Multiple Mechanisms of Ketamine Blockade of N-methyl-D-aspartate Receptors 

1997 ◽  
Vol 86 (4) ◽  
pp. 903-917 ◽  
Author(s):  
Beverley A. Orser ◽  
Peter S. Pennefather ◽  
John F. MacDonald
Keyword(s):  
Hippocampal Neurons ◽  
Open Channel ◽  
Single Channel ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Open Time ◽  
Channel Opening ◽  
Single Channel Currents ◽  
A Site ◽  
Change In Mean

Background The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor is blocked by ketamine, and this action likely contributes to ketamine's anesthetic and analgesic properties. Previous studies suggest that ketamine occludes the open channel by binding to a site located within the channel pore. This hypothesis was examined by investigating the effects of ketamine on single-channel currents from NMDA receptors. Methods The cell-attached and outside-out configurations of the patch clamp technique were used to study NMDA-activated currents recorded from cultured mouse hippocampal neurons. Results In cell-attached patches, NMDA evoked currents that had an apparent mean open time (tau o) of 3.26 ms. The probability of at least one channel being open (Po') was 0.058. The addition of ketamine (0.1 microM or 1 microM) to the pipette solution decreased Po' to 53% and 24% of control values, respectively. At 1 microM ketamine, this reduction was due to a decrease in both the frequency of channel opening and the mean open time (44% and 68% of control values, respectively). Ketamine did not influence channel conductance and no new components were required to fit the open- or closed-duration distributions. Ketamine (50 microM), applied outside the recording pipette, reduced the opening frequency of channels recorded in the cell attached configuration. This observation suggests that ketamine gained access to a binding site by diffusing across the hydrophobic cell membrane. In outside-out patches, ketamine potency was lower than that observed in cell-attached patches: 1 microM and 10 microM ketamine reduced Po' to 63% and 34% of control values, respectively, and this reduction was due primarily to a decrease in the frequency of channel opening with little change in mean open time. Conclusions These observations are consistent with a model whereby ketamine inhibits the NMDA receptor by two distinct mechanisms: (1) Ketamine blocks the open channel and thereby reduces channel mean open time, and (2) ketamine decreases the frequency of channel opening by an allosteric mechanism.

Single voltage-dependent potassium channels in cultured rat hippocampal neurons

1986 ◽  
Vol 56 (2) ◽  
pp. 481-493 ◽  
Author(s):  
M. A. Rogawski
Keyword(s):  
Hippocampal Neurons ◽  
Single Channel ◽  
Reversal Potential ◽  
Resting Potential ◽  
Moderate Degree ◽  
Patch Clamp Technique ◽  
Membrane Pore ◽  
Channel Opening ◽  
Voltage Dependent ◽  
Single Channel Currents

Single-channel recordings using the gigohm seal patch-clamp technique were carried out on the somatic membranes of dissociated embryonic rat hippocampal neurons grown in cell culture. The recording medium contained tetrodotoxin to block the voltage-dependent Na+ conductance and Cd2+ to block Ca2+ and Ca2+-activated conductances. In the cell-attached configuration, depolarizing voltage steps activated outward directed single-channel currents with conductance 15-20 pS. The channel openings exhibited a moderate degree of flickering. The mean burst lifetimes ranged from 5 to 13 ms with a tendency to increase slightly at more depolarized potentials (T = 21-25 degrees C). Reversal potential measurements using excised membrane patches indicated that the channels behaved as expected of a K+-selective membrane pore. Channel opening occurred in Ca2+-free EGTA-containing solutions but was never observed in the presence of tetraethylammonium (TEA; 20 mM). The frequency of channel opening increased as the membrane was depolarized by up to 50 mV from resting potential; the fraction of time spent in the open state during the first 300 ms following a step depolarization increased e-fold for a 8-25 mV change in potential. First-latency histograms and simulations of the macroscopic current based on channel data obtained during repeated depolarizing voltage steps indicated that the probability of the channel being in the open state increases gradually with time after a step depolarization. During repeated depolarizing steps the channels appeared to randomly enter and exit a long-lived inactive state. It is concluded that these channels may underly the slowly activating, very slowly inactivating, TEA-sensitive voltage-dependent K+ current (IK) in cultured hippocampal neurons.

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.

scholarly journals On the Mechanism by which 4-Aminopyridine Occludes Quinidine Block of the Cardiac K+ Channel, hKv1.5

1998 ◽  
Vol 111 (4) ◽  
pp. 539-554 ◽  
Author(s):  
Fred S.P. Chen ◽  
David Fedida
Keyword(s):  
Open Channel ◽  
Channel Blocker ◽  
Single Channel ◽  
K Channel ◽  
Gating Currents ◽  
Channel Activation ◽  
Gating Charge ◽  
Channel Opening ◽  
Conformational Rearrangements ◽  
A Site

4-Aminopyridine (4-AP) binds to potassium channels at a site or sites in the inner mouth of the pore and is thought to prevent channel opening. The return of hKv1.5 off-gating charge upon repolarization is accelerated by 4-AP and it has been suggested that 4-AP blocks slow conformational rearrangements during late closed states that are necessary for channel opening. On the other hand, quinidine, an open channel blocker, slows the return or immobilizes off-gating charge only at opening potentials (>−25 mV). The aim of this study was to use quini-dine as a probe of open channels to test the kinetic state of 4-AP-blocked channels. In the presence of 0.2–1 mM 4-AP, quinidine slowed charge return and caused partial charge immobilization, corresponding to an increase in the Kd of ∼20-fold. Peak off-gating currents were reduced and decay was slowed ∼2- to 2.5-fold at potentials negative to the threshold of channel activation and during depolarizations shorter than normally required for channel activation. This demonstrated access of quinidine to 4-AP-blocked channels, a lack of competition between the two drugs, and implied allosteric modulation of the quinidine binding site by 4-AP resident within the channel. Single channel recordings also showed that quinidine could modulate the 4-AP-induced closure of the channels, with the result that frequent channel reopenings were observed when both drugs were present. We propose that 4-AP-blocked channels exist in a partially open, nonconducting state that allows access to quinidine, even at more negative potentials and during shorter depolarizations than those required for channel activation.

scholarly journals Gating of Acid-sensitive Ion Channel-1: Release of Ca2+ Block vs. Allosteric Mechanism

2006 ◽  
Vol 127 (2) ◽  
pp. 109-117 ◽  
Author(s):  
Ping Zhang ◽  
Fred J. Sigworth ◽  
Cecilia M. Canessa
Keyword(s):  
Open Channel ◽  
Channel Blocker ◽  
Single Channel ◽  
Allosteric Mechanism ◽  
Open Time ◽  
Channel Opening ◽  
Voltage Dependent ◽  
High Affinity Binding Site ◽  
The Mean ◽  
High Concentrations

The acid-sensitive ion channels (ASICs) are a family of voltage-insensitive sodium channels activated by external protons. A previous study proposed that the mechanism underlying activation of ASIC consists of the removal of a Ca2+ ion from the channel pore (Immke and McCleskey, 2003). In this work we have revisited this issue by examining single channel recordings of ASIC1 from toadfish (fASIC1). We demonstrate that increases in the concentration of external protons or decreases in the concentration of external Ca2+ activate fASIC1 by progressively opening more channels and by increasing the rate of channel opening. Both maneuvers produced similar effects in channel kinetics, consistent with the former notion that protons displace a Ca2+ ion from a high-affinity binding site. However, we did not observe any of the predictions expected from the release of an open-channel blocker: decrease in the amplitude of the unitary currents, shortening of the mean open time, or a constant delay for the first opening when the concentration of external Ca2+ was decreased. Together, the results favor changes in allosteric conformations rather than unblocking of the pore as the mechanism gating fASIC1. At high concentrations, Ca2+ has an additional effect that consists of voltage-dependent decrease in the amplitude of unitary currents (EC50 of 10 mM at −60 mV and pH 6.0). This phenomenon is consistent with voltage-dependent block of the pore but it occurs at concentrations much higher than those required for gating.

The LQT-associated calmodulin mutant E141G induces disturbed Ca2+-dependent binding and a flickering gating mode of the CaV1.2 channel

2020 ◽  
Vol 318 (5) ◽  
pp. C991-C1004
Author(s):  
Jingyang Su ◽  
Qinghua Gao ◽  
Lifeng Yu ◽  
Xuanxuan Sun ◽  
Rui Feng ◽  
...  
Keyword(s):  
Single Channel ◽  
Missense Variant ◽  
Inhibitory Effect ◽  
Channel Activity ◽  
Peptide Fragments ◽  
Patch Clamp Technique ◽  
Open Time ◽  
Channel Opening ◽  
Lqt Syndrome ◽  
Calmodulin Mutant

Calmodulin (CaM) mutations are associated with congenital long QT (LQT) syndrome (LQTS), which may be related to the dysregulation of the cardiac-predominant Ca2+ channel isoform CaV1.2. Among various mutants, CaM-E141G was identified as a critical missense variant. However, the interaction of this CaM mutant with the CaV1.2 channel has not been determined. In this study, by utilizing a semiquantitative pull-down assay, we explored the interaction of CaM-E141G with CaM-binding peptide fragments of the CaV1.2 channel. Using the patch-clamp technique, we also investigated the electrophysiological effects of the mutant on CaV1.2 channel activity. We found that the maximum binding (Bmax) of CaM-E141G to the proximal COOH-terminal region, PreIQ-IQ, PreIQ, IQ, and NT (an NH2-terminal peptide) was decreased (by 17.71–59.26%) compared with that of wild-type CaM (CaM-WT). In particular, the Ca2+-dependent increase in Bmax became slower with the combination of CaM-E141G + PreIQ and IQ but faster in the case of NT. Functionally, CaM-WT and CaM-E141G at 500 nM Ca2+ decreased CaV1.2 channel activity to 24.88% and 55.99%, respectively, compared with 100 nM Ca2+, showing that the inhibitory effect was attenuated in CaM-E141G. The mean open time of the CaV1.2 channel was increased, and the number of blank traces with no channel opening was significantly decreased. Overall, CaM-E141G exhibits disrupted binding with the CaV1.2 channel and induces a flickering gating mode, which may result in the dysfunction of the CaV1.2 channel and, thus, the development of LQTS. The present study is the first to investigate the detailed binding properties and single-channel gating mode induced by the interaction of CaM-E141G with the CaV1.2 channel.

Oxantel-activated single channel currents in the muscle membrane of Ascaris suum

Parasitology ◽  
1995 ◽  
Vol 110 (4) ◽  
pp. 437-448 ◽  
Author(s):  
V. M. E. Dale ◽  
R. J. Martin
Keyword(s):  
Single Channel ◽  
Ascaris Suum ◽  
Muscle Membrane ◽  
Kinetic Properties ◽  
Voltage Sensitivity ◽  
Channel Block ◽  
Patch Clamp Technique ◽  
Open Time ◽  
Single Channel Currents ◽  
Channel Currents

SUMMARYThe patch clamp technique was used to investigate the action of the anthelmintic drug, oxantel, on nicotinic acetylcholine receptor (nAChR) currents recorded from vesicles of the somatic muscle cells of the nematode parasite Ascaris suum. The amplitudes of the currents were analysed at different membrane potentials to determine the single channel conductance. Also the open and closed durations were measured to determine the kinetic properties of the activated channel. Oxantel activated single nAChR currents throughout a concentration range 10–100 μM, these currents were not observed with oxantel-free pipette solutions. The mean open time of the activated channels at a membrane potential of –75 mV and a concentration of 10 μM was 1·34 ms. At higher concentrations the open times were shorter and voltage sensitive, decreasing in duration on hyperpolarization, thus suggesting open channel block. The kinetics were analysed using a simple channel block model. The forward block rate, K + B, increased with increasing oxantel concentration but showed little increase as the membrane was hyperpolarized. K + B was 2·41×107 M−1s−1 – 50 mV and 2·64 × 107 M−1s−1 at – 100mV. The unblocking rate constant, K – B, did exhibit voltage sensitivity being 443·6 s−1 at – 50 mV and 86·8 s−1 at –100 mV. Thus the blocking dissociation constant KB (= K – B/K + B) was 18·5 μM at –50 mV and 3·3 μM at –100 mV. The simple channel block scheme was found to be insufficient to explain fully the observations made; reasons for this are discussed.

Whole cell and single channel properties of a new GABA receptor transiently expressed in the Hippocampus

1995 ◽  
Vol 73 (2) ◽  
pp. 902-906 ◽  
Author(s):  
M. Martina ◽  
F. Strata ◽  
E. Cherubini
Keyword(s):  
Postnatal Development ◽  
Hippocampal Neurons ◽  
Single Channel ◽  
Gamma Aminobutyric Acid ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Slow Kinetics ◽  
Inward Currents ◽  
Single Channel Currents ◽  
Channel Properties

1. The patch-clamp technique was used to characterize, in acutely dissociated CA3 rat hippocampal neurons, the whole cell and single channel properties of a novel response to gamma-aminobutyric acid (GABA) present only during a restricted period of postnatal development. 2. At postnatal days 0-10 (P0-P10), both GABA (100 microM) and isoguvacine (50 microM) evoked at a holding potential of -50 mV, in symmetrical chloride solution, whole cell inward currents. Bicuculline blocked the response to isoguvacine but only reduced the response to GABA (from 512 +/- 137 pA to 60 +/- 13 pA, mean +/- SE). After P12, bicuculline abolished the response to GABA. 3. The bicuculline-insensitive GABA currents were Cl- mediated and antagonized by picrotoxin. The desensitization rate was slower than the conventional bicuculline-sensitive response. The peak to plateau ratio induced by 0.1 or 1 mM of GABA shifted from 4.6 +/- 0.4 and 17.7 +/- 2.6 to 1.5 +/- 0.1 and 3.1 +/- 0.5 in the absence or in the presence of bicuculline, respectively. The recovery from desensitization was significantly faster for the bicuculline-insensitive responses. 4. In excised outside-out patches, GABA (20 microM) activated, in the presence of bicuculline (100 microM), single channel currents having conductances of 14, 22, and 31 pS. These values were similar to those obtained in the same preparation, in the absence of bicuculline. 5. These findings suggest that this new receptor type, which mediates bicuculline-insensitive responses with slow kinetics, may potentiate the depolarizing action of GABA during a critical period of postnatal development and therefore play a crucial role in synaptogenesis.

Negative shift of cardiac Na+ channel kinetics in cell-attached patch recordings

1990 ◽  
Vol 258 (1) ◽  
pp. H247-H254 ◽  
Author(s):  
T. Kimitsuki ◽  
T. Mitsuiye ◽  
A. Noma
Keyword(s):  
Patch Clamp ◽  
Single Channel ◽  
Resting Potential ◽  
Na Channel ◽  
Channel Kinetics ◽  
Patch Clamp Technique ◽  
Inactivation Curve ◽  
Pipette Solution ◽  
Single Channel Currents ◽  
Channel Currents

Na+ channel kinetics were studied by recording single-channel currents in the cell-attached patch configuration of the patch-clamp technique in single ventricular cells isolated from guinea pig hearts. The inactivation time course of ensemble currents was accelerated, and the peak amplitude increased temporarily and then decreased within a few minutes after the gigaohm seal formation. After reaching a new steady state, the inactivation-voltage relation was found to have shifted to more negative potentials. The potential of half-maximal inactivation was more negative by 20–31 mV from the resting potential or between -96 and -112 mV. The voltage dependency of the channel activation also shifted. Although the cell membrane was depolarized using the whole cell patch-clamp electrode and single-channel currents were recorded with an independent cell-attached electrode, the shift of the inactivation curve was also evident. Complete removal of Ca2+ using 5 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid in the pipette solution failed to prevent the shift. Increasing Ca2+ to 10 mM, however, reduced magnitude of the shift significantly. Involvement of an increased membrane fluidity and surface potential of the glass pipette to the shift is discussed.

Ion channel block by acetylcholine, carbachol and suberyldicholine at the frog neuromuscular junction

1985 ◽  
Vol 225 (1240) ◽  
pp. 329-355 ◽  
Keyword(s):  
Open Channel ◽  
Single Channel ◽  
Equilibrium Constants ◽  
Channel Noise ◽  
Muscle Fibres ◽  
Frog Neuromuscular Junction ◽  
Open Time ◽  
The Mean ◽  
Single Channel Currents ◽  
Channel Currents

Three nicotinic agonists, suberyldicholine, acetylcholine and carbachol, have been investigated by single channel recording at the endplates of adult frog muscle fibres. All three agonists can block the channels that they open. Suberyldicholine is the most potent blocker; it has an equilibrium constant for binding to the open channel of about 6 μM and blockages last for about 5 ms on average, at —105 mV. A plot of the mean number of blockages per unit open time against concentration (‘blockage frequency plot’) suggests that suberyldicholine does not produce long-lived blocked states such as might occur, for example, if it could be trapped within a shut channel. The characteristics of the 'blockage frequency plot’ are analysed in Appendix 2. Block by acetylcholine and carbachol has much lower affinity (the equilibrium constants being a few millimolar for both), and blockages are much briefer, so that blockage appears to produce noisy single channel currents of reduced amplitude. A method based on the spectral density of the excess ‘open’ channel noise has been used to investigate the rate of blocking and unblocking. The basis of this method is discussed in Appendix 1. It is estimated that the mean duration of a blockage is about 18 μs for acetylcholine and 9 μs for carbachol.

scholarly journals Gating of Na channels in the rat cortical collecting tubule: effects of voltage and membrane stretch.

1996 ◽  
Vol 107 (1) ◽  
pp. 35-45 ◽  
Author(s):  
L G Palmer ◽  
G Frindt
Keyword(s):  
Voltage Dependence ◽  
Single Channel ◽  
Apical Membrane ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Time ◽  
Collecting Tubule ◽  
Excised Patches ◽  
The Mean ◽  
Cortical Collecting Tubule

The gating kinetics of apical membrane Na channels in the rat cortical collecting tubule were assessed in cell-attached and inside-out excised patches from split-open tubules using the patch-clamp technique. In patches containing a single channel the open probability (Po) was variable, ranging from 0.05 to 0.9. The average Po was 0.5. However, the individual values were not distributed normally, but were mainly < or = 0.25 or > or = 0.75. Mean open times and mean closed times were correlated directly and inversely, respectively, with Po. In patches where a sufficient number of events could be recorded, two time constants were required to describe the open-time and closed-time distributions. In most patches in which basal Po was < 0.3 the channels could be activated by hyperpolarization of the apical membrane. In five such patches containing a single channel hyperpolarization by 40 mV increased Po by 10-fold, from 0.055 +/- 0.023 to 0.58 +/- 0.07. This change reflected an increase in the mean open time of the channels from 52 +/- 17 to 494 +/- 175 ms and a decrease in the mean closed time from 1,940 +/- 350 to 336 +/- 100 ms. These responses, however, could not be described by a simple voltage dependence of the opening and closing rates. In many cases significant delays in both the activation by hyperpolarization and deactivation by depolarization were observed. These delays ranged from several seconds to several tens of seconds. Similar effects of voltage were seen in cell-attached and excised patches, arguing against a voltage-dependent chemical modification of the channel, such as a phosphorylation. Rather, the channels appeared to switch between gating modes. These switches could be spontaneous but were strongly influenced by changes in membrane voltage. Voltage dependence of channel gating was also observed under whole-cell clamp conditions. To see if mechanical perturbations could also influence channel kinetics or gating mode, negative pressures of 10-60 mm Hg were applied to the patch pipette. In most cases (15 out of 22), this maneuver had no significant effect on channel behavior. In 6 out of 22 patches, however, there was a rapid and reversible increase in Po when the pressure was applied. In one patch, there was a reversible decrease. While no consistent effects of pressure could be documented, membrane deformation could contribute to the variation in Po under some conditions.

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