current of glomus cells. The reversible inhibition of KO2 channel activity by low PO2 does not desensitize and is not related to the presence of F-, ATP, and GTP-gamma-S at the internal face of the membrane. These results indicate that KO2 channels confer upon glomus cells their unique chemoreceptor properties and that the O2-K+ channel interaction occurs either directly or through an O2 sensor intrinsic to the plasma membrane closely associated with the channel molecule.

Single-channel basis for conductance increase induced by isoflurane in Shaker H4 IR K+ channels

2001 ◽  
Vol 280 (5) ◽  
pp. C1130-C1139 ◽  
Author(s):  
Jichang Li ◽  
Ana M. Correa
Keyword(s):  
Time Course ◽  
Xenopus Oocytes ◽  
Single Channel ◽  
Noise Analysis ◽  
Single Channels ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Open Probability ◽  
Slowing Down ◽  
Conductance Increase

Volatile anesthetics modulate the function of various K+ channels. We previously reported that isoflurane induces an increase in macroscopic currents and a slowing down of current deactivation of Shaker H4 IR K+ channels. To understand the single-channel basis of these effects, we performed nonstationary noise analysis of macroscopic currents and analysis of single channels in patches from Xenopus oocytes expressing Shaker H4 IR. Isoflurane (1.2% and 2.5%) induced concentration-dependent, partially reversible increases in macroscopic currents and in the time course of tail currents. Noise analysis of currents (70 mV) revealed an increase in unitary current (∼17%) and maximum open probability (∼20%). Single-channel conductance was larger (∼20%), and opening events were more stable, in isoflurane. Tail-current slow time constants increased by 41% and 136% in 1.2% and 2.5% isoflurane, respectively. Our results show that, in a manner consistent with stabilization of the open state, isoflurane increased the macroscopic conductance of Shaker H4 IR K+ channels by increasing the single-channel conductance and the open probability.

scholarly journals Gating of Recombinant Small-Conductance Ca-activated K+ Channels by Calcium

1998 ◽  
Vol 111 (4) ◽  
pp. 565-581 ◽  
Author(s):  
Birgit Hirschberg ◽  
James Maylie ◽  
John P. Adelman ◽  
Neil V. Marrion
Keyword(s):  
Time Course ◽  
Single Channel ◽  
Cell Types ◽  
K Channel ◽  
Open Probability ◽  
K Channels ◽  
Sensitive Clone ◽  
Single Channel Currents ◽  
Open States ◽  
Small Conductance

Small-conductance Ca-activated K+ channels play an important role in modulating excitability in many cell types. These channels are activated by submicromolar concentrations of intracellular Ca2+, but little is known about the gating kinetics upon activation by Ca2+. In this study, single channel currents were recorded from Xenopus oocytes expressing the apamin-sensitive clone rSK2. Channel activity was detectable in 0.2 μM Ca2+ and was maximal above 2 μM Ca2+. Analysis of stationary currents revealed two open times and three closed times, with only the longest closed time being Ca dependent, decreasing with increasing Ca2+ concentrations. In addition, elevated Ca2+ concentrations resulted in a larger percentage of long openings and short closures. Membrane voltage did not have significant effects on either open or closed times. The open probability was ∼0.6 in 1 μM free Ca2+. A lower open probability of ∼0.05 in 1 μM Ca2+ was also observed, and channels switched spontaneously between behaviors. The occurrence of these switches and the amount of time channels spent displaying high open probability behavior was Ca2+ dependent. The two behaviors shared many features including the open times and the short and intermediate closed times, but the low open probability behavior was characterized by a different, long Ca2+-dependent closed time in the range of hundreds of milliseconds to seconds. Small-conductance Ca- activated K+ channel gating was modeled by a gating scheme consisting of four closed and two open states. This model yielded a close representation of the single channel data and predicted a macroscopic activation time course similar to that observed upon fast application of Ca2+ to excised inside-out patches.

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