scholarly journals Mutations within the P-Loop of Kir6.2 Modulate the Intraburst Kinetics of the Atp-Sensitive Potassium Channel

2001 ◽  
Vol 118 (4) ◽  
pp. 341-353 ◽  
Author(s):  
Peter Proks ◽  
Charlotte E. Capener ◽  
Phillippa Jones ◽  
Frances M. Ashcroft
Keyword(s):  
Katp Channel ◽  
Single Channel ◽  
Katp Channels ◽  
Burst Duration ◽  
Open Probability ◽  
Closed Intervals ◽  
Open Time ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Kinetics Of

The ATP-sensitive potassium (KATP) channel exhibits spontaneous bursts of rapid openings, which are separated by long closed intervals. Previous studies have shown that mutations at the internal mouth of the pore-forming (Kir6.2) subunit of this channel affect the burst duration and the long interburst closings, but do not alter the fast intraburst kinetics. In this study, we have investigated the nature of the intraburst kinetics by using recombinant Kir6.2/SUR1 KATP channels heterologously expressed in Xenopus oocytes. Single-channel currents were studied in inside-out membrane patches. Mutations within the pore loop of Kir6.2 (V127T, G135F, and M137C) dramatically affected the mean open time (τo) and the short closed time (τC1) within a burst, and the number of openings per burst, but did not alter the burst duration, the interburst closed time, or the channel open probability. Thus, the V127T and M137C mutations produced longer τo, shorter τC1, and fewer openings per burst, whereas the G135F mutation had the opposite effect. All three mutations also reduced the single-channel conductance: from 70 pS for the wild-type channel to 62 pS (G135F), 50 pS (M137C), and 38 pS (V127T). These results are consistent with the idea that the KATP channel possesses a gate that governs the intraburst kinetics, which lies close to the selectivity filter. This gate appears to be able to operate independently of that which regulates the long interburst closings.

scholarly journals How ATP Inhibits the Open KATP Channel

2008 ◽  
Vol 132 (1) ◽  
pp. 131-144 ◽  
Author(s):  
Tim J. Craig ◽  
Frances M. Ashcroft ◽  
Peter Proks
Keyword(s):  
Binding Site ◽  
Single Channel ◽  
Katp Channels ◽  
Burst Duration ◽  
Atp Binding ◽  
Atp Binding Site ◽  
Open Time ◽  
Mechanism Of Interaction ◽  
The Mean ◽  
Kinetics Of

ATP-sensitive potassium (KATP) channels are composed of four pore-forming Kir6.2 subunits and four regulatory SUR1 subunits. Binding of ATP to Kir6.2 leads to inhibition of channel activity. Because there are four subunits and thus four ATP-binding sites, four binding events are possible. ATP binds to both the open and closed states of the channel and produces a decrease in the mean open time, a reduction in the mean burst duration, and an increase in the frequency and duration of the interburst closed states. Here, we investigate the mechanism of interaction of ATP with the open state of the channel by analyzing the single-channel kinetics of concatenated Kir6.2 tetramers containing from zero to four mutated Kir6.2 subunits that possess an impaired ATP-binding site. We show that the ATP-dependent decrease in the mean burst duration is well described by a Monod-Wyman-Changeux model in which channel closing is produced by all four subunits acting in a single concerted step. The data are inconsistent with a Hodgkin-Huxley model (four independent steps) or a dimer model (two independent dimers). When the channel is open, ATP binds to a single ATP-binding site with a dissociation constant of 300 μM.

Potassium channels activated by GABAB agonists and serotonin in cultured hippocampal neurons

1994 ◽  
Vol 71 (6) ◽  
pp. 2570-2575 ◽  
Author(s):  
L. S. Premkumar ◽  
P. W. Gage
Keyword(s):  
Potassium Channels ◽  
Hippocampal Neurons ◽  
Single Channel ◽  
Gamma Aminobutyric Acid ◽  
Kinetic Behavior ◽  
Open Time ◽  
Voltage Dependent ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Cultured Hippocampal Neurons

1. Single-channel currents were recorded in cell-attached patches on cultured hippocampal neurons in response to gamma-aminobutyric acid-B (GABAB) agonists or serotonin applied to the cell surface outside the patch area. 2. The channels activated by GABAB agonists and serotonin were potassium selective but had a different conductance and kinetic behavior. Channels activated by GABAB agonists had a higher conductance, longer open-time, and longer burst-length than channels activated by serotonin. 3. The kinetic behavior of channels activated by GABAB agonists varied with potential whereas channels activated by serotonin did not show voltage-dependent changes in kinetics. 4. In a few cell-attached patches, both types of channel were activated when the cell was exposed to GABA together with serotonin. 5. It was concluded that GABAB agonists and serotonin activate different potassium channels in the soma of cultured hippocampal neurons.

Regulation of ATP-sensitive K+ channels by ATP and nucleotide diphosphate in rabbit portal vein

1994 ◽  
Vol 266 (5) ◽  
pp. H1687-H1698 ◽  
Author(s):  
M. Kamouchi ◽  
K. Kitamura
Keyword(s):  
Portal Vein ◽  
Katp Channel ◽  
Single Channel ◽  
Katp Channels ◽  
K Channel ◽  
Channel Activity ◽  
Rabbit Portal Vein ◽  
Internal Side ◽  
The Right ◽  
Single Channel Currents

The modulation of ATP-sensitive K+ (KATP)-channel activity was investigated by recording single-channel currents in isolated smooth muscle cells from rabbit portal vein. K(+)-channel openers (KCOs; pinacidil, lemakalim, and nicorandil) induced burstlike openings of single KATP channels in the cell-attached configuration. After patch excision, KATP channels showed "run-down" phenomenon in the presence of KCOs, but subsequent application of Mg-ATP (1 mM) restored KATP-channel activity. Removal of Mg-ATP resulted in transient augmentation of KATP currents, which eventually decayed out. Nucleotide diphosphates (NDPs; GDP, ADP, UDP, IDP, and CDP) also induced channel reopening in the presence of KCOs, which was markedly enhanced by addition of Mg2+ in millimolar concentrations at the internal side of the membrane. The dose-response relation between ATP and the UDP-induced KATP-channel activity was shifted to the right in the presence of Mg2+ (2 mM). These results suggest that intracellular ATP, NDPs, and Mg2+ regulate the channel state of KATP channels (operative and inoperative states) and that KCOs open KATP channels only in the operative state.

Blockade of Adenosine Triphosphate–sensitive Potassium Channels by Thiamylal in Rat Ventricular Myocytes

2000 ◽  
Vol 92 (4) ◽  
pp. 1154-1159 ◽  
Author(s):  
Yasuo Tsutsumi ◽  
Shuzo Oshita ◽  
Hiroshi Kitahata ◽  
Yasuhiro Kuroda ◽  
Takashi Kawano ◽  
...  
Keyword(s):  
Adenosine Triphosphate ◽  
Katp Channel ◽  
Single Channel ◽  
Ventricular Myocytes ◽  
Katp Channels ◽  
Dependent Manner ◽  
Open Probability ◽  
Rat Ventricular Myocytes ◽  
Inside Out ◽  
Cell Attached Configuration

Background The adenosine triphosphate (ATP)-sensitive potassium (KATP) channels protect myocytes during ischemia and reperfusion. This study investigated the effects of thiamylal on the activities of KATP channels in isolated rat ventricular myocytes during simulated ischemia. Methods Male Wistar rats were anesthetized with ether. Single, quiescent ventricular myocytes were dispersed enzymatically. Membrane currents were recorded using patch-clamp techniques. In the cell-attached configuration, KATP channel currents were assessed before and during activation of these channels by 2,4-dinitrophenol and after administration of 25, 50, and 100 mg/l thiamylal. The open probability was determined from current-amplitude histograms. In the inside-out configuration, the current-voltage relation was obtained before and after the application of thiamylal (50 mg/1). Results In the cell-attached configuration, 2,4-dinitrophenol caused frequent channel opening. 2,4-Dinitrophenol-induced channel activities were reduced significantly by glibenclamide, suggesting that the channels studied were KATP channels. Open probability of KATP channels was reduced by thiamylal in a concentration-dependent manner. KATP channels could be activated in the inside-out configuration because of the absence of ATP. Thiamylal inhibited KATP channel activity without changing the single-channel conductance. Conclusions The results obtained in this study indicate that thiamylal inhibits KATP channel activities in cell-attached and inside-out patches, suggesting a direct action of this drug on these channels.

Dihydropyridine-sensitive calcium channels expressed in canine colonic smooth muscle cells

1993 ◽  
Vol 264 (3) ◽  
pp. C745-C754 ◽  
Author(s):  
A. Rich ◽  
J. L. Kenyon ◽  
J. R. Hume ◽  
K. Overturf ◽  
B. Horowitz ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Calcium Channels ◽  
Smooth Muscle Cells ◽  
Calcium Current ◽  
Single Channel ◽  
Muscle Cells ◽  
Open Probability ◽  
Boltzmann Function ◽  
Single Channel Currents ◽  
Channel Currents

Experiments were performed to identify and characterize the types of calcium channels that regulate inward calcium current in canine colonic smooth muscle. Freshly dispersed smooth muscle cells from the circular layer of the canine proximal colon were used. Single-channel currents were measured with 80 mM Ba2+ as the charge carrier. Small-conductance (10 +/- 2 pS, EBa = 46 +/- 11 mV, n = 9) and large-conductance (21 +/- 1 pS, EBa = 52 +/- 3 mV, n = 19) single-channel currents were observed during depolarizing voltage steps positive to -30 mV. Both types of single-channel currents were inhibited by the addition of 10(-6) M nifedipine to the bath solution. The smaller current was infrequently observed and therefore was not further characterized. Open probability (P(o)) of the larger current amplitude was strongly dependent on voltage. Activation curves were well described by a Boltzmann function with half activation occurring at 4 mV, and a 5-mV increase in membrane potential resulted in an e-fold increase in P(o). BAY K 8644 (1 microM) shifted the activation curve to the left while nifedipine (1 microM) resulted in a right shift. Molecular analysis showed that only the C class of Ca2+ channel alpha 1-subunit is expressed in this tissue. Furthermore, only a single splice variant (rbc-II) was observed. The results suggest that a single class of dihydropyridine-sensitive calcium channels regulates inward calcium current in canine colonic smooth muscle cells.

Regulation of low-amiloride-affinity sodium channels in alveolar type II cells

1994 ◽  
Vol 267 (1) ◽  
pp. L94-L100 ◽  
Author(s):  
G. Yue ◽  
R. L. Shoemaker ◽  
S. Matalon
Keyword(s):  
Single Channel ◽  
Alveolar Type ◽  
Type Ii ◽  
Channel Conductance ◽  
Open Probability ◽  
Pipette Solution ◽  
Ionic Substitution ◽  
Beta Agonists ◽  
Single Channel Currents ◽  
Channel Currents

We determined the mechanisms by which beta-agonists increase sodium (Na+) currents across rat alveolar type II (ATII) cells grown in primary culture. When ATII cells were patched in the cell-attached mode using symmetrical Na+ solutions (150 mM Na(+)-glutamate), single-channel currents were observed for holding potentials between -80 and 30 mV (referenced to the pipette solution) with a single-channel conductance of 27 +/- 3 pS, a mean open time (tau 1) of 3.3 +/- 0.15 ms and an open probability (Po) of 0.36 +/- 0.06 (n = 7). Addition of 10 microM terbutaline into the bath increased tau 1 to 6.43 +/- 0.5 ms and Po to 0.62 +/- 0.06 (n = 7) without affecting channel conductance. Single-channel currents with a conductance of 25 +/- 2 pS were also recorded across ATII cells patched in the inside-out mode. Addition of 250 U/ml of protein kinase A (PKA), 1 mM ATP, and 5 mM MgCl2 in the bath solution (150 mM Na(+)-glutamate) increased the single channel tau 1 from 3.26 +/- 0.15 to 7.38 +/- 0.38 and Po from 0.41 +/- 0.06 to 0.72 +/- 0.07 (n = 6) without altering conductance. Addition of 1 microM amiloride or ethylisopropylamiloride (EIPA) in the pipette solution (150 mM Na(+)-glutamate) blocked single-channel activity almost completely. Ionic substitution experiments showed the relative permeability of Na+ to K+ and Na+ to Cl- to be 7:1 and 8:1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

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 Tail End of the S6 Segment

2002 ◽  
Vol 120 (1) ◽  
pp. 87-97 ◽  
Author(s):  
Shinghua Ding ◽  
Richard Horn
Keyword(s):  
Potassium Channels ◽  
Energy Barrier ◽  
Single Channel ◽  
Noise Analysis ◽  
Selectivity Filter ◽  
Open Probability ◽  
Cytoplasmic Extension ◽  
Activation Gate ◽  
Single Channel Currents ◽  
Channel Currents

The permeation pathway in voltage-gated potassium channels has narrow constrictions at both the extracellular and intracellular ends. These constrictions might limit the flux of cations from one side of the membrane to the other. The extracellular constriction is the selectivity filter, whereas the intracellular bundle crossing is proposed to act as the activation gate that opens in response to a depolarization. This four-helix bundle crossing is composed of S6 transmembrane segments, one contributed by each subunit. Here, we explore the cytoplasmic extension of the S6 transmembrane segment of Shaker potassium channels, just downstream from the bundle crossing. We substituted cysteine for each residue from N482 to T489 and determined the amplitudes of single channel currents and maximum open probability (Po,max) at depolarized voltages using nonstationary noise analysis. One mutant, F484C, significantly reduces Po,max, whereas Y483C, F484C, and most notably Y485C, reduce single channel conductance (γ). Mutations of residue Y485 have no effect on the Rb+/K+ selectivity, suggesting a local effect on γ rather than an allosteric effect on the selectivity filter. Y485 mutations also reduce pore block by tetrabutylammonium, apparently by increasing the energy barrier for blocker movement through the open activation gate. Replacing Rb+ ions for K+ ions reduces the amplitude of single channel currents and makes γ insensitive to mutations of Y485. These results suggest that Rb+ ions increase an extracellular energy barrier, presumably at the selectivity filter, thus making it rate limiting for flux of permeant ions. These results indicate that S6T residues have an influence on the conformation of the open activation gate, reflected in both the stability of the open state and the energy barriers it presents to ions.

scholarly journals Inhibitors of asparagine-linked oligosaccharide processing alter the kinetics of the nicotinic acetylcholine receptor.

1989 ◽  
Vol 93 (5) ◽  
pp. 765-783 ◽  
Author(s):  
M Covarrubias ◽  
C Kopta ◽  
J H Steinbach
Keyword(s):  
Single Channel ◽  
Response Curves ◽  
Nicotinic Acetylcholine ◽  
Low Concentrations ◽  
Oligosaccharide Processing ◽  
Processing Step ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Kinetics Of ◽  
Ach Receptors

We used selective inhibitors of the asparagine-linked oligosaccharide processing pathway to study the effect of sugar trimming on the functional properties of the nicotinic acetylcholine (ACh) receptor expressed in clonal mammalian BC3H-1 cells. Inhibitors of initial steps of the processing pathway (1-deoxynojirimycin[DNJ] and castanospermine[CS]) reduced the density of ACh receptors on the cell surface (3- to 5-fold) but their responsiveness to ACh was more reduced (5- to 10-fold). These results suggest that the function of the ACh receptor was altered. When the ACh receptors were expressed in the presence of DNJ or CS, analysis of ACh-evoked single-channel currents (-100 mV and 11 degrees C) revealed an approximate threefold reduction in the opening rate (control: 600-650 s(-1)), treated: 130-250 s(-1)) and an approximate twofold reduction in the rate of agonist dissociation (control: 900-1,000 s(-1), treated: 400-500 s(-1)). In addition, the proportion of brief duration bursts (tau = 50-100 microseconds) was increased (1.5- to 2-fold) by treatments with DNJ or CS. In contrast, an inhibitor of a late processing step (swainsonine) did not produce such alterations. The single-channel conductance was not altered by any of the three inhibitors, and the slopes of log-log dose-response curves at low concentrations and desensitization did not appear to be affected. Each inhibitor altered the electrophoretic mobility of the ACh receptor subunits. We conclude that early sugar trimming can influence the kinetics of the nicotinic ACh receptor in BC3H-1 cells.

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