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.

Zinc Potentiates Neuronal Nicotinic Receptors by Increasing Burst Duration

2008 ◽  
Vol 99 (2) ◽  
pp. 999-1007 ◽  
Author(s):  
Bernard Hsiao ◽  
Karla B. Mihalak ◽  
Karl L. Magleby ◽  
Charles W. Luetje
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Single Channel ◽  
Large Fraction ◽  
Burst Duration ◽  
Dependent Manner ◽  
Neuronal Nicotinic Acetylcholine Receptors ◽  
Open Probability ◽  
Neuronal Nicotinic Receptors ◽  
Channel Current

Micromolar zinc potentiates neuronal nicotinic acetylcholine receptors (nAChRs) in a subtype-dependent manner. Zinc potentiates receptor function even at saturating agonist concentrations, without altering the receptor desensitization rate. Potentiation could occur through an increase in the number of available receptors, an increase in single-channel current amplitude, or an increase in single-channel open probability. To distinguish among these possibilities, we examined rat neuronal nAChRs expressed in Xenopus oocytes. Blockade of a large fraction of ACh activated α4β4 or α4β2 receptors by the open channel blocker hexamethonium failed to change the extent of potentiation by zinc, suggesting that zinc does not change the number of available receptors. The single-channel amplitudes of ACh (1 μM) activated α4β4 receptors in outside-out patches were similar in the absence and the presence of 100 μM zinc (3.0 ± 0.1 and 2.9 ± 0.1 pA, respectively). To determine the effect of zinc on single-channel open probability, we examined α4β4 receptors in cell-attached patches. The open probability at 100 nM ACh (0.011 ± 0.002) was increased 4.5-fold by 100 μM zinc (0.050 ± 0.008), accounting for most of the potentiation observed at the whole cell level. The increase in open probability was due to an increase in burst duration, which increased from 207 ± 38 ms in the absence of zinc to 830 ± 189 ms in the presence of zinc. Our results suggest that potentiation of neuronal nAChRs by zinc is due to a stabilization of the bursting states of the receptor.

scholarly journals The Na+/Ca2+ Exchanger Inhibitor, KB-R7943, Blocks a Nonselective Cation Channel Implicated in Chemosensory Transduction

2009 ◽  
Vol 101 (3) ◽  
pp. 1151-1159 ◽  
Author(s):  
A. Pezier ◽  
Y. V. Bobkov ◽  
B. W. Ache
Keyword(s):  
Transient Receptor Potential ◽  
Single Channel ◽  
Receptor Potential ◽  
Trpc Channels ◽  
Dependent Manner ◽  
Open Probability ◽  
Olfactory Transduction ◽  
Voltage Dependent ◽  
Chemosensory Transduction ◽  
Transient Receptor

The mechanism(s) of olfactory transduction in invertebrates remains to be fully understood. In lobster olfactory receptor neurons (ORNs), a nonselective sodium-gated cation (SGC) channel, a presumptive transient receptor potential (TRP)C channel homolog, plays a crucial role in olfactory transduction, at least in part by amplifying the primary transduction current. To better determine the functional role of the channel, it is important to selectively block the channel independently of other elements of the transduction cascade, causing us to search for specific pharmacological blockers of the SGC channel. Given evidence that the Na+/Ca2+ exchange inhibitor, KB-R7943, blocks mammalian TRPC channels, we studied this probe as a potential blocker of the lobster SGC channel. KB-R7943 reversibly blocked the SGC current in both inside- and outside-out patch recordings in a dose- and voltage-dependent manner. KB-R7943 decreased the channel open probability without changing single channel amplitude. KB-R7943 also reversibly and in a dose-dependent manner inhibited both the odorant-evoked discharge of lobster ORNs and the odorant-evoked whole cell current. Our findings strongly imply that KB-R7943 potently blocks the lobster SGC channel and likely does so directly and not through its ability to block the Na+/Ca2+ exchanger.

scholarly journals Intracellular calcium strongly potentiates agonist-activated TRPC5 channels

2009 ◽  
Vol 133 (5) ◽  
pp. 525-546 ◽  
Author(s):  
Nathaniel T. Blair ◽  
J. Stefan Kaczmarek ◽  
David E. Clapham
Keyword(s):  
Single Channel ◽  
Reversal Potential ◽  
Fold Increase ◽  
Receptor Activation ◽  
Brain Regions ◽  
Repetitive Firing ◽  
Dependent Manner ◽  
Open Probability ◽  
Current Voltage ◽  
Voltage Dependent

TRPC5 is a calcium (Ca2+)-permeable nonselective cation channel expressed in several brain regions, including the hippocampus, cerebellum, and amygdala. Although TRPC5 is activated by receptors coupled to phospholipase C, the precise signaling pathway and modulatory signals remain poorly defined. We find that during continuous agonist activation, heterologously expressed TRPC5 currents are potentiated in a voltage-dependent manner (∼5-fold at positive potentials and ∼25-fold at negative potentials). The reversal potential, doubly rectifying current–voltage relation, and permeability to large cations such as N-methyl-d-glucamine remain unchanged during this potentiation. The TRPC5 current potentiation depends on extracellular Ca2+: replacement by Ba2+ or Mg2+ abolishes it, whereas the addition of 10 mM Ca2+ accelerates it. The site of action for Ca2+ is intracellular, as simultaneous fura-2 imaging and patch clamp recordings indicate that potentiation is triggered at ∼1 µM [Ca2+]. This potentiation is prevented when intracellular Ca2+ is tightly buffered, but it is promoted when recording with internal solutions containing elevated [Ca2+]. In cell-attached and excised inside-out single-channel recordings, increases in internal [Ca2+] led to an ∼10–20-fold increase in channel open probability, whereas single-channel conductance was unchanged. Ca2+-dependent potentiation should result in TRPC5 channel activation preferentially during periods of repetitive firing or coincident neurotransmitter receptor activation.

scholarly journals Charybdotoxin selectively blocks small Ca-activated K channels in Aplysia neurons.

1987 ◽  
Vol 90 (1) ◽  
pp. 27-47 ◽  
Author(s):  
A Hermann ◽  
C Erxleben
Keyword(s):  
Single Channel ◽  
Delayed Rectifier ◽  
Pacemaker Activity ◽  
Dependent Manner ◽  
Open Probability ◽  
Apparent Dissociation Constant ◽  
External Application ◽  
K Channels ◽  
Voltage Dependent ◽  
K Current

The action of charybdotoxin (ChTX), a peptide component isolated from the venom of the scorpion Leiurus quinquestriatus, was investigated on membrane currents of identified neurons from the marine mollusk, Aplysia californica. Macroscopic current recordings showed that the external application of ChTX blocks the Ca-activated K current in a dose- and voltage-dependent manner. The apparent dissociation constant is 30 nM at V = -30 mV and increases e-fold for a +50- to +70-mV change in membrane potential, which indicates that the toxin molecule is sensitive to approximately 35% of the transmembrane electric field. The toxin is bound to the receptor with a 1:1 stoichiometry and its effect is reversible after washout. The toxin also suppresses the membrane leakage conductance and a resting K conductance activated by internal Ca ions. The toxin has no significant effect on the inward Na or Ca currents, the transient K current, or the delayed rectifier K current. Records from Ca-activated K channels revealed a single channel conductance of 35 +/- 5 pS at V = 0 mV in asymmetrical K solution. The channel open probability increased with the internal Ca concentration and with membrane voltage. The K channels were blocked by submillimolar concentrations of tetraethylammonium ions and by nanomolar concentrations of ChTX, but were not blocked by 4-aminopyridine if applied externally on outside-out patches. From the effects of ChTX on K current and on bursting pacemaker activity, it is concluded that the termination of bursts is in part controlled by a Ca-activated K conductance.

Quinidine blocks adenosine 5'-triphosphate-sensitive potassium channels in heart

1990 ◽  
Vol 259 (5) ◽  
pp. H1609-H1612 ◽  
Author(s):  
A. I. Undrovinas ◽  
N. Burnashev ◽  
D. Eroshenko ◽  
I. Fleidervish ◽  
C. F. Starmer ◽  
...  
Keyword(s):  
Single Channel ◽  
Dependent Manner ◽  
Open Probability ◽  
Pipette Solution ◽  
Channel Current ◽  
Closed Intervals ◽  
Ventricular Cells ◽  
Voltage Dependent ◽  
Inside Out ◽  
Ischemic Arrhythmias

The ATP-sensitive potassium channel current (IK-ATP) was studied in excised inside-out patches from rat ventricular cells at 20-23 degrees C. The bath solution contained 140 mM KF, and the pipette solution contained 140 mM KCl and 1.2 mM MgCl2. ATP (0.5 mM) in the bath inhibited IK-ATP. In the absence of ATP, 10 microM quinidine decreased open probability 67 +/- 1% (n = 6) at -50 mV and 28 +/- 12% at -130 mV (n = 5) without affecting single channel conductance (48-52 pS). The block increased with 25 and 50 microM quinidine and could be reversed on washing quinidine for several minutes. Interburst (closed) intervals were increased by quinidine, whereas open and closed time distributions within bursts were not changed. We conclude that quinidine blocks IK-ATP in a "slow" and voltage-dependent manner in clinically relevant concentrations. Because of the postulated role for IK-ATP in cardiac ischemia, quinidine block of this channel may play a role in ischemic arrhythmias.

scholarly journals Mechanism of activation of the prokaryotic channel ELIC by propylamine: A single-channel study

2014 ◽  
Vol 145 (1) ◽  
pp. 23-45 ◽  
Author(s):  
Alessandro Marabelli ◽  
Remigijus Lape ◽  
Lucia Sivilotti
Keyword(s):  
Ion Channel ◽  
Nicotinic Acetylcholine Receptors ◽  
Intermediate State ◽  
Acetylcholine Receptors ◽  
Time Course ◽  
Single Channel ◽  
Structural Information ◽  
Kinetic Properties ◽  
Open Probability ◽  
Single Channel Currents

Prokaryotic channels, such as Erwinia chrysanthemi ligand-gated ion channel (ELIC) and Gloeobacter violaceus ligand-gated ion channel, give key structural information for the pentameric ligand-gated ion channel family, which includes nicotinic acetylcholine receptors. ELIC, a cationic channel from E. chrysanthemi, is particularly suitable for single-channel recording because of its high conductance. Here, we report on the kinetic properties of ELIC channels expressed in human embryonic kidney 293 cells. Single-channel currents elicited by the full agonist propylamine (0.5–50 mM) in outside-out patches at −60 mV were analyzed by direct maximum likelihood fitting of kinetic schemes to the idealized data. Several mechanisms were tested, and their adequacy was judged by comparing the predictions of the best fit obtained with the observable features of the experimental data. These included open-/shut-time distributions and the time course of macroscopic propylamine-activated currents elicited by fast theta-tube applications (50–600 ms, 1–50 mM, −100 mV). Related eukaryotic channels, such as glycine and nicotinic receptors, when fully liganded open with high efficacy to a single open state, reached via a preopening intermediate. The simplest adequate description of their activation, the “Flip” model, assumes a concerted transition to a single intermediate state at high agonist concentration. In contrast, ELIC open-time distributions at saturating propylamine showed multiple components. Thus, more than one open state must be accessible to the fully liganded channel. The “Primed” model allows opening from multiple fully liganded intermediates. The best fits of this type of model showed that ELIC maximum open probability (99%) is reached when at least two and probably three molecules of agonist have bound to the channel. The overall efficacy with which the fully liganded channel opens was ∼102 (∼20 for α1β glycine channels). The microscopic affinity for the agonist increased as the channel activated, from 7 mM for the resting state to 0.15 mM for the partially activated intermediate state.

scholarly journals Intracellular Mg2+ Enhances the Function of Bk-Type Ca2+-Activated K+ Channels

2001 ◽  
Vol 118 (5) ◽  
pp. 589-606 ◽  
Author(s):  
Jingyi Shi ◽  
Jianmin Cui
Keyword(s):  
Binding Sites ◽  
Neurotransmitter Release ◽  
Single Channel ◽  
Bk Channels ◽  
Bk Channel ◽  
Dependent Manner ◽  
Physiological Conditions ◽  
Channel Function ◽  
Open Conformation ◽  
Voltage Dependent

BK channels modulate neurotransmitter release due to their activation by voltage and Ca2+. Intracellular Mg2+ also modulates BK channels in multiple ways with opposite effects on channel function. Previous single-channel studies have shown that Mg2+ blocks the pore of BK channels in a voltage-dependent manner. We have confirmed this result by studying macroscopic currents of the mslo1 channel. We find that Mg2+ activates mslo1 BK channels independently of Ca2+ and voltage by preferentially binding to their open conformation. The mslo3 channel, which lacks Ca2+ binding sites in the tail, is not activated by Mg2+. However, coexpression of the mslo1 core and mslo3 tail produces channels with Mg2+ sensitivity similar to mslo1 channels, indicating that Mg2+ sites differ from Ca2+ sites. We discovered that Mg2+ also binds to Ca2+ sites and competitively inhibits Ca2+-dependent activation. Quantitative computation of these effects reveals that the overall effect of Mg2+ under physiological conditions is to enhance BK channel function.

Ammonium ion enhances the calcium-dependent gating of a mammalian large conductance, calcium-sensitive K+ channel

2001 ◽  
Vol 79 (11) ◽  
pp. 919-923 ◽  
Author(s):  
Andrew P Braun
Keyword(s):  
Single Channel ◽  
Ammonium Ion ◽  
K Channel ◽  
Free Calcium ◽  
Dependent Manner ◽  
Open Probability ◽  
Concentration Dependent Manner ◽  
Hek 293 ◽  
Calcium Dependent ◽  
Single Channel Currents

We observed that the current amplitude and activation of expressed, mouse brain large conductance, calcium-sensitive K+ channels (BKCa channels) may be reversibly enhanced following addition of low concentrations of the weakly permeant cation NH4+ to the cytoplasmic face of the channel in excised, inside-out membrane patches from HEK 293 cells. Conductance-voltage relations were left-shifted along the voltage axis by addition of NH4Cl in a concentration-dependent manner, with an EC50 of 18.5 mM. Furthermore, this effect was observed in the presence of cytosolic free calcium (~1 µM), but was absent in a cytosolic bath solution containing nominally zero free calcium (e.g., 5 mM EGTA only), a condition under which these channels undergo largely voltage-dependent gating. Recordings of single BKCa channel events indicated that NH4+ increased the channel open probability of single channel activity ~3-fold, but did not alter the amplitude of single channel currents. These findings suggest that the calcium-sensitive gating of mammalian BKCa channels may be modified by other ions present in cytosolic solution.Key words: potassium channel, calcium, modulation, electrophysiology.

Isoflurane Modulation of Neuronal Nicotinic Acetylcholine Receptors Expressed in Human Embryonic Kidney Cells

2005 ◽  
Vol 102 (1) ◽  
pp. 76-84 ◽  
Author(s):  
Megumi Yamashita ◽  
Takashi Mori ◽  
Keiichi Nagata ◽  
Jay Z. Yeh ◽  
Toshio Narahashi
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Single Channel ◽  
Single Channels ◽  
Kidney Cells ◽  
Neuronal Nicotinic Acetylcholine Receptors ◽  
Open Probability ◽  
Human Embryonic Kidney Cells ◽  
Open Time ◽  
The Mean

Background It is well established that neuronal nicotinic acetylcholine receptors (nAChRs) are sensitive to inhalational anesthetics. The authors previously reported that halothane potently blocked alpha4beta2-type nAChRs of rat cortical neurons. However, the effect of isoflurane, which is widely used clinically, on nAChRs largely remains to be seen. The authors studied the effects of isoflurane as compared with sevoflurane and halothane on the human alpha4beta2 nAChRs expressed in human embryonic kidney cells. Methods The whole-cell and single-channel patch clamp techniques were used to record currents induced by acetylcholine. Results Isoflurane, sevoflurane, and halothane suppressed the acetylcholine-induced currents in a concentration-dependent manner with 50% inhibitory concentrations of 67.1, 183.3, and 39.8 microM, respectively, which correspond to 0.5 minimum alveolar concentration or less. When anesthetics were coapplied with acetylcholine, isoflurane and sevoflurane decreased the apparent affinity of receptor for acetylcholine, but halothane, in addition, decreased the maximum acetylcholine current. When isoflurane was preapplied and coapplied, its inhibitory action was independent of acetylcholine concentration. Isoflurane blocked the nAChR in both resting and activated states. Single-channel analyses revealed that isoflurane at 84 microM decreased the mean open time and burst duration without inducing "flickering" during channel openings. Isoflurane increased the mean closed time. As a result, the open probability of single channels was greatly reduced by isoflurane. Conclusions Isoflurane, sevoflurane, and halothane potently blocked the alpha4beta2 nAChR. Isoflurane suppression of whole-cell acetylcholine currents was a result of decreases in the open time, burst duration, and open probability and an increase in the closed time of single channels. The high sensitivity of neuronal nAChRs to inhalational anesthetics is expected to play an important role in several stages of anesthesia.

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